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https://f1000research.com/articles/10-921/v1	15 Sep 21	{ "type": "Research Article", "title": "Lightning safety awareness level in Malaysia", "authors": [ "Khairul Nazri", "Siow Chun Lim", "Chandima Gomes", "Khairul Nazri", "Chandima Gomes" ], "abstract": "Introduction: Malaysia is one of the countries with the highest lightning flash density globally. While sufficiency of lightning protection system is crucial to ensure human safety against lightning strikes, the public awareness towards lightning safety is also equally important in Malaysia. Hence, this study was conducted to understand the current lightning safety awareness level of the Malaysian population. Methods: An online questionnaire survey which consists of 22 scientific statements of lightning was first developed in Malay and English. The questionnaire allows the respondent to also check their own score upon completion of the questionnaire. It was then distributed to the public for data collection. The sample size comprised of both genders, all layers of society from various educational level and social background. Results: Overall, the awareness on lightning safety amongst Malaysian is at moderate level with an average score of slightly above 50%. Urbanites scored marginally better than their rural counterparts. One’s education level does not dictate their awareness level of lightning safety. Discussion: In conclusion, the public in Malaysia needs to be better educated on lightning safety. Similar studies should be replicated in other countries experiencing similar levels of lightning activity to better understand the public’s perception on lightning.", "keywords": [ "Lightning", "lightning safety", "public belief", "Malaysia", "lightning myth" ], "content": "Introduction\n\nMalaysia is in the top three in the world with high lightning density experiencing an annual mean lightning ground flash density of 13.9 flashes per square kilometre yearly.1,2 A recent study stated that a factor that probably contributes to the high numbers of thunderstorm and lightning events in Peninsular Malaysia is due to its geographical position being encircled by the Andaman Sea, Sulu Sea, Straits of Malacca and South China Sea.3 Undeniably, the other substantial factors are the massive increment of factories, deforestation and other development progress. All these activities and factors are contributing towards heating of the Earth thus increasing the severity and number of thunderstorms.\n\nAs many as 131 deaths and injuries have been reported due to lightning strikes, with 92 death injury rates per million per year. There were 22 fatalities per year from 2008–2011 reported in.4,5 A study recently stated that lightning had killed an average of one in 10 victims in Malaysia and 235 were either killed or injured from 2008 to 2015.2 These unfortunate statistics could be attributed to the weak public awareness of lightning among Malaysians. Thus, understanding lightning safety is necessary to keep them safe during the phenomena.\n\nTwo recent research were conducted to understand the public awareness level of lightning safety.1,6 These studies have considered numerous sociological characteristics. However, the sample size of the previous study in1 is not representative of the Malaysian population. Furthermore, it would be advantageous for the participants in the survey to also know their misconception towards lightning safety upon completion of the survey. Thus, this research was conducted on a larger scale to not only understand the Malaysian public’s conception of lightning safety but also attempt to educate the respondents on their misconceptions towards lightning.\n\n\nMethods\n\nFirstly, the questionnaire was designed online in Google Form and was made bilingual, i.e. in Malay and English, to provide optimum understanding to respondents from different backgrounds. The questionnaire was adapted from recent surveys and interview questions in.1,6 However, they have been further enhanced to consist of 22 questions which are grouped into two general knowledge questions, eight scientifically unaccepted statements and 12 scientifically accepted statements about lightning awareness. Respondents had to select one answer from three choices of answers namely disagree, undecided and agree. Unlike the previous studies in,1 respondents would now be able to view their scores and correct their misconceptions upon completion of the survey.\n\nNext, the survey was distributed to the Malaysian public without bias using a probability sampling approach so that everyone has an equal possibility to be selected. This approach is critical to prevent population sample size bias. A minimum of 1000 respondents is targeted as sample size based on the methodology in https://news.gallup.com/poll/101872/how-does-gallup-polling-work.aspx.7,8 The questionnaire was distributed randomly and was kept active until the minimum respondents is received. Each respondent was only allowed to attempt the survey once. A total of 1062 responses were received from 9th December 2020 until 6th January 2021. The survey was distributed to citizens aged above 18 from various social and educational backgrounds with their anonymity preserved. Their responses were analysed by organising the data into three parts namely age, level of education, and residency. There are three levels of age, seven levels of education, and four types of residency.\n\n\nResults\n\nThe questionnaire started with three questions to understand the level of exposure of the respondents to lightning effects. From Table 1, only 3.3% responded that they have been injured by lightning before and 9.3% have met person injured by lightning. However, 38% of the respondents reported that their home has been affected by lightning. This number seems to complement the findings in1 in that the damage due to lightning is significant in Malaysia. Note that only 31.5% of the respondents consistently follow weather forecasting on television and radio; 55.5% only occasionally, and 14.9% do not follow the weather forecast at all.\n\nThe rest of the questionnaire is divided into sections A, B and C. Section A which consists of two general knowledge statements with the aim to gauge the basic understanding of lightning among the respondents. The remaining Sections B and C aim to gauge the respondents’ awareness on the nature and safety aspects of lightning. There are eight scientifically unaccepted statements in Section B and 12 scientifically accepted statements in Section C as shown in Table 2. Scientifically accepted statements means scientifically acceptable facts based on present day knowledge and understanding of lightning. In the questionnaire, the sequence of these 18 statements are randomised to ensure that the respondents could not “guess” the grouping of the statements. The participants have to select either disagree, undecided or agree for each statement.\n\nIn section B, the first three statements were adopted from1 Over 50% of respondents believed a supernatural power is behind a lightning strike.1 However, in the present study with a much larger sample size, only 27% has similar suspicion. The responses were evenly distributed for statements 4 and 6. Majority of the respondents is aware that they should immediately cease their outdoor activities when there is thunderstorm as reflected in statement 7. In section C, statements 9–15 were adopted from.1 About 28% of the respondents are confused about the lightning’s electrical nature and this seems to concur with.1 Statement 10 came from a famous slogan from the United States and statement 14 is based on the 30–30 rule.9\n\nOverall, the majority of the respondents agreed with the scientifically accepted statements except for statement 11, 17, 18, and 19. The fact that the majority did not believe CPR can help lightning victims is worrying because it seems to suggest that the public is not prepared for any emergency arising from lightning struck victims. Statements 18 and 19’s results show that respondents are not aware of lightning issues in Malaysia.\n\n\nDiscussion\n\nIn this section, the respondents’ awareness level will be analysed according to their age group, education level and residency. This awareness level is quantified by the marks that they scored. Note that the respondent will be given 1 mark for every correct response to the statements in Table 2. Hence, the maximum mark that they can score is 22.\n\nTable 3 shows the responses which are categorized according to the respondents’ age. There is only slight difference in their understanding level when observed across the three age groups.\n\nTable 4 shows the responses which are categorized according to the respondents’ education level. The findings suggest that a higher education level does not necessarily means a higher level of awareness and lightning safety knowledge.\n\nTable 5 illustrates the responses grouped according to the residencies of the respondents. As observed here, respondents living in metropolitan areas have the highest awareness of lightning safety. However, the difference is only marginal.\n\nAll in all, on the average, the respondents could only get half of the maximum score which clearly indicates the lack of awareness. Finally, Table 6 summarises the common misconceptions on lightning safety among the respondents. This could perhaps serve as a guide for relevant parties promoting lightning safety awareness in Malaysia.\n\n\nConclusions\n\nTo summarize, the public awareness of lightning safety in Malaysia is moderate, proven by the number of misconceptions that existed through their responses. In the same context, their knowledge of dealing with the lightning situation is worrying. Many did not believe in the capability of CPR to save a lightning victim. From here, note that the majority will be confused about what to do if a lightning incidence happens. Furthermore, one’s level of education has little impact on their awareness of lightning safety. Moreover, urbanites in particular metropolis citizens have a better awareness of lightning safety than others.\n\nOn the average, 53% agreed with the scientifically accepted statements, and 54% disagreed with the scientifically unaccepted. The fact that the average mark of all respondents is barely half of the maximum mark means that the awareness level is still unsatisfactory. Relevant parties such as the Energy Commission and perhaps the Ministry of Education can collaborate to enhance national lightning safety education and promotion by utilising the findings in this paper. Lightning safety education campaign in Malaysia should ideally be as progressive as those in Sri Lanka, Colombia and the United States. It would also be interesting for similar studies to be replicated in other countries as well to gain a better understanding at the global level.\n\n\nData availability\n\nSiow, Dr S.C. LIM (Multimedia University) (2021): Lightning Safety Awareness Level in Malaysia. DANS. https://doi.org/10.17026/dans-zut-4u2s.\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\nEthics and consent\n\nThis survey had obtained approval number of EA2152021 from Research Ethics Committee of Multimedia University.", "appendix": "Acknowledgements\n\nThe authors would like to thank the Faculty of Engineering, Multimedia University (MMU) for supporting this study.\n\n\nReferences\n\nA S, C G, E T, et al.: Public Beliefs about Lightning in Malaysia. 2019 Int Sympo Lightning Protection (XV SIPDA). Sao Paulo, Brazil; 2019. Publisher Full Text\n\nAb-Kadir MZA: Lightning Severity in Malaysia and Some Parameters. Thermal Sci . 2016; 20: S437–S450. Publisher Full Text\n\nRufus S, Ahmad N, Abdul Malek Z, et al.: Characteristics of Lightning Trends in Peninsular.2019. Publisher Full Text\n\nM H, M Z, A A-K, et al.: A Comparison of Lightning Human Fatalities. 33rd Int Conf Lightning Protection. Estoril, Portugal; 2016. Publisher Full Text\n\nSierra DEV: Characterization of The Lightning Safety Education Programs in the World as a First Step for The Creation of A Lightning Safety Policy in Colombia.Bogota D.C., Colombia: Universidad Distrital Francisco Jose De Caldas; 2017.\n\nIslam MS: Lightning hazard safety measures and awareness in Bangladesh. Natural Hazards; 2020.\n\nThe Secretariat: Citizens’ Perception Survey Component of The State Peer Review Mechanism, Practical Considerations in Implementing the Survey.June 2014. Reference Source\n\nGALLUP: How does Gallup polling work? GALLUP. Accessed 25 January 2021.Reference Source\n\nM A, C M, R LH: Lightning Safety Campaign – USA Experience. 2012 Int Conf Lightning Protection (ICLP). Vienna, Austria; 2012." }	[ { "id": "94361", "date": "04 Oct 2021", "name": "Helio Eiji Sueta", "expertise": [ "Reviewer Expertise I am currently the deputy head of the Scientific Division of Energy Planning", "Analysis and Development at the Institute of Energy and Environment at the University of São Paulo. My main research area is the protection of structures and people against lightning strikes. I am currently the secretary of the Brazilian Committee that reviews the lightning protection standard." ], "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 principle, it is not a “purely “scientific article with dozens of formulas and/or complex mathematical simulations, but nevertheless it is a very important article. I comment on this because the vast majority of technical papers published in scientific journals bring theoretical or experimental developments with great mathematical formulations and models. In this paper we find a very well done analysis of the responses to a very well-designed survey for a given population. An advantage of not being “purely” scientific is that it can be read by any type of person, regardless of their area of expertise, as in addition to having a very accessible language, the subject is of general interest to all areas. I don't see any disadvantages in this fact.\nI fully agree with the author that in many countries, especially developing ones, the general knowledge of the population about the dangers of lightning is very limited and, many times, surrounded by myths. The work presents the results of a survey to the population about general aspects of lightning, mixing some scientifically accepted statements with others not accepted. The results were quite interesting, showing that in Malaysia, where the research was applied, the general population still has many failures in awareness of the dangers of lightning. It also showed that this awareness is a little better in urban centers than in rural ones, but that better school education is not significant for a better awareness of this issue.\nThe work fulfills what was proposed and can be replicated in other countries. As a suggestion, it would be interesting to compare the results of the same survey in several countries, including well-developed countries, in order to better understand the general awareness of the world population about the dangers involved in lightning.\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": "7291", "date": "04 Nov 2021", "name": "Chun Lim Siow", "role": "Author Response", "response": "Thank you for the comments. Indeed, we need more similar kind of work to be done especially in developing or less-developed countries to gain a holistic understanding of how the public perceives lightning. With these findings, relevant stakeholders (policymakers, academia etc) can then strategise a more targeted lightning awareness promotion approach to minimise unacceptable loss of human lives due to a natural phenomenon which we have already heavily researched on for more than a century." } ] }, { "id": "94364", "date": "04 Oct 2021", "name": "Norhidayu Binti Rameli", "expertise": [ "Reviewer Expertise Lightning and High Voltage Power Cable." ], "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 is about understanding the current lightning safety awareness level of the Malaysian population. The methodology surveyed 1062 respondents and included 22 questions about their level of lightning safety awareness. Additionally, the conclusion drawn from Malaysia's population is that it is of a moderate awareness level. The author and team have made an admirable effort. Nonetheless, the following points may be considered for improvement;\n1. The work's methodology should be more detailed and justified.\nIt is necessary to justify the minimum number of respondents. Is this enough to represent the Malaysian population? Kindly show the evidence to support the number of respondents.\n\nIs there any other evidence to support the choice of the 22 questions?\n\nPlease demonstrate how the probability sampling method is implemented.\n\nAny software tools that authors use to analyse the result?\n2. To provide a more meaningful view, the result should be presented in an infographic such as a pie chart or histogram.\n3. The interpretation of the results discussed in this section should be based on statistical inference to draw conclusions about a population (Please take note that the Malaysian population is about 32 million). As a result, the conclusion should be revised appropriately.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7292", "date": "04 Nov 2021", "name": "Chun Lim Siow", "role": "Author Response", "response": "Comment: This paper is about understanding the current lightning safety awareness level of the Malaysian population. The methodology surveyed 1062 respondents and included 22 questions about their level of lightning safety awareness. Additionally, the conclusion drawn from Malaysia's population is that it is of a moderate awareness level. The author and team have made an admirable effort. Nonetheless, the following points may be considered for improvement; 1. The work's methodology should be more detailed and justified. It is necessary to justify the minimum number of respondents. Is this enough to represent the Malaysian population? Kindly show the evidence to support the number of respondents. Response: Thank you for the comment. We followed the approach in the Gallup poll where the typical sample size is 1000 national adults to represent the opinion of the population of a given country with a margin of error of ±4%. We have also verified the sample size using the methodology proposed by Krejcie and Morgan (1970) to determine sample size based on a confidence level of 95% and a variability of 50%. Comment: Is there any other evidence to support the choice of the 22 questions? Response: Thank you for the comment. Based on available literature, the most comprehensive questionnaire is those available in Syakura et al. (2019), which we have adapted from. However, we have slightly enhanced the questionnaire by adding 5 more questions. In addition, the respondents are also able to know their score upon completion of the questionnaire. This is a side objective of this work to also educate the respondents on lightning safety. Comment: Please demonstrate how the probability sampling method is implemented. Response: Thank you for the comment. The questionnaire was randomly distributed to citizens aged above 18 years old. Respondents are only allowed to respond once. Comment: Any software tools that authors use to analyse the result? Response: Thank you for the comment. Microsoft Excel is used to analyse the result. Comment: 2. To provide a more meaningful view, the result should be presented in an infographic such as a pie chart or histogram. Response: Thank you for the comment. We totally agree with your suggestion that a pie chart or histogram enhances the clarity. However, presenting the data in a table also has its own merit as it allows readers to conveniently extract the raw data. To provide a meaningful view and further enhance the visibility of the findings, we have included 2 infographics (Figure 1 and Figure 2) which are also accessible here. Comment: 3. The interpretation of the results discussed in this section should be based on statistical inference to draw conclusions about a population (Please take note that the Malaysian population is about 32 million). As a result, the conclusion should be revised appropriately. Response: Thank you for the comment. We have explained how we arrived at the sample size of 1000 in our earlier response to your earlier comment above. While we did not claim that the findings reflect the opinion of the Malaysian population, the sample size used in this study is actually statistically valid assuming 95% confidence interval for a 32 million population." } ] } ]	1	https://f1000research.com/articles/10-921
https://f1000research.com/articles/10-1113/v1	03 Nov 21	{ "type": "Genome Note", "title": "Isolation and sequencing of three RB49-like bacteriophages infecting O antigen-producing E. coli strains", "authors": [ "Alexander Efimov", "Eugene Kulikov", "Alla Golomidova", "Ilya Belalov", "Vladislav Babenko", "Andrey Letarov", "Alla Golomidova", "Ilya Belalov", "Vladislav Babenko" ], "abstract": "E. coli strains 4s, F5 and F17, whose O antigens are structurally characterized and shown to effectively shield the cell surface from bacteriophage attack, were used as the hosts to isolate novel RB49-like bacteriophages. Three novel phage isolates were obtained, and their genomes were sequenced and annotated. Despite high overall identity levels of these genomic sequences, the variants of large distal tail fiber subunit, orthologous to the bacteriophage T2 long tail receptor recognition protein gp38, were unique for each phage, suggesting their role in host range determination. The annotated genomes are available via NCBI Genbank, acc. numbers MZ504876-MZ504878.", "keywords": [ "Bacteriophage genomes", "RB49-like bacteriophages", "coliphages", "O antigens", "receptor recognition proteins." ], "content": "Introduction\n\nThe threat of rapidly spreading microbial resistance to antibacterial drugs has revitalized the interest to the usage of bacteriophages as biocontrol agents for agriculture (Allen et al. 2013), aquaculture (D'Accolti et al. 2021), phage therapy in humans and animals (Abedon 2018, Clokie and Mary Ann Liebert Inc. 2020) and other applications.\n\nOne of the main limitations of phage-based biocontrol technologies is narrow host range of most of bacteriophages. T-even-related bacteriophages, including RB49-like viruses, are widely spread in nature and feature significant sequence flexibility in the receptor recognition proteins (RBPs), coupled with an high overall homology of the other regions in their genomes (Comeau et al. 2007). This makes them attractive platforms for developing systems of artificial host range management.\n\nThe paradigm of the host cell recognition by T-even-like phages was studied in great detail on the bacteriophage T4 model system. The infection of the host cell by T4 is initiated by binding of the tips of the phage long tail fibers (LTFs) to their cognate receptor molecules (OmpC) (Hu et al. 2015). This initial binding triggers the baseplate rearrangement and deployment of the short tail fibers (Taylor et al. 2016) that bind to different receptors tightly fixing the virion on the cell surface to prevent it from been pushed out by the contraction of the phage tail.\n\nThe bioinformatic and experimental evidence suggest that in other T-even-like phages the outline of the early stages of infection is essentially the same as in T4, however in most of these viruses (including RB49 and related phages) the recognition of LTF receptors is performed not by the C-terminal domain of the trimeric gp37 protein forming the distal moiety of the LTF, but by a tiny monomeric protein gp38 attached to the end of the gp37 trimer (Trojet et al. 2011, Hyman and van Raaij 2018).\n\nInterestingly, the above-described host recognition scenario was mainly evaluated by studying the phage T4 infection of the laboratory E. coli strains that are depleted of the O antigen biosynthesis, such are the derivatives of K-12 or B strains (Tetart et al. 2001). At the same time evidence accumulates that many types of E. coli O antigen serve as an effective shields restricting the access of the phage receptor-recognition structures to the receptors, located at the outer membrane surface (Peng et al. 2007, Golomidova et al. 2021) (Broeker and Barbirz 2017). The penetration through this shield requires specific recognition and, in some cases, enzymatic degradation of the O polysaccharide, by the phage RBPs (Letarov and Kulikov 2017, Kulikov et al. 2019) . In order to evaluate the mechanisms employed by T-even-like bacteriophages to attack the O antigen-producing E. coli strains it is necessary to obtain such phages infective against the host strains in which the protective features of the O antigens were studied in some detail. We previously investigated the structure and protectivity of the O antigens of several wild type isolates of E. coli, including the strains 4s (Knirel et al. 2015, Kulikov et al. 2017), F5 (Golomidova et al. 2019), and F17 (Knirel et al. 2019). Here we report the isolation and sequencing of three RB49-like bacteriophages able to infect these strains. Novel viruses with high degree of overall similarity but featuring different RBP variants serve as useful model systems for in-depth analysis of T-even phage strategies used to circumvent O antigen protection of the host cell, efficiently shielded otherwise.\n\n\nMethods\n\nThree previously characterized E. coli strains featuring different O antigen types were used for bacteriophage isolation. These were: O28 strain F5 (Golomidova et al. 2019), E. coli F17, producing a novel O antigen type (Knirel et al. 2019) and O22 strain 4s (Knirel et al. 2015). To prepare concentrated phage stocks the the relevant host culture was propagated at 37°C with agitation up to OD600 = 0.2, and inoculated with phage at multiplicity of infection (moi) = 0.1. The culture was incubated under the same conditions until the lysis was observed, then 0.05 volume of chlorophorm was added and the lysates were cleared by centrifugation at 10 000 g for 10 min.\n\nThe bacteriophages were isolated by direct plating of the commercial therapeutical bacteriophage preparation “Intesti-bakteriofag” (Mikrogen, Russia) that is marketed as a broad range therapeutic phage cocktail (active against Shigella flexneri, S. sonnei, S. paratyphi A, S. paratyphi B, Salmonella typhimurium, S. infantis, S. choleraesuis, S. oranienburg, S. enteritidis, E. coli, P. vulgaris, P. mirabilis, Enterococcus sp., Staphylococcus, P. aeruginosa) (https://www.microgen.ru/products/bakteriofagi/intesti-bakteriofag/). This preparation was titrated to single plaques by the conventional double-layer plating technique using three above-mentiones E. coli strains as the hosts. All the methods for phage isolation were as described in Kulikova et al. (2007). PCR assay with RB49 g38 specific primers cttgctggatgagccaattcgccggacgctctaaagagattcattatagca-gp38C(RB49)-F and ccttttaagggtatttattcacattacacgcgagcaccgtaga-gp38C(RB49)-R was used to pick RB49-like phage candidates for further study.\n\nTransmission electron microscopy study was performed using uranyl acetate negative contrast as described in (Kulikov et al. 2014).\n\nTo extract the DNA the lysates were treated with DNAse (0,01 mg mL−1) for 30 min at room temperature and the phages were collected by ultracentrifugation (1 h, 75000 g). Viral genomic DNA was extracted from the pellets with CTAB (cetyltrimethylammonium bromide) as described in (Kulikov et al. 2020) using. DNA quality and quantity was assessed by agarose gel analysis and Qubit dsDNA HS fluorometer assay (Qubit, USA). The phage genomic DNA libraries were prepared and sequenced using an Ion Torrent sequencer system (Applied Biosystems, USA) with 400-fold coverage and a median read length of 185 bp. The raw reads from the run were then combined and filtered using the spectral alignment error correction tool SAET 3 (Pevzner et al. 2001). This yielded 157,407-163,568 reads per library with average 190-fold coverage. Primary assembly was conducted with Newbler version 3.0 (Roche Diagnostics, USA), resulting in a single contigs.\n\nAnnotation was performed using Prokka (Seemann 2014) with further manual curation. Potential open reading frames (ORFs) were detected by use of SnapGene (GSL Biotech, USA) and subsequently analyzed with HMMER, HHPRED (Soding et al. 2005) (MPI Bioinformatics Toolkit), BLAST (Altschul et al. 1990), tRNAscan-SE (Lowe and Eddy 1997).\n\nA subset of 25 highly similar RB49-like phages using with blastN search with RB49 genome sequence as a query was retrieved from GenBank. The thresholds used were 93% of the query coverage and the average nucleotide identity over aligned segments 92%. Full genome nucleotide alignment and phylogenetic tree reconstruction were performed with NGPhylogeny.fr web services. The genome of RB49 and the bacteriophage genomes obtained in this work were aligned and visualized with Clinker v0.0.21 and clustermap.js (Gilchrist and Chooi 2021).\n\n\nResults\n\nThe direct plating of the commercial therapeutical phage mixture on the lawns of the E. coli strains 4s, F5 and F17 yielded phage plaques. PCR screening with the primers hybridizing to bacteriophage RB49 g38 sequence was performed and about half of the plaques were found positive. One PCR-positive phage isolate per host strain was selected and purified by repeated single plaque isolation. The obtained bacteriophage strains were named as follows: F5-derived phage – Cognac49, F17-derived phage – Whisky49 and 4s-derived phage – Brandy49. The concentrated phage stocks were grown and analyzed by transmission electron microscopy. The bacteriophages were morphologically undistinguishable (Figure 1) having typical appearance of T-even-like viruses (Ackermann and Krisch 1997, Comeau et al. 2007) that was compatible with their expected relatedness to the phage RB49.\n\nThe genomic DNA was extracted from the phage stocks and sequenced using the Ion Torrent technology. The assembly procedure yielded a single contig for each of the bacteriophages with the coverage level > 190. Since T-even related phages feature unlimited circular permutations (Comeau et al. 2007), the start of each genomes was set arbitrary according to the standard representation of the RB49 genome. The main features of the genomes assembled are listed in the Table 1.\n\nGene functions were predicted based on the existing annotations of RB49-like phages producing the best blastN hits for each of the genomes for Brandy49: Phi1 (nucleotide identity 95.77%), vB_EcoM_011D4 (95.57%), kaaroe (96.86%); for Cognac49: Sf20 (98.04%), RB49 (97.59%), KFS-EC (97.79%); for Whisky49: vB_EcoM_011D4 (97.76%), Sf20 (98.38%), GEC-3S (98.17%); query cover >95% for all comparisons. The annotations were then manually curated.\n\nScan by tRNAscan-SE detected no tRNAs in phage genomes.\n\nThe comparison of three genomes to the bacteriophage RB49 (Figure 2) revealed that 42 ORFs shared identical aa sequences in all four genomes, another 140 ORFs were also very similar with the aa identity ≥ 0.90. The phages share also 71 more distantly related ORFs with aa identity levels ≥ 0,50 but ≤ 0.90. This result indicates that the isolated bacteriophages are closely related to each other.\n\nThe grayscale indicates the level of the amino acid identity. The ORF colors reflect the groups of homology as revealed by Clinker software.\n\nAbout 56 ORFs making the difference between these three phages (Figure 2), mostly encode for hypothetical proteins with unknown function. Not all RB49 ORFs or their homologues are present in all phages. A trace of recombination event was detected in genome of Cognac49 phage. An integration of foreign genetic material occurred within DNA polymerase gene, which is essential for phage viability. This insertion contains an 825 bp ORF predicted to be an HNH homing nuclease, often present within self-splicing introns. Rfam search (Kalvari et al. 2021) yielded no significant hits, while the analysis of putative secondary structures of corresponding RNA revealed a complex pattern of the stem-loop structures suggesting that insertion could be a bacterial type I intron containing homing nuclease. Ten ORFs with unknown function present in RB49, Brandy49 and Whisky49 are missing in genome of Cognac49, spanning the region homologous to NP_891764.1-NP_891775.1 in RB49.\n\nHigh similarity of the studied phages to RB49 allows us to classify them as follows: Realm: Duplodnaviria, Kingdom: Heunggongvirae, Phylum: Uroviricota, Class: Caudoviricetes, Order: Caudovirales, Family: Myoviridae, Subfamily: Tevenvirinae, Genus: Krischvirus; Escherichia virus RB49 type viruses, just as expected from g38 PCR and TEM results. The genomes of 25 bacteriophages closely related to RB49 were extracted from GenBank. Overall nucleotide phylogeny tree of this subset of the phage genomes with the addition of three genomes sequenced during this work is shown on the Figure 3.\n\nDespite their close relatedness, the isolated bacteriophages showed marked differences in their host specificity. When they were cross-tested for the infectivity on all three host strains used, all three phages were found infective towards F5 strain, however, phage Whisky49 could form plaque on both E. coli F5 and F17 strains, while Brandy49 had the broadest host range infecting all three host strains (been the only one forming plaques on the E. coli 4s lawns). In all the cases the efficiency of plating was similar that observed on the respective isolation host strain.\n\nThe specificity of host cell recognition by most of the T-even-like phages is determined by two proteins – the LTF adhesin gp38 and by the short tail fiber protein gp12 (PMID: 21746838). The comparison of gp12 aa sequences from the bacteriophages under the study revealed them to be nearly identical to gp12 from RB49 phage (data not shown). At the same time the sequences of gp38 were found more diverged (Figure 4). Gp38 from the phage Brandy49 was more distant from the consensus sequence compared to other two viruses (Figure 4) that is correlated with a broader host range of this phage. Some of the aa polymorphisms were observed within the loops located between polyglycine stretches and predicted to be responsible for interactions with the receptor (Trojet et al. 2011, Dunne et al. 2018). Therefore, differences in the particular regions of gp38 sequence may explain different host specificities of these bacteriophages.\n\nThe aa residues that differ from the consensus are highlighted in red. Blue bars indicate polyglycine stretches and yellow bars the loops according to the structural data on phage S16 gp38 (Dunne et al. 2018).\n\nThe characterization of three new viruses that been closely related to each other show distinct patterns of the infectivity towards E. coli strain producing different types of the O antigen provides a useful model system for in-depth analysis of the strategies employed by T-even like phages to infect the host cells featuring effective non-specific protection of the outer membrane surfaces by this polysaccharide structure. The close relatedness of these phages to each other will facilitate the comparison of the results of experiments aiming at deciphering fine mechanisms employed by RB49-like phages to penetrate the O antigen shields of different E. coli strains.\n\n\nData availability\n\nRepository: NCBI Nucleotide database\n\nAccession number: MZ504876 for Brandy49\n\nRoot URL: https://www.ncbi.nlm.nih.gov/nuccore\n\nAccession number URL: https://www.ncbi.nlm.nih.gov/nuccore/MZ504876\n\nAccession number: MZ504877 for Cognac49\n\nRoot URL: https://www.ncbi.nlm.nih.gov/nuccore\n\nAccession number URL: https://www.ncbi.nlm.nih.gov/nuccore/MZ504877\n\nAccession number: MZ504878 for Whisky49\n\nRoot URL: https://www.ncbi.nlm.nih.gov/nuccore\n\nAccession number URL: https://www.ncbi.nlm.nih.gov/nuccore/MZ504878\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\nAbedon ST: Phage Therapy: Various Perspectives on How to Improve the Art. Host-Pathogen Interactions: Methods and Protocols. 2018; 1734: 113–127. Publisher Full Text\n\nAckermann HW, Krisch HM: A catalogue of T4-type bacteriophages. Arch. Virol. 1997; 142(12): 2329–2345. PubMed Abstract \| Publisher Full Text\n\nAllen HK, Levine UY, Looft T, et al.: Treatment, promotion, commotion: antibiotic alternatives in food-producing animals. Trends Microbiol. 2013; 21(3): 114–119. PubMed Abstract \| Publisher Full Text\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J. Mol. Biol. 1990; 215(3): 403–410. PubMed Abstract \| Publisher Full Text\n\nBroeker NK, Barbirz S: Not a barrier but a key: How bacteriophages exploit host's O-antigen as an essential receptor to initiate infection. Mol. Microbiol. 2017; 105(3): 353–357. PubMed Abstract \| Publisher Full Text\n\nClokie MRJMary Ann Liebert Inc: PHAGE: therapy, applications, and research. New Rochelle, NY: Mary Ann Liebert, Inc; 2020.\n\nComeau AM, Bertrand C, Letarov A, et al.: Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery. Virology. 2007; 362(2): 384–396. PubMed Abstract \| Publisher Full Text\n\nD'Accolti M, Soffritti I, Mazzacane S, et al.: Bacteriophages as a Potential 360-Degree Pathogen Control Strategy. Microorganisms. 2021; 9(2): Publisher Full Text\n\nDunne M, Denyes JM, Arndt H, et al.: Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition. Structure. 2018; 26(12): 1573–1582.e4. PubMed Abstract \| Publisher Full Text\n\nGolomidova AK, Efimov AD, Kulikov EE, et al.: O antigen restricts lysogenization of non-O157 Escherichia coli strains by Stx-converting bacteriophage phi24B. Sci. Rep. 2021; 11(1): 3035. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGolomidova AK, Naumenko OI, Senchenkova SN, et al.: The O-polysaccharide of Escherichia coli F5, which is structurally related to that of E. coli O28ab, provides cells only weak protection against bacteriophage attack. Arch. Virol. 2019; 164(11): 2783–2787. PubMed Abstract \| Publisher Full Text\n\nHu B, Margolin W, Molineux IJ, et al.: Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc. Natl. Acad. Sci. U. S. A. 2015; 112(35): E4919–E4928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHyman P, van Raaij M : Bacteriophage T4 long tail fiber domains. Biophys. Rev. 2018; 10(2): 463–471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalvari I, Nawrocki EP, Ontiveros-Palacios N, et al.: Rfam 14: expanded coverage of metagenomic, viral and microRNA families. Nucleic Acids Res. 2021; 49(D1): D192–D200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnirel YA, Ivanov PA, Senchenkova SN, et al.: Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup. Int. J. Biol. Macromol. 2019; 124: 389–395. PubMed Abstract \| Publisher Full Text\n\nKnirel YA, Prokhorov NS, Shashkov AS, et al.: Variations in O-antigen biosynthesis and O-acetylation associated with altered phage sensitivity in Escherichia coli 4s. J. Bacteriol. 2015; 197(5): 905–912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulikov E, Golomidova A, Babenko V, et al.: A Simple Method for Extraction of the Horse Feces Virome DNA, Suitable for Oxford Nanopore Sequencing. Microbiology. 2020; 89(2): 246–249. Publisher Full Text\n\nKulikov EE, Golomidova AK, Letarova MA, et al.: Genomic sequencing and biological characteristics of a novel Escherichia coli bacteriophage 9g, a putative representative of a new Siphoviridae genus. Viruses. 2014; 6(12): 5077–5092. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulikov EE, Golomidova AK, Prokhorov NS, et al.: High-throughput LPS profiling as a tool for revealing of bacteriophage infection strategies. Sci. Rep. 2019; 9(1): 2958. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulikov EE, Majewska J, Prokhorov NS, et al.: Effect of O-acetylation of O antigen of Escherichia coli lipopolysaccharide on the nonspecific barrier function of the outer membrane. Microbiology. 2017; 86(3): 310–316. Publisher Full Text\n\nKulikova EE, Isaeva AS, Rotkina AS, et al.: Diversity and dynamics of bacteriophages in horse feces. Mikrobiologiia. 2007; 76(2): 271–278. PubMed Abstract \| Publisher Full Text\n\nLetarov AV, Kulikov EE: Adsorption of Bacteriophages on Bacterial Cells. Biochemistry (Mosc). 2017; 82(13): 1632–1658. PubMed Abstract \| Publisher Full Text\n\nLowe TM, Eddy SR: tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25(5): 955–964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng D, Hu WG, Choudhury BP, et al.: Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane. FEBS J. 2007; 274(20): 5350–5359. Publisher Full Text\n\nPevzner PA, Tang H, Waterman MS: An Eulerian path approach to DNA fragment assembly. Proc. Natl. Acad. Sci. U. S. A. 2001; 98(17): 9748–9753. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeemann T: Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014; 30(14): 2068–2069. PubMed Abstract \| Publisher Full Text\n\nSoding J, Biegert A, Lupas AN: The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 2005; 33(15980461): W244–W248. Publisher Full Text\n\nTaylor NM, Prokhorov NS, Guerrero-Ferreira RC, et al.: Structure of the T4 baseplate and its function in triggering sheath contraction. Nature. 2016; 533(7603): 346–352. Publisher Full Text\n\nTetart F, Desplats C, Kutateladze M, et al.: Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. J. Bacteriol. 2001; 183(1): 358–366. Publisher Full Text\n\nTrojet SN, Caumont-Sarcos A, Perrody E, et al.: The gp38 adhesins of the T4 superfamily: a complex modular determinant of the phage's host specificity. Genome Biol. Evol. 2011; 3: 674–686. PubMed Abstract \| Publisher Full Text" }	[ { "id": "114759", "date": "16 Mar 2022", "name": "Marcin Los", "expertise": [ "Reviewer Expertise Bacteriophage biology", "phage-bacterial host interaction", "prophage induction" ], "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 sequencing and analysis of three T4-like phages capable of degradation of O-antigens of E. coli. The degradation of O-antigen and use of surface receptors by phage may play a crucial role in the host range of T4-like phages and their ability to be potential components of phage cocktails for phage therapy. The comparison of the sequences of isolated phages shed a light on their potential mechanisms involved in the broadening of host range among this group of phages.\n\nThe manuscript is well written and the data is clear. Also, the interpretation of the data seems to be justified. I have a request for minor revision - since Brandy49 had the broadest host range, but also it contained additional genes not present in other phages studied, can you please explain if the function of these additional genes can be predicted and if these genes can play a role in broader host range?\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] }, { "id": "138503", "date": "25 May 2022", "name": "Mikael Skurnik", "expertise": [ "Reviewer Expertise Molecular microbiology", "bacterial genetics", "bacteriophages", "phage therapy" ], "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 of Efimov et al. describes three new phages isolated against three E. coli strains expressing structurally different LPS O-antigens. While I find the manuscript interesting, I have a few major and many minor comments that I think the authors could consider:\nMajor comments\nIt would be nice to have a picture of the O-unit structures of the three LPSs, just to see how they differ structurally.\n\np.4: “Potential open reading frames (ORFs) were detected by use of SnapGene (GSL Biotech, USA) and subsequently analyzed with HMMER, HHPRED (Soding et al. 2005) (MPI Bioinformatics Toolkit), BLAST (Altschul et al. 1990), tRNAscan-SE (Lowe and Eddy 1997).” When describing the annotation of the genomes, I personally prefer using “predicted genes” or “predicted gene products” instead of ORFs. The genomes are full of ORFs in all six reading frames but only a small fraction of them contain potential genes. In addition, the predicted gene products were not analyzed with tRNAscan-SE, but the phage genomes were.\n\nThe authors should discuss what role the different O-antigens play, if any, for the three phages. Is the LTF receptor an OMP, or is it an LPS structure? If it is OmpC or OmpF (as we identified for three Yersinia phages1), for example, would it be possible to compare the sequences of these proteins of the three E. coli strains?\nMinor comments\nPage 3\nChlorophorm ==> chloroform\n\nOD600 ==> OD600\n\nCheck the usage of italics for bacterial names, specifically for E. coli.\nPage 4\nThe sentence, “Viral genomic DNA was extracted from the pellets with CTAB (cetyltrimethylammonium bromide) as described in (Kulikov et al. 2020) using.” is not finished.\n\nThe sentence, “A subset of 25 highly similar RB49-like phages using with blastN search with RB49 genome sequence as a query was retrieved from GenBank.” needs attention. Perhaps like this: “A subset of 25 highly similar RB49-like phages identified with blastN search using the RB49 genome sequence as a query was retrieved from GenBank.”\n\nAlso, the next sentence is clumsy: “The thresholds used were 93% of the query coverage and the average nucleotide identity over aligned segments 92%.” Perhaps like this: “A threshold of 93% was used for the query coverage, and 92% for the average nucleotide identity over the aligned segments.”\n\n“Gene functions were predicted based on…”, perhaps like this: “The functions of the gene products were predicted based on…”\nPage 5\nTable 1 column: “Number of ORFs annotated” ==> Number of predicted genes.\n\nFigure 2 caption: “The ORF colors”==> The colors of the predicted genes.\nPage 6\nquery cover ==> query coverage\n\n“Scan by tRNAscan-SE detected no tRNAs in phage genomes.” ==> tRNAscan-SE detected no tRNAs encoding genes in phage genomes.\n\nThis text is problematic: “The comparison of three genomes to the bacteriophage RB49 (Figure 2) revealed that 42 ORFs shared identical aa sequences in all four genomes, another 140 ORFs were also very similar with the aa identity ≥0.90. The phages share also 71 more distantly related ORFs with aa identity levels ≥0,50 but ≤0.90. This result indicates that the isolated bacteriophages are closely related to each other.” If you compare genomes, you use nucleotide sequences, if you compare predicted gene products, you use amino acid sequences. ORFs ==> predicted gene products. aa identity ==> aa sequence identity. Perhaps it would be more clear to use identity percentages instead of a 0-1 scale.\n\n“About 56 ORFs making the difference between these three phages (Figure 2), mostly encode…“ ==> \"The three phages differed based on the presence or absence of altogether 56 predicted genes (Figure 2), most of which encode…\"\nPage 7\nwere extracted from GenBank ==> were downloaded from GenBank\n\nefficiency of plating was similar that observed ==> efficiency of plating was similar to that observed\n\nSome of the aa polymorphisms ==> Some of the aa sequence polymorphisms\n\nThe sentence, “The characterization of three new viruses that been closely related to each other show distinct patterns of the infectivity towards E. coli strain producing different types of the O antigen provides a useful model system for in-depth analysis of the strategies employed by T-even like phages to infect the host cells featuring effective non-specific protection of the outer membrane surfaces by this polysaccharide structure.” is far too long and confusing. Better to split it into 2-3 shorter sentences.\n\nFigure 4 caption. “Blue bars indicate polyglycine stretches and yellow bars the loops”. The colors are mixed up: blue indicates the loops and yellow the polyglycines.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-1113
https://f1000research.com/articles/10-1112/v1	03 Nov 21	{ "type": "Case Report", "title": "Case Report: Asymptomatic COVID-19 patient with a subtle hypercoagulable state and fluctuating D-dimer level", "authors": [ "Jefferson Caesario", "Decsa M. Hertanto", "Kukuh D. Hernugrahanto", "Dwikora N. Utomo", "Nicolaas C. Budhiparama", "Djoko Santoso", "Pancras C.W. Hogendoorn", "Jefferson Caesario", "Decsa M. Hertanto", "Kukuh D. Hernugrahanto", "Dwikora N. Utomo", "Nicolaas C. Budhiparama", "Pancras C.W. Hogendoorn" ], "abstract": "Background: COVID-19 can infect an asymptomatic person silently without any overt symptoms despite diffuse blood clots throughout the body. Clot formation is induced by COVID-19 associated coagulopathy that can cause a high mortality rate. D-dimer, a fairly decisive marker for the coagulopathy event, is physiologically a marker of the fibrinolysis process. The increase of D-dimers in COVID-19 cases must be followed up because it relates to the initiation of a cytokine storm. Case presentation: We report an asymptomatic patient with sudden D-dimer elevation who received anticoagulant therapy. After three days of heparin administration, D-dimer results became normal and anticoagulant therapy was stopped. However, on the 12th day, the D-dimer level rebounded back and was followed by an increase of hs-C-reactive protein, erythrocyte sedimentation rate, IL-6, although SARS-CoV-2 PCR result became negative. A hyperglycaemic reaction and a sudden increase of HbA1C was observed in the patient. After three weeks D-dimer had returned to normal levels, and so did the other markers. The patient recovered fully and still no symptoms were obvious. Conclusion: COVID-19 patients without symptoms may be at risk of an asymptomatic coagulopathy process. The decreasing level of D-dimer erroneously cannot ensure that the coagulopathy process stops.", "keywords": [ "COVID-19", "Coagulopathy", "Asymptomatic", "D-dimer", "Anticoagulant Therapy" ], "content": "Introduction\n\nCoronavirus Disease 2019 (COVID-19), following SARS-Cov-2 infection, silently attacks various organs in the human body including the lung.1 Silent hypoxia and blood clots throughout the body are sometimes not followed by any symptoms.2,3 COVID-19 associated coagulopathy leading to massive thrombosis is the cause of high mortality rates and adds complexity in treating the disease.4,5 It has been shown to be present in up to 31% of critically ill intensive care unit (ICU) patients.5 COVID-19 associated coagulopathy has a fairly-decisive marker, namely D-dimer, which is a physiological marker of the fibrinolysis process.6 D-dimer elevation in Disseminated Intravascular Coagulation (DIC) generally shows a highly active fibrinolysis process to oppose clot formation. However, the fibrinolysis process in COVID-19 is very minimal when compared to the clot formation.7 This imbalance has the potential to increase mortality.8 Therefore, the increase in D-dimer in COVID-19 cases must be monitored more seriously because it is related to the initiation of a cytokine storm, which causes worsening conditions in COVID-19. D-dimer monitoring is necessary because subclinical thrombosis tends to be missed in asymptomatic cases.7,8 Here we present an asymptomatic case in which the elevation of D-dimer was present before the cytokine-storm.\n\n\nCase report\n\nAn Asian male patient, 59-years-old and working as an employee, was found to have a positive result of SARS-CoV-2 virus detection by active tracing after attending a mass event in a closed hall for three hours. The patient had a 15-year history of controlled hypertension, one year of pre-diabetes, and dyslipidaemia, for which he received medical treatment of 16 mg Candesartan, 10 mg Atorvastatin since first diagnosed, and 5 mg bisoprolol after routine medical check-up five years ago. There was a family history of hypertension and diabetes from the patient’s mother, but no psycho-social issues were found.\n\nPeriodical Polymerase Chain Reaction (PCR) tests were monitored and COVID-19-positive tests were found for two weeks while no symptoms were felt by the patient (Table 1). During the two weeks, a complete exploration of laboratory results such as hematological test, liver function, renal function, and urinalysis was observed, including markers of coagulopathy and cytokine-storm.\n\nSince dealing with a very unpredictable disease, once the original PCR result was obtained, aggressive therapy, as stipulated by previous studies,9 was given immediately. The therapy, received by the patient at a private clinic, included 5 g intravenous immune globulin for three days and 10 g for the next three days (with premedication of 5 mg dexamethasone intravenous for six days), 2 g intravenous meropenem for six days, and 2400 mg favipiravir (first day) and 1200 mg for ten days.\n\nOn the third day, anticoagulant heparin (9000 IU) was given since there was a sudden spike of D-dimer from 160 to 1015 ng/mL in a day that was beyond the normal value (<500 ng/mL) (Figure 1). After three days of heparin administration, on the seventh day, the D-dimer result decreased to 467 ng/mL and heparin was stopped. However, after four days, on the 12th day D-dimer level monitoring showed a second spike of 1416 ng/mL and was followed up by 1728 ng/mL two days after. Enoxaparin (6000 IU) was given for three days, and anticoagulant treatment continued with rivaroxaban 10 mg for three weeks. Eventually, after three days, D-dimer results showed continuously normal levels below 500 ng/mL. The findings, however, became a particular concern since the D-dimer second spike coincided with the conversion of the COVID-19 PCR result to negative.\n\nNote: bold numbers including those in boxes indicate batches of therapy regimens.\n\nIn general, D-dimer elevation is closely associated with DIC so other laboratory tests such as Activated Partial Thromboplastin Time (aPTT), Prothrombin Time (PT), fibrinogen, and platelets were performed from the first day of treatment for COVID-19. These markers showed unremarkable changes. The patient showed normal or slightly elevated levels of aPTT and PT in the range of 23.1-34.8 s and 10.1-15.4 s, respectively (normal value: 23.0-30.2 s for APTT and 10.1-11.9 s for PT) (Figure 2). The PT level increase was still less than 3 seconds, so the elevation had not fulfilled the criteria as in guidelines of The International Society for Thrombosis and Haemostasis (ISTH).10\n\nSince DIC is also associated with excessive use of fibrinogens and platelets for clot formation, in this patient its levels should have declined. Fibrinogen levels of the patient (235-480 mg/dL) were found in or with a slight increase to the normal range (200-400 mg/dL). According to the ISTH classification, the decrease of fibrinogen in this case, is insignificant, unless <100 mg/dL. Platelet levels of the patient (183,000-269,000 cells/uL) were also in the normal range (150-450 × 103 cells/uL), whereas significant platelet levels, according to the ISTH criteria, are <100,000 cells/uL.\n\nMarkers of a cytokine-storm were observed, such as Interleukin-6 (IL-6), Ferritin, Erythrocyte Sedimentation Rate (ESR), and C-Reactive Protein (CRP). The Neutrophil-Lymphocyte Ratio (NLR) was also examined to predict the progression of COVID-19. When D-dimer increased for the second time, there was a surge of hs-CRP and ESR (Figure 3) of 0.56 mg/dL and 15-25 mm/hour, respectively, which were above the normal range of <0.5 mg/dL and < 10 mm/hour, respectively. IL-6 examination was carried out three times and showed an increased level of 5.52-10.85 pg/mL above normal level (<7 pg/mL) (Table 2). On the contrary, the patient's ferritin level increased to normal levels while the NLR was relatively stable. These unique findings strongly displayed the abnormalities in laboratory results on the 12th day, tending to a profile of cytokine storms.\n\nIn addition to laboratory results, lung radiological examination and chest X-rays were performed and showed normal results on the 1st, 7th, and 12th days. On the 12th day, when D-dimer increased for the second time, accompanied by an increase in other inflammatory markers, the patient underwent a chest CT scan, which showed a minimal ground glass appearance in the right lobe of the lung (Figure 4).\n\nCircles indicate ground-glass appearance on the right lobe lung.\n\nOther laboratory examinations were examined to rule out the causes of increased D-dimers due to non-COVID-19 causes, such as autoimmune, endocrine disorders, and malignancy. Complement proteins (C3 and C4), free-T4, T3, and TSH, Alpha-Fetoprotein (AFP), Carbohydrate Antigen 19-9 (CA 19-9), and Carcinoembryonic Antigen (CEA) were all normal. The patient's blood glucose profile was also observed due to a history of prediabetes. HbA1C level increased from 5.9% to 6.3% within one week. The data of random plasma glucose increased inconsistently despite insulin administration (Figure 5).\n\nAfter four weeks of treatment, the patient fully recovered and still did not feel any symptoms of COVID-19 and completed four weeks of rivaroxaban.\n\n\nDiscussion\n\nCOVID-19 has two silent killer mechanisms: silent hypoxia and massive thromboembolism, where a considerable number of SARS-CoV-2 infected individuals can be asymptomatic.1–3 Silent hypoxia can be detected using pulse oximetry. Post-mortem reports indicate that thromboembolism is a more complex process involving a complicated interaction of SARS-CoV-2 virus invasion with platelets, endothelial cells, leukocytes, inflammation, and immune response. Reports revealed that 59.6% of severe COVID-19 cases showed an increase in D-dimer level (threshold as 0.5 mg/L), decreased platelet count (thrombocytopenia), and prolonged prothrombin time.11\n\nA thrombo-embolism event in an asymptomatic individual frequently happens and can be fatal.9,12 Cui et al investigated thrombosis events that occurred in asymptomatic ICU patients using ultrasonography imaging showing an incidence of 25% (20/81).13 Del Nonno et al also discovered the presence of a large thrombus clogging the bifurcation of the main pulmonary artery in autopsy despite asymptomatic history. The asymptomatic thrombosis process led to sudden death and surprisingly can possibly occur after the swab results becoming negative.12 Moreover, pulmonary thromboembolism should be considered as the cause of the sudden onset of oxygenation degradation and hypoxia.10,12\n\nOur case highlighted D-dimer's importance as a screening tool for a thromboembolism event since D-dimer could indicate COVID-19 associated coagulopathy.6 The D-dimer elevation of patients infected by SARS-CoV-2 is strongly related to poor prognostic results and the need for intensive care management.5,8,14 Poor prognosis included patients with mild and no symptoms.12 D-dimer early spike detection, related to COVID-19, can signal the need for additional treatments such as anticoagulant therapy before the coagulopathy process develops massively.7,8\n\nIn general, such as in DIC-sepsis cases, D-dimer shows fibrinolysis’ escalation to balance the clotting process that occurs throughout the body.7 On the contrary, in COVID-19 coagulopathy, the fibrinolysis process remains inefficiently 0.02% times its normal capability to encounter clotting formation.7,8 Due to that unique characteristic of coagulopathy, physicians should treat their patients carefully since a small D-dimer elevation above 500 ng/mL could represent a severe clotting process that occurs quickly and demands immediate treatment. If this condition is not handled correctly, patients can suddenly fall into a fatal hypercoagulability state.8,15 The administration of anticoagulant agents, such as enoxaparin, heparin, and apixaban, demonstrate a significant reduction in the mortality rate of COVID-19 coagulopathy.16\n\nIn this case report, the patient showed relatively normal values of fibrinogen, platelet, APTT, and PT which was different from common DIC related to sepsis. APTT prolongation, fibrinogen depletion, and platelet deprivation can be found in COVID-19 in the later stage due to sepsis.7 However, in contrast, in this case, normal values of the markers were probably due to the therapeutic management applied to the patient.\n\nPCR tests for this patient had been negative on day 15, but on day 12 until 15 the patient experienced a D-dimer rebound after discontinuing anticoagulation therapy, which happened simultaneously with the elevation of ESR, CRP, and IL-6 as cytokine storm markers. Studies showed D-dimer elevation appeared approximately five days before the cytokine storm and probably induced the cytokine storm.10 This case showed that anticoagulant discontinuation after normal D-dimer value could lead to a misjudgement that affects the therapeutic result.\n\nCOVID-19 and hyperglycaemia are clinical problems. According to the CORONADO study (Coronavirus SARS-CoV-2 and Diabetes Outcomes), people with diabetes that have a hypoglycaemic condition at the time of hospital admission seem to worsen the prognosis of COVID-19.17 People without diabetes but having hyperglycaemia also have a poorer prognosis of COVID-19 than people with diabetes.18 HbA1c is also associated with inflammation, hypercoagulability, low SaO2, and mortality in COVID-19 patients. Early determination of HbA1c in hospital admission can help assess inflammation, hypercoagulability, and prognosis of COVID-19 patients.19 Initial therapy to lower the hyperglycaemic conditions is essential, besides management of infection, inflammation, and supportive care. This management can prevent a prolonged poor condition and subsequent poor prognosis.18\n\nThose clinical manifestations were undoubtedly related to our patient's medical history of hypertension and prediabetes, which affected the coagulation process's quality. The sudden changes of HbA1C and the hyperglycaemic reaction showed unusual findings since erythrocytes survive for three months.19,20 These blood glucose impairments do not seem related to the thromboembolism process.20\n\nAnother note from this case was that the progression to cytokine storm could happen even after the conversion of PCR to negative result.12 If the management of COVID-19 is only focused on the PCR result, then the coagulopathy and cytokine storm can be missed by clinicians, which may lead to a false assumption that the cause of death is other factors, instead of COVID-19. A post-COVID-19 physical and laboratory follow-up is propagated to objectify the recovery of COVID-19 infection patients, especially focussing on functional impairment.20 Within this scale, our patient would have fitted in the grade 0 cohort. It would be very interesting to have long term follow-up of a cohort of these grade 0 patients.\n\n\nConclusion\n\nWhen facing COVID-19, with its diverse clinical spectrum, clinicians should be cautious. Coagulopathy associated with COVID-19 can occur without any symptoms but probably leads to worse results. D-dimer can become a reliable screening tool against COVID-19 thromboembolism to anticipate the asymptomatic presence of COVID-19 to provide early necessary treatments for preventing fatality.\n\n\nConsent\n\nWritten informed consent for the publication of this case report and any associated images was obtained from the patient.\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": "Acknowledgments\n\nWe would like to thank Faculty of Medicine, Universitas Airlangga and Leiden University Medical Center for the support of the study\n\n\nReferences\n\nTobin M, Laghi F, Jubran A: Why COVID-19 Silent Hypoxemia Is Baffling to Physicians. Am. J. Respir. Crit. Care Med. 2020; 202: 356–360. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan B, Umapathi T, Chua K, et al.: Delayed catastrophic thrombotic events in young and asymptomatic post COVID-19 patients. J. Thromb. Thrombolysis. 2020; 51: 971–977. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarner K, Cox E: COVID-19 as the cause of thrombosis: recognising COVID-19 infection in apparently asymptomatic patients. BMJ Case Rep. 2021; 14: e241027. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaughety M, Morgan A, Frost E, et al.: COVID-19 associated coagulopathy: Thrombosis, hemorrhage and mortality rates with an escalated-dose thromboprophylaxis strategy. Thromb. Res. 2020; 196: 483–485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalas M, Naazie I, Elsayed N, et al.: Thromboembolism risk of COVID-19 is high and associated with a higher risk of mortality: A systematic review and meta-analysis. EClinicalMedicine. 2020; 29-30: 100639. Publisher Full Text\n\nUmemura T, Kondo H, Ohta H, et al.: D-dimer level elevation can aid in detection of asymptomatic COVID-19 presenting with acute cerebral infarction. eNeurologicalSci. 2021; 22: 100294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsakura H, Ogawa H: COVID-19-associated coagulopathy and disseminated intravascular coagulation. Int. J. Hematol. 2021; 113: 45–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKruse J, Magomedov A, Kurreck A, et al.: Thromboembolic complications in critically ill COVID-19 patients are associated with impaired fibrinolysis. Crit. Care. 2020; 24: 676. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanders J, Monogue M, Jodlowski T, et al.: Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19). JAMA. 2020. Publisher Full Text\n\nThachil J, Tang N, Gando S, et al.: ISTH interim guidance on recognition and management of coagulopathy in COVID-19. J. Thromb. Haemost. 2020; 18(5): 1023–1026. PubMed Abstract \| Publisher Full Text\n\nHernugrahanto KD, Utomo DN, Hariman H, et al.: Thromboembolic involvement and its possible pathogenesis in COVID-19 mortality: lesson from post-mortem reports. Eur. Rev. Med. Pharmacol. Sci. 2021; 25: 1670–1679. Publisher Full Text\n\nDel Nonno F, Colombo D, Nardacci R, et al.: Fatal pulmonary arterial thrombosis in a COVID-19 patient, with asymptomatic history, occurred after swab negativization. Thromb. J. 2021; 19: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui S, Chen S, Li X, et al.: Prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia. J. Thromb. Haemost. 2020; 18(6): 1421–1424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYao Y, Cao J, Wang Q, et al.: D-dimer as a biomarker for disease severity and mortality in COVID-19 patients: a case control study. J. Intensive Care. 2020; 8: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColling M, Kanthi Y: COVID–19-associated coagulopathy: An exploration of mechanisms. Vasc. Med. 2020; 25: 471–478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBillett H, Reyes-Gil M, Szymanski J, et al.: Anticoagulation in COVID-19: Effect of Enoxaparin, Heparin, and Apixabanon Mortality. Thromb. Haemost. 2020; 120: 1691–1699. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCariou B, Hadjadj S, Wargny M, et al.: Phenotypic characteristic and prognosis of inpatients with COVID-19 and diabetes: the CORONADO study. Diabetologia. 2020; 63: 1500–1515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGianchandani R, Esfandiari NH, Ang L, et al.: Managing Hyperglycemia in the COVID-19 Inflammatory Storm. Diabetes-Journal. 2020; 69: 2048–2053. PubMed Abstract \| Publisher Full Text\n\nWang Z, Du Z, Zhu F: Glycosylated hemoglobin is associated with systemic inflammation, hypercoagulability, and prognosis of COVID-19 patients. Diabetes Res ClinPract. 2020; 164: 108214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMathis A, Villiger L, Reiner MF, et al.: Elevated HbA1c is not associated with recurrent venous thromboembolism in the elderly, but with all-cause mortality– the SWEETCO 65+ study. Sci. Rep. 2020; 10: 2495. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "99043", "date": "29 Nov 2021", "name": "Bayu Satria Wiratama", "expertise": [ "Reviewer Expertise epidemiology", "injury", "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\nThis study provided a useful insight into the clinical characteristics of thromboembolism risk on asymptomatic patients. There are few questions that need to be answered about this study:\nIn figure 2, it will be more informative if the authors added the D-dimer level on each of the figures to give more useful information.\n\nIn this study, please use consistent date information, for example if authors use day 1, day 12 then in the chart please stick to the same information. If authors use exact date such as 16/01/2021 then please use the same one in the text.\n\nIn the last sentence of Introduction, authors mentioned that \".. the elevation of D-dimer was present before the cytokine-storm\". It would be interesting if authors could elaborate and describe more using the clinical data about D-dimer elevation and cytokine storm. For example, in figure 3, authors could try adding D-dimer inside each charts about cytokine storm parameters.\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "121288", "date": "21 Feb 2022", "name": "Najib Alwi", "expertise": [ "Reviewer Expertise Neuroscience", "mental health", "impacts of Covid-19 on society." ], "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 has been clearly written, highlighting the otherwise possibly missed rise in D-dimer level in asymptomatic Covid-19 positive patients. The main strength of the report is a comprehensive workout of the patient, including all relevant lab and radiological investigations to support the issue that is being highlighted. The case has also been sufficiently discussed, including comparing the D-dimer elevation phenomena with DIC. Strongly supported for acceptance for indexing.\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/10-1112
https://f1000research.com/articles/10-678/v1	28 Jul 21	{ "type": "Research Article", "title": "Correlation between CD44+ cancer stem cell expression and histopathological types of nasopharyngeal carcinoma", "authors": [ "Muhtarum Yusuf", "Indriyadevi Indra", "Sri Herawati Juniati", "Yussy Afriani Dewi", "Indriyadevi Indra", "Sri Herawati Juniati", "Yussy Afriani Dewi" ], "abstract": "Background: Nasopharyngeal carcinoma (NPC) recurrency rate is still high despite patients receiving complete treatment. The response to treatment may vary depending on the type of histopathology and Epstein-Barr virus, however the mechanism remains unclear. Recent studies have found that there is a relationship between response to treatment and the presence of cancer stem cells (CSCs). CD44+ cancer stem cells may cause cancer cells to be resistant to treatment. Therefore, this cross-sectional study aims to determine the correlation between CD44 + cancer stem cell expression and the histopathological types of NPC. Method: Samples were obtained from NPC biopsies of type I, II, III patients (based on WHO histopathology criteria), who had not received prior treatment. CD44+ expression was examined using immunohistochemistry methods by staining CD44+ monoclonal antibodies. The degree of CD44+ cell membrane expression was based on the immunoreactive score scale or the Remmele index scale. Results: Most histopathological types were WHO type III (21 patients, 50%), followed by type II (18 patients, 42.86%), and type I (3 patients, 7.14%). CD44 + expression on type I showed one patient had moderate positive and two patients had a high-positive expression. In type II, 10 were moderate positive and eight were high-positive. In type III, one patient was low-positive, 11 were moderate positive and nine patients were high-positive. Statistical analysis showed that the CD44+ expression difference between the three histopathology types were not statistically significant. Conclusion: There were no correlations between CD44 + expression and histopathological type of NPC.", "keywords": [ "NPC stem cells", "CD44 +", "nasopharyngeal carcinoma", "histopathology", "cancer" ], "content": "Introduction\n\nNasopharyngeal carcinoma (NPC) recurrency rate is still high despite patients receiving complete treatment, such as radiotherapy, chemotherapy, or a combination of both. The responses of NPC to therapy varies greatly and might be associated with the type of histopathology. The histological classification of NPC issued by WHO in 1978 includes: (i) type I as keratinizing squamous carcinoma, (ii) type II as non keratinizing squamous carcinoma, (iii) type III as undifferentiated carcinoma.1 Type II and III World Health Organization (WHO) histopathology types respond more effectively to radiotherapy and have a better locoregional control than type I.2 The difference in therapeutic response was associated with the presence of Epstein-Barr virus (EBV) as a causative factor of NPC, but the mechanism remains unclear.3 Recent studies have shown that there is a relationship between therapeutic response and the presence of cancer stem cells (CSCs). The presence of CSCs can cause cancer cells to be resistant to treatment4,5 Type II and III WHO histopathology of NPC are closely related to EBV based on the etiology factor of NPC. This was supported by immunoglobulin (IgG and IgA) viral capsid antigen titer increase, early antigen, EBV nuclear antigen (EBNA), however increased titer was not found in type I histopathology of NPC.6 A recent study has shown the important role of EBV in NPC stem cells related to the cancer development.7\n\nCD44+ molecule is an identification marker of NPC stem cells. The study on NPC with positive EBV cell line C666-1 showed that CD44+ cancer stem cells have the capacity to self-renew, differentiate, initiate tumor cells in vivo, and they are highly resistant to 5-fluorouracil therapy.8 Ebstein-Barr virus can potentially induced CSCs in EBV-positive NPC by altering the Notch and hedgehog signaling pathways through the latent membrane protein-1 (LMP1), LMP2A.9,10\n\nThe purpose of this study was to determine the correlation between CD44+ CSCs expression and the histopathological type in the tissue sample of NPC patients who had received treatment at the department of otolaryngology, head, and neck surgery in the general hospital Dr Soetomo Surabaya (RSUD Dr. Soetomo Surabaya). At present this correlation has not been investigated in this teaching hospital. In a clinical setting the results of this study can be used as a predictor biomarker to assess therapeutic response.\n\n\nMethods\n\nIn this cross-sectional study 43 tumor tissue samples (n = 43) that were embedded in paraffin blocks were obtained from NPC patients at the otolaryngology, head, and neck surgery department of the RSUD Dr Soetomo Surabaya. These samples were collected from July to September 2016.\n\nWe collected subject, age, ethnicity, stage, histopathology and immunoreactive score (IRS) data consecutively according to the inclusion and exclusion criteria (see underlying data: https://figshare.com/s/cd689f9f2918d71671c6).11 After the patient received the explanation and agreed to participate in the study, we collected the histopathological examination numbers from the nasopharyngeal biopsy. We also obtained the paraffin blocks of the NPC patients for further examination of immunohistochemical staining with CD44 monoclonal antibody (Cell marque, USA, Inteli PATH HLX, model number: IPS0001INTL) at the Anatomical Pathology Laboratory, Faculty of Medicine, Airlangga University, Surabaya. To avoid bias, we used positive controls with paraffin block breast carcinoma. CD44 expression was assessed by an anatomical pathologist from the Department of Anatomical Pathology, Faculty of Medicine, Airlangga University, Surabaya.\n\nTissue samples included in this study were obtained from patients that had not received radiotherapy, chemotherapy, or a combination of both treatments, in addition to the biopsy material having to be sufficient for immunohistochemistry (IHC) examination. The examination of the IHC method was done with a 400× magnification microscope by staining CD44+ monoclonal antibodies (Cell marque, USA, Inteli PATH HLX, model number: IPS0001INTL).12 The assessment of CD44+ cell membrane expression levels were based on the IRS scale or the Remmele scale index (Table 1).13\n\nOf the 43 samples obtained from the anatomical pathology laboratory, 42 samples met the inclusion criteria, and one damaged sample did not meet the requirements during the immunohistochemical staining process. The population of this study had, 3 people (7.14%) aged 20-29 years, 4 people (9.52%) aged 30-39 years, 14 people (33.33%) aged 40-49 years, 15 people (35.72%) aged 50-59 years and 6 people (14.29%) aged 60-69 years. The male: female ration is 1.3:1, with 24 (57.14%) male patients in the population of the study.\n\nThe final score (immunoreactive score/IRS) is the result of multiplying the percentage score of positive cells with the intensity score of brownish-red color on immunoreactive cells.\n\nThe degree of expression of CD44 + was categorized as follows:\n\nNegative expression (-), if the final score is 0\n\nLow expression (+), if the final score is 1 to 3\n\nModerate expression (++), if the final score is 4 to 6\n\nHigh expression (+++), if the final score is 7 to 12\n\nNPC histopathology, was divided into three types namely squamous cell carcinoma (WHO type I), nonkeratinizing carcinoma (WHO type II), and undifferentiated carcinoma (WHO type III).3\n\n\nData analysis\n\nSpearman’s statistical test was done to determine the correlation between CD44+ expression and NPC type of histopathology. CD44+ expression and histopathological type were assessed as an ordinal scale with the significance level <0.05.\n\n\nEthical considerations\n\nPatient from the ear nose and throat outpatient unit Dr. Soetomo hospital were given explanation about the research objectives, benefits and examinations carried out. If they were willing to take part in the study, patients were asked to sign a letter of informed consent for the study. Ethical approval (522/Panke.KKE/IX/2016) for this study was obtained by the ethics committee of RSUD Dr Soetomo Surabaya.\n\n\nResults\n\nThe NPC patients’ distribution data were based on the type of histopathology (Table 2).\n\nOur results show 21 patients (50%) with WHO-III NPC, followed by 18 (42.86%) with WHO-II, and 3 (7.14%) with WHO-I NPC.\n\nStage IV was found in 26 (61.90%) patients, followed by stage III in nine (21.43%) patients, stage II in six (14.29%) patients, and stage I in one (2.38%) patient.\n\nCD44 + expression based on the histopathological type of NPC can be seen in Table 4. CD44 + on type I tissue exhibited one moderate positive and two high-positive expressions. In type II tissue, 10 samples showed a moderate positive and eight had high-positive expressions. In type III tissue, one patient had low-positive, 11 had moderate positive and nine had high-positive expressions.\n\nCD44+ expression with IHC staining with the use of 400x magnification is shown in Figure 1.\n\nA. The appearance of a brownish-red color with weak intensity which shows a low-positive. B. Medium intensity brownish-red tint indicating a mid-positive. C. A brownish-red high intensity representing a high-positive (arrows).\n\nStatistical analysis of the correlation between CD44+ expression and the histopathology type of NPC was assessed by Spearman’s correlation test (p = 0.925) with (r) = 0.015. This showed that the high expression of CD44 + was obtained in the histopathology of NPC WHO types I, II, III, however the difference was not statistically significant with p > 0.05.\n\n\nDiscussion\n\nThe purpose of this study was to explain the relationship between CD44+ cancer stem cell expression with the histopathological type of NPC. This relationship can have an important clinical impact since CD44 can be used as a predictor biomarker for assessment of therapeutic response in NPC.\n\nThe findings of this study indicate that the majority (15 patients (35.72%)) of NPC were aged 50-59 years. Research from another center at Dr. RSUP. Kariadi Semarang, majority of the NPC patients, 32.80% (128 people), were in the age group 40-49 years, followed by 31.30% that were aged 50-59 years.14As the majority of NPC patients in these studies are above the age of 40, it brings into focus that the theory of carcinogenesis is a multifactorial, multistep process with a delay in diagnosis.15\n\nPatients diagnosed with NPC at a young age tend to experience occupational carcinogenesis. Cancer cells arise from normal cells that undergo transformation to become malignant, due to spontaneous mutation or carcinogen induction. Research has shown that exposure to carcinogens until the emergence of cancer cells can take up to 15-30 years.16\n\nPatients’ sex may be a contributing factor to the incidence of NPC. A total of 24 samples (57.14%) were from male patients in this study, with a ratio of 1.3:1. Research by Roezin (1995) shows that the ratio of NPC sufferers throughout Indonesia was 2.3:1.17 Research in China indicates similar results (2.5:1).18 In Asia, high incidence of NPC in men is thought to be caused by differences in living and work habits, as men are more prone to carcinogen exposure such as smoking, use of firewood, exposure to industrial heat and combustion products, which can cause NPC.19\n\nThis research was conducted at Dr. Soetomo Surabaya Hospital, East Java, which is a referral tertiary hospital from Eastern Indonesia, especially the Java region. Therefore, the majority of the research sample are from the Javanese individuals. The research findings also support that the majority of the NPC sufferers (28 samples (66.67%)) were Javanese. Additionally, 61.90% (26 samples) of the samples were NPC stage IV. Research in England also found that stage IV was 52.20%, stage III was 31.30% and stage II was 14.90%, while stage I was not obtained by the study.20 Patients with NPC often seek treatment when this disease is at an advanced stage. Early diagnosis of NPC is challenging as the tumor site is located in a painless area, therefore patients are late to notice this disease.21 Additionally the lack of knowledge about the risks of NPC, along with the distance from the place of treatment, and socioeconomic levels adds to the difficulties of early diagnosis.\n\nCancer stem cells are a subset of tumor cells that have the ability to self-renew and produce new cells to form tumors.22 The function of cancer stem cells as a progenitor and multipotent cell renewal is for proliferation, differentiation and increasing resistance to cancer therapy.23,24 NPC has similarities in the type of histopathology and the origin of tumor development between head and neck cancers, including cancer of the oral cavity, pharynx and larynx. In NPC 90% of the histopathological type is squamous cell carcinoma (SCC), which is aggressive and has a high recurrence rate.25 Cluster of differentiation 44 (CD44) is a transmembrane adhesion receptor which is an intermediary for cellular interactions with the tumor microenvirontment that plays a role in the invasion and spread of tumor cells.26,27 CD44 expression is associated with cellular invasion and metastasis of cancer cells caused by reorganization of the cytoskeleton to facilitate active migration. The expression of CD44 increases the invasion of cancer cells by increasing in proteases and decreasing the expression and enzymatic activity.26\n\nThe IHC findings of this study indicate that the increase in CD44 expression is not in parallel with the histopathological types of WHO in NPC, as one WHO type I sample had moderate positive expression (++), and two samples had strong positive expression (+++). In WHO II NPC type 10 samples showed moderate positive expression (++), and eight samples had strong positive expression (+++). In WHO III NPC type one sample had weak positive expression, 11 samples had moderate positive expression (+++), and nine samples had strong positive expression (+++), no negative CD44 expression was found.\n\nStatistical analysis indicated that the correlations between the expression of CD44 cancer stem cells with the histopathological types of NPC is not significant (p = 0.925). This could possibly be due to the sonic hedgehog (Shh) pathway regulation of the CSCs activities, such as self-renewal.7 Positive CD44 expression was found in WHO type I NPC in two patients, WHO II type NPC in ten patients, and WHO III type NPC in nine patients. This suggests that toll-like receptor-3 (TLR3) activates a signaling cascade via the Toll-interleukin-1 receptor [(TIR)] domain-containing-adapter-inducing interferon-β (TRIF), that leads to the activation of the nuclear factor- κB (NF-κB) signaling pathway.28 LMP-1 (latent membrane protein-1) also activates NF-κB and signal transducer and activator of transcription (STAT) signals in EBV-infected epithelial cells via transcription and secretion of interleukin-6 (IL-6).29 Activation of the NF-B signal induces an increase in CD44 expression, which plays a role in the differentiation of NPC cells in WHO I, II and III histopathological types.28,29\n\nThe role of CD44 on cancer development has been found in several studies. Ozman et al. (2014) reported that there was a significant relationship between high CD44 expression and perineural invasion and positive lymph nodes in gastric cancer. Research by Simiuniscu et al., (2012) found a weak staining of CD44 expression in poorly differentiated oral SCC, while moderate and strong expressions were markers of moderately and well differentiated carcinoma. This study found weak positive in one undifferentiated carcinoma sample (WHO type III). In the histopathological type of NPC, 90% is aggressive SCC, with a high recurrence cancer rate.25 In the case of oropharyngeal SCC, it was reported that there was a 39.4% decrease in CD44 isoform expression against the CD44 immunoreactivity criteria, and direct relationship between CD44 expression and cervical lymph node metastases, survival, and recurrence was not found in patients with significantly weak/negative CD44 expression. Those who had weak expression of CD44 had worse free survival compared with positive CD44 expression.30\n\nThe limitation of this study was in the sample size. Additionally, when IHC staining with CD44 antibody was performed, the paraffin block specimens that were obtained were neither of sufficient nor good quantity to make sample preparations. Finally, this study was not able to evaluate the factors that influence the stage and aggressiveness of NPC.\n\n\nConclusion\n\nThis study has shown that there is no correlation between CD44+ CSCs expression and the histopathological type of NPC. CD44+ expression can not be used as a predictor marker of therapeutic response. Therefore, there is a need for further studies that take into consideration the stage and metastasis factors, as well as other variables such as CD133 expression.\n\n\nData availability statement\n\nFigshare: dataset for The Correlation between CD44+ cancer stem cell expression and histopathological type of nasopharyngeal carcinoma.\n\nDOI: https://figshare.com/s/cd689f9f2918d71671c6.11\n\nThe project contains the following underlying data:\n\nCD44_PA_data: Data include subject, age, ethnicity, stage, histopathology and IRS (immunoreactive score).\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\nRobinson M, Suh Y, Paleri V, et al.: Oncogenic human papillomavirus-associated nasopharyngeal carcinoma: an observational study of correlation with ethnicity, histological subtype and outcome in a UK population. Infect. Agents Can. 2013; 8: 30–36. 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Airlangga University Pess; 2000.\n\nRoezin A: Deteksi dan pencegahan karsinoma nasofaring. Dalam: Pencegahan dan deteksi dini penyakit kanker.pdf. 1995: 274–288.\n\nKe K, et al.: Epstein-Barr virus-encoded RNAs as a survival predictor in nasopharyngeal carcinoma. Chin Med J. 2014; 127: 294–299. PubMed Abstract\n\nChang ET, Adami HO: The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2006; 15: 1765–1777. PubMed Abstract \| Publisher Full Text\n\nRobinson M, et al.: Oncogenic human papillomavirus-associated nasopharyngeal carcinoma: an observational study of correlation with ethnicity, histological subtype and outcome in a UK population. Infect. Agent. Cancer. 2013; 8: 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTulalamba W, Janvilisri T: Nasopharyngeal Carcinoma Signaling Pathway: An Update on Molecular Biomarkers. Int. J. Cell Biol. 2012; 2012: 594681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonroe MM, Anderson EC, Clayburgh DR, et al.: Cancer stem cells in head and neck squamous cell carcinoma. J Oncol. 2011; 2011: 762780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun L, Cabarcas SM, Farrar WL: Radioresistance and cancer stem cells: survival of the fittest. Cancer stem cell section, loboratory of cancer prevention, center for cancer research. 2016; 2016.\n\nZang Z, Filh MS, Nor JE: The biology of head and neck cancer stem cells. Oral Oncol 2012; 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChai L, et al.: CD44 Expression Is Predictive of Poor Prognosis in Pharyngolaryngeal Cancer: Systematic Review and Meta-Analysis. Tohoku J. Exp. Med. 2014; 232: 9–19. PubMed Abstract \| Publisher Full Text\n\nChun JW, et al.: The utility of CD44 and D2-40 as a prognostic predictor in invasive carcinomas of the breast. Korean J Clin Oncol. 2013; 9: 59–65. Publisher Full Text\n\nNoroozinia F, Fahmideh A, Yekta Z, et al.: Expression of CD44 and P53 in renal cell carcinoma: Association with tumor subtypes. Saudi J. Kidney Dis. Transplant. 2014; 25: 79–84. PubMed Abstract \| Publisher Full Text\n\nLi XJ, et al.: As an independent unfavorable prognostic factor, IL-8 promotes metastasis of nasopharyngeal carcinoma through induction of epithelial-mesenchymal transition and activation of AKT signaling. Carcinogenesis. 2012; 33: 1302–1309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung LS, Dawson CW: Epstein-Barr virus and nasopharyngeal carcinoma. Chin J Cancer. 2014; 33: 581–590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMostaan LV, et al.: Correlation between E-cadherin and CD44 adhesion molecules expression and cervical lymph node metastasis in oral tongue SCC: Predictive significance or not. Pathol. Res. Pract. 2011; 207: 448–451. PubMed Abstract \| Publisher Full Text" }	[ { "id": "90636", "date": "02 Aug 2021", "name": "Farhat Farhat", "expertise": [ "Reviewer Expertise ENT oncology" ], "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 topic for this research is interesting. For now, the recurrency of cancer including NPC is still challenging for clinicians. Exploration about the etiology of the recurrency should be done and the idea to analyze the CD44+ cancer stem cell is interesting. Associating the CD44+ cancer stem cell with the histopathologic type is interesting due to the variation of relation with EBV and sensitivity with radiotherapy. It is good to be focused on population or geographical distribution of the NPC patients. We all know that geographical distribution has high relation with NPC incidence. It can help for mapping the cases.\n\nThe method of this research is quite clear and complete. The antibody monoclonal that is used for IHC is mentioned. The effort to decrease bias and the references for determining CD44 expression scores are also done and mentioned. Statistical analysis is also clear enough. The ethic approval from ethical committee is also mentioned in the method.\n\nTable and Figure are quite representative. It will be good if the p value is also put on the table.\n\nThe insignificant result may be caused by several factors, and I agree that the sample size may also contribute to the result.\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": "6992", "date": "04 Aug 2021", "name": "Muhtarum Yusuf", "role": "Author Response", "response": "Thank you to the reviewer for the suggestion. Authors think about the possibility of being insignificant for the result of this publication." } ] }, { "id": "90635", "date": "02 Aug 2021", "name": "Mohd Razif Mohamad Yunus", "expertise": [ "Reviewer Expertise clinical head and neck oncology" ], "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 a good research for the local Indonesian area and data. Reference to their own local data is excellent. The authors have done up to their capabilities at their centre.\nRecommendations:\nThe authors may expand about the limitations of the study and why - possibly due to budget, poor response from the patients etc. as the incidence of NPC in Indonesia is high.\n\nMay expand the use of having these markers in treatment strategies, possibly as follow up measures.\n\nThe target sample size should be calculated and included in the methods. This is important to get the significant results of the study. Based on the NPC incidence in Indonesia, the sample size for significant results can be calculated.\n\nMultiple centres - collection of sample from other hospital can be considered to get the numbers.\n\nIt is difficult to give conclusion as stated because the number of sample is not representative of the NPC incidence in Indonesia.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6993", "date": "04 Aug 2021", "name": "Muhtarum Yusuf", "role": "Author Response", "response": "1. The authors may expand about the limitations of the study and why -possibly due to budget, poor response from the patients etc. as the incidence of NPC in Indonesia is high. Because the research is limited in time so that the fulfillment of the minimum number of samples to pass the IHC staining on paraffin blocks is quite difficult. In addition, the researchers only examined the expression of CD44 cancer stem cells without examining CD44 activity. Therefore, the explanation and role of CD44 in NPC stem cells will be more detailed, especially when assessed based on the activity of Shh and Bmi-1 proteins. 2. May expand the use of having these markers in treatment strategies, possibly as follow up measures. We might want to use this marker as follow up measures for NPC patients who have undergone chemotherapy. But from this study it has insignificant result so we cannot recommend CD44 as a targeted therapy yet. Therefore, further research is needed, especially those that represent each type of WHO representatively 3. The target sample size should be calculated and included in the methods. This is important to get the significant results of the study. Based on the NPC incidence in Indonesia, the sample size for significant results can be calculated. The sample size in this study was calculated using the cross sectional research formula: Total sample size = [(Za).2p . q] : d2 = (1,962x 0,5 x 0,5) : 0,152 = 42,38 (rounded to 43 sample) note: n = total sample size Z = Standard deviation (α = 0,05 with Z = 1,96) p = 0,5 From these calculations, the sample size is sufficient 4. Multiple centres - collection of sample from other hospital can be considered to get the numbers. We are thinking of doing research from multicentre in order to get better external validity 5. It is difficult to give conclusion as stated because the number of sample is not representative of the NPC incidence in Indonesia. This research would be better if it was conducted with a larger number of samples and multicentre. In addition, the sample in this study did not have an equivalent distribution of the number of WHO types. The largest sample in this study was WHO types III-IV with WHO types I and II samples not being adequately represented Thanks to reviewer for better advice." } ] } ]	1	https://f1000research.com/articles/10-678
https://f1000research.com/articles/10-1110/v1	03 Nov 21	{ "type": "Research Article", "title": "COVIDSpread: real-time prediction of COVID-19 spread based on time-series modelling", "authors": [ "Siroos Shahriari", "Taha Hossein Rashidi", "AKM Azad", "Fatemeh Vafaee", "Siroos Shahriari", "AKM Azad" ], "abstract": "A substantial amount of data about the COVID-19 pandemic is generated every day. Yet, data streaming, while considerably visualized, is not accompanied with modelling techniques to provide real-time insights. This study introduces a unified platform, COVIDSpread, which integrates visualization capabilities with advanced statistical methods for predicting the virus spread in the short run, using real-time data. The platform uses time series models to capture any possible non-linearity in the data. COVIDSpread enables lay users, and experts, to examine the data and develop several customized models with different restrictions such as models developed for a specific time window of the data. COVIDSpread is available here: http://vafaeelab.com/COVID19TS.html.", "keywords": [ "COVID-19", "SARS-CoV-2", "prediction of spread", "time series modelling" ], "content": "Introduction\n\nThe pandemic of coronavirus disease 2019 (COVID-19), caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents the most serious public health threat during the last century.1 The global impact of COVID-19 has been profound. As of 12 September 2021, over 224 million cases and 4 million deaths have been confirmed globally.2 Forecasting the imminent spread of COVID-19 informs policymaking and enables an evidence-based allocation of medical resources, arrangement of production activities and economic development.3 Therefore, it is urgent to establish efficient trend prediction models, on the latest available data, to provide a point of reference for the governments to formulate adaptive responses based on reliable predictions on the impending progress of the pandemic.\n\nThe classical Susceptible-[Exposed]-Infected-Recovered (SEIR/SIR) epidemic models,4 have been widely developed to simulate the transmission dynamics of COVID-195,6 and the impact of non-therapeutic interventions – e.g., travel and border restrictions,7,8 quarantines and isolations,5,9–11 or social distancing and closure of facilities – on the spread of the pandemic, and in some cases, on the healthcare demand.5,9,11–13 These studies have been mostly focused on calibrating models for a specific country/region based on the data at the time of the model-development and assuming a multitude of parameters initialized upon prior knowledge such as social contact structure, rate of compliance with the policy and incubation or infection period among others. Complementing upon SEIR mathematical models, and owing to the increased amount of data and consistency of reports, some recent efforts have been focused on developing statistical3,14 or machine learning methods15 to predict the near-future spread of COVID-19 (in terms of the number of confirmed cases or deaths) based on the historical data.\n\nWhile reliable predictions of the pandemic trend are essential for policymaking and resource-allocation, there is a lack of an adaptive real-time modelling platform which evolves as new data arrives. Here, we present, COVIDSpread, a time-series online platform for real-time modelling of the progression of COVID-19 using the Autoregressive Integrated Moving Average (ARIMA)16 statistical analyses combined with different non-linear transformation approaches.17 Our platform offers an interactive online dashboard which efficiently generates country-wise predictive models, in real-time, based on the latest report of COVID-19 cases worldwide.\n\nThe proposed modelling approach neither relies on strict modelling assumptions (e.g., linearity, stationarity, or existence of an epidemic steady state) nor on any initial parameters requiring a priori knowledge. It offers a transparent mathematical function to better understand the trend and to predict future points in the series. Different types of transformation have been examined to capture the nonlinearity in the time-series data followed by multiple differencing steps to eliminate the non-stationarity status.\n\nThe main objective of this study is to introduce an easy-to-use and readily available statistical tool to develop rigor models for time series data of COVID-19 as data becomes available on a real time basis. In this article, it is demonstrated that the proposed modelling tool is reliable to estimate accurate model parameters and predict the short-term spread of COVID-19 across different countries.\n\n\nMethods\n\nThe structure of the data and the autocorrelation between daily reported instances makes an intuitive case for time series analyses. Autocorrelation occurs when there is a correlation between ordered observations in time or space resulting in the covariance between the error terms being nonzero. In the context of the ordinary least square method, where the dependent variable (even if it is observed for multiple times) is regressed only against the explanatory variables rather than previous observation of the dependent variable, the estimated parameters remain unbiased (their expected value is equal to the true values), and asymptotically normally distributed. However, the coefficients are not any-more efficient (not having the minimum variance) which means that, the commonly used, t and F statistics are not any more reliable.\n\nIf we define Yt as the dependent variable, i.e., the number of confirmed cases, and Xt as the explanatory variables, i.e., an intervention, γ being a parameter to be estimated, and εt as the residual at time t; in a typical linear regression models, we have Yt=γXt+εt which can be simply written as Yt−1=γXt−1+εt−1 for one time interval earlier than t. This can be extended for s time intervals earlier as Yt−s=γXt−s+εt−s. To examine the first order autocorrelation (s = 1, i.e., only the correlation between residuals are considered which can be simply translated to the dependency between Yt and Yt−1), the unobserved part of the error terms are correlated and denoted by εt=ρεt−1+ϵt, whereϵt is white noise series and ρ can be estimated which is the main factor for examining the strength of autocorrelation as well as correcting the estimated coefficients.\n\nTime series models implicitly assume that the stochastic process is stationary which loosely implies that mean and variance of data do not change over time. Then by using autoregressive regression (AR, as explained in the previous paragraph) and a moving average (MA, as explained in the next paragraph) mechanism, unbiased parameters can be estimated. If the data is not stationary, there are ways to transform it to stationary data such as by differencing i times which is denoted as I(i), integrated of order i.\n\nWhen Yt is defined for one side, i.e., dependent on the past, and it is weighted by parameters, say θi for the earlier s time intervals), a moving average model is constructed as:\n\nWhen the data includes both the impact of scaled white noise and previous instances of the dependent variable affecting the current situation and ARIMA model is used. Unlike the compartmental models in epidemiology (e.g., SIR/SIER), ARIMA does not require exogeneous information about the susceptible population and recovery patterns. Instead, it captures the declining or increasing pattern of the data by extracting information from the nonlinear trends observed in the previous time intervals.\n\nThe platform retrieves daily number of confirmed cases from Coronavirus Resource Centre at John Hopkins University using coronavirus R package. Yet, it is independent of data source and can incorporate other major COVID-19 reporting parties (on countries, provinces and territories time-series), and can be readily extended to model number of deaths or recovered cases. John Hopkins reports latest available public data on COVID-19 on daily bases for all affected countries; latest data can be directly accessed from R environment. Countries with more than 30 reporting days from >50 cases were retained by the dashboard assuming that filtered countries do not hold enough data for reliable modelling/forecasting. At the time of submission 195 counties pass this constraint and modelled by COVIDSpread. The number of countries modelled increases continuously, as number of observations increases daily.\n\nThe data driven approach of this study employs three transformation operations including Ratio transformation (the ratio of observations in two consecutive days), power transformation (nth root) and logarithmic (natural log) transformation to stabilize variance in raw data and adjust the historical data for a simpler forecasting task. Models on transformed data were compared against the models developed for the non-transformed data and the best overall model were selected.\n\nTime series models often assume stationary time-series which implies that statistical properties such as mean, variance, autocorrelation, etc. are constant over time Other than transformation, differencing once or twice (i.e., differencing between the values of consecutive data points) helps estimating the speed of growth or the acceleration/deceleration of growth. If data is non-stationary based on augmented Dickey–Fuller (ADF) test, the differencing step were applied one or more times to eliminate the non-stationarity (ADF p-value > 0.05). The differencing step allows to develop a model that is comparable to models developed based on in the SIR\\SIER models. The differencing will continue until the stationary status is obtained.\n\nOnce stationary data is obtained, the best ARIMA (p, i, q) model were fitted to each time-series by searching through different combinations of p which is the order of the autoregressive model, i is the degree of differencing (as previously discussed), and q is the order of the moving-average model. The ‘auto.arima’ function in the R ‘forecast’ package has been used for model optimisation using non-stepwise selection. The selection of the best model is based on the root mean square error (RMSE) value estimated based on an out of sample estimation process on the latest 20% part of the observations (as a rough estimate of out-sample RMSE). The best model would be then used for prediction of the disease spread in the next N days (defined by the user) using the parameters of the model. The developed models can be then used to forecast changes in the number of infected cases. The prediction algorithm simulates the expected total number of infected cases as well as a bandwidth around the expected values reflecting the 80%-95% confidence level which is estimated based on the significance of the estimated parameters.\n\nThe whole pipeline including automated data retrieval, pre-processing and modelling, has been implemented in R, providing a unified platform for ease of reuse and maintenance. The online dashboard has been developed using R Shiny. Scheduled data updates were automated via a reactive file reader. Interactive line plots and maps were visualised using R-integrated Plotly and Leaflet JavaScript graphing libraries, respectively. We recommend clearing the browser autocomplete history to delete previous selections from the date-picker. The shiny web server can run in any modern web browser including Google Chrome, Mozilla Firefox, and Safari. Moreover, the COVIDSpread source code can be run using the RStudio IDE on a standard workstation (Windows/Linux/Mac) with an i5 processor and 8 GB RAM.\n\n\nResults\n\nMultiple transformation operations are investigated to stabilise variance, coupled with recursive differencing until eliminating non-stationarity in the time-series data, i.e., p-value < 0.05 based on augmented Dickey–Fuller test.16 Upon each transformation, the best ARIMA model is obtained for each country, according to Akaike information criterion (AIC) value using maximum likelihood estimation. The optimal model for each transformation is then recorded based on the overall model Root Mean Square Error (RMSE) on the last 20% of observations reported as a surrogate estimate of out-sample prediction performance where models are trained on the first 80% of data. The predictive power of the best model per country is compared against estimations provided through exponential growth in number of cases including, 1) doubling time of two days, 2) doubling time of three days and 3) doubling time of one week, as well as a conventional linear univariate regression on log-transformed data. Extended Table 1 shows the parameters of the optimal ARIMA model per country and the corresponding RMSE measures (of the last 20% of observations) compared with conventional trends using data obtained on 23rd April 2021 from Coronavirus Resource Centre at John Hopkins University (https://coronavirus.jhu.edu). While, the purpose of this study is not to develop the most accurate time-series predictive model, statistics of the Extended Table (Extended Table 1 shows that using a more sophisticated statistical model significantly improves the prediction accuracy of COVID-19 spread in the near future (Wilcoxon test p-value << 0.001 comparing distributions of residuals).\n\nDifferent time-series transformation operations, namely power transformation, logarithmic transformation and ratio transformation, have been applied to pre-process the data prior to the differencing step. We have observed that the type of transformation can significantly improve the performance of a model (in terms of the estimated out-sample RSME) as there is no a priori knowledge about the best-performing transformation (except that power transformation always performs poorly). Figure 1 shows some countries, as case studies, whose ARIMA models (as of April 23, 2021) are significantly affected by the type of transformation. As Figure 1 shows models on countries such as Zimbawe and Burundi has better performance with logarithmic transformation. For Nepal, Argentina and Greece, ratio transformations provide superior results. The case of Eswatini, interestingly, demonstrates that the ratio and logarithmic transformations outperform without transformation because the model can capture rapid fluctuation better with those transformations. Overall, the results signify the value of a performance-driven transformation selection approach upon trying multiple operations, as implemented in this platform.\n\nSix countries were selected as case studies to demonstrate the effect of ratio and logarithmic transformations on the model performance as measured by RMSE on last (most recent) 20% of time-series data. The solid line shows the observed trend and the dashed lines shows model fitted values without transformation (red) and after ratio (green) or logarithmic (blue) transformation. The bar plot beside each trend graph shows the corresponding RMSE estimations.\n\nThe nonlinear dynamic system underlying COVID-19 spread is producing a regularly disrupted pattern making static predictions increasingly unreliable. Accordingly, a powerful feature of the platform is dynamic model estimation, that is, all models are re-optimised temporally with availability of new daily observations. Accordingly, the latest reports on COVID-19 case numbers are reflected in model estimation which accounts for the impact of new interventions improving the reliability of the future forecasts. As a case study, we have chosen to show the value of this feature on prediction of future case numbers in Iran. Iran’s trend shows significant fluctuations in the last 10 days (as of April 13, 2020) offering an interesting case study. We assumed that the model has access to data up to April 03, and then reported the next 10 days predictions and the RMSE of predicted number on April 13th. This procedure was repeated 9 times, where new observations became available to the model, one at the time. Figure 2 shows how such dynamic re-estimation adjusts the model with emerging pattern in time-series trend and improves prediction accuracy.\n\nA. Predictions of next 10 days for COVID trend in Iran, assuming that the last available date was April 03 2020 to April 11 2020 as marked on the plot (last obsereved date at the time of analysis: April 13). Solid line shows observed cases. B. RMSE comparing predictions with obsereved data at April 13.\n\nWe have developed an interactive online dashboard to facilitate real-time model development for lay users as well as data scientists (Figure 3). Users can select the country of interest from the left panel and observe an interactive visualisation on cumulative counts of confirmed cases in the middle panel. Upon pressing the ‘Predict’ button’, the platform provides users with optimal models fitted to the latest reports of COVID-19 spared as provided by John Hopkins University Coronavirus Resource Centre. For any country of interest, the interactive user-interface enables users to re-estimate models by customising the range of days to be included in the model. The right panel visualises the cumulative number of confirmed cases since the 1000th case of top 10 countries in terms of total number of cases, plus predictions of growth trajectories in the next 10 days. Similarly, the middle-button panel shows the world map color-coded with predicted number of cases per 100K, together providing a global comparative view of the forthcoming COVID-19 spread. The dashboard back-end, i.e., data mining, pre-processing, and model development were implemented in R with several R packages including forecast, tseries, tsir, imputeTS, and coronavirus. The front-end of the dashboard was implemented in R shiny with several R packages, including rplotly, ggplot2, ggiraph, leaflet, DT, sparkline, data.table, survival, tidyr, and shinyWidgets. Having a single codebase for the whole framework is useful, especially in the context of reproducibility and ongoing maintenance. This dashboard offers users to not only view information in an interactive manner (e.g., on mouse hover), but also allows to download the parameters of the selected model used for the forecasting.\n\n\nDiscussion\n\nReal-time COVID-19 data analytics have been mainly focused on visualizing the spread18 with comparatively less effort in developing models to dynamically analyze the data. Epidemiological models, i.e., SIR/SIER models, have a strong foundation in analyzing epidemic growth/decline, and have been substantially explored for modelling the speed of infectious disease progression. Yet, such models are often offline/static, require assumptions for the parametric formulation of the model and rely on multitude of initial parameters.\n\nAside from SIR/SIER models, several models have been used to predict COVID-19 cases, including ARIMA, nonlinear autoregression neural network (NARNN), support vector regression (SVR), Prophet, and different deep neural network-based models such as long short-term memory (LSTM)/Stacked LSTM, Convolutional LSTM, and Bidirectional LSTM.\n\nTomar and Gupta19 and Chimmula and Zhang20 are two recent studies that concentrate on the LSTM model. In a similar vein, Shastri, et al.21 developed LSTM, Stacked LSTM, Bidirectional LSTM, and Convolutional LSTM models for COVID-19 cases prediction. Another area of study is the use of a recurrent neural network (RNN) to predict possible COVID-19 cases which was followed by Arora, et al.22 with proposed models based on RNN, LSTM, Bi-LSTM. Similarly, Hawas23 used RNN for daily COVID-19 infection predictions.\n\nAside from LSTM and RNN, one of the most commonly used models is the ARIMA time series model. Several studies focused on ARIMA models for predicting future cases including Alzahrani, et al.24 with forecasting the spread of COVID-19 cases based on the ARIMA model and Shahid, et al.25 focusing on ARIMA, SVR, LSTM and Bi-LSTM models.\n\nAlong the same vein, Ribeiro, et al.26 compared ARIMA, cubist regression (CUBIST), random forest (RF), ridge regression (RIDGE), SVR, and stacking-ensemble learning for the prediction of COVID-19 cumulative cases. Similarly, Kırbaş, et al.27 focused on ARIMA, nonlinear autoregression neural network (NARNN) and LSTM for future case prediction. Likewise, Papastefanopoulos, et al.28 provided a comparison on six different forecasting methods including ARIMA, the Holt-Winters additive model (HWAAS), TBAT, Facebook’s Prophet and deep AR for active case prediction and Devaraj, et al.29 evaluated ARIMA, LSTM and SLSTM for COVID-19 prediction.\n\nWe developed a time-series based statistical model to dynamically predict the future trend of COVD-19 spread. It is coupled with the capacity of time-series models in 1) considering higher orders of derivatives of the number of cases in previous time intervals, 2) accounting for the impact of residuals of the previous time intervals. The literature demonstrates a diverse set of short-term forecasting models, although the focus of this research is on ARIMA models, other models may be considered if the developed model does not perform adequately. For example, as shown by the results, ARIMA (0,2,0) was the best-fitting ARIMA model for India, with high RMSE values. This means that the model does not include any autoregressive or moving average terms and does not provide a suitable representative of COVID-19 cases in India at the investigated time period.\n\nDifferent models for analyzing COVID-spread in India can be used as an alternative as several studies have been conducted to model COVID-19 spread in India using other models. For instance, Tomar and Gupta19 used the LSTM model and Arti and Bhatnagar30 proposed a tree-based model to model spread the disease in the community. Bherwani, et al.31 used the SEIR model to model the spread of COVID-19 while Mahajan, et al.32 used the compartmental epidemic model SIPHERD for modelling spread COVID-19 cases in India. In the same line, Roy and Roy Bhattacharya33 proposed A mathematical model based on a differential equation to demonstrate how the number of asymptomatic patients grows over time and Kumari, et al.34 proposed multiple linear regression with autoregression used to predict the possible number of cases in the future.\n\nSimilarly, models based on countries like Brazil and Italy do not work well with high RMSE values, meaning that other models can be used instead. Other studies with focus on Brazil used models such as SIR,35 Holt36 and artificial intelligence (AI) models.37 Studies on Italy focused on extended SIR (eSIR) models38 and mathematical models with a Gaussian error function type.39\n\nAs shown in the results, ARIMA (5,2,5) was the best-fitting ARIMA model for Panama, Iran, and Spain when data was not transformed. Despite having the same model structure, the established model for Panama outperforms Iran and Spain with a lower RMSE values. The explanation may be due to a fluctuation in the number of cases in Iran, such as the rapid spread of COVID-19 in Iran at the start of the pandemic, and misunderstandings that led to ignoring the issue of social distance while eliminating travel restrictions one by one in April 2020 resulting in the reappearance of the virus.40 Another reason may be that the Iran COVID-19 data behavior is more complicated due to the geographical correlation of cases in Iran41 and the variation caused by sudden rises in the number of cases in different parts of the country at different times.\n\nAnother reason for the models’ disparities in performance could be the effect of weather factors on COVID-19 cases, which is being investigated by Fernández-Ahúja and Martínez.42 While Panama has a tropical maritime climate, Spain is home to four distinct climates. Climate variables clarify some key aspects of COVID-19 spread in Spain42 that are not captured by ARIMA models, whereas the influence of such variables in Panama will be less due to more consistent weather. In the same line, Gupta, et al.43 investigated the impact of weather on COVID-19 spread in the United States, while the derived model for the United States of America has a high RMSE value. Other models has been used for Iran such as LSTM,44 Recursive-based prediction model, Boltzmann function-based prediction model and Beesham’s prediction model.45 LSTM46 and SEIR47 have been employed to model the cases in USA and Spain.\n\nWhile the developed model works well in African countries like Uganda, Congo, and Algeria, the model for South Africa has a high RMSE value, indicating that different models should be used for this country which is investigated by Ding, et al.48 with SIR model and Reddy, et al.49 with a set of nonlinear growth models and Nadim and Chattopadhyay50 with mathematical model considering the imperfect lockdown effect. Similarly, the developed model for Ethiopia shows high RMSE value. Other studies researched on COVID-19 cases in Ethiopia with machine learning algorithms51 and mathematical models based on susceptible, exposed, symptomatically infected, asymptomatically infected, hospitalized and recovered/immune compartments.52\n\nAs results show, developing models for a number of countries, including China and Eswatini, performs better on transformed results. Both countries have seen a rapid fluctuation in the number of cases, which can be due to a change in how the virus is diagnosed and the number of diagnostic tests conducted. While data transformation in ARIMA models can help to improve model performance by removing skewness and fluctuation from the original data, other data processing methods such as machine learning can be used for data preprotein as discussed by Pinter, et al.53 to improve the performance of models such as SIR models. In the same line, Other models for predicting new cases in China and Brazil that are developing AI and data-driven models include a data pre-processing phase.54,55 Table 1 summarises the section and the studies conducted on countries where the developed ARIMA model does not provide adequate performance.\n\n\nConclusion\n\nIn this study, we presented an automated modelling platform that delves into multiple layers of information in the COVID-19 time series data to find the best fit with the aim of providing robust forecasts. COVIDSpread was shown to be effective in estimating the trend of the pandemic for each country. We elaborated the importance of data transformation as a preprocessing step and shown that there is no transformation operation which consistently provides the best fit to the data. Hence, exploring multiple options are recommended to stabilize variations prior to modelling using conventional econometrics formulations. A unique aspect of the presented platform is that it facilitates real-time model development incorporating latest reported data into modelling. We have shown that such adaptive model estimation significantly improves the prediction power and therefore, forecasting reliability.\n\n\nData availability\n\nThe platform retrieves daily number of confirmed cases from Coronavirus Resource Centre at John Hopkins University using coronavirus R package.\n\nZenodo: VafaeeLab/COVIDSpread: First release of COVIDSpread, https://doi.org/10.5281/zenodo.5587835.56\n\nThis project contains the following extended data:\n\n- Extended-Table-1.xlsx (represents model specifications and performance across 162 countries as of the time of submission)\n\n\nSoftware availability\n\nCOVIDSpread is available online: http://vafaeelab.com/COVID19TS.html\n\nAll the codes, including Shiny app is available at the project GitHub Repository: https://github.com/VafaeeLab/COVIDSpread\n\nArchived code as at time of publication: https://doi.org/10.5281/zenodo.558783556\n\nLicense: Apache License, Version 2.0", "appendix": "Acknowledgments\n\nSS and THR acknowledge the support from the Australian Research Council under Linkage Scheme (LP160100450). THR acknowledges the support from the Australian research Council under the DECRA Scheme (DE170101346). An earlier version of this article can be found on medRxiv (doi: https://doi.org/10.1101/2020.04.24.20078923).\n\n\nReferences\n\nFetzer T, et al.: Pandemics and social capital: From the Spanish flu of 1918-19 to COVID-19.2020.\n\nCOVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). ArcGIS. Johns Hopkins University;12 September 2021.\n\nPetropoulos F, Makridakis S: Forecasting the novel coronavirus COVID-19. PLoS One. 2020; 15: e0231236. PubMed Abstract \| Publisher Full Text \| Free Full Text .\n\nLiu Y, Gayle AA, Wilder-Smith A, et al.: The reproductive number of COVID-19 is higher compared to SARS coronavirus. J. Travel Med. 2020; 27. 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Publisher Full Text\n\nDevaraj J, et al.: Forecasting of COVID-19 cases using deep learning models: Is it reliable and practically significant?. Results in Physics. 2021; 21: 103817. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArti M, Bhatnagar K: Modeling and predictions for COVID 19 spread in India.2020.no. April.\n\nBherwani H, Gupta A, Anjum S, et al.: Exploring dependence of COVID-19 on environmental factors and spread prediction in India. npj Climate and Atmospheric Science. 2020; 3: 1–13. Publisher Full Text\n\nMahajan A, Sivadas NA, Solanki R: An epidemic model SIPHERD and its application for prediction of the spread of COVID-19 infection in India. Chaos, Solitons Fractals. 2020; 140: 110156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoy S, Roy Bhattacharya K: Spread of COVID-19 in India: A mathematical model.2020.SSRN 3587212.\n\nKumari R, et al.: Analysis and predictions of spread, recovery, and death caused by COVID-19 in India. Big Data Mining and Analytics. 2021; 4: 65–75. Publisher Full Text\n\nBastos SB, Cajueiro DO: Modeling and forecasting the early evolution of the Covid-19 pandemic in Brazil. Sci. Rep. 2020; 10: 1–10. Publisher Full Text\n\nMartinez EZ, Aragon DC, Nunes AA: Short-term forecasting of daily COVID-19 cases in Brazil by using the Holt’s model. Rev. Soc. Bras. Med. Trop. 2020; 53. Publisher Full Text\n\nda Silva RG , Ribeiro MHDM, Mariani VC, et al.: Forecasting Brazilian and American COVID-19 cases based on artificial intelligence coupled with climatic exogenous variables. Chaos, Solitons Fractals. 2020; 139: 110027. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWangping J, et al.: Extended SIR prediction of the epidemics trend of COVID-19 in Italy and compared with Hunan, China. Front. Med. 2020; 7: 169. Publisher Full Text\n\nCiufolini I, Paolozzi A: Mathematical prediction of the time evolution of the COVID-19 pandemic in Italy by a Gauss error function and Monte Carlo simulations. The European Physical Journal Plus. 2020; 135: 355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhanbari B: On forecasting the spread of the COVID-19 in Iran: The second wave. Chaos, Solitons Fractals. 2020; 140: 110176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamírez-Aldana R, Gomez-Verjan JC, Bello-Chavolla OY: Spatial analysis of COVID-19 spread in Iran: Insights into geographical and structural transmission determinants at a province level. PLoS Negl. Trop. Dis. 2020; 14: e0008875. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández-Ahúja JML, Martínez JLF: Effects of climate variables on the COVID-19 outbreak in Spain. Int. J. Hyg. Environ. Health. 2021; 234: 113723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta S, et al.: Effect of weather on COVID-19 spread in the US: A prediction model for India in 2020. Sci. Total Environ. 2020; 728: 138860.\n\nWang P, et al.: Time series prediction for the epidemic trends of COVID-19 using the improved LSTM deep learning method: Case studies in Russia, Peru and Iran. Chaos, Solitons Fractals. 2020; 140: 110214.\n\nNiazkar M, et al.: Assessment of Three Mathematical Prediction Models for Forecasting the COVID-19 Outbreak in Iran and Turkey. Comput. Math. Methods Med. 2020. 2020.\n\nKumar M, et al.: Spreading of COVID-19 in India, Italy, Japan, Spain, UK, US: A prediction using ARIMA and LSTM model. Digital Government: Research and Practice. 2020; 1(4): 1–9.\n\nEfimov D, Ushirobira R: On an interval prediction of COVID-19 development based on a SEIR epidemic model. Annu. Rev. Control. 2021.\n\nDing W, et al.: Analysis and prediction of COVID-19 epidemic in South Africa. ISA Trans. 2021.\n\nReddy T, et al.: Short-term real-time prediction of total number of reported COVID-19 cases and deaths in South Africa: A data driven approach. BMC Med. Res. Methodol. 2021; 21(1): 1–11.\n\nNadim SS, Chattopadhyay J: Occurrence of backward bifurcation and prediction of disease transmission with imperfect lockdown: A case study on COVID-19. Chaos, Solitons Fractals. 2020; 140: 110163.\n\nKhakharia A, et al.: Outbreak prediction of COVID-19 for dense and populated countries using machine learning. Annals of Data Science. 2021; 8(1): 1–19.\n\nDeressa CT, Duressa GF: Modeling and optimal control analysis of transmission dynamics of COVID-19: The case of Ethiopia. Alex. Eng. J. 2021; 60(1): 719–732.\n\nPinter G, Felde I, Mosavi A, et al.: COVID-19 pandemic prediction for Hungary; a hybrid machine learning approach. Mathematics. 2020; 8: 890.\n\nPereira IG, et al.: Forecasting Covid-19 dynamics in Brazil: A data driven approach. Int. J. Environ. Res. Public Health. 2020; 17: 5115.\n\nZheng N, et al.: Predicting COVID-19 in China using hybrid AI model. IEEE Transactions on Cybernetics. 2020; 50: 2891–2904.\n\nVafaeeLab: VafaeeLab/COVIDSpread: First release of COVIDSpread (Version v1). Zenodo. 2021. Publisher Full Text" }	[ { "id": "123128", "date": "31 Mar 2022", "name": "Mahdieh Allahviranloo", "expertise": [ "Reviewer Expertise Transportation", "Big data", "Machine learning" ], "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 presents an interactive dashboard which models the real-time spread of COVID-19 using time-series analysis, specifically Autoregressive Integrated moving average (ARIMA). The analysis does not rely on heavy assumption and simply generates the spread which can be utilized to predict short term spread. The paper is very well-written and easy to read. It also addresses an ongoing problem in the world, which is a plus. The design of the platform is very well thought and is user-friendly.\nHowever, three main comments can be made to the paper:\nThe first comment is related to the spread of COVID-19. Spread of COVID depends on the proximity of the people and also the density of the population in a given area (crowdedness). How spatial parameters are factored in here? In fact, the impacts of density and concentration of crowd in areas, the attributes of the built environment (e.g. percentage of indoor, outdoor areas in the analysis site) are other factors that can be taken into account or at least being factored as a coefficient in the types of models. A discussion on further expansion of the study to incorporate these parameters is preferred.\n\nI think depending on the scale of the regions, (neighborhood level, to city level to regional level), a tuning step is necessary in the model to account for these changes. Can the spread be illustrated and updated in different spatial granularity level? What modifications to the models are required to address different scenarios?\n\nAs the time has passed, the size of collected data on COVID-19 and its variants has increased. I would suggest authors to revise their models according to latest data from the available data repositories.\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": "8512", "date": "20 Jul 2022", "name": "Fatemeh Vafaee", "role": "Author Response", "response": "We would like to thank the reviewer for her time and valuable comments. The comments are addressed below: \"The first comment is related to the spread of COVID-19. Spread of COVID depends on the proximity of the people and also the density of the population in a given area (crowdedness). How spatial parameters are factored in here? In fact, the impacts of density and concentration of crowd in areas, the attributes of the built environment (e.g. percentage of indoor, outdoor areas in the analysis site) are other factors that can be taken into account or at least being factored as a coefficient in the types of models. A discussion on further expansion of the study to incorporate these parameters is preferred.\" Response: We acknowledge the reviewer’s comment. We would like to stress that due to the nature of time series models each prediction is based on the previous lagged observations and residual error from a moving average model applied to lagged observations. One difference from standard linear regression is that the data are not necessarily independent. As a result, the impact of spatial parameters such as density, the concentration of crowd in areas, and the attributes of the built environment (e.g. percentage of indoor, and outdoor areas in the analysis site) are included in the model for each prediction, spatial parameters had their impact on the previous observations (as long as no changes applied to these variables). The model should be altered using exogenous regressors to account for the influence of each parameter in the event that any change occurs for the parameters (such as concentration of crowd in particular places). \"I think depending on the scale of the regions, (neighborhood level, to city level to regional level), a tuning step is necessary in the model to account for these changes. Can the spread be illustrated and updated in different spatial granularity level? What modifications to the models are required to address different scenarios?\" Response: We appreciate receiving this comment. As mentioned in the previous comment, due to the nature of time series models, the scale of spread would be incorporated into the model (regressor of lagged observations). However, for converting the spread (calculated for a specific region) to another region, scaling factors could be defined based on variables (such as the ratio of population portion of two regions). \"As the time has passed, the size of collected data on COVID-19 and its variants has increased. I would suggest authors to revise their models according to latest data from the available data repositories.\" Response: There are examples provided in the manuscript regarding the model application (such as the effect of transformation and dynamic model estimation). The focus of such examples is to show model performance and features using a subset of data. As a result, updating the model with the latest data would not alter the point of those examples. On the other hand, the dashboard is constantly updated with the latest collected data on COVID-19." } ] }, { "id": "247250", "date": "04 Mar 2024", "name": "Ross Gore", "expertise": [ "Reviewer Expertise data science", "predictive analytics", "modeling and simulation" ], "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\nWhile reliable predictions of the pandemic trend are essential for policymaking and resource-allocation, there is a lack of an adaptive real-time modelling platform which evolves as new data arrives. … Our platform offers an interactive online dashboard which efficiently generates country-wise predictive models, in real-time, based on the latest report of COVID-19 cases worldwide .. The main objective of this study is to introduce an easy-to-use and readily available statistical tool to develop rigor models for time series data of COVID-19 as data becomes available on a real time basis.” This motivation could be improved by describing exactly how this platform will fit into a policymaking and resource-allocation workflow so that decisions can be made regularly by policy makers. Specifically, what current unanswered or ambiguous questions have been posed by policymakers that this platform actionably answers and what does the work/action flow look like for policy makers in the specific modeled countries when these questions/issues arise.\nThe paper would benefit from additional statistical analysis of the results presented in the paper. While the most effective model for each country is identified it is unclear if the best performing model is statistically significantly different from the presented alternatives. Conducting statistical tests to identify which model is the statistically significantly superior (or at least those with the same level of statistically significant performance) as the alternatives would benefit the paper.\nThe paper would benefit from a discussion of the limitations of the analysis. As it currently stands all the results at are the same geographic level (i.e. country). Identifying research (Ref [1,2,3,4]) that has shown that statistics related to the prevalence of respiratory viruses can depend on the geographic level of evaluation (i.e country vs. city vs. county vs. neighborhood). For example, different geographic areas experience different transmission rates of respiratory viruses. Similarly, there can be stark differences in the vaccination levels at the country vs. city vs. county vs. neighborhood level. Furthermore, the vaccines (and their associated efficacy) available to individuals in different countries can differ. Identifying exactly which the context in which the authors expect these results to generalize and would improve the validity of the paper and make it more actionable for public health professionals. References related to the importance of geographic context in understanding respiratory virus transmission (including SARS-CoV-2) are added to the review.\nThere are a nontrivial number of readers who suffer from red/green color blindness. Using a color-blindness safe color palette in the graphics in the web application, and figures 1, 3, would improve the paper. A series of color blind safe palettes are available here (https://davidmathlogic.com/colorblind/#%23D81B60-%231E88E5-%23FFC107-%23004D40) and larger text fonts would improve the readability of figures.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1110
https://f1000research.com/articles/10-906/v1	09 Sep 21	{ "type": "Research Article", "title": "Lightning Behaviour during the COVID-19 Pandemic", "authors": [ "Fazandra Yusfiandika", "Siow Chun Lim", "Chandima Gomes", "Aravind Chockalingam", "Lee Cheng Pay", "Fazandra Yusfiandika", "Chandima Gomes", "Aravind Chockalingam", "Lee Cheng Pay" ], "abstract": "Background COVID-19 has drastically dampened human activities since early 2020. Studies have shown that this has resulted in changes in air temperature and humidity. Since lightning activities are dependent on air temperature and humidity, this study is conducted to evaluate the correlation between the intensity of lightning activities with the atmospheric changes, and investigates the changes, in lightning activities due to atmospheric changes during the COVID-19 pandemic. Methods The hypothesis was tested through a t-test and Pearson’s correlation study. The variation trend of lightning strikes count (LSC) in Europe and Oceania during the five months COVID-19 lockdown period (March – July) compared to the same period in the previous five years from 2015 to 2019 is investigated. Results Statistical analysis shows the LSC in Europe and Oceania during the lockdown period dropped significantly by more than 50% and 44% respectively compared to the same period in previous five years. Furthermore, LSC was found to be positively correlated with air temperature and relative humidity in Europe. However, in Oceania, LSC seems to be only positively correlated with air temperature but negatively correlated with relative humidity. Conclusions This study seems to suggest that lightning activities have significantly changed during this pandemic due to reduction in human activities.", "keywords": [ "lightning", "air temperature", "relative humidity", "COVID-19", "thunderstorm" ], "content": "Introduction\n\nMany countries have enforced lockdown since the beginning of the COVID-19 pandemic.1-3 Energy-intensive human activities such as travelling and the hospitality sector were drastically reduced resulting in reduced emissions of greenhouse gases.4 The global CO2 emission is estimated to drop by 8.8% (−1551 Mt) in the first half-year of 2020 compared to the same period in 2019. Moreover, almost 18% of CO2 emissions in recent years were produced from ground transportation.5\n\nTherefore, the trend of temperature is expected also to be reduced. A significant positive correlation between the atmospheric temperature and CO2emission is reported in.6 Furthermore, COVID-19 lockdown has caused micro-climate changes such as localized variations in air temperature and relative humidity.7 The pandemic is also having an effect on NOX, causing a decline that could possibly lead to short-term cooling.8 Air humidity will also be affected as global warming are dependent on both temperature and humidity.9\n\nLightning, a natural atmospheric discharge, is affected by various environmental factors. Lightning brings about hazards to human life and appropriate risk assessment has to be conducted for any habitable structure.10,11 Atmospheric variables such as climate change, humidity, aerosol level, and wind motion can affect the cloud charge distribution, electric field and threshold electromagnetic fields that give rise to air breakdown. It is predicted that lightning may strike more frequently as a result of the ongoing climate change.12 The lightning intensity may also increase due to the high greenhouse gases in the atmosphere.\n\nLightning could also be triggered by aerosols released by industrial processes and transportation activities.13,14 During the lockdown period, many industrial sectors stopped operating. Thus, human activities have considerably reduced during the COVID-19 pandemic which may affect the rate of lightning. Lightning ground flash density tends to increase with drier and warmer surface air.15 The frequency of thunderstorms in Denver and Colorado shows a major peak during summer time.17 Previous studies have also found a strong relationship between relative humidity and lightning occurrence.17,18 Hence, it is of interest to investigate the correlation between the environmental changes that happened during the period of COVID-19 related restriction of human activities and the lightning occurrence density. This study is an attempt to analyse this situation. This study investigates the trend of five months of lightning occurring from March to July in 2020 compared with the same period (March-July) in 2015-2019 in Europe and Oceania. The outcomes of this work could yield interesting insights into the correlation between human activities and lightning frequency.\n\n\nMethods\n\nLightning stroke counts (LSC) and two atmospheric factors namely air temperature and relative humidity are considered as the variables in this study. The relationship between LSC with respect to air temperature and relative humidity will be statistically analysed via the dependent t-test and Pearson correlational studies.\n\nFrom March until July in Europe and Oceania, the total LSC from the year 2015 to 2020 were extracted from LightningMaps.org.19 LightningMaps.org provides historical data of LSC and has been widely used in previous studies.20,21 The distribution of LSC data is presented in Tables 1 and 2.\n\nThe air temperature and relative humidity data from March until July in Europe and Oceania from year 2020 are extracted from the Physical Sciences Laboratory using Panoply Version 4.12.0.22 Europe is divided into seven sub-regions such as North Europe, West Europe, Central Europe, East Europe, South Europe, Southeast Europe, and the British Isles. After that, eight points (57.5°N, 10.0°E; 42.5°N, 12.5°E; 50.0°N, 25.0°E; 50.0°N, 5.0°E; 50.0°N, 10.0°E; 50.0°N, 20.0°E; 52.5°N, 0.0°; 42.5°N, 22.5°E) of around the sub-regions of Europe were selected in this study. For the Oceania region, five points (−12.5°N, 132.5°E; −37.5°N, 142.5°E; −27.5°N, 152.5°E; −30.0°N, 115.0°E; −27.5°N, 135.0°E) covering the North, South, East and West of Australia; Three points (−37.5°N, 175°E; −45.0°N, 167.5°E; −42.5°N, 170.0°E) covering the North, South and Centre of New Zealand; one point (−10.0°N, 147.5°E) from Papua were considered. Tables 3 and 4 show the average value of air temperature and relative humidity in Europe and Oceania in year 2020.\n\nA dependent t-test is was conducted using Microsoft Excel 2016 (Microsoft Excel, RRID:SCR_016137) to determine whether there is a statistically significant difference between the LSC during the lockdown period in the year 2020 and the LSC in the same period (March-July) in year 2015 until 2019. The LSC is measured from a single population (Europe or Oceania) and two different timelines (before and during). Period A represents the lightning activities before lockdown period i.e. March to July in year 2015 to 2019. Period B represents the lightning activities during the lockdown i.e. March to July in the year 2020.\n\nThe t-test is conducted by comparing the data from Period B and Period A. The null hypothesis, H0 and the alternative hypothesis, Ha is defined as below:\n\nH0: There is no significant difference in lightning frequency in between Period A and Period B.\n\nHa: There is a significant difference in lightning frequency in between Period A and Period B.\n\nThe confidence level of 95% at a significant level, α=0.05 is used. This approach tests the hypothesis and calculates the probability of determining whether there is evidence to reject the null hypothesis. When the P value < 0.05, the null hypothesis is rejected, and vice versa.\n\nNext, the Pearson correlation coefficient is used to evaluate the correlation between the frequency of lightning activities with the atmospheric changes. The Pearson’s correlation coefficient, r, is computed to measure the strength of the relationship between total lightning strikes, air temperature, and relative humidity in Period B.\n\nFurthermore, the correlation between the variables was analysed using regression and correlation analyses. The significant level, P value can be obtained from the regression data analysis. The null hypothesis, H0 and the alternative hypothesis, Ha is defined as below:\n\nNull hypothesis, H0: P = 0, There is no significant relationship between lightning strikes with air temperature or relative humidity.\n\nAlternative hypothesis, HA: P ≠ 0, There is a significant relationship between lightning strikes with air temperature or relative humidity\n\nBy using the P-value method (α=0.05), the decision on rejection or acceptance of the null hypothesis can be made. There is sufficient evidence to conclude that there is a significant correlation between lightning strikes and air temperature or relative humidity as the correlation coefficient is significantly different from zero. Exact P values are provided in Table 5.\n\n\nResults\n\nFigure 1 shows the LSC has dropped significantly in the year 2020 when the lockdown started. The dependent t-test shows a statistically significant (P-value <0.05) difference between 2020 and each previous year as shown in Table 5. Notably, LSC in Europe during the five-month lockdown period were reduced by more than 50% compared to the same period in the year 2019, 2018, and 2017.\n\nFigure 2 illustrates the variation of LSC against air temperature levels in Europe. Figure 3. illustrates the relationship between LSC and relative humidity in Europe. Table 6 shows that the correlation of lightning strikes with air temperature and relative humidity in Europe are statistically significant. The Pearson correlation between lightning strikes and air temperature is 0.92, indicating a strong positive relationship between the variables. Pearson correlation between LSC and relative humidity is 0.52, indicating a moderate positive relationship between the variables. The positive correlation between lightning strikes with air temperature and relative humidity in Europe concurs with the findings of.15,17,18,23,24\n\nThere was a 44% drop in LSC from 2019 to 2020 as shown in Figure 4. Table 7 shows there is statistically significant difference between the year 2020 with all previous years except 2017. Figure 5 and Table 8 indicates a moderate positive correlation between LSC and air temperature in Oceania during the lockdown period. Unlike Europe, Figure 6 and Table 8 shows that the relationship between LSC and relative humidity in Oceania is negatively correlated. The positive correlation of LSC and air temperature is consistent with previous studies.23,24 The negative correlation of LSC and relative humidity in Oceania obtained in this study contradicted the study of.18\n\n\nConclusions\n\nIn conclusion, there was a drastic drop in LSC in Europe and Oceania during the first lockdown period in 2020. A dependent t-test confirmed that a statistically significant difference in LSC between Period A and Period B. There is a positive relationship between LSC and air temperature in Europe (r = 0.92) and Oceania (r = 0.55). Furthermore, there is a positive relationship between LSC and relative humidity in Europe (r = 0.52) but a negative relationship between LSC and relative humidity in Oceania (r = −0.54).\n\nThe differences in correlation between lightning, air temperature, and relative humidity in Europe and Oceania may also be due to other possible factors such as aerosol level, wind motions, and particulate matter. Future work should be replicated in other geographical regions such as America and Asia.\n\n\nAuthor roles\n\nFazandra Y: Conceptualization, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing;\n\nSiow C.L.: Conceptualization, Supervision, Writing – Review & Editing\n\nChandima G.: Conceptualization, Writing – Review & Editing\n\nAravind C.: Methodology, Validation\n\nLee C.P.: Validation, Supervision\n\n\nData availability statement\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nThe authors would like to thank LightningMaps.org and National Centers for Environmental Prediction (NCEP) and National Centers for Atmospheric Research (NCAR) for providing the data for this research, and Faculty of Engineering, MMU for providing the necessary support for this study.\n\n\nReferences\n\nCucinotta D, Vanelli M: WHO declares COVID-19 a pandemic. Acta Biomedica. 2020; vol. 91, no. 1. Mattioli 1885, pp. 157–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlfano V, Ercolano S: The Efficacy of Lockdown Against COVID-19: A Cross-Country Panel Analysis. Appl. Health Econ. Health Policy . 2020; 18(4): 509–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGautam AS, et al.: Pandemic induced lockdown as a boon to the Environment: trends in air pollution concentration across India. Asia Pac J Atmos Sci. 2021; no. March 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Z, et al.: Near-real-time monitoring of global CO2 emissions reveals the effects of the COVID-19 pandemic. Nat. Commun. 2020; 11(1): 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi Z, Wang HC, Tan YB, et al.: Influence of aerosols on lightning activities in central eastern parts of China. Atmos. Sci. Lett. 2020; 21(2): 1–10. Publisher Full Text\n\nLee H, Cheong HW: Effects of Carbon Dioxide and Clouds on Temperature. Procedia Computer Science. Jan. 2018, vol. 139, pp. 95–103. Publisher Full Text\n\nSingh M, Vikas V, Sharma C, et al.: Effect of Lockdown amid COVID-19 Pandemic on Weather Parameters of Mid Hill Region of Jammu District of J&K, UT. Int. J. Environ. Clim. Chang. Jul. 2020: 53–77. Publisher Full Text\n\nForster PM, et al.: Current and future global climate impacts resulting from COVID-19. Nat. Clim. Chang. Oct. 2020; 10(10): 913–919. Publisher Full Text\n\nMcKinnon KA, Poppick A: Estimating Changes in the Observed Relationship Between Humidity and Temperature Using Noncrossing Quantile Smoothing Splines. J. Agric. Biol. Environ. Stat. Sep. 2020; 25(3): 292–314. Publisher Full Text\n\nAbulaban H, Siow C: Recent Progress on Lightning Risk Assessment and its Applications in Malaysia. Int Rev Electrical Engineering (IREE). 2021; 16 (1): 41–49. Publisher Full Text\n\nFathi MAA, Lim SC, Pay ILC: Development of a Template for the Risk Assessment for Lightning Protection System Design. 2018 34th Int Conf Lightning Protection (ICLP). 2018, pp. 1–5.\n\nRomps DM, Seeley JT, Vollaro D, et al.: Projected increase in lightning strikes in the united states due to global warming. Science (80-.). 2014; 346(6211): 851–854. Publisher Full Text\n\nThornton JA, Virts KS, Holzworth RH, et al.: Lightning enhancement over major oceanic shipping lanes. Geophys. Res. Lett. 2017; 44(17): 9102–9111. Publisher Full Text\n\nPal J, Chaudhuri S, Chowdhury AR, et al.: Cloud — Aerosol interaction during lightning activity over land and ocean: Precipitation pattern assessment. Asia-Pacific J. Atmos. Sci. 2016; 52(3): 251–261. Publisher Full Text\n\nDiaz-Ortiz F, Roman F: Correlation between air surface temperature and lightning events in Colombia during the last 15 years. Asia Electromagn. Conf. ASIAEM. 2015, no. August, pp. 112–115.\n\nChaudhuri S, Middey A: Comparison of tropical and midlatitude thunderstorm characteristics anchored in thermodynamic and dynamic aspects. Asia-Pacific J. Atmos. Sci. 2014; 50(2): 179–189. Publisher Full Text\n\nWestermayer AT, Groenemeijer P, Pistotnik G, et al.: Identification of favorable environments for thunderstorms in reanalysis data. Meteorol. Zeitschrift . 2017; 26(1): 59–70. Publisher Full Text\n\nShi Z, et al.: Effects of relative humidity on electrification and lightning discharges in thunderstorms. Terr. Atmos. Ocean. Sci. 2018; 29(6): 695–708. Publisher Full Text\n\n“LightningMaps.org.” (accessed Mar. 06, 2021).Reference Source\n\nHarvey BA: Long-Term Field Study of Lightning Surges Through Traffic Monitoring In-Pavement Sensors. IEEE Trans. Ind. Appl. 2015; 51(4): 2797–2803. Publisher Full Text\n\nGarkusha IN, Hnatushenko VV, Vasyliev VV: Research of influence of atmosphere and humidity on the data of radar imaging by Sentinel-1. 2017 IEEE 37th Int. Conf. Electron. Nanotechnology, ELNANO 2017 - Proc. 2017; no. September 2018, pp. 405–408. Publisher Full Text\n\nDatasets- NCEP/NCAR Reanalysis: Pressure: NOAA Physical Sciences Laboratory. (accessed Jan. 24, 2021). Reference Source\n\nPrice C, Asfur M: Can lightning observations be used as an indicator of upper-tropospheric water vapor variability? Bull. Am. Meteorol. Soc. 2006; 87(3): 291–298. Publisher Full Text\n\nWilliams ER: Lightning and climate: A review. Atmos. Res. 2005; 76(1–4): 272–287. Publisher Full Text" }	[ { "id": "94352", "date": "16 Sep 2021", "name": "Francisco Javier Pérez Invernón", "expertise": [ "Reviewer Expertise Atmospheric electricity", "meteorology", "plasma physics." ], "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 manuscript reports an analysis of the changes in lightning, temperature and relative humidity between 2015 and 2020, including the COVID-19 lockdown period. The authors find a significant change in these variables during the lockdown. They perform a statistical analysis to find possible relationships between the temperature, relative humidity and lightning. They find a possible correlation between them.\nThe analysis of these variables is interesting, as well as finding possible relationships between them. However, the authors do not report a solid connection between the COVID-19 pandemic and the variables.\nThe topic is in the scope of F1000 Research. The manuscript is well written and organized. However, references are not updated, the method needs some more clarification, and the conclusions are not supported by the results. In addition, I miss a Discussion.\nI think the authors have to make a major revision before this manuscript can be accepted for indexing. If the authors cannot explain the possible relationship between the reduction of lightning and the pandemic, they may consider excluding the attribution of the decrease in lightning activity to the pandemic.\nI provide specific comments below:\nIntroduction\nSecond paragraph: Please mention the reduction in aerosols during the lockdown.\nThe reference provided by the authors for the relationship between CO2 emissions and temperature reports a significant positive correlation between the atmospheric temperature and CO2 emission at a scale of years. Please provide some reference for a possible correlation between CO2 and temperature at a time period similar to the lockdown (weeks-months). Jones et al. (2021)1 did not find any relationship between the CO2 reduction and changes in temperature.\nThird paragraph: Some authors have reported a possible decrease in lightning activity due to climate change (Finney et al., 2018)2.\nFourth paragraph: Please explain how aerosols are involved in lightning activity.\n\"Lightning ground flash density tends to increase with drier and warmer surface air\": Please provide some reference. In general, lightning does not tend to increase with drier surface.\nWhy mention Denver and Colorado in this study?\nPrevious studies have already investigated the possible relationship between the lockdown and lightning activity. Please cite and explain them in the introduction: Chowdhuri et al. (2020)3, Pinto Neto et al. (2020)4, Pérez-Invernón et al. (2021)5.\nMethods\nThe authors have to estimate the temporal evolution of the lightning Detection Efficiency of the sensors used by LightningMaps.org, as you will study the temporal evolution of lightning in the data set. Lightning can increase or decrease year by year due to changes in the Detection Efficiency. The Detection Efficiency of the data set can be investigated by comparison with other lightning data set, such as LIS.\nResults\nFigures 2, 3, 5 and 6: What does each point represent?\nGeneral comments and conclusions\nThis study shows that there could be a possible relationship between temperature, relative humidity and lightning in the studied regions (Europe and Oceania). However, the influence of the COVID-19 pandemic is not clear. There were significant changes in both temperature and relative humidity during the pandemic that could influence lightning. However, such changes could not be connected to the pandemic. See for example Jones et al. (2021)1, who used 12 models to produced over 300 simulations. They did not find any associated impact of the reduction of CO2 on temperature or rainfall. Changes observed in temperature and relative humidity can be due to other factors instead of the pandemic.\nIn addition, the authors find an opposite correlation between relative humidity and lightning in Europe and Oceania. The reasons for this opposite relationship are not explained.\nFinally, previous studies have found a connection between the reduction of lightning and the reduction in the concentration of aerosols during the pandemic. However, this study does not investigate the role of aerosols in lightning activity.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7281", "date": "27 Oct 2021", "name": "Chun Lim Siow", "role": "Author Response", "response": "General Comments: This manuscript reports an analysis of the changes in lightning, temperature and relative humidity between 2015 and 2020, including the COVID-19 lockdown period. The authors find a significant change in these variables during the lockdown. They perform a statistical analysis to find possible relationships between the temperature, relative humidity and lightning. They find a possible correlation between them. The analysis of these variables is interesting, as well as finding possible relationships between them. However, the authors do not report a solid connection between the COVID-19 pandemic and the variables. The topic is in the scope of F1000 Research. The manuscript is well written and organized. However, references are not updated, the method needs some more clarification, and the conclusions are not supported by the results. In addition, I miss a Discussion. I think the authors have to make a major revision before this manuscript can be accepted for indexing. If the authors cannot explain the possible relationship between the reduction of lightning and the pandemic, they may consider excluding the attribution of the decrease in lightning activity to the pandemic. Response to General Comments: Thank you for the comment. The focus of this manuscript is more on examining any statistical relationship between the lightning activities and the changes in atmospheric temperature and humidity during the pandemic. Discussion is partially included with the results and in the conclusion. At this point of research, we could only discuss it statistically and refer to any concurring literature. We have addressed the comments and incorporated the recommendations at our level best. I provide specific comments below: Comment: Introduction The reference provided by the authors for the relationship between CO2 emissions and temperature reports a significant positive correlation between the atmospheric temperature and CO2 emission at a scale of years. Please provide some reference for a possible correlation between CO2 and temperature at a time period similar to the lockdown (weeks-months). Jones et al. (2021) did not find any relationship between the CO2 reduction and changes in temperature. Response: Thank you for the comment. The recommended reference has been cited. However, we could not find any reference for similar time period to the lockdown. Here is our response: The release of carbon dioxide (CO2) from the combustion of coal, oil, and natural gas is the primary driver of rising global temperatures. These activities, as can be seen, tend to decrease during the lockdown period. As a result, the rate of temperature rise may be delayed during the lockdown period. This, however, is insufficient to cause long-term effects on CO2 levels and climate change on a worldwide scale. Comment: Third paragraph: Some authors have reported a possible decrease in lightning activity due to climate change (Finney et al., 2018). Response: Thank you for the comment. The recommended reference has been cited. Here is our response: (Finney et al., 2018) stated that many previous studies found a positive correlation between lightning and temperature, and one previous study that found lightning decreases with an increase in temperature. This may explain that such relationships become highly uncertain on longer timescales. Comment: Fourth paragraph: Please explain how aerosols are involved in lightning activity. Response: Thank you for the comment. The following has been added into fourth paragraph: Aerosol could affect lightning activity through modification of cloud micro-physics. Aerosol particles serve as cloud condensation nuclei and ice nuclei, and the amount of this particles could affect the formation of cloud droplets and ice particles. More aerosol will suppress the coalescence and making the average size of cloud droplet to be reduced as well as inhibiting precipitation. Therefore, the process enables the water droplets to rise further to upper layers of the clouds and may enhance the lightning processes. Comment: \"Lightning ground flash density tends to increase with drier and warmer surface air\": Please provide some reference. In general, lightning does not tend to increase with drier surface. Response: Thank you for the comment. We have provided reference based on the study from Diaz-Ortiz reference number 17 into third paragraph: Yes, in general lightning does not tend to increase with warmer and drier surface, but the presence of high air temperature can quickly rise and cause powerful updrafts. These updrafts carried water droplets and quickly freeze and begin collision with ice crystals and graupel which causing charge transfer process. Therefore, low temperature may slow down this process. So high temperature can be a driving factor to lightning formation. Comment: Why mention Denver and Colorado in this study? Response: Thank you for the comment. Denver and Colorado have stated that lightning peaked during summer time, which may possibly explain the high temperature leads to high lightning activity. Comment: Previous studies have already investigated the possible relationship between the lockdown and lightning activity. Please cite and explain them in the introduction: Chowdhuri et al. (2020), Pinto Neto et al. (2020), Pérez-Invernón et al. (2021). Response: Thank you for the comment. All the recommended articles are relevant and have been cited and explained accordingly. Comment: Methods The authors have to estimate the temporal evolution of the lightning Detection Efficiency of the sensors used by LightningMaps.org, as you will study the temporal evolution of lightning in the data set. Lightning can increase or decrease year by year due to changes in the Detection Efficiency. The Detection Efficiency of the data set can be investigated by comparison with other lightning data set, such as LIS. Response: Thank you for the comment. Currently, this is beyond our scope of work. We will consider this for our future work. Comment: Results Figures 2, 3, 5 and 6: What does each point represent? Response: Thank you for the comment. Each point represents the lightning strikes count (LSC). Figure 2 and 5 is the scatter plots of the LSC against air temperature. Figure 3 and 6 is the scatter plots of the LSC against humidity. General comments and conclusions This study shows that there could be a possible relationship between temperature, relative humidity and lightning in the studied regions (Europe and Oceania). However, the influence of the COVID-19 pandemic is not clear. There were significant changes in both temperature and relative humidity during the pandemic that could influence lightning. However, such changes could not be connected to the pandemic. See for example Jones et al. (2021), who used 12 models to produced over 300 simulations. They did not find any associated impact of the reduction of CO2 on temperature or rainfall. Changes observed in temperature and relative humidity can be due to other factors instead of the pandemic. Response: Thank you for the comment. We acknowledged the findings by Jones et al. (2021) as highlighted. However, we have supplemented our argument with a few supporting references. Comment: In addition, the authors find an opposite correlation between relative humidity and lightning in Europe and Oceania. The reasons for this opposite relationship are not explained. Response: Thank you for the comment. Unfortunately, it is beyond our current level of understanding to provide the exact explanation on the opposite relationship found. Higher relative humidity will lead to stronger updraft and increased lightning occurrence. However, too much vapor may weaken the updraft by blocking the vapor to rise up to complete the phase transformation. Comment: Finally, previous studies have found a connection between the reduction of lightning and the reduction in the concentration of aerosols during the pandemic. However, this study does not investigate the role of aerosols in lightning activity. Response: Thank you for the comment. Yes, we have not considered in this in this study. However, we have noted your recommendation and have since enhanced the literature review of this study based on your feedback. We will consider the role of aerosols in lightning activity in our future work." } ] }, { "id": "94274", "date": "23 Sep 2021", "name": "Wooi Chin Leong", "expertise": [ "Reviewer Expertise Lightning" ], "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 study on the lightning activities during the covid-19 pandemic periods. This article also relates lightning to air temperature and humidity. However, there are some parts of the article that can be further justified in order to improve the paper quality. Some suggestions have been made here:\nWhat is the definition of the mean value shown in table 5?\n\nPerhaps the authors can further explain the relationship between temperature and LSC. Why does low temperature mean less LSC etc?\n\nPerhaps the authors can further explain the relationship between humidity and LSC. Why does low temperature mean less LSC etc?\n\nA suggestion for the authors to discuss the relationship between months and lightning events/LSC, whether the lockdown or economic slow down affects the lightning occurrence. The authors may provide reasons to justify it 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? 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": "7282", "date": "27 Oct 2021", "name": "Chun Lim Siow", "role": "Author Response", "response": "Comment: This is an interesting study on the lightning activities during the covid-19 pandemic periods. This article also relates lightning to air temperature and humidity. However, there are some parts of the article that can be further justified in order to improve the paper quality. Some suggestions have been made here: What is the definition of the mean value shown in table 5? Response: Thank you for the comment. It actually means the mean value of lightning strikes from March-July. We have added the following to clarify: Exact P values and the mean value of lightning strikes from May to July are provided in Table 5 Comment: Perhaps the authors can further explain the relationship between temperature and LSC. Why does low temperature mean less LSC etc? Response: Thank you for the comment. We have added the following explanation in paragraph 3: When warm, wet air rises into cold air, thunderstorms form. As the warm air cools, moisture in the form of water vapour condenses into water droplets, a process known as condensation. Cooled air descends through the atmosphere, warms up, and rises again. A convection cell is a circuit of rising and descending air. A cloud will form if this happens in a small amount. A thunderstorm can arise if this happens with a lot of air and moisture. The presence of high air temperature can quickly rise and cause powerful updrafts. These updrafts carried water droplets and quickly freeze and begin collision with ice crystals and graupel which causing charge transfer process. Therefore, low temperature may slow down this process. Comment: Perhaps the authors can further explain the relationship between humidity and LSC. Why does low temperature mean less LSC etc? Response: Thank you for the comment. Studies found that higher relative humidity may enhance the upward updraft and easing the particle collision in the cloud. Lower relative humidity may lead to weaker updraft and decreased the chance of lightning occurrence. This trend has been demonstrated in Europe but not in Oceania. We have added the explanation in the Results and Discussion section under Europe. Comment: A suggestion for the authors to discuss the relationship between months and lightning events/LSC, whether the lockdown or economic slow down affects the lightning occurrence. The authors may provide reasons to justify it as well. Response: Thank you for the suggestion. The introduction section has been further enhanced by incorporating additional references to support our argument." } ] } ]	1	https://f1000research.com/articles/10-906
https://f1000research.com/articles/10-1107/v1	02 Nov 21	{ "type": "Data Note", "title": "The CKD.QLD data linkage framework: chronic kidney disease and health services utilisation in Queensland, Australia", "authors": [ "Jianzhen Zhang", "Zaimin Wang", "Anne Cameron", "P Marcin Sowa", "Vishal Diwan", "Sree Krishna Venuthurupalli", "Helen G. Healy", "Luke B Connelly", "Wendy E Hoy", "Zaimin Wang", "Anne Cameron", "P Marcin Sowa", "Vishal Diwan", "Sree Krishna Venuthurupalli", "Helen G. Healy", "Luke B Connelly", "Wendy E Hoy" ], "abstract": "Chronic kidney disease (CKD) is one of the most common chronic diseases in the western world. In Australia, around 1.7 million Australians aged 18 years and over (about one in ten) have indicators of CKD, and 1.8 million hospitalisations were associated with CKD in 2017–18. There is currently very little understanding of the impact of CKD on health service utilisation and costs. Understanding the disease pathways of CKD and its effects on service utilisation and patient outcomes is essential to predicting the course of the disease in the future, its effects on health services utilisation and capacity to better manage the burden of premature deaths or the need for dialysis that results from CKD. We describe the establishment of a data linkage framework to study hospital admissions of CKD patients in the public renal services in the Australian state of Queensland, and its potential to advance understanding of their course and outcomes. Seven years of retrospective data (2011–2018) on hospital-based health services utilisation were provided by Queensland Health for all 7,341 patients who enrolled in the CKD.QLD Registry up to Jan 2019. The data were supplied from three datasets: the Queensland Hospital Admitted Patient Data Collection, the Queensland Registrar General deaths, and the Activity Based Funding Model Output data. In addition, data were supplied from two cohorts of de-identified patients admitted to hospital in the same interval (22,023 patients each), who were not in the CKD.QLD Registry, the first with CKD and the second without CKD as indicated by International Classification of Diseases and Related Health Problems, Tenth Revision, Australian Modification. The comprehensive and multifaceted data via the data linkage will enable us to identify opportunities for efficiencies in management of patients with CKD and for interventions that improve their outcomes.", "keywords": [ "chronic kidney disease", "CKD.QLD Registry", "data linkage", "framework", "health service utilisation", "Queensland Health" ], "content": "Introduction\n\nChronic kidney disease (CKD) is a condition characterised by a constant loss of kidney function over time. It may eventually lead to kidney failure, which requires dialysis or a kidney transplant to maintain life. Recent findings from the Australian Health Survey 2014–2015 showed that 203,400 Australians, or 0.9% of the population, self-reported that they had kidney disease,1 and biochemical testing revealed that 10% of adults (approximately 1.7 million people in 2011–12) had markers of CKD (aihw.gov.au). However, nearly 90% of people with CKD (about 1.5 million in Australia) are unaware they have indicators of the disease.2 Rates of CKD increase with age, and are higher in Indigenous Australians, in other minority groups and in people who are socioeconomically disadvantaged.3 Around 1.8 million hospitalisations in Australia were associated with CKD (principal and/or other diagnosis) in 2017–18 or 16% of all hospitalisations.4 Thirteen percent of all hospitalisations were for scheduled dialysis as a principal diagnosis, performed as a same-day day procedure, which was the most common reason for hospitalisation in Australia. There were 16,800 CKD-related deaths, which contributed to 11% of all deaths in 2018.5 In 2019, 26,746 Australians were receiving kidney replacement therapy (KRT) for end-stage kidney failure, including 12,815 who had a functioning kidney transplant and 13,931 who were receiving dialysis.6 The number of people receiving KRT continues to climb, increasing from 23,111 in 2015 to 26,746 in 2019.6 KRT places a large burden on the Australian health-care system, particularly dialysis, including frequent and regular hospitalisations.7\n\nCKD shares many common risk factors with other chronic conditions such as cardiovascular disease and type 2 diabetes, including being overweight and obesity, physical inactivity, poor diet, tobacco smoking and hypertension.8 Kidney disease increases the risk of having heart and blood vessel disease, as well as develops complications like high blood pressure, anaemia, weak bones, poor nutritional health and nerve damage. These problems may happen slowly over a long period of time. CKD may be caused by diabetes, high blood pressure and other disorders.5 Early detection and treatment can often keep chronic kidney disease from getting worse. The better understanding, prevention, and modification of CKD is the potential to reduce the escalating burden of kidney disease. The techniques and the costs of dialysis for people with terminal kidney failure are well delineated, but the appropriate streams of care and cost for the pre-terminal phase of CKD are not well defined and studied.\n\nThere is no system for systematic ambulatory CKD surveillance in Australia. In 2011, we established the CKD Queensland (CKD.QLD) Registry, which is a program for surveillance, practice improvement and research of adult patients with CKD referred to the renal practice network in the public health system within Queensland. This program, which we have described elsewhere,9 is unique within Australia. Queensland has the second-largest land area and is the third-most populous Australian state, with over 5.1 million people in March 2020.10 The state's population is multicultural, with 28.9% of inhabitants classified as immigrants. Four percent of the population identified as Indigenous Australians (Aboriginal and/or Torres Strait Islander Australians) in 2016.11 Public health services in Queensland are provided through 16 Hospital and Health Services (HHSs), distributed across different regions in Queensland. These are statutory bodies, each governed by a Hospital and Health Board. Some public health services are also provided by private providers. Queensland Health operates and administers the state's public health system, and monitors the performance of HHSs.12\n\nThe key strength of the CKD.QLD Registry is its framework of data linkage. To date, with ethics approvals and an opt-in patient consent process, the registry has developed a core data set of demographic and clinical information of CKD patients, generated from the participating kidney health services at the time of patient consent. Unique patient identifiers were assigned in the Registry and linked with relevant person-specific data derived from a variety of site-specific databases, which have historically included Ferret, Audit4, ERIC, Kidney Health Service-BigR, and from larger multi-site data platforms such as The Viewer, the integrated electronic Medical Record (ieMR) being progressively deployed across the state, and the laboratory information system AUSLab, all operated by Queensland Health, as previously described.9\n\nThere is currently very little understanding of the impact of CKD on health service utilisation and costs. Understanding the multiple components of the resource bundles consumed in the care pathways of CKD and their interactions in service utilisation and impact on patient outcomes is essential to predicting future need and identifying opportunities to better manage the burden of premature deaths and morbidities. In this manuscript we describe the establishment of a data linkage framework to study hospital admissions of CKD patients in the public renal services in the Australian state of Queensland, and its potential to advance understanding of their course and outcomes.\n\n\nMethods\n\nSeven years of retrospective data for persons with any hospital admissions with a discharge date from 1 May 2011 to 30 June 2018 were sourced from Queensland Health (QH) for two cohorts of patients with CKD and one cohort without CKD. The specific data sources were the Queensland Hospital Admitted Patient Data Collection (QHAPDC), the Queensland Registrar General (RG) deaths and the Activity Based Funding (ABF) Model Output data for Queensland. The data linkage framework is illustrated in Figure 1.\n\nThe first cohort of people with CKD (Cohort 1) had consented and enrolled in the CKD.QLD Registry until January 2019. As for all admitted patients, the Data Linkage Team of the Queensland Department of Health assigned each a unique identifier within the QH system.13,14\n\nA second cohort of people with CKD (Cohort 2), but not in the CKD.QLD Registry, were identified by the QH Data Linkage Team through International Classification of Diseases and Related Health Problems, Tenth Revision, Australian Modification (ICD-10-AM) codes related to their first hospital admission that were compatible with CKD (see Table 1). They were matched by HHS with Cohort 1 patients in a ratio of 3 to 1. The second cohort reflects a broader group of people with CKD than those in the CKD.QLD Registry and includes many managed by non-nephrologists.\n\nThe third cohort (Cohort 3) consisted of people without an ICD-10-AM diagnosis compatible with CKD in their first hospital admission, as identified by the QH Data Linkage Team, who were matched by age, sex and HHS to Cohort 1 in a ratio of 3 to 1.\n\nData were generated for the discharge from hospital admission period of 1 May 2011 to 30 June 2018. For all members of Cohort 2, and Cohort 3 the same scope and data items were requested from QHAPDC, Deaths and the ABF Model Output datasets, as for as Cohort 1. In addition, patient details including date of birth, sex, country of birth and Indigenous status were provided.\n\nCKD.QLD is an initiative of the University of Queensland’s Centre for Chronic Disease, in partnership with Queensland Health and the Queensland University of Technology.\n\nThere are 12 adult public renal services across the 16 regional Queensland Health Hospital and Health Services (see Figure 2). The renal services operate varied models of CKD community and satellite clinics. Participants are pre-dialysis CKD patients who were referred or within these services are eligible for inclusion in the Registry, via informed consent, and with ethics and governance oversight. Patients with CKD from both the Registry and the matched cohorts in Queensland are age 18 years and over.\n\nQHAPDC and RG deaths\n\nThe Queensland Hospital Admitted Patient Data Collection (QHAPDC) receives demographic and clinical information on all admitted patients separated (an inclusive term meaning discharged, died, transferred or statistically separated) from both public and licensed private hospitals and licenced day surgery units in Queensland.15\n\nThe linked data included the following QHAPDC data items and death Registry data from the Queensland RG deaths for participants in the three cohorts (see Table 2).\n\nQueensland ABF model output data\n\nQH allocate funds to the HHSs on their activity using the ABF Model.16 The model calculates number of episodes of care and case complexity to derive a weighted unit of activity (WAU). The methodology of deriving WAU standardises resource utilisation across episodes of care that are diverse in resource usage and geography. The ABF model calculates a Queensland Efficient Price using Queensland WAUs (QWAU) and the Queensland base price per WAU, noting the QWAU may differ from the national WAU (NWAU) reflecting State-Government specific costs and priorities.17 HHSs report their activities through the National Hospital Cost Data Collection (NHCDC) and the data are also used as inputs into the ABF model to derive output data.18 Person service level costs are determined using the methodology of the NHCDC and are reported in NHCDC summation and format. The data in scope for the NHCDC includes all patient level activity for all public hospital facilities across Australia, and the costs incurred by the hospital with the activity in each financial year. For this data linkage study, we had the following ABF data items (see Table 3), linked to the cohort file for each CKD.QLD Registry participant and participants in the matched cohorts 2 and 3.\n\nThe data linkage process for this study is illustrated in Figure 3. The process is adapted from the “Overview of Application Process for Access to Confidential Health Information held by Queensland Health for Research Purposes” published by the State of Queensland (Queensland Health), January 2016.14 We adopted the process, contacting the Statistical Analysis and Linkage Team (SALT), Statistical Services Branch, Queensland Health for initial advice and consulted data custodians. The work was conducted with the ethical approval of the QH Health Innovation, Investment and Research Office (HIIRO) under the Public Health Act (PHA), a pathway if researchers are requesting identifiable or potentially re-identifiable health information but are unable to obtain participant consent. The Public Health Act 200519 establishes the process for accessing health information held by Queensland Health for approved research projects.\n\nAfter receiving PHA approval, CKD.QLD provided a unique identifier – Registry ID – and associated patient key demographic variables (see Table 4) to the Data Linkage Unit in a password protected file. Using this unique identifier, data outlined in this framework were linked from data custodian sources to the affiliated individual.\n\nThe information on CKD.QLD participants to enable data linkage was provided to Queensland Health by the research Chief Investigator (WH) at the University of Queensland. The data were stored electronically with an encrypted and password protected file and sent to the Linkage Team for linkage to the requested cohort and extraction of their data. The data linkages were carried out in compliance with QH data management guidelines, under the governance of the Statistical Services Branch, the Queensland Department of Health.14 Participant privacy was paramount. Upon the collation of the data via the data linkage protocols, a zipped file was loaded by SALT onto a secure web portal and downloaded by one of the approved investigators in this study (JZ) who stored it in a secure database at the University of Queensland.\n\nThe linked data are being analysed by the CKD.QLD research team and by the health economics team using statistical software (Stata Statistical Software: Release 16. StataCorp LLC (RRID:SCR_012763)). (An open-access alternative that can provide an equivalent function is the R stats package (R Project for Statistical Computing, RRID:SCR_001905)). The analysis methods including descriptive statistics, comparations, and multivariable analysis, e.g., cox regressions are or will be applied. The results will be transformed into reports of de-identified data for user groups and collaborators, which will include, but are not be limited to, Queensland Health, the Australian Institute of Health and Welfare, The University of Queensland, the Australasian and New Zealand Society of Nephrology, the Renal Society of Australasia, and Kidney Health Australia. In addition, outcomes will be published in academic journals and on the CKD.QLD (www.ckdqld.org) and CKD.CRE website (https://cre-ckd.centre.uq.edu.au) addresses for professional and patient access and dissemination. Data will be published in a form that does not identify, or allow the re-identification, of any individual participant or health service provider.\n\nThis study under the data linkage framework is considered a sub-study of the CKD.QLD Registry (HREC/15/QRBW/294), which has been approved by Metro North Human Research Ethics Committee and University of Queensland Medical Research Ethics (Number: 2011000029). The study of the QH held datasets of CKD patients, both those in the CKD.QLD Registry and the comparison patient cohort, listed in this study is through an ethically approved waiver of consent according to the National Health and Medical Research Council (NHMRC) National Statement on Ethical Conduct in Human Research.20 In addition, a Public Health Act application (RD006802) was approved to access all hospitalisation information of the CKD.QLD Registry patients.\n\nThe framework linked 7,341 CKD patients from the CKD.QLD Registry (Cohort 1) to health service data including QHAPDC, RG Deaths and Queensland ABF Model Output Data. In addition, a cohort of 22,023 participants with CKD (Cohort 2) and a cohort of 22,023 without CKD (Cohort 3) were matched. This dataset embraces 51,387 persons and 974,700 admissions over seven years of retrospective data, with admitted persons divided into three groups, as follows.\n\nIn the CKD.QLD cohort (Cohort 1), 45.9% were female and 54.1% were males, and 8.4 % were Indigenous Australians. The mean age (SD; IQR) on their first admission was 65.2 (21.0; 18.8). They had 221,347 admissions, of which 77,822 admissions (35.2%) were for KRT for people who ultimately required that treatment. Of the 7,341 CKD.QLD patients who had at least one hospitalisation episode, 831 patients (11.3%) started KRT and 1,797 died (23.1%) during the study period. The person years of the study period (from their first admission to death or censor date) was 27,900.8 years.\n\nOf the 22,023 people in the non-CKD.QLD CKD cohort (Cohort 2), 45.9% were female and 54.1% were males, and 4.3 % were Indigenous Australians. The mean age (SD; IQR) on their first admission was 73.8 (13.7; 17.0). They had 626,554 admissions, of which 277,994 admissions (44.4%) were eventually for KRT. Of the 22,023 participants in this cohort, 1,233 (5.6%) started KRT and 9,494 died (43.1%). The person years of the study interval was 128,570.6 years.\n\nOf the 22,023 age and gender matched non-CKD cohort (cohort 3), 45.8% were female and 54.2% were males, and 1.8 % were Indigenous Australians. The mean age (SD; IQR) on their first admission was 63.5 (14.9; 18.6). They had 126,799 total admissions, only three eventually had dialysis, and 2,444 patients died (11.1%). The person years of the study interval was 149,439.3 years.\n\nLinking established large datasets is a powerful approach to identifying patterns and interaction between multiple variables without bias whilst limiting the costs of data acquisition. Our first objective in establishing this framework is to better understand hospital admissions and outcomes among persons with preterminal CKD seen in public renal services across the Australian state of Queensland. The second objective is to compare them with persons from the same regions who had CKD documented in one or more hospital admissions who were not in the CKD.QLD cohort, to understand the characteristics and outcomes of CKD in other community and practice settings. The third objective is to compare these findings with persons admitted to hospital without a diagnosis of CKD, but who are matched age, gender, place of residence and HHSs with the CKD cohort in order to assess the degree which a diagnosis of CKD and its accompaniments might influence hospital usage.\n\nThe data on CKD.QLD patients will allow evaluation of health service utilisation for individual patients, covering all admissions, not limited to renal admissions, both public and private, across all disciplines. Many facts can be illuminated, including assessment of the extent to which CKD-related conditions drive admissions vs conditions not clearly related to CKD, characterisation of high cost users of hospital services, potentially avoidable hospital admissions and readmissions, trajectory over time of persons of different baseline characteristics, processes, costs and outcomes with specific interventions, different models of care and changes in policy, for example, strengthening supportive/palliative care pathways for patients with end stage kidney failure, and for persons who ultimately go onto KRT, the changes in resource utilisation and costs. Additionally, patterns of hospital utilisation can be inspected by postcode of residence, by HHS, by ethnic group, and other variables, to identify gaps and opportunities for service modification to improve outcomes.\n\nThe data from the comparator CKD group show the broader view of persons with CKD interacting with all hospitals in Queensland. These include patients in private renal practices, in nonrenal public specialty practices, in primary care and general practice, in Indigenous health services, and people in whom CKD was not recognised prior to their hospital admission within the study period.\n\nThe data from the non-CKD matched cohort will help clarify to what extent the presence of CKD influences the frequency, causes, costs and outcomes of hospital admissions in persons who are otherwise matched for age, gender, region and hospital services.\n\nThere is extensive information on the health and outcomes of patients on KRT in Australia and elsewhere, but hospital usage by preterminal CKD patients is not well defined. This study is the first in Australia aiming to define this issue in preterminal CKD in public renal practices, to compare it with CKD patients in other practice and community settings and to estimate how much CKD amplifies hospital usage over the level of matched persons without CKD. The Queensland population is similar to the aggregate Australian populations in demographics, ethnic mix,21 and rates of CKD,1–4 so our findings are likely to be generalizable across Australia. In addition, hospital admission data include all (licensed) hospital facilities across the state of Queensland, so that all episodes of care were captured even if patients used both private and public facilities.\n\nThere are several limitations to this study. First, whilst most public renal practices participated in CKD.QLD, patient enrolment depended on availability of discretionary time of study coordinators who were busy with other clinical duties, so not all patients were captured. Second, the final CKD.QLD cohort does not include one major renal specialty practice, for which all 998 enrolees were dropped from the database after discrepancies in outcomes data could not be resolved. Third, renal patients who received care exclusively in private specialty practices are not included; patient profiles and service utilisation in private renal services might be somewhat different than described here. Finally, people with CKD who were not admitted to the hospital during the observation interval were not included. For example, 11.6% of CKD.QLD patients did not have hospital admissions after their recruitment to the registry. Finally, the framework does not capture encounters and resource consumption in the outpatient setting. This usage is under study in another framework.22\n\nWe have already used this framework to describe hospital admissions for patients at each individual registry site (ongoing), to define impacts of anaemia in CKD patients,23 frequency and consequences of acute kidney injury in patients with CKD,24 the characteristics of high-cost users,25,26 and to explore factors involved with readmissions by Machine Learning,27 and several other studies are planned. Beyond these, the datasets can inform CKD health practitioners, Queensland Health, the Federal government, the national and international research communities, and local and national advocacy groups like Kidney Health Australia. The outcomes of this linkage study can assist the Australian Institute of Health and Welfare in defining the broader implications of CKD in terms of service consumption and its associated costs.\n\n\nConclusion\n\nThis study will provide the first detailed overview of the utilisation of health care resources, affiliated activity-based funding, and death of patients with CKD in Australia, who are in the public renal specialty system and those in other streams of care. Analysis of the data will provide opportunities for better understanding of CKD, early diagnosis of renal disease and its attendant cardiovascular risks and interventions to improve the outcomes of patients, whilst improving the value of provided health care. The outcomes from this study will subsequently inform other bodies of work to support best practice throughout the CKD practice network. Finally, we expect our research to identify opportunities for efficiencies of CKD management and for interventions to improve the outcomes of patients.\n\n\nData availability\n\nThe CKD.QLD Registry is managed according to legislative requirements including the Information and Privacy Act 2009, and as per the National Statement on Ethical Conduct in Human Research.20 This study utilises an established data linkage methodology to ensure the security and confidentiality of all participants’ data, and to minimise any risk to participants, family members and the community.\n\nResearchers (including PhD students) interested in using the data, should contact Jenny Zhang (jenny.zhang@uq.edu.au) and an amendment PHA will be submitted to Queensland Health adding the researcher’s name in the application. Researchers must write a letter requesting access to specific data and attach their CV. Once the amendment PHA is approved, a policy agreement regarding data access must be signed by the researchers and then a deidentified dataset will be sent.", "appendix": "Acknowledgements\n\nThe study is operated under the NHMRC CKD Centre of Research Excellence and Chronic Kidney Disease (CKD.CRE) in Queensland Research Collaborative (CKD.QLD). We thank the clinical and academic teams that have enabled the linkage study and wholeheartedly thank all the patients who are participating in the CKD.QLD Registry Study. Sincere thanks also to the Queensland Health data custodians for providing the linked data and data linkage officers from the Statistical Analysis and Linkage Team of Queensland Health for conducting the data linkage.\n\n\nReferences\n\nAustralian Bureau of Statistics: National Health Survey: First Results, 2014-15. Canberra: Australian Bureau of Statistics; 2015.\n\nAustralian Bureau of Statistics: Australian health survey: First results 2011-12. Canberra: ABS; 2012.\n\nAustralian Bureau of Statistics: Australian Health Survey: Biomedical Results for Chronic Diseases, 2011-12. Canberra: ABS; 2013.\n\nAustralian Bureau of Statistics: National Health Survey: First results, 2017-2018. Australian Bureau of Statistics; 2018.\n\nAIHW: Chronic kidney disease Canberra: AIHW; 2020; 2021. .\n\nANZDATA Registry: 43rd Annual Report 2020 (Data to 2019). Adelaide: Australia and New Zealand Dialysis and Transplant Registry; 2020.\n\nAustralian Institute of Health and Welfare: Dialysis and kidney transplantation in Australia: 1991–2010. Cat. no. PHE 162. Canberra: AIHW; 2012.\n\nAustralian Institute of Health and Welfare: Cardiovascular disease, diabetes and chronic kidney disease—Australian facts: Risk factors. Cardiovascular, diabetes and chronic kidney disease series no 4 Cat no CDK 4. Canberra: AIHW; 2015.\n\nVenuthurupalli SK, Hoy WE, Healy HG, et al.: CKD.QLD: Establishment of a chronic kidney disease [CKD] registry in Queensland, Australia. BMC Nephrol. 2017; 18(1): 189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAustralian Bureau of Statistics: National, state and territory population – June 2020. Canberra: Australian Bureau of Statistics; 2020.\n\n2016 Census of Population and Housing: General Community Profile.\n\nQueensland Health: Handbook for Queensland Hospital and Health Boards (second edition). Brisbane © State of Queensland (Queensland Health); 2018.\n\nKelman CW, Bass AJ, Holman CDJ: Research use of linked health data — a best practice protocol. Aust. N. Z. J. Public Health. 2002; 26(3): 251–255. PubMed Abstract \| Publisher Full Text\n\nDepartment of Health: Queensland Data Linkage Framework. Brisbane The State of Queensland (Queensland Health); 2016.\n\nQueensland Health: The Queensland Hospital Admitted Patient Data Collection (QHAPDC) Manual 2016-2017. Brisbane, QLD: State of Queensland (Queensland Health); 2017.\n\nQueensland Health: Health funding principles and guidelines 2016-17 financial year. Brisbane: QLD State of Queensland (Queensland Health); 2015.\n\nQueensland Health: Health funding principles and guidelines 2019-20 financial year. Brisbane: QLD State of Queensland (Queensland Health); 2019.\n\nIndependent Hospital Pricing Authority: National Hospital Cost Data Collection, Public Hospitals Cost Report, Round 19 (Financial year 2014-15). National Hospital Cost Data Collection, Public Hospitals Cost Report. Sydney: IHPA; 2017.\n\nQueensland Health: Public Health Act 2005. Queensland Health: Brisbane: Queensland Govenment; 2017.\n\nThe Australian Research Council and the Australian Vice-Chancellors’ Committee: National Statement on Ethical Conduct in Human Research (Updated May 2015). The National Health and Medical Research Council: Canberra: Commonwealth of Australia; 2007.\n\nAustralian Bureau of statistics: Census of Population and Housing: Reflecting Australia - Stories from the Census, 2016. statistics ABo: Canberra: Australian Bureau of statistics2017.\n\nByrnes J, Nghiem S, Afoakwah C, et al.: Queensland Cardiovascular Data Linkage (QCard): A population-based cohort study [version 1; peer review: awaiting peer review]. F1000Res. 2020; 9(282). Publisher Full Text\n\nZhang J, Diwan V, Wang Z, et al.: The Impact of Anaemia on Outcomes and Costs in Patients with Chronic Kidney Disease in Two Public Nephrology Practices in Queensland: A CKD.QLD Registry Study. ANZSN. Sydney: Australian and New Zealand Society of Nephrology; 2020.\n\nZhang J, Healy HG, Baboolal K, et al.: Frequency and Consequences of Acute Kidney Injury in Patients With CKD: A Registry Study in Queensland Australia. Kidney Medicine. 2019; 1(4): 180–190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSowa PM, Zhang J, Hoy WE, et al.: Intensive Users of Hospital Care in Chronic Kidney Disease. ANZSN. Sydney Australian and New Zealand: Society of Nephrology; 2019.\n\nSowa M, Venuthurupalli S, Hoy W, et al.: Factors associated with high cost use of hospital care in pre-end stage chronic kidney disease. The 55th ANZSN ASM: 2020; Online. Australian and New Zealand: Society of Nephrology; 2020.\n\nIm SJ, Xu Y, Watson J, et al.: Hospital Readmission Prediction using Discriminative patterns. 2020 IEEE Symposium Series on Computational Intelligence (SSCI): 1-4 Dec. 2020 2020. 2020; 50–57." }	[ { "id": "98786", "date": "15 Nov 2021", "name": "Hong Nghiem", "expertise": [ "Reviewer Expertise Health economics" ], "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 present the description of CKD.QLD data linkage framework. The article is clearly written and almost can be indexed without changes. I have only a couple of minor recommendations:\nProvide a rationale for the choice of matching ratio of 1:3 for both second and third data cohorts relatively to the first cohort.\n\nWas CKD in the second cohort identified using primary or other diagnosis codes?\n\nWhat is the rationale to limit the matching variable to only sex, age and HHS?\n\nWhat method was used to match data across cohort? What criteria was used to identified match records?\n\nAdding a table to summarize three cohorts and test for difference based on matching variables would be useful.\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? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "276972", "date": "07 Jun 2024", "name": "Wubshet Hailu Tesfaye", "expertise": [ "Reviewer Expertise I am a pharmacist and academic with interest in kidney 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\nThis is a great initiative potentially creating further research opportunities in the future.\nI was wondering if any of the linked data will have any information on medication use pattern and medication-related hospitalisations.\nI was also wondering what the considerations were when selecting a matching non-CKD cohort. And I noticed the following re this cohort: \"They had 126,799 total admissions, only three eventually had dialysis, and 2,444 patients died (11.1%).\" Does this mean there could be people with some form of CKD even in the non-CKD cohort?\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/10-1107
https://f1000research.com/articles/10-570/v1	15 Jul 21	{ "type": "Software Tool Article", "title": "PEGS: An efficient tool for gene set enrichment within defined sets of genomic intervals", "authors": [ "Peter Briggs", "A. Louise Hunter", "Shen-hsi Yang", "Andrew D. Sharrocks", "Mudassar Iqbal", "Peter Briggs", "A. Louise Hunter", "Shen-hsi Yang", "Andrew D. Sharrocks" ], "abstract": "Many biological studies of transcriptional control mechanisms produce lists of genes and non-coding genomic intervals from corresponding gene expression and epigenomic assays. In higher organisms, such as eukaryotes, genes may be regulated by distal elements, with these elements lying 10s–100s of kilobases away from a gene transcription start site. To gain insight into these distal regulatory mechanisms, it is important to determine comparative enrichment of genes of interest in relation to genomic regions of interest, and to be able to do so at a range of distances. Existing bioinformatics tools can annotate genomic regions to nearest known genes, or look for transcription factor binding sites in relation to gene transcription start sites. Here, we present PEGS (Peak set Enrichment in Gene Sets). This tool efficiently provides an exploratory analysis by calculating enrichment of multiple gene sets, associated with multiple non-coding elements (peak sets), at multiple genomic distances, and within topologically associated domains. We apply PEGS to gene sets derived from gene expression studies, and genomic intervals from corresponding ChIP-seq and ATAC-seq experiments to derive biologically meaningful results. We also demonstrate an extended application to tissue-specific gene sets and publicly available GWAS data, to find enrichment of sleep trait associated SNPs in relation to tissue-specific gene expression profiles.", "keywords": [ "Genomic data integration", "ChIP-seq", "RNA-seq", "gene set enrichment", "genomic intervals" ], "content": "Introduction\n\nGene expression control in higher organisms is achieved through a complex hierarchical process involving opening of chromatin, histone modifications, and binding of transcription factors (TFs). Experimental approaches to understand transcriptional regulatory mechanisms in a biological context involve large-scale measurement of gene expression. Depending on the design of the experiment, these analyses produce differentially expressed gene sets or clusters for further analysis. These studies are often complemented by assays which map, on a genome-wide scale, TF binding sites (ChIP-seq) or regions of chromatin accessibility (DNase-seq, ATAC-seq). Analyses of these data produce a collection of genomic intervals (peak sets). An important computational task is then to integrate these data to produce meaningful results; i.e. to relate gene sets to peak sets to aid functional interpretation. Bearing in mind distal regulation, an important consideration here is to be able to calculate gene set enrichment at multiple genomic distances from peak sets, and to be able to do this efficiently within the same analysis.\n\nWe present a new tool – PEGS (Peak set Enrichment in Gene Sets)1 – which calculates mutual enrichment of multiple gene sets associated with multiple peak sets, simultaneously and efficiently. This can be at user-defined peak-to-TSS (transcription start site) distances, as well as constraining to topologically associated domains (TADs). Thus, PEGS quickly produces an overall picture of gene set enrichment in relation to peaks, and shows at what genomic distances this is most pronounced. It is applicable to gene sets derived from any source, and peak sets derived from different epigenomic assays, as well as single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS).\n\n\nMethods\n\nIn PEGS, input peaks are extended in both directions for a given distance or constrained within known TAD boundaries, if provided (Figure 1). Subsequently, the enrichment of the input gene set is calculated among the genes whose TSSs overlap with the extended peaks. These tasks are performed in PEGS as follows:\n\n1. Creating a gene interval file in BED (Browser Extensible Data) format for all genes in the given genome using refGene from UCSC Table Browser. This reference TSSs BED file only needs to be created once (human hg38 and mouse mm10 are provided with the tool; a utility is provided to create these for other genomes).\n\n2. For a given peak set, peaks are extended to specified genomic distances in both directions, and up to overlapping TAD boundaries. Intersection of these extended peaks with the gene intervals BED file from step 1 is calculated using BEDTools (RRID:SCR_006646)2, leading to a gene set whose TSSs overlap with extended peaks.\n\n3. Using the intersection of the input gene set, and unique genes from step 2, a Hypergeometric test is performed to calculate the p-value using Equation 1, similar to GREAT (RRID:SCR_00580)3. Here, M is the total number of genes in the genome, Nc is the number of genes in the input cluster/set c, Np is the number of unique genes overlapping the peaks for given distance and npc is the intersection of two gene sets.\n\nCartoon showing peak expansion and overlapping TSSs in PEGS, with a specified genomic distance λ from the centre of the peak in both directions (a) where TSS2 and TSS3 are included and a TAD overlapping with the left peak in (b) where all four TSSs within the TAD are included\n\n\n\nStep 2 and 3 are repeated for every combination of gene cluster, peak set and distance and/or TADs. The final combined heatmap shows −log10 of the resulting p-values.\n\nPEGS is implemented in Python 3, where we have reused functions from existing Python packages included with Python distributions, or available from the Python Package Index (PyPI). We also make use of BEDTools2 for working with genomic intervals. We provide online documentation (https://pegs.readthedocs.io/en/latest/), and an example analysis with input data at the PEGS GitHub repository.\n\nPEGS works with Python >= 3.6 and, when installed through pip, automatically installs all the dependencies. These are listed in requirements.txt file in our PEGS GitHub repository. We provide extensive documentation online at https://pegs.readthedocs.io which includes easy-to-follow instructions about:\n\nInstallation and system requirements\n\nFormat of input files, output files, and graphics\n\nPEGS commands for standard operations, as well as running PEGS with additional input options, e.g. TAD definition files\n\nCreating customised reference TSSs files for new genomes\n\n\nResults\n\nHere, we present three use cases where we apply PEGS to different publicly available data sets. The format of input files is the same for all use cases below. Gene clusters are provided as text files with one gene symbol on each line; genomic region coordinates are provided in standard BED format. These input files for Use Case 1, as well as example analysis reproducing Figure 2A, are provided in our GitHub repository (https://github.com/fls-bioinformatics-core/pegs).\n\nPEGS applications: (A) gene expression, ChIP-seq and DNase I data on mouse liver, (B) gene clusters derived from scRNA-seq and intergenic putative enhancer clusters from bulk ATAC-seq from three matching early stem cell differentiation time-points. In both plots, numbers in the cells show common genes among the input genes (x-axis) and genes overlapping with expanded peaks (y-axis) and the colour shows −log10 of p-value (Hypergeometric test)\n\nThe first application (Figure 2A) uses up- and down-regulated glucocorticoid target genes obtained by an RNA-seq study of liver samples from mice treated acutely with synthetic glucocorticoid dexamethasone or vehicle4. Corresponding GR ChIP-seq and chromatin accessibility data (DNase I hypersensitive (DHS) regions) were obtained from 5, and 6 respectively, whilst the mouse liver TAD boundaries were obtained from 7. Raw published datasets were downloaded from GEO Sequence Read Archive (RRID:SCR_005012) using sratoolkit v2.9.2 (http://ncbi.github.io/sra-tools/). Reads were aligned to the reference genome (mouse mm10), sorted and indexed using Bowtie2 (v2.3.4.3, RRID:SCR_005476,8) and SAMtools (v1.9, RRID:SCR_002105,9). MACS2 (v2.1.1.20160309, RRID:SCR_013291,10) was used to call peaks, using default settings. PEGS analysis shows strong association of dexamethasone up-regulated genes with dexamethasone-induced GR peaks at distances up to 500kbp from these peaks, but no enrichment of down-regulated genes, indicating distinct mechanisms of gene activation and repression by glucocorticoids. At the same time, there is promoter proximal enrichment for both up- and down-regulated genes in the DHS regions.\n\nNext, using PEGS, we calculated enrichment of gene clusters derived from single-cell RNA-seq and open chromatin regions defined by bulk ATAC-seq at three matching stages (ESCs- embryonic stem cells, day1 EpiLCs - epiblast-like cells, day2 EpiLCs) of early embryonic stem cell differentiation11. The intergenic regions (peak sets) were defined as those with differential accessibility between any two time points and were clustered into four profiles based on z-score of tag densities, as described in 11. Similarly, differentially expressed genes were identified from pseudo-bulk gene expression data at each time point, and were clustered into four patterns. As shown in Figure 2B, strong association is observed between the matching gene expression (x-axis) and chromatin opening profiles (y-axis) at intergenic enhancers, reflecting correspondence between differential accessibility and gene expression changes.\n\nFurthermore, we present an extended application of PEGS to GWAS data and find associations of SNPs for different sleep phenotypes with sets of tissue-specific genes from the Genotype-Tissue Expression (GTEx) Portal, RRID:SCR_013042). For this purpose, we downloaded GWAS data from the Sleep Disorder Knowledge Portal (RRID:SCR_016611) for certain sleep associated phenotypes (with genome wide p-value cutoff <=5e − 8) and calculated enrichment of tissue-specific gene lists defined using the GTEx Portal. Using median transcripts per million (TPM) data for different tissues in GTEx, a gene list for a tissue was defined as genes with 5x median TPM compared to the average in the remaining tissues.\n\nIn Figure 3, we show enrichment of SNPs from three sleep related phenotypes, namely chronotype, daytime sleepiness adjusted for BMI, and sleep duration. These enrichments are calculated for tissue-specific genes lists created from GTEx for 22 tissues, the majority of them from the brain. Application of PEGS to these data reveals some strong associations, e.g. chronotype SNPs strongly enriched for genes expressed in liver and blood, while daytime sleepiness SNPs are enriched in gene sets for different brain tissues. Some of these associations are reported in the literature, e.g. daytime sleepiness SNPs in brain tissue12, others may warrant further investigation.\n\nThe x-axis shows different tissue-specific gene lists, and y-axis shows three sets of sleep related SNPs, expanded to multiple genomic distances. The colour of the cells show −log10 of p-value of enrichment of corresponding gene list (x-axis) in the genes identified through overlap with expanded SNP intervals, the numbers in the cells show the common genes among the two (used in the calculation of Hypergeometric p-value)\n\n\nConclusions\n\nThrough the three different applications above, we demonstrate that PEGS is a versatile and highly efficient tool to integrate different genomic data, and is able to generate hypotheses for further analysis. The implementation of PEGS is highly efficient and as an example of computational efficiency, with pre-created reference TSS files, it only took 7.6 seconds to produce the output for Figure 2A on a laptop with Intel(R) Core(TM) i5-7200U CPU @ 2.50GHz processor with 16GB RAM.\n\nFurthermore, the user can adjust the background population and control for bias. For example, depending on the scientific question at hand, the reference gene interval file (TSSs BED file) could be limited to include only those genes known to be expressed in the tissue of interest. The efficiency of PEGS allows multiple gene and peak input files (e.g. with varying false discovery rate or fold-change cut-offs) to be tested quickly.\n\nPEGS builds on some aspects of, and is complementary to, GREAT3, an existing tool, which performs functional enrichment of regulatory regions using annotations of nearby genes. PEGS could also be used in conjunction with other tools to gain further mechanistic understanding (e.g. by finding enriched transcription factors with TFEA.ChIP13, ranking of their target genes with Cistrome-GO14 or BETA15, or predicting which TFs might regulate differentially expressed gene sets with Lisa16).\n\n\nData availability\n\nAll data underlying the results are available as part of the article or available publicly.\n\n\nSoftware availability\n\nSoftware available from Zenodo: https://zenodo.org/record/5012058\\#.YNG7xmhKiUl. It is easily installable through the Python Package Index (PyPI).\n\nSource code available from Github: https://github.com/fls-bioinformatics-core/pegs.\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.50120581\n\nLicense: PEGS is distributed under BSD 3-Clause license.\n\nOnline manual: https://pegs.readthedocs.io", "appendix": "Acknowledgements\n\nThe authors thankfully acknowledge useful discussions with Magnus Rattray and Leo Zeef.\n\n\nReferences\n\nBriggs P, Hunter AL, Yang S, et al.: Pegs: An efﬁcient tool for gene set enrichment within deﬁned sets of genomic intervals. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.5012058\n\nQuinlan AR, Hall IM: BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLean CY, Bristor D, Hiller M, et al.: GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol. 2010; 28(5): 495–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaratti G, Iqbal M, Hunter L, et al.: REVERBa couples the circadian clock to hepatic glucocorticoid action. J Clin Invest. 2018; 128(10): 4454–4471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrøntved L, John S, Baek S, et al.: C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements. EMBO J. 2013; 32(11): 1568–1583. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSobel JA, Krier I, Andersin T, et al.: Transcriptional regulatory logic of the diurnal cycle in the mouse liver. PLoS Biol. 2017; 15(4): e2001069. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim YH, Marhon SA, Zhang Y, et al.: Rev-erbα dynamically modulates chromatin looping to control circadian gene transcription. Science. 2018; 359(6381): 1274–1277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with bowtie 2. Nat Methods. 2012; 9(4): 357–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Liu T, Meyer CA, et al.: Model-based analysis of chip-seq (macs). Genome Biol. 2008; 9(9): R137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang SH, Andrabi M, Biss R, et al.: ZIC3 Controls the Transition from Naive to Primed Pluripotency. Cell Rep. 2019; 27(11): 3215–3227.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang H, Lane JM, Jones SE, et al.: Genome-wide association analysis of self-reported daytime sleepiness identifies 42 loci that suggest biological subtypes. Nat Commun. 2019; 10(1): 3503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuente-Santamaria L, Wasserman WW, Del Peso L: TFEA.ChIP: A tool kit for transcription factor binding site enrichment analysis capitalizing on ChIP-seq datasets. Bioinformatics. 2019; 35(24): 5339–5340. PubMed Abstract \| Publisher Full Text\n\nLi S, Wan C, Zheng R, et al.: Cistrome-GO: A web server for functional enrichment analysis of transcription factor ChIP-seq peaks. Nucleic Acids Res. 2019; 47(W1): W206–W211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang S, Sun H, Ma J, et al.: Target analysis by integration of transcriptome and ChIP-seq data with BETA. Nat Protoc. 2013; 8(12): 2502–2515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQin Q, Fan J, Zheng R, et al.: Lisa: inferring transcriptional regulators through integrative modeling of public chromatin accessibility and ChIP-seq data. Genome Biol. 2020; 21(1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "89906", "date": "02 Aug 2021", "name": "Nicolae Radu Zabet", "expertise": [ "Reviewer Expertise Computational biology", "bioinformatics", "chromatin and epigenetics" ], "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\nBriggs and co-authors present a new tool called PEGS to generate gene set enrichment for ChIP-seq and DNase-seq datasets. In fact, the tool can be applied to any set of genomic intervals, including SNPs datasets. Generation of gene set enrichment for genomic intervals is a very important task and the authors propose an interesting approach to address it. Particularly, I appreciate the use of TADs to limit the expansion of genomic intervals. They also provide three use cases with different datasets and prove the applicability of this tool.\nI have the following comments:\nDo you consider alternative TSS? Would a gene with multiple TSSs be overrepresented or not?\n\nDo you think that distal loops connecting TSS with enhancers residing outside of TADs would affect your results?\n\nWhile readable, the resolution of figure 2 is low. I would advise the authors to upload a higher resolution figure.\n\nFor case1, maybe I missed it, but I think it would be interesting to interpret the results with or without TADs. This would allow us to see the impact of TAD annotation on the analysis.\n\nI think the authors should add more explanations in the text about the results of their three cases.\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": "7341", "date": "02 Nov 2021", "name": "Mudassar Iqbal", "role": "Author Response", "response": "We thank the reviewer for constructive comments, here we will address their points one by one. 1 - Do you consider alternative TSS? Would a gene with multiple TSSs be overrepresented or not? We consider all TSSs defined in the given genome build, which can include multiple TSSs for some genes. When calculating the enrichment for gene sets obtained through overlap of TSSs with expanded peaks, we remove duplicates. Therefore, genes with multiple TSSs are not over-represented. 2 - Do you think that distal loops connecting TSS with enhancers residing outside of TADs would affect your results? We have two scenarios for enrichment calculations in PEGS. First, the user can provide genomic distances which are not constrained to TADs. Hence enhancers residing in a separate TAD to the TSS could be included. Secondly, we provide an option to the user to constrain the peak expansion to TAD/subTAD boundaries, if available. This will exclude enhancers outside the TAD boundaries, but the distances option can still be utilised to test multiple distances within and beyond TAD boundaries. 3 - While readable, the resolution of figure 2 is low. I would advise the authors to upload a higher resolution figure. We agree, and have added a high-resolution version of Fig. 2 4 - For case1, maybe I missed it, but I think it would be interesting to interpret the results with or without TADs. This would allow us to see the impact of TAD annotation on the analysis. This is related to point 2. In Fig.2A, we do provide the analysis with and without TADs for case 1. Enrichment for multiple distances is at the top two panels, and enrichment calculations using TADs are at the bottom. We have improved Fig 2 to make this clear. 5 - I think the authors should add more explanations in the text about the results of their three cases. We have added more text in the manuscript, further explaining the three cases." } ] }, { "id": "89907", "date": "26 Aug 2021", "name": "Aziz Khan", "expertise": [ "Reviewer Expertise bioinformatics", "gene regulation", "regulatory genomics", "epigenomics" ], "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 paper, the authors presented a Python-based command-line tool, PEGS, for gene set enrichment in association with genomic regions. PEGS computes the enrichment of gene sets with proximity-based association with genomic region sets. These associations are further restricted within the Topologically Associated Domains (TADs), which is good. The manuscript is moderately written and it provides three use cases of the tool.\nThe tool itself is very useful but it lacks several key options to give users the flexibility to customize the input data and also the output heatmap.\nI have the following comments for the authors to address:\nIt is useful to restrict peak-gene association within the TAD boundaries, but it is not the case that all the interactions, such as enhancer-gene interactions occur within the TAD boundaries. The enhancer–gene communication can also occur outside topological domains or in-between TADs. Do authors plan to provide an optional feature to integrate chromatin interaction data, such as HI-C?\n\nThe command-line tool can be further improved by providing additional options to improve user experience and its usage. Below are some recommendations.\nCurrently, the peaks sets and gene lists inputs arguments are positional and the tools can only scan files available in the provided folders. Instead of looking into provided folders for BEDs files and gene lists, the argument should also allow chaining a list of bed files with a path. This is because in real analysis scenarios BEDs can be spread across multiple folders or a single folder can have other visible/hidden files. For example, I was testing the tool on a Mac machine, and PEGS started processing peaks for .DS_Store, which is the default directory structure and a hidden file.\n\nThe tool arguments could be: pegs --peaks peaks/.bed --genes genes/.txt\nand also pegs --peaks A.bed B.bed --genes A.txt B.txt\n\nThe output heatmap should also have an option to generate vector-based plots, such as PDF or SVG.\n\nAuthors may consider adding additional options to adjust the heatmap, such as setting labels, dimensions, colors, and gene/peak set names.\n\nFigures can be further improved.\n\nPlease highlight the limitations of the tool such as the enhancer–gene associations are solely based on proximity.\n\nProviding an installation option through Conda using the bioconda channel (https://bioconda.github.io/) will be useful and it will increase the usage/availability of the tool.\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": "7342", "date": "02 Nov 2021", "name": "Mudassar Iqbal", "role": "Author Response", "response": "We thank the reviewer for constructive review and useful suggestions. We have updated the software taking into account the reviewer’s suggestions, improved the manuscript overall and added more text. Here we will address their individual comments. 1 - It is useful to restrict peak-gene association within the TAD boundaries, but it is not the case that all the interactions, such as enhancer-gene interactions occur within the TAD boundaries. The enhancer–gene communication can also occur outside topological domains or in-between TADs. Do authors plan to provide an optional feature to integrate chromatin interaction data, such as HI-C? We would like to emphasise that the genomic distances are not restricted to TAD boundaries. PEGS provides the user with the option of supplying any distances. In addition, we also provide the user with the option to restrict peak expansion to TADs boundaries (if available), as a separate analysis (Fig 2A). We have revised the relevant text and we hope this will address reviewer’s main concerns and clarify any confusion. Integration of HiC data is beyond the scope of this work, but we will think of ways to incorporate that in future developments of PEGS. 2 - The command-line tool can be further improved by providing additional options to improve user experience and its usage. Below are some recommendations. Currently, the peaks sets and gene lists inputs arguments are positional and the tools can only scan files available in the provided folders. Instead of looking into provided folders for BEDs files and gene lists, the argument should also allow chaining a list of bed files with a path. This is because in real analysis scenarios BEDs can be spread across multiple folders or a single folder can have other visible/hidden files. For example, I was testing the tool on a Mac machine, and PEGS started processing peaks for .DS_Store, which is the default directory structure and a hidden file. The tool arguments could be: pegs --peaks peaks/.bed --genes genes/.txt and also pegs --peaks A.bed B.bed --genes A.txt B.txt The output heatmap should also have an option to generate vector-based plots, such as PDF or SVG. Authors may consider adding additional options to adjust the heatmap, such as setting labels, dimensions, colors, and gene/peak set names. We thank the reviewer as these are very useful suggestions and we have updated PEGS to a new version (please see latest version 0.6.2), which includes all of the above command line options. We also output the heatmap data as an excel file, so the user can customise their heatmaps/plots according to their choice/requirements. We have updated the documentation accordingly. 3 - Figures can be further improved. We have improved and updated all of the figures (please see new version of the manuscript) 4 - Please highlight the limitations of the tool such as the enhancer–gene associations are solely based on proximity. We have updated the manuscript text making it clear that our enrichment calculations are based on genomic proximity, expanding peaks (in both directions) with given distances (and/or TADs), and obtaining genes whose TSSs overlap with the expanded peaks. 5 - Providing an installation option through Conda using the bioconda channel (https://bioconda.github.io/) will be useful and it will increase the usage/availability of the tool. We thank the reviewer; our tool is now installable through Conda." } ] } ]	1	https://f1000research.com/articles/10-570
https://f1000research.com/articles/10-434/v1	02 Jun 21	{ "type": "Opinion Article", "title": "Is Tourette syndrome a rare disease?", "authors": [ "Andreas Hartmann", "Natalia Szejko", "Nanette Mol Debes", "Andrea E. Cavanna", "Kirsten Müller-Vahl", "Natalia Szejko", "Nanette Mol Debes", "Andrea E. Cavanna", "Kirsten Müller-Vahl" ], "abstract": "Based on its prevalence, Tourette syndrome cannot be considered a rare disease. However, in this opinion article, we make the claim that it should nonetheless be considered as an orphan or neglected disease.", "keywords": [ "Tourette syndrome", "tics", "rare disease", "orphan disease" ], "content": "What is a rare disease?\n\nRare diseases are diseases which affect a small number of people compared to the general population. In Europe, a disease is considered to be rare when it affects 1 person per 2,000.1 In contrast, the European Commission on Public Health2 defines rare diseases not only based on low prevalence (<1 in 2,000 people), but in addition as “life-threatening or chronically debilitating diseases which are of such low prevalence that special combined efforts are needed to address them.” Accordingly, diseases that are statistically rare, but not also life-threatening, chronically debilitating, or inadequately treated, are excluded from this definition. In the United States, the Rare Diseases Act of 20023 also defines rare disease strictly according to prevalence, but on the basis of different rates, as follows: “Any disease or condition that affects fewer than 200,000 people in the United States”, or about 1 in 1,500 people.4\n\nBecause of definitions that include reference to treatment availability, a lack of resources, and severity of the disease, the term “orphan disease” has been introduced as a synonym for “rare disease”. Interestingly, in the United States and the European Union, orphan diseases have a distinct legal meaning. Originally, the orphan drug movement began in the United States. The United States Orphan Drug Act5 summarizes under the term “orphan diseases” both rare diseases and any non-rare diseases “for which there is no reasonable expectation that the cost of developing and making available in the United States a drug for such disease or condition will be recovered from sales in the United States of such drug”. Similarly, the European Organization for Rare Diseases (EURORDIS6) also includes both rare diseases and neglected diseases into a larger category of “orphan diseases”.\n\n\nDefinition(s) of Tourette syndrome\n\nw?>Tourette syndrome (TS) is defined by the DSM-5 as a chronic tic disorder with the presence of at least two motor and one vocal tics over a period >12 months in someone under the age of 18 after excluding secondary causes (APA, 2013). This definition is not substantially different from that given by the previous version, the DSM-IV-TR, on which the current epidemiological literature is based. However, there was a major shift between the initial version of the DSM-IV and its revision: it was no longer a requirement that tics must be debilitating. This essentially descriptive vision of TS has its merits as it is difficult if not impossible to define operational criteria for what can be considered debilitating. Also, the waxing and waning nature of tics, both phenomenologically and with regard to severity, means that handicap may vary over time, even if the overall condition can be considered chronic. However, this very broad definition of TS also means that likely many people fall under this diagnostic umbrella category who do not at all require medical attention at any time during their life. The need to achieve a balance between the gains and losses resulting from the removal of the “impairment” criterion could be fruitfully reassessed a few decades after the change took place. The positive repercussions have been particularly manifest in the domain of research (e.g. inclusion of milder cases in genetic studies, cf. Müller-Vahl et al., 2019). However, over time many clinicians working in specialist settings have noticed that a substantial degree of overlap between the definition of TS and what is observed in everyday clinical practice has inevitably been lost.\n\n\nEpidemiology of Tourette syndrome\n\nRecent epidemiological studies of TS have estimated its prevalence between 0.3 to 0.7% in school-aged children (Knight et al., 2012; Scharf et al., 2015). A conservative estimate would be around 0.5%, that is one child in 200, 10 times the accepted rate for a rare disease. In adults there is no solid epidemiological data although we may extrapolate for the pediatric findings. Assuming that two thirds of patients with TS remit when entering adulthood (Leckman et al., 1998), around 0.2% of adults might still suffer from TS, which still does not fulfill any of the diagnostic criteria for rare diseases given above. A recent meta-analysis based on only three studies, however, suggested a prevalence rate of 0.012%, which would make adult TS indeed a rare disease (Levine et al., 2019). Clearly, more epidemiological research is warranted in the field of adult TS.\n\n\nThe problem\n\nWhat experts on TS agree on is that the condition is underdiagnosed, and the delay between onset of symptoms (tics) and diagnosis is too long (Mol Debes et al., 2008; Shilon et al., 2008). Yet, they also acknowledge that a substantial number of people, regardless of age (but likely more adults) remain unbothered by their tics and live perfectly normal lives; in other words, they never seek medical attention and have no reason to do so. Based on this fact, the term “tic spectrum disorder” has been suggested including all variants and severity levels of the disease (Müller-Vahl et al., 2019).\n\nHowever, in an ideal world, how many people with TS according to DSM-5 criteria might legitimately be called patients? We speculate the percentage to be around 10-20%. One of the strategies to tackle this problem could be division of patients with TS in clinical subgroups mainly dependent on tic severity. According to this premise, severe TS is indeed, and thankfully, a rare condition. But here also, tolerance to tics and their sequalae, both social and functional, differ enormously from patient to patient, so defining operational criteria or some sort of cut-off (i.e., on the Yale Global Tic Severity Scale) will most likely be impossible. A further strategy could be pursued by focusing on the complexity or phenotype – with and without comorbidity – of the clinical picture of TS. This strategy might involve a return to the origin, namely to Gilles de la Tourette’s original definition of TS, which encompasses the symptom triad of tics, echolalia, and coprolalia. The condition presenting with both simple and complex tics is sometimes referred to as “full-blown TS” and would arguably qualify for the “rare disease” category (Cavanna, 2018). The abovementioned strategies would lead to the identification of a subgroup of patients whose condition bears a clinically relevant impact on quality of life. This pathway could compensate for the information lost with the removal of the “impairment” criterion from the revised set of diagnostic criteria for TS in the DSM-IV-TR (2000).\n\n\nA proposal\n\nAlthough tics are a common symptom, we wish to officially continue considering TS a rare (or orphan) disease because of certain public health (and other) benefits this implies. Clearly, TS is underrecognized (or misrepresented – Fat et al., 2012) and underdiagnosed, so, de facto, rare for treating physicians and other health care professionals. There is very little targeted drug research for the treatment of tics to date, and in most patients off-label prescriptions are made. For these reasons, we might reasonably call TS a neglected disease, needing further support as a “rare disease” by policy makers until things change. Those insisting on high prevalence of TS to increase support/awareness of this condition may, ironically, render a disservice to patients. Similar to patients with essential tremor, we will then be left to our own devices in the no man’s land between public and industrial support, and progress may slowly grind to a halt.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Competing interests\n\n\n\nAndreas Hartmann has received consultant's honoraria from Lundbeck and Noema Pharma.\n\nNatalia Szejko: None.\n\nNanette Mol Debes: None.\n\nAndrea E. Cavanna: None.\n\nKirsten Müller-Vahl: Financial or material research support from EU (FP7-HEALTH-2011 No. 278367, FP7-PEOPLE-2012-ITN No. 316978) DFG: GZ MU 1527/3-1 and GZ MU 1527/3-2, BMBF: 01KG1421, National Institute of Mental Health (NIMH), Tourette Gesellschaft Deutschland e.V. Else-Kröner-Fresenius-Stiftung, GW pharmaceuticals, Almirall Hermal GmbH, Abide Therapeutics, and Therapix Biosiences. She has received consultant's honoraria from Abide Therapeutics, Boehringer Ingelheim International GmbH, Bionorica Ethics GmbH, CannaMedical Pharma GmbH, Canopy Grouth, Columbia Care, CTC Communications Corp., Demecan, Eurox Deutschland GmbH, Global Praxis Group Limited, IMC Germany, Lundbeck, Sanity Group, Stadapharm GmbH, Synendos Therapeutics AG, and Tilray. She is an advisory/scientific board member for CannaMedical Pharma GmbH, Bionorica Ethics GmbH, CannaXan GmbH, Canopy Growth, Columbia Care, IMC Germany, Leafly Deutschland GmbH, Sanity Group, Syqe Medical Ltd., Therapix Biosciences Ltd., and Wayland Group. She has received speaker’s fees from Aphria Deutschland GmbH, Almirall, Cogitando GmbH, Emalex, Eurox Deutschland GmbH, Ever pharma GmbH, Meinhardt Congress GmbH, PR Berater, Spectrum Therapeutics GmbH, Takeda GmbH, Tilray, Wayland Group. She has received royalties fromDeutsches Ärzteblatt, Der Neurologie und Psychiater, Elsevier, Medizinisch Wissenschaftliche Verlagsgesellschaft Berlin, and Kohlhammer. She served as a guest editor for Frontiers in Neurology on the research topic “The neurobiology and genetics of Gilles de la Tourette syndrome: new avenues through large-scale collaborative projects”, is an associate editor for “Cannabis and Cannabinoid Research” and an Editorial Board Member of “Medical Cannabis and Cannabinoids” und “MDPI-Reports” and a Scientific board member for “Zeitschrift für Allgemeinmedizin”.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAmerican Psychiatric Association: DSM-5 Diagnostic Classification. In: Diagnostic and Statistical Manual of Mental Disorders. 2015.\n\nCavanna AE: The neuropsychiatry of Gilles de la Tourette syndrome: The état de l’art. Rev Neurol (Paris). 2018 Nov; 174(9): 621–627. PubMed Abstract \| Publisher Full Text\n\nFat MJ, Sell E, Barrowman N, et al.: Public perception of Tourette syndrome on YouTube. J Child Neurol. 2012 Aug; 27(8): 1011–1016. PubMed Abstract \| Publisher Full Text\n\nKnight T, Steeves T, Day L, et al.: Prevalence of tic disorders: a systematic review and meta-analysis. Pediatr Neurol. 2012 Aug; 47(2): 77–90. PubMed Abstract \| Publisher Full Text\n\nLeckman JF, Zhang H, Vitale A, et al.: Course of tic severity in Tourette syndrome: the first two decades. Pediatrics. 1998 Jul; 102(1 Pt 1): 14–19. PubMed Abstract \| Publisher Full Text\n\nLevine JLS, Szejko N, Bloch MH: Meta-analysis: Adulthood prevalence of Tourette syndrome. Prog Neuropsychopharmacol Biol Psychiatry. 2019 Dec 20; 95: 109675. PubMed Abstract \| Publisher Full Text\n\nMol Debes NM, Hjalgrim H, Skov L: Limited knowledge of Tourette syndrome causes delay in diagnosis. Neuropediatrics. 2008 Apr; 39(2): 101–105. PubMed Abstract \| Publisher Full Text\n\nMüller-Vahl KR, Sambrani T, Jakubovski E: Tic disorders revisited: introduction of the term “tic spectrum disorders”. Eur Child Adolesc Psychiatry. 2019 Aug; 28(8): 1129–1135. Epub 2019 Jan 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScharf JM, Miller LL, Gauvin CA, et al.: Population prevalence of Tourette syndrome: a systematic review and meta- analysis. Mov Disord. 2015 Feb; 30(2): 221–228. Epub 2014 Dec 8. PubMed Abstract \| Publisher Full Text\n\nShilon Y, Pollak Y, Benarroch F, et al.: Factors influencing diagnosis delay in children with Tourette syndrome. Eur J Paediatr Neurol. 2008 Sep; 12(5): 398–400. Epub 2007 Dec 4. PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 https://en.wikipedia.org/wiki/Rare_disease.\n\n2 https://en.wikipedia.org/wiki/European_Commission.\n\n3 https://en.wikipedia.org/wiki/Rare_Diseases_Act_of_2002 .\n\n4 https://rarediseases.info.nih.gov/diseases/pages/31/faqs-about-rare-diseases .\n\n5 https://en.wikipedia.org/wiki/Orphan_Drug_Act .\n\n6 www.eurordis.org." }	[ { "id": "86525", "date": "18 Jun 2021", "name": "Peter Nagy", "expertise": [ "Reviewer Expertise neurodevelopmental disorders", "psychopharmacology" ], "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 an important problem Tourette's experts face: by demonstrating how Tourette's syndrome (TS) is not in fact the rare curiosity people had long thought it to be, TS was actually removed from the category of rare diseases, stripping patients of the opportunities rare disease research could bring them. They argue that even though prevalence rates are higher than rare disease definition requirements, the number of patients, especially adult patients with a degree of impairment calling for treatment will be considerably lower, making this subgroup qualify for the rare disease category.\n\nThe need for intensive research is painfully obvious to experts of the field: with the enormous advances in research technologies over the past decades, it is almost embarrassing to admit that the \"exact pathophysiology remains elusive\" to this day (Cavanna, 2018), and it is then no surprise that we are far from being able to confidently offer safe and effective treatment options for our patients (the most recent AAN guideline found only moderate confidence for the efficacy of most therapies, except for CBIT, Pringsheim et al., 2019). However, the main argument (TS should be considered a rare disease) needs some careful consideration. It appears from the available data that TS based on its current definition is definitely not a rare disease, so, based on evidence, classifying the diagnosis per se as rare will not be possible. Careful and evidence-based definition of subgroups, however, as suggested by the authors, could be a useful route to take, but specific criteria based on evidence will need to be defined. Although for research on certain aspects, like pathogenesis, the rationale for excluding some more frequent presentations of the disorder (e.g. pediatric patients or patients without coprolalia and echolalia) seems difficult to support, the study of other aspects, like pharmacological treatment, will have to be limited to substantially smaller populations, very likely to hit rare disease definitions on one hand, and very unlikely to motivate companies to invest in such research on the other.\n\nIn conclusion, the accurate definition of evidence-based subgroups could be a promising way forward towards understanding and hopefully better treating this elusive, but very real and sometimes very impairing disorder.\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": [ { "c_id": "6963", "date": "27 Jul 2021", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr Nagy for his thoughtful comments. We agree that reclassifying TS is impossible or actually helpful. In the same vein, we have no wish to officially move TS into the rare disease category. However, we wish to alert the public and policy makers that, in real life, TS shares many features with rare diseases, i.e., a certain neglect and underfunding – especially when taking into account its prevalence, which represents a mismatch in our opinion. Therefore, benefitting from certain advantages inherent to rare diseases seems legitimate to us – for the time being. One example given is participating in the European Organization for Rare Diseases (EURORDIS) network, and apply for national or EU funding in this field. As to the pharmaceutical industry, it would, as Dr Nagy points out, rather be demotivated to invest into research for TS if it was indeed rare (and again, it isn’t based on prevalence), so that obtaining orphan drug status for TS cannot be considered neither an achievable or desirable aim. However, we agree again with Dr Nagy that more research is needed in defining subgroups in TS; given the fluctuation in symptomatology, it would theoretically be quite possible for an individual to be part of different subgroups within her lifetime! Especially adult TS remains very much of black box in terms of prevalence, evolution and long-term prognosis: here, major research efforts (longitudinal and epidemiological) are needed." }, { "c_id": "7274", "date": "02 Nov 2021", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr Nagy for his thoughtful comments. We agree that reclassifying TS might not be possible or actually helpful. In the same vein, we have no wish to officially move TS into the rare disease category. However, we wish to alert the public and policy makers that, in real life, TS shares many features with rare diseases, i.e., a certain degree of neglect and underfunding – especially when taking into account its prevalence, which represents a mismatch in our opinion. Therefore, benefitting from certain advantages inherent to rare diseases seems legitimate to us – for the time being. One example given is participating in the European Organization for Rare Diseases (EURORDIS) network, and applying for national or EU funding in this field. As Dr Nagy points out, the pharmaceutical industry would be rather demotivated to invest into research for TS if it was indeed rare (and again, it isn’t based on prevalence), so that obtaining orphan drug status for TS cannot be considered neither an achievable nor desirable aim. However, we agree again with Dr Nagy that more research is needed in defining subgroups in TS; given the fluctuation in symptomatology, it would theoretically be quite possible for an individual to be part of different subgroups within her lifetime! Especially adult TS remains very much of black box in terms of prevalence, evolution, and long-term prognosis: here, major research efforts (both longitudinal and epidemiological) are needed. This point has been expanded and highlighted in the revised version of the manuscript, and two references added." } ] }, { "id": "87458", "date": "16 Jul 2021", "name": "Tammy Hedderly", "expertise": [ "Reviewer Expertise Paediatric Movement Disorders and Neurodevelopmental 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\nThis viewpoint article, with its interesting challenge to consider Tourette syndrome (TS) as a rare (or orphan) disease, has come during an unfortunate moment in time where we are experiencing a Covid associated pandemic of tic and tic-like-movements referrals (Heyman, Liang and Hedderly 2021). We are reflecting on these concepts whilst also sitting amongst an ever-increasing pile of new referrals for children and adolescents with a dramatic increase in tics and functional tic-like attacks, making the current prevalence rate far from ‘rare’.\nThe viewpoint paper opens with a title that, in itself, raises controversy. Should TS even be considered a disease at all? When engaging with some of the children and families with tics we often embrace terms such as developmental differences and neuro diversity so as to reduce the stigma sometimes associated with tics. We often encourage children to re-frame their tics and co-morbidities as diversity and difference in order to focus on some of the benefits this neurodiversity brings (for example heightened attention to detail, perfectionist traits or special abilities to switch attention)We routinely do this when the situation allows accepting, of course, for some of the ‘patients’ that more detailed therapeutic strategies are needed to enable individuals to achieve a therapeutic goal. Should the term disease be therefore removed from the discussion?\nThe proposed postulate should probably remind us all of the lively (and fun) debate held at the European Society of Tourette Syndrome (ESSTS) in Copenhagen in 2018 (?) when we were all fortunate enough to meet face to face. At this meeting, we asked if ‘Syndrome’ within Tourette should be replaced with ‘spectrum’ instead, a discussion which helpfully involved those with lived experience of Tourette syndrome. To our unit’s surprise the vote was in favour of keeping the term syndrome, as we had personally changed to using the term Tic/Tourette Spectrum within our own clinical service in 2012. We published our rationale for this in a paediatric journal (Malik and Hedderly 2018) and our unit’s patient surveys revealed a preference for this term. We were pleased to see that our colleagues in Germany (Muller-Vahl 2019) support this preference. We feel this user-led preference for less pathologizing language supports not viewing Tourette syndrome as a disease. It would be interesting to hear the views of individuals with lived experience of Tourette syndrome about the palatability of the term “rare disease” to describe their symptoms.\nThis viewpoint paper provides an engaging discussion around how definitions of rarity of disease and orphan status vary depending on which area the reader sits in, whether in UK, USA or in the European Commission of Public Health. An informative point is raised about the lack of operational criteria for applying the term ‘debilitating’ and the removal if this criteria in DSM -V. The authors also point out helpfully that when using the term ‘wax and wane’ the clinicians should remember this does not just apply to the tic phenomenology but also to how impairing the symptoms are at any given time, although personally we recommend avoiding the use of the term handicap. This is so that we can fully respect people presenting with difficulties or with disability as individuals who can still have control over their own lives.\nIn this article, the term TS is highlighted to be used very much as an umbrella term including people with no presenting complaints and those who do not seek medical attention. The authors appear to advocate a re-evaluation of the term ‘impairment’ from the diagnostic criteria, whilst at the same time acknowledging that removing this term has had some positive benefits such as allowing for more cases to be included in genetic studies.\nThe final position is not entirely clear and we would like to propose the need to consider the views of a user group-the children, adolescents and adults experiencing tics and symptoms or signs of Tourette Spectrum conditions. We would propose again the need to identify different clusters and subgroups of ‘Tourette experiencers’ and, although tics will be at the core of each cluster, we would propose that the defining features of different groups should not involve markers of tic severity at all moving away from the importance placed on using the ‘tic count’.\nThe presence of the co-occuring conditions with a detailed formulation around driving forces to the tics are probably much more relevant (i.e. cluster groups). For example, in the novel currently exploding phenotype which (in our UK experience) has a higher representation of teenage girls with marked social anxiety and self-harm rates reaching 44%, we also see other groups with toe walking and autism traits and the emotionally dysregulated inattentive boys. The clinical cluster view involves making a patient-led, hierarchy of important ‘wishes’ to prioritise for potential modification.\nWe propose that the social and functional difficulties described following tics may be better viewed as primary drivers of tics in some families and not just sequalae, as mentioned in the viewpoint paper. We understand that potentially histaminergic, serotoninergic, dopaminergic, glutaminergic genes and pathways are involved in the generation of tics and tic-like behaviour and in the co-occuring mental health associations. The decision about how rare or not a Tic cluster may be will probably depend on the old dilemma of ‘lumping vs splitting’. Some of the newer bioinformatic approaches of research will ultimately answer the question of which of the pathogenetic mechanisms underpinning the Tourette Spectrum are rarer that the others.\nIn conclusion, we would propose that Tourette syndrome should not be considered a rare disease due to the current prevalence rates, the potential impact on the user group and a need to consider the often frequent co-occurring symptoms. We prioritise an approach which includes the provision of a strength- and needs-based formulation. It is of course imperative to hold user-led consultations regarding these discussions. In the meantime, we would propose not calling TS rare or an orphan ‘disorder or disease’ and instead highlight that a major problem in the field is the ‘rarity’ of available and accessible treatments. In the UK the CAMHS services are overloaded and our movement disorder clinics have long waiting lists to even get to the point of diagnosis. Therapists trained in the management of tics or functional tic like episodes are scarce. Working together we must aim to address the orphan-ness or rarity of the resources to help provide solutions to the users with ever-increasing problems that we are all seeing in our clinics.\nReferences:\nMalik O, Hedderly T. Childhood tic disorders: diagnosis and management. Symposium: Neurology\| Volume 28, Issue 10, P445-453, October 01, 2018\nMüller-Vahl KR, Sambrani T, Jakubovski E. Tic disorders revisited: introduction of the term \"tic spectrum disorders\". Eur Child Adolesc Psychiatry. 2019 Aug;28(8):1129-1135.\nHeyman I, Liang H, Hedderly T. COVID-19 related increase in childhood tics and tic-like attacks. Arch Dis Child. 2021 Mar 6:archdischild-2021-321748. doi: 10.1136/archdischild-2021-321748. Epub ahead of print. PMID: 33677431.\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": "6964", "date": "27 Jul 2021", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr Hedderly for her thoughtful comments. To begin with, we acknowledge that there is an ongoing debate on whether to ground TS – among other neurodevelopmental conditions such as autism spectrum disorder (ASD) or attention deficit hyperactivity disorder (ADHD) – in the neurodiversity field. Which means, among other things, letting go of terms like “disease” or “disorder”, at least for a subset of concerned people, as these may be considered stigmatizing. And, as also mentioned by Dr Hedderly, refrain for using terms like “handicap” or “disability” in relation to these conditions. We consider this debate to be important, but it is not specifically the subject of our article as we depart from three premises: (i) that TS is listed in the DSM-5 and thus, by definition, a medical condition; and (ii) that various health care professionals are following people with tics; and (iii) most importantly – that people with tics seek medical attention out of their own volition (there is no need to advertise our services and waiting lists are, unfortunately, much too long, see below). Therefore, we posit that terms such as “disease” of “disorder” are not entirely out of place, even though we fully acknowledge that impairment related to tics and/or comorbidities can fluctuate significantly, both inter- and intra-individually, as also stated in our article. Because of that fleetingness, we are actually not in favour of reintroducing the ‘impairment’ criterion to the DSM – we simply point out that removing it has created problems of its own. However, in our opinion, there is no perfect solution to these dilemmas, just compromises. At the end of the day, classifications (DSM, ICD and others) and the way medical departments are constructed are rather irrelevant when compared to the demands people in need – suffering people – place on us. And, calling these people “patients” carries no stigma in our opinion: one wonders why any medical diagnosis should carry stigma, to begin with. Moreover, it can be argued that some bullying and harassment people with TS experience stems precisely from the fact that their tics are not recognized as a movement disorder; rather, they find themselves accused of “doing it on purpose”, of trying to attract attention, etc. As we may agree that some people may be incapacitated from tics and comorbidities, Dr Hedderly indeed points out that our departments are in high demand, with severe case loads and long waiting lists. And this is where our points of view merge: neither chronic tics nor TS are rare, numerically speaking (again, we simply conform to official definitions of rarity – 1:2000 prevalence rate – which can be debated in itself); but there are not enough trained experts, providers, and infrastructure around to adequately take care of these patients. Which, in effect, makes TS rare which is, for us, a matter-of-fact statement – but also a call for action. This unsatisfactory situation may be an opportunity to secure support and funding from regional, national or European bodies to help the TS community – both patients and healthcare providers. One example is COST action BM905 (2010-2014) which was instrumental in creating a European network on TS and gave a tremendous boost to the European Society for the Study of Tourette Syndrome (ESSTS); which, since its inception, has always entertained close ties to patient associations which have now organized in an international network (Tics and Tourette across the Globe - TTAG). So, to rephrase: TS is not rare but should be supported as such by public bodies as long as adequate research and medical care is not available in so many countries around the globe. Once TS has become more “mainstream” and is on the map of the general public, government agencies, but also the private sector, and across countries and continents, we’ll be happy to join the ranks of non-rare (common) diseases." }, { "c_id": "7275", "date": "02 Nov 2021", "name": "Andreas Hartmann", "role": "Author Response", "response": "We thank Dr Hedderly for her thoughtful comments. To begin with, we acknowledge that there is an ongoing debate on whether to ground TS – among other neurodevelopmental conditions such as autism spectrum disorder (ASD) or attention deficit hyperactivity disorder (ADHD) – in the neurodiversity field. Which means, among other things, letting go of terms like “disease” or “disorder”, at least for a subset of concerned people, as these may be considered stigmatizing. For the same reason, as also mentioned by Dr Hedderly, we refrain from using terms like “handicap” or “disability” in relation to these conditions. We consider this debate to be important, but it is not specifically the subject of our article as we endorse three premises: (i) that TS is listed in the DSM-5 and thus, by definition, a medical condition; (ii) that various health care professionals are involved in the care of people with tics; and (iii) most importantly – that people with tics typically seek medical attention out of their own volition (there is no need to advertise our services and waiting lists are, unfortunately, much too long - see below). Therefore, we posit that terms such as “disease” of “disorder” are not entirely out of place, even though we fully acknowledge that impairment related to tics and/or comorbidities can fluctuate significantly, both inter- and intra-individually, as also stated in our article. In consideration of such fleetingness, we are actually not in favour of reintroducing the ‘impairment’ criterion to the DSM – we simply point out that its removal has created problems of its own. However, in our opinion, there is no perfect solution to these dilemmas, just compromises. At the end of the day, classifications (DSM, ICD and others) and the way medical departments are structured are rather irrelevant when compared to the demands people in need – suffering people – place on us. Calling these people “patients” carries no stigma in our opinion: one wonders why any medical diagnosis should carry stigma, to begin with. Moreover, it can be argued that some bullying and harassment people with TS experience stems precisely from the fact that their tics are not recognized as a movement disorder; rather, they find themselves accused of “doing it on purpose”, of trying to attract attention, etc. We have incorporated these points in the revised version of the manuscript. We have also changed the title by using “condition” instead of “disease”, even though this term cannot be avoided entirely when speaking of research initiatives and networks in this domain. Finally, we would like to remind of the question mark in the title and the ”proposal” in the end: this is an opinion paper supposed to stimulate discussion on an admittedly complicated topic, not scripture. As we agree that some people may be incapacitated by their tics and comorbidities, Dr Hedderly indeed points out that our departments are in high demand, with severe caseloads and long waiting lists. And this is where our points of view merge: neither chronic tics nor TS are rare, numerically speaking (again, we simply conform to official definitions of rarity – 1:2000 prevalence rate – which can be debated in itself); but there are not enough trained experts, providers, and infrastructure around to adequately take care of these patients. This, in practical terms, makes TS rare which is, for us, a matter-of-fact statement – but also a call for action. This unsatisfactory situation may be an opportunity to secure support and funding from regional, national, or European bodies to help the TS community – both patients and healthcare providers. One example is COST action BM905 (2010-2014) which was instrumental in creating a European network on TS and gave a tremendous boost to the European Society for the Study of Tourette Syndrome (ESSTS). Since its inception, ESSTS has consistently entertained close ties to patient associations which have now organized in an international network (Tics and Tourette across the Globe - TTAG). As to the recent surge in functional tics, these are not the subject of our article, and we do not consider them to be a variant of TS but rather a separate entity (Müller-Vahl et al., 2021). However, we agree that these patients put an additional strain on our already overstretched capacities. Concerning definition of evidence-based clusters, this point has also been raised by Dr Nagy, and modifications were made to the manuscript. We agree that this is an important endeavour and, of course, these clusters precisely incorporate comorbidities and move beyond what Dr Hedderly refers to as the ‘tic count’. As TS professionals, we have neither felt nor stated that tics are the sole source of impairment. Likewise, instead of arguing that tics drive social and functional difficulties, we have acknowledged a bidirectional relationship. So, to rephrase: TS is not rare but should be supported as such by public bodies, as long as adequate research and medical care is not available in so many countries around the globe. Once TS has become more “mainstream” and features more prominently in the radar of the general public, government agencies, but also the private sector, and across countries and continents, we’ll be prepared to join the ranks of non-rare (common) conditions. Müller-Vahl KR, Pisarenko A, Jakubovski E, Fremer C. Stop that! It's not Tourette's but a new type of mass sociogenic illness. Brain. 2021 Aug 23:awab316. doi: 10.1093/brain/awab316. Epub ahead of print. PMID: 34424292." } ] } ]	1	https://f1000research.com/articles/10-434
https://f1000research.com/articles/10-231/v1	24 Mar 21	{ "type": "Systematic Review", "title": "SARS-CoV-2 and the role of orofecal transmission: a systematic review", "authors": [ "Carl J. Heneghan", "Elizabeth A. Spencer", "Jon Brassey", "Annette Plüddemann", "Igho J. Onakpoya", "David H. Evans", "John M. Conly", "Tom Jefferson", "Carl J. Heneghan", "Jon Brassey", "Annette Plüddemann", "Igho J. Onakpoya", "David H. Evans", "John M. Conly", "Tom Jefferson" ], "abstract": "Background: Mode of transmission of SARS-CoV-2 is of key public health importance. SARS-CoV-2 has been detected in the feces of some COVID-19 patients, suggesting the possibility that the virus could, in addition to droplet and fomite transmission, be transmitted via the orofecal route. Methods: This review is part of an Open Evidence Review on Transmission Dynamics of COVID-19. We conduct ongoing searches using WHO COVID-19 Database, LitCovid, medRxiv, and Google Scholar; assess study quality based on five criteria and report important findings on an ongoing basis. Where necessary, authors are contacted for further details on the content of their articles. Results: We include searches up until 20 December 2020. We included 110 relevant studies: 76 primary observational studies or reports, and 35 reviews (one cohort study also included a review) examining the potential role of orofecal transmission of SARS-CoV-2. Of the observational studies, 37 were done in China. A total of 48 studies (n=9,081 patients) reported single cases, case series or cohort data on individuals with COVID-19 diagnosis or their contacts and 46 (96%) detected binary RT-PCR with 535 out of 1358 samples positive for SARs-CoV-2 (average 39.4%). The results suggest a long duration of fecal shedding, often recorded after respiratory samples tested negative, and symptoms of gastrointestinal disease were reported in several studies. Twenty-nine studies reported finding SARS-CoV-2 RNA in wastewater, river water or toilet areas. Six studies attempted viral culture from COVID-19 patients’ fecal samples: culture was successful in 3 of 6 studies, and one study demonstrated invasion of the virus into the intestinal epithelial cells. Conclusions: Varied observational and mechanistic evidence suggests SARS-CoV-2 can infect and be shed from the gastrointestinal tract, including some data demonstrating viral culture in fecal samples. Future studies should test this hypothesis rigorously to allow the development of appropriate public health measures.", "keywords": [ "Orofecal", "transmission", "COVID-19", "SARS-CoV-2", "systematic review" ], "content": "Introduction\n\nUnderstanding how, when and in what types of settings SARS-CoV-2 spreads between people is critical to developing effective public health and infection prevention and control measures to break the chains of transmission. Current evidence suggests SARS-CoV-2 is primarily transmitted via respiratory droplets and fomites between infected individuals and others in close contact1.\n\nSARS-CoV-2 has been shown to contaminate and survive on certain surfaces, but currently, no reports have directly demonstrated fomite to human transmission. SARS-CoV-2 has also been detected in the feces of some patients which suggests the possibility of fomite transmission and that SARS-CoV-2 could transmit via the orofecal route. It is well recognized that coronaviruses are major pathogens in many mammalian species and predominantly target epithelial lining cells in the respiratory and gastrointestinal (GI) tracts. Many animal coronaviruses are transmitted by the fecal-oral route and there are many reports of intestinal disease associated with SARS-CoV-1 and other human coronaviruses. Main causes include lack of adequate sanitation and poor hygienic practices. Fecal contamination of food is another form of orofecal transmission. Therefore, we aimed to systematically review the evidence on orofecal SARS-CoV-2 transmission. Terminology for this article can be found in Box 1.\n\n\n\nOrofecal: describes a route of transmission where the virus in fecal particles can pass from one person to the mouth of another.\n\nViral load:A measure of the number of viral particles present in an individual.\n\nCycle threshold: The number of cycles required for the fluorescent signal to cross the threshold. Ct levels are inversely proportional to the amount of target nucleic acid in the sample.\n\n\nMethods\n\nWe are undertaking an open evidence review investigating factors and circumstances that impact on the transmission of SARS-CoV-2, based on our protocol (see Extended data: Appendix 12). For the original protocol, see https://www.cebm.net/evidence-synthesis/transmission-dynamics-of-covid-19/. In brief, this review aims to identify and evaluate relevant articles (peer-reviewed or awaiting peer review) that examine the mode of viral transmission and ecological variables influencing the mode of transmission. We conduct an ongoing search using WHO COVID-19 Database, LitCovid, medRxiv and Google Scholar for keywords and associated synonyms. Results are reviewed for relevance and for articles that looked particularly relevant forward citation matching was undertaken and relevant results were identified. Studies with modelling are only included if they report transmission outcome data and not predicted outcomes (see further details of the search strategy in the Extended data: Appendix 22). Searches are updated every two weeks.\n\nWe extracted data on the type of study, setting, sample source and methods, fecal PCR positive samples for SARS-CoV-2 RNA including cycle threshold (including methods), symptom chronology in relation to PCR testing and/or taking samples and viral culture. We tabulated the data and summarised data narratively by mode of sample. We assessed quality using a modified QUADAS-2 risk of bias tool3. We simplified the tool as the included studies were not designed as primary diagnostic accuracy studies and assessed study quality based on five criteria. Where necessary we write to authors of included studies for further details or clarification on the content of their articles. Meta-analyses were not performed, due to the variability of available data. The protocol was last updated on 1 December 2020 (Version 3: 1 December 2020).\n\n\nResults\n\nThis update includes searches up until 20 December 2020 (see Figure 1). We identified 110 relevant studies (see Extended data: Appendix 3 for references of included studies2): 76 primary studies or reports, and 35 reviews, one of which also reported primary study results from a cohort study [Cheung K 2020].\n\nThe included reviews summarised a range of observational studies including studies of detection of SARS-CoV-2 RNA in fecal samples of individuals testing positive for SARS-CoV-2 in respiratory samples, frequency of GI symptoms among those with COVID-19, and observations of SARS-CoV-2 RNA in toilets and wastewaters. The reviews included overlapping studies and must therefore not be considered as entirely additional information. Five followed systematic review methodology and reporting [Edwards 2020, Karia 2020, Pamplona 2020, Parasa 2020, Santos 2020]. The quality of the other reviews was low to moderate, with none assessing included study quality, and with reporting of methods often missing or very limited.\n\nNone of these reviews focussed on the viability (and hence transmission potential) of SARS-CoV-2 identified in fecal or wastewater samples. A review on the potential for foodborne transmission of SARS-CoV-2 found no published studies of SARS-CoV-2 survival in or on food products. The totality of the reviews’ evidence shows that the SARS-CoV-2 RNA is commonly present in stool samples of COVID-19 patients but it is unknown if this represents primary invasion of enterocytes or simply saliva and sputum that has been swallowed and is transiting it way through the GI tract. The presence of viral RNA in the feces does not allow any conclusions to be drawn about infectiousness. The contribution of orofecal transmission to viral spread in the pandemic has not been established or quantified.\n\nQuality of included studies. Overall the quality of the evidence was low to moderate mainly due to a lack of standardisation of techniques, omissions in reporting and a failure to account for biases in the research process (see Table 3; Figure 2). Sample sources were clear in two-thirds of studies (65.8%). Several studies mention the possibility of bias influencing their findings but did not use strategies (design or analysis) to deal with bias, and as such have been recorded as unclear risk of bias.\n\nResults. A total of 48 (n=9,081 patients) studies reported single cases, case series or cohort data on individuals with COVID-19 diagnosis or their contacts and 46 studies (96%) detected binary RT-PCR with 535 out of 1358 samples RT-PCR positive for SARS-CoV-2 (average 39.4%). All but five studies were in hospitalized patients; 31 were done in China; the others were in East Asia, South East Asia, South Asia, USA and Europe (see Table 1).\n\nPCR testing for SARS-CoV-2 RNA in fecal samples in hospitalised COVID-19 patients. Of the 43 hospital studies, 41 (95%) detected binary RT-PCR, with 522 positive tests out of 1293 fecal samples (average 40.4%) from COVID-19 patients (see Table 4). These studies were mainly small case series, they included patients of a range of ages from infant to elderly and with widely varying severity of the disease, and the proportion of fecal samples varies from 1 (nine studies) to 258 [Zhang Y, Chen C], and the proportion testing positive for SARS-CoV-2 RNA varied from 14% to 100% across studies. One study that identified SARS-CoV-2 RNA in fecal samples among 39 of 73 hospitalised COVID-19 patients, also studied the gastric, duodenal and rectal epithelia of one patient using specimens collected via endoscopy [Xiao F, Tang M 2020].\n\nImmunofluorescence data showed that ACE2 protein, proven to be a cell receptor for SARS-CoV-2, was abundantly expressed in the glandular cells of gastric, duodenal, and rectal epithelia, supporting entry of SARS-CoV-2 into the host cells. Intracellular staining of viral nucleocapsid protein in gastric, duodenal, and rectal epithelium showed that SARS-CoV-2 infects these GI glandular epithelial cells. Viral RNA was also detected in esophageal mucous tissue, but a lack of viral nucleocapsid protein staining in esophageal mucosa suggested low viral infection there. Viral nucleocapsid protein in rectal epithelial cells was detected in specimens from some additional COVID-19 patients, suggesting that infectious SARS-CoV-2 can survive the GI environment.\n\nReporting of GI symptoms among COVID-19 patients is frequent but not consistent within these studies (diagnosis is typically based on fever, respiratory symptoms and the results of PCR testing in respiratory swabs, so recording GI symptoms may not be routine). However, several observational studies report the presence of GI symptoms among COVID-19, including Chan 2020, Cheung 2020 and Han C 2020. GI symptoms do not necessarily correlate in severity or time with other COVID-19 disease symptoms.\n\nFecal shedding of SARS-CoV-2 has been reported throughout the disease course and also continuing after respiratory samples tested negative. A five-person family with a confirmed COVID-19 case was hospitalized and observed: the parents and two children aged two and five years became infected but the youngest child was not infected. These children shed infectious virus via the respiratory system, and this shedding observed in the nasopharynx cleared after five to 6 days; however, viral RNA was continuously detected in the children’s stool for more than four weeks [Wolf 2020]. Tang 2020 et al. reported an apparently asymptomatic (no fever or cough) 10-year-old child, from whom, 17 days after the last close contact with individuals testing positive for SARS-CoV-2, a fecal sample was positive for SARS-CoV-2 RNA. A retrospective study of 133 hospitalised COVID-19 patients identified 22 whose sputum or fecal samples tested positive after pharyngeal swabs became negative [Chen C 2020]. A study of 59 hospitalised COVID-19 patients reported that fecal discharge of SARS-CoV-2 RNA continued long after respiratory shedding had ceased [Cheung 2020]. Among fecal samples from 69 hospitalized COVID-19 patients, 20 tested PCR positive; SARS-CoV-2 RNA persisted for significantly longer in fecal samples than in oropharyngeal swabs [Wang X, Zheng J 2020].\n\nThe role of aerosol-generating procedures in relation to orofecal transmission. One study reported the results of PCR tests among participants attending for upper GI endoscopy at a UK hospital [Hayee 2020]. Only individuals testing negative in nasopharyngeal swab PCR underwent their booked procedure. Post-procedure follow-up of 6,208 patients at one and two weeks identified no incident case of COVID-19.\n\nViral culture using fecal samples was attempted in 6 studies [Jeong 2020, Kim JM 2020, Wang W, Xu Y 2020; Wölfel 2020; Xiao F 2020 and Zhang Y 2020], three reported culture of SARS-CoV-2 from stool samples [Wang W, Xu Y 2020; Xiao F 2020, Zhang Y 2020], and three did not [Jeong 2020, Kim JM 2020 and Wölfel 2020].\n\nWang W 2020 et al. reported two of four viral culture samples, however, no culture methods were reported; electron microscopy was performed suggesting the presence of the virus, but this observation does not show that the virus was viable. In Xiao F Sun J 2020, viral culture was attempted from an unreported number of specimens and cases and the cytopathic effect in Vero E cells was observed 2 days after a second-round passage. Zhang Y 2020 et al. reported isolating the virus from the stools of one severe hospitalised COVID-19 pneumonia case. The number of samples taken was unclear and while Vero cells were used for viral isolation from stool samples, culture methods were not described.\n\nAdditional evidence of SARS-CoV-2 replication activity was observed within the intestine: an inpatient for treatment of a rectal adenocarcinoma had samples taken from enteric sections, and the mucosa of rectum and ileum analysed [Qian 2020]. The rectal swab sample tested negative for SARS-CoV-2 RNA by PCR. However, typical coronavirus virions in rectal tissue were observed under electron microscopy with abundant lymphocytes and macrophages (some SARS-CoV-2 positive) infiltrating the lamina propria.\n\nMethodological issues across these studies including variability in sample selection and methods of viral culture, reported in Table 5, mean the results may not be comparable and should be interpreted with caution.\n\nStudies of wastewater and sewage. 22 global studies investigated SARS-CoV-2 in wastewater or sewage, four looked at the role of toilets or sewage, and one reported observation in river water [Ahmed 2020, Ampuero 2020, Arora 2020, Chavarria-Miro 2020, Curtis 2020, Fongaro 2020, Fernandez-de-Mera 2020, Haramoto 2020, Hata 2020, Lara 2020, La Rosa 2020b, Medema 2020, Neault 2020, Ong 2020, Peccia 2020, Sharif 2020, Shutler 2020, Trottier 2020, Wang J 2020, Wang XW, Li J 2020, Wurtzer 2020, Zhao 2020].\n\nAll reported the detection of SARS-CoV-2 RNA and/or SARS-CoV-2 viral proteins; a number of studies suggested the potential of detection in sewage to be used as a public health monitoring system.\n\nWastewater surveillance may anticipate outbreaks: a Brazilian study identified SARS-CoV-2 RNA from sewage samples collected in November 2019 [Fongaro 2020]; a Spanish study detected sewage samples from 41 days prior to the declaration of the first COVID-19 case in Spain, and additionally in frozen samples dating back to 12 March 2019 [Chavarria-Miró 2020]. Virus concentrations found in wastewaters may correlate with prevalence in the local community. At very low community levels of circulation, sewage surveillance could detect SARS-CoV-2 RNA presence [La Rosa b 2020, Medema 2020].\n\nA study of hospital surfaces and sewage areas in China reported no viable virus was detected by culture of surface swabs or sewage samples [Wang J 2020]. Another study in China tested hospital sewage and found SARS-CoV-2 RNA but no viral culture of SARS-CoV was present in the sewage in their assays [Wang XW, Li J 2020].\n\nToilets. An additional four studies investigated SARS-CoV-2 in toilets (Del Brutto 2020, Ding Z 2020, Kang 2020, Ong 2020]. A study in rural Guatemala reported the sole predictive variable was the use of the open latrines [Del Brutto 2020]. Ding 2020 et al. collected surface samples from a COVID-19 hospital in China reported toilet areas represented the most contaminated areas. Kang et al. reported the possibility of fecal aerosol spread but with little discussion of alternate pathways for transmission. They did not find any evidence of virus -laden bioaerosols. Ong and co-workers reported that 3 of 5 toilet sites (toilet bowl, sink, and door handle) in hospital settings were PCR-positive; anteroom and corridor samples were negative [Ong 2020].\n\nRiver water. One study tested river water for SARS-CoV-2: taking samples from three urban river locations in a low sanitation urban context (i.e. highly impacted by raw sewage) in Quito, Ecuador, during a peak of COVID-19 cases. SARS-CoV-2 RNA was detected in all samples, at levels similar to those in wastewater from cities during outbreaks [Guerreo-Latorre 2020]. Evidence from reviews can be found in Table 2.\n\n\nDiscussion\n\nThe evidence from 110 relevant studies supports a potential role of orofecal transmission of SARS-CoV-2. Fecal shedding of SARS-CoV-2 RNA has been reported in 96% of the included observational studies, often for relatively long durations. Three studies reported the culture of SARS-CoV-2 using fecal samples, but requisite methods to confirm viral growth were lacking. One study demonstrated viral isolation from rectal tissue of a COVID-19 patient. Studies in hospitals show the presence of SARS-CoV-2 RNA at and around toilets and toilet rooms; there is evidence that disinfectant cleaning leaves no SARS-CoV-2 RNA detectable. Many studies report identifying SARS-CoV-2 RNA in sewage and wastewaters, but viral culture from such sources has not been demonstrated, and therefore the detection in sewage and wastewaters can be useful as a surveillance tool.\n\nExperimental models of the human intestinal epithelium show that SARS-CoV-2 can infect this tissue and replicate, supporting the rationale for the human GI tract as a possible transmission route4–6). Zang 2020 demonstrated that human enterocytes express high ACE2 receptor levels, supporting viral invasion at these sites4. Zang 2020 and Lamers 2020 et al. showed that SARS-CoV-2 productively infected human small intestinal organoids4,5. Zhou J 2020 et al. and co-workers showed active replication of SARS-CoV-2 in human intestinal organoids, and isolated infectious virus from the stool specimen of a patient with diarrheal COVID-196. Yao et al. investigated the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived SARS-CoV-2 isolates in diverse cell lines; the authors report that \"three of our viral isolates were extracted from stool samples (two of which were very potent) indicating that viable SARS-CoV-2 particles could be found in stool samples\"7.\n\nMERS-CoV has been shown to infect human primary intestinal epithelial cells, small intestine explants and intestinal organoids8. MERS-CoV has been detected in 42% of milk samples collected from lactating camels where it can survive for a prolonged period. A study of human primary intestinal epithelial cells and small intestine explants of MERS-CoV patterns identified the viral replication intermediates in stool specimens. MERS-CoV was found to be resistant to fed-state gastrointestinal fluids but less tolerant to the high acidic fasted-state gastric fluid.\n\nProlonged excretion of coronaviruses in feces was first observed in 19779. In the SARS-CoV-1 outbreak in 2002-03, a significant portion of patients had enteric involvement. In the Toronto outbreak in 2003, 6% of 144 patients had diarrhoea on presentation10. Among 138 patients with SARS in Hong Kong, 20% presented with watery diarrhoea and 38% had symptoms of diarrhoea during the illness. Intestinal biopsy specimens showed the presence of active viral replication, and SARS-CoV RNA was detected in the stool of some patients for more than ten weeks after symptom onset11. A retrospective study on specimens from 154 patients in Hong Kong with laboratory-confirmed SARS found the viral load to be the highest in stool specimens12. Up to 70% of 75 patients in a community outbreak in Hong Kong developed watery diarrhoea13. This outbreak was linked to a faulty sewage system in the Amoy Gardens apartment complex, further suggesting orofecal transmission might be a route for transmission14.\n\nThe human gastrointestinal tract can act as a primary infection site for SARS-CoV. Ding et al. used a monoclonal antibody specific for the SARS‐CoV nucleoprotein, and probes for the RNA polymerase gene fragment in four patients who died from SARS‐CoV-115. Virus was detected in the stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal, liver and cerebrum. The authors discussed that viruses in contaminated food and water may enter the human body through epithelial cells covering the surface of the gastrointestinal tract, although there was no direct evidence to show that food‐borne transmission had occurred. A study from the sewage of two hospitals receiving SARS patients in Beijing found no infectious SARS-CoV contamination in any of the samples collected but did detect the nucleic acid in the sewage from the two hospitals before disinfection - providing further evidence that SARS-CoV-1 can be excreted by feces into the sewage system16.\n\nTransmission of coronaviruses via the feces is established among animals: feline coronavirus, for instance, is typically shed in feces of healthy cats and transmitted by the orofecal route to other cats17. Pigs are also infected by the transmissible gastroenteritis coronavirus via the orofecal18. Bat coronavirus infects the gastrointestinal and respiratory tracts of bats, seemingly without causing disease19. Transmission following exposure to camel feces has also been considered biologically plausible20.\n\nThere is evidence that SARS-CoV-2 can survive adverse conditions in the gastrointestinal system. It has been identified in endoscopic specimens of the oesophagus, stomach, duodenum, and rectum of COVID-19 patients; substantial amounts of SARS-CoV-2 RNA have been consistently detected in stool specimens, and evidence suggests that SARS-CoV-2 can survive the adverse conditions in the gastrointestinal system. Heavy glycosylation of the large spike S protein has been shown to lead to resistance to the proteases, the low pH and bile salts found in the gastrointestinal system. Some gastric processes may actually facilitate viral entry into the enterocytes: in bovine coronavirus, one specific site on the S glycoprotein has to be cleaved by an intracellular protease or trypsin to activate viral infectivity and cell fusion21.\n\nEvidence of ingestion, penetration of enterocytes and excretion of live SARS-CoV-2 is possible; however, this working hypothesis requires testing by conducting case-control studies during the investigation of outbreaks, following a set protocol. For such investigations, cases of COVID-19 (categorised by symptom presence and severity) either fecally excreting virions or not (cases and contacts) and controls would be healthy matches. Exposure to potentially fecally contaminated materials and protective measures taken would be elicited at interview. To minimise the play of recall and ascertainment bias, interviewers should be blind to fecal excretion status and the interview should take place as soon as possible after the event. Viability of fecal isolates and their possible pathogenicity should be tested in outbreaks.\n\nStrengths and limitations. This review is limited by the quality of included studies: many were small and did not provide a protocol that established a priori methods. Studies were often poorly reported and often did not take biases into account. Reporting is often heterogeneous and essential information such as symptom onset and cycle threshold values were often missing. We do not have information on publication bias, but the current urgency to understand SARS-CoV-2 may have an impact on research, with unknown implications and a tendency to publish those studies with positive results. It is likely we missed some studies, but we plan to keep updating this review. Furthermore, our judgments of quality are to some extended subjective and open to disagreement. This does not, however, undermine our overall assessment of the quality of the included studies. We perceive that standardization of methods in this area would improve the quality of the research. Some of these limitations increase uncertainties and prevent firm conclusions being drawn; however, this body of research provides largely consistent evidence on the main conclusions that SARS-CoV-2 is excreted fecally, is found in sewage and can be cultured from fecal samples.\n\n\nConclusion\n\nObservational and mechanistic evidence as well as established animal orofecal transmission of coronaviruses suggests SARS-CoV-2 can infect and be shed from the human gastrointestinal tract. Future studies should test this hypothesis rigorously to allow the development of appropriate public health measures.\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 for SARS-CoV-2 and the role of orofecal transmission: a systematic review, https://doi.org/10.6084/m9.figshare.14247470.v12.\n\nThis project contains the following extended data:\n\n- Appendix 1: Updated protocol\n\n- Appendix 2: Search strategy\n\n- Appendix 3: References to included studies.\n\nFigshare: PRISMA checklist for ‘SARS-CoV-2 and the role of orofecal transmission: a systematic review’, https://doi.org/10.6084/m9.figshare.14247470.v12.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nA previous version of this review is available on medRxiv: https://doi.org/10.1101/2020.08.04.20168054\n\n\nReferences\n\nWorld Health Organisation. Accessed 2 January 2021. Reference Source\n\nHeneghan C, Spencer E, Brassey J, et al.: Extended data for SARS-CoV-2 and the role of orofecal transmission: a systematic review. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14247470.v1\n\nWhiting PF, Rutjes AW, Westwood ME, et al.: QUADAS-2 Group. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med. 2011; 155(8): 529–36. PubMed Abstract \| Publisher Full Text\n\nLamers MM, Beumer J, van der Vaart J, et al.: SARS-CoV-2 productively infects human gut enterocytes. Science. 2020; 369(6499): 50–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZang R, Castro MFG, McCune BT, et al.: TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol. 2020; 5(47): eabc3582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou J, Li C, Liu X, et al.: Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med. 2020; 26(7): 1077–1083. PubMed Abstract \| Publisher Full Text\n\nYao H, Lu X, Chen C, et al.: Patient-derived mutations impact pathogenicity of SARS-CoV-2. Reference Source\n\nZhou J, Li C, Zhao G, et al.: Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus. Sci Adv. 2017; 3(11): eaao4966. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaul EO, Egglestone SI: Further studies on human enteric coronaviruses. Arch Virol. 1977; 54(1–2): 107–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth CM, Matukas LM, Tomlinson GA, et al.: Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. JAMA. 2003; 289(21): 2801–2809. PubMed Abstract \| Publisher Full Text\n\nLeung WK, To KF, Chan PK, et al.: Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology. 2003; 125(4): 1011–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHung IF, Cheng VC, Wu AK, et al.: Viral loads in clinical specimens and SARS manifestations. Emerg Infect Dis. 2004; 10(9): 1550–1557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeiris JS, Chu CM, Cheng VC, et al.: Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet. 2003; 361(9371): 1767–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO environmental health team reports on Amoy gardens. http\n\nDing Y, He L, Zhang Q, et al.: Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol. 2004; 203(2): 622–630. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang XW, Li JS, Guo TK, et al.: Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital. [published correction appears in J Virol Methods. 2005 Dec; 130(1–2): 210]. J Virol Methods. 2005; 128(1-2): 156–161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHartmann K: Feline infectious peritonitis. Vet Clin North Am Small Anim Pract. 2005; 35(1): 39–79, vi. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Q, Yoo D: Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling. Virus Res. 2016; 226: 128–141. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFan Y, Zhao K, Shi ZL, et al.: Bat Coronaviruses in China. Viruses. 2019; 11(3): 210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKillerby ME, Biggs HM, Midgley CM, et al.: Middle East Respiratory Syndrome Coronavirus Transmission. Emerg Infect Dis. 2020; 26(2): 191–198. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolmes KV: Enteric infections with coronaviruses and toroviruses. Novartis Found Symp. 2001; 238: 258–69; discussion 269-75. PubMed Abstract \| Publisher Full Text" }	[ { "id": "82122", "date": "07 Apr 2021", "name": "Mark D. Sobsey", "expertise": [ "Reviewer Expertise I am an environmental and public health virologist and microbiologist with expertise in infection protection and control and water-sanitation and hygiene", "as well as in infectious disease epidemiology in the context of environmental exposure risks and disease transmission of human pathogens ." ], "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 of generally high quality, but improvements are suggested in some areas, including in the conclusions. Starting from the beginning of the article here are some points that should be clarified or improved:\nIn Box 1. Terminology, Viral load is defined in a rather vague and insufficiently quantitative way. Viral load can be quantified on the basis of virions (physical particles (as seen by electron microscopy), gene copies or equivalents based on RNA units or CT values, antigen units such viral proteins quantified in different ways, including immunofluorescence intensity observed in infected tissues and as infectious units for replication competent virus based on titers in susceptible cell cultures or experimental animals. There is value in focusing on quantified \"viral load\" in human gastrointestinal tissues, intestinal contents and fecally contaminated materials such as fomites, wastewater (e.g., sewage) and water (such as recreational, agricultural irrigation and drinking waters). These nuances in \"viral load\" are important in interpreting and communicating the transmission-relevant results reported in the research studies reviewed..\n\nThe data found in the study and provided in Table 1 indicate that only a few studies (5 according to table 1) attempted to detect infectious or replication-competent SARS-CoV-2 in either feces, sewage or a related sample, of which only 3 reported the presence of infectious virus. Further, infectious virus results are reported as \"yes\" or \"no\" (presence or absence) per sample. The data for infectious virus are not quantified in typical units such as plaque-forming units or TCID-50 units. Therefore, the concentrations of the detected viruses are unknown. Such lack of quantitative data on infectious virus concentration makes it impossible to determine if sufficient infectious virus is present to pose a transmission risk from human exposure. This point should be mentioned in the overall presentation of the infectious virus results. Furthermore, we do not know the infectivity dose-response relationship of SARS-CoV-2 for human hosts, such an estimate of 50% infectious dose. Unless we know what virus concentration is in samples such as feces or sewage/wastewater and unless we know what the likelihood of human infection is from exposure to known concentrations of infectious virus, it is probably unjustified to suggest that the presence of an unknown concentration of infectious virus in a very small number of reported virus-positive fecal samples poses a human health risk from exposure. While infectious virus presence in a few samples is noteworthy, the lack of quantitative infectivity data makes it impossible to judge that there is plausible human health exposure risk from this infectious virus-positive fecal matter of unknown concentration. The best one can say is that the risk of infection or other health effects from exposure is not zero, but is also not quantifiable due to lack of data on virus concentration in feces and lack of knowledge of human infectivity dose-response for infection or other health endpoints.\n\nFor sewage and water, the lack of data on virus infectivity in such samples makes it impossible to determine if these samples pose risks of human infection from exposure. Although some data on concentrations of viral RNA are reported based on estimated gene copies or CT values, the quantitative relationship of virus infectious units to RNA concentration units is highly variable and therefore uncertain This is especially so for samples that have been in the environment for unknown and variable periods of time since the feces were shed from a human host. This is due to the variable and difficult-to-quantify effects of exposure time, temperature, microbial activity, predation and other environmental stressors on virus survival. These factors are not mentioned in the paper, even though we know they are important for determining the quality and validity of the results for infectious virus presence and concentrations. The last sentence of the first paragraph of the Discussion section does not adequately address the issue of lack of evidence of virus culture in wastewater and sewage and its implications for human health risks from sewage. Instead it sidesteps to the potential value using SARS-CoV-2 RNA in sewage and wastewater as a surveillance tool. This seems to miss the main point of assessing the evidence for human health risk from exposure to sewage or wastewater.In the \"strengths and limitations section of the Discussion section, the authors state \"however, this body of research provides largely consistent evidence on the main conclusions that SARS-CoV-2 is excreted fecally, is found in sewage and can be cultured from fecal samples.\" While there there is some evidence that unknown but probably low concentrations of infectious SARS-CoV-2 have been occasionally found in excreted feces, they have not been found in sewage or wastewater. The wording of the above sentence conflates feces with wastewater and sewage, even though there is only limited and poor evidence for infectious virus in the latter and no evidence for it in sewage or wastewater. I recommend that the sentence be revised to avoid conflating these rather separate exposure sources of feces versus sewage and wastewater.\n\nThe Conclusion sentence is short and limited in scope. It does not adequately address the issue of whether sufficient infectious SARS-CoV-2 is or is not likely to be present in feces or wastewater and sewage to pose human health risks from exposure. The available evidence seems to indicate that such risks are not possible to assess or quantify without better and more rigorous and as well as more quantitative data that is still not available.\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": "7239", "date": "02 Nov 2021", "name": "Elizabeth Spencer", "role": "Author Response", "response": "Thank you for these helpful suggestions to improve the review. Please see our changes made, noted below. 1. Added: typically quantified by the number of virions, gene copies or equivalents, or by antigen indicators of viral load. 2. Added to the Discussion: these studies do not provide an estimate of infectious virus concentration; nor has the infectious dose for humans been established, so at this point it is unwarranted to deduce a human transmission risk based on this small number of virus-positive fecal samples. 3. Changed Discussion sentence: Many studies report identifying SARS-CoV-2 RNA in sewage and wastewaters, but viral culture from such sources has not been demonstrated, so there is no evidence of infection risk from those sources; however, detection of SARS-CoV-2 RNA in sewage and wastewaters can be useful as a surveillance tool. 4. Added: However, quantitative data on infectiousness and the consequent likelihood of transmission from orofecal contamination is not available. Whilst SARS-CoV-2 RNA is observed in sewage and wastewaters, there is no evidence of infectiousness in these sources and a transmission risk from these sources is therefore unlikely. To properly assess these risks, quantitative data on infectious virus are needed, along with information on likely infectious dose in humans." } ] }, { "id": "89625", "date": "12 Aug 2021", "name": "Jennifer Audsley", "expertise": [ "Reviewer Expertise I am a Clinical Research Fellow with a major research interest in HIV-Hepatitis co-infection", "and since 2020 also COVID-19 infection" ], "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\nHeneghan et al. present a systematic review of SARS-CoV-2 and the role of orofecal transmission. They included 110 relevant studies (including 35 reviews) from searches up until 20th December 2020. Potential orofecal transmission of SARS-CoV-2 is an important public health issue. They found the observational evidence suggested SARS-CoV-2 infects and is shed from the gastrointestinal tract (GIT) and with further testing could allow the development of appropriate public health measures. This systematic review has followed the PRISMA guidelines including developing a review protocol and completing the PRISMA checklist. All underlying data is available and extended data has been uploaded onto Figshare. This review is very well-written and the references relevant. The authors have undertaken this review thoroughly, with the largely low-moderate data available. There are, however, some points that should be addressed to strengthen the review and conclusions.\nThe aim was to systematically review evidence on orofecal SARS-CoV-2 transmission while the conclusion focuses on SARS-CoV-2 fecal excretion and presence in sewage, which is not evidence of transmission. Detection of SARS-CoV-2 in stool samples and in ecological settings is important but it should be noted that was not the aim of this review. Viral culture data from six studies is included but this is a small minority of studies, and as the authors note due to methodological issues should be interpreted with caution.\n\nExamining the ecological studies and review is very worthwhile but none address the issues of orofecal SARS-CoV-2 transmission. Perhaps ecological factors are a secondary outcome?\n\nWhile the authors do note in the results that some reviews included overlapping studies, there is no figure/percentage given for this. How much overlap in total is there, is the overlap greater for cohort/case series or ecological studies? What is the quality of the overlapping studies – are they over-representing the low-quality data?\n\nWhat is the breakdown for included reviews between cohort/case series and ecological studies? This is provided for primary studies in Figure 1 but I can’t find it for the reviews.\n\nHave you considered a sub analysis of the studies in children?\n\nIs gender reported routinely across the studies?\n\nHow many/percentage of the studies are only on MedRxiv? Consider including breakdown by primary/review, cohort-case series/ecological. This could be a source of bias.\n\nMinor:\nTable 1 pages 10, 11 & 12 – total patient number from the cohort/case series (9081) appears in the “sewage” header rows.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "7240", "date": "02 Nov 2021", "name": "Elizabeth Spencer", "role": "Author Response", "response": "Thank you for these helpful suggestions to improve the review. Please see our changes made, noted below. 1. Added: typically quantified by the number of virions, gene copies or equivalents, or by antigen indicators of viral load. 2. Added to the Discussion: these studies do not provide an estimate of infectious virus concentration; nor has the infectious dose for humans been established, so at this point it is unwarranted to deduce a human transmission risk based on this small number of virus-positive fecal samples. 3. Changed Discussion sentence: Many studies report identifying SARS-CoV-2 RNA in sewage and wastewaters, but viral culture from such sources has not been demonstrated, so there is no evidence of infection risk from those sources; however, detection of SARS-CoV-2 RNA in sewage and wastewaters can be useful as a surveillance tool. 4. Added: However, quantitative data on infectiousness and the consequent likelihood of transmission from orofecal contamination is not available. Whilst SARS-CoV-2 RNA is observed in sewage and wastewaters, there is no evidence of infectiousness in these sources and a transmission risk from these sources is therefore unlikely. To properly assess these risks, quantitative data on infectious virus are needed, along with information on likely infectious dose in humans. 5. Added to Discussion: We note the possibility that some cases may be reported in more than one publication, but there is no adequate means by which to identify this. 6. Unclear reporting in the reviews did not allow full analysis of their included studies. To do this we could revisit the full text of their included studies, but this is beyond the scope of this review. Inclusion of these reviews represents a level of interest in the field but necessarily they will overlap in scope and therefore we do not analyse their findings in detail. 7. Thank you for the suggestion to further sub analyse the data; at this stage there are insufficient data for such sub analyses including among children. 8. Unfortunately there are several studies in which sex of participants is not reported and so we were limited by this. 9. This was a very interesting suggestion to sub-analyse according to preprint publication and examine possible publication biases; this is unfortunately beyond the scope of this review, not least because this changes over time. The next time we update this review we hope to have a better breakdown of those fully published, and hopefully a better bank of evidence to analyse. 10. Thank you for noting the anomaly in the table, we have checked and rectified and hopefully this will all be in the right place now. Thank you for these comments." } ] } ]	1	https://f1000research.com/articles/10-231
https://f1000research.com/articles/9-1037/v1	25 Aug 20	{ "type": "Case Report", "title": "Case Report: Pediatric pharmacobezoar with subacute intestinal obstruction", "authors": [ "Mahnaz Hakeem", "Heeramani Lohana", "Sarwat Urooj", "Sheraz Ahmed", "Mahnaz Hakeem", "Sarwat Urooj", "Sheraz Ahmed" ], "abstract": "Bezoars are an undigested mass causing an intraluminal obstruction in children. Pharmacobezoars are formed from medicines or their vehicle, considered as a less frequent type observed in children. Our objective is to report a relatively rare entity as a potential cause of intestinal obstruction in children. Here we report a case of 13-year-old girl with a history of herbal medicine intake who presented with persistent vomiting and abdominal distension. She was diagnosed with acute intestinal obstruction and managed conservatively without any complications. The patient became stable within two days so was discharged home. We found that ineffective history could lead to a delay in diagnosis and management. Clinicians should have a high index of suspicion for pica and psychiatric disorders, especially in adolescent children.", "keywords": [ "bezoar", "pharmacobezoar", "intestinal obstruction", "children." ], "content": "Introduction\n\nThe word bezoar is derived from the Persian word, or Arabic word Badzehr, which both refer to antidote1. It is defined as the accumulation of undigested ingested material leading to the formation of a mass. Its most common site is the stomach followed by the intestines. The risk of bezoars is higher in children with altered gastrointestinal anatomy, altered motility, and psychiatric disorders. It is further classified by the composition of accumulated material into phytobezoar containing food particles from plant origin, trichobezoar made of hair, lactobezoar formed from milk concretion, and pharmacobezoar formed from medicines or their carrier material2. Phytobezoars are the most common among all. Pharmacobezoars are a rare cause of mechanical obstruction of the gastrointestinal tract, and are hence difficult to diagnose. Drugs commonly observed causing bezoar formation are antacids, such as aluminum hydroxide and sucralfate, but they may be caused by other medications, including nifedipine and cholestyramine. Here, we report a case of young girl presenting with intestinal obstruction secondary to excessive intake of herbal medicine. Her diagnosis was delayed for a week despite visiting multiple hospitals due to the paucity of proper history intake.\n\n\nCase report\n\nA 13-year-old girl presented to the emergency room of our hospital with a complaint of abdominal pain for six days. She had a history of eating 30 ayurvedic digestive tablets to relieve her indigestion 1 day prior to the onset of abdominal pain. The pain was moderate to severe in intensity, more in the umbilical region, non-radiating, associated with vomiting, and refusal to feed. The patient was taken to a local hospital where intravenous antiemetic dimenhydrinate 50mg stat was given, along with intravenous fluids. An X-ray of the abdomen was done at that time showing distended bowel loops with significant fecal impaction. The patient was given laxative sodium picosulfate 7.5mg stat and discharged after 4 hours.\n\nTwo days later, the patient developed persistent vomiting along with constipation. She was taken to another local hospital where an X-ray of the abdomen was performed showing multiple air-fluid levels. The patient was restricted to oral intake both solids and liquids, an intravenous line was maintained, intravenous fluids and antibiotic ceftriaxone (2 gram once daily) were given, and a nasogastric tube was passed. Surgical opinion was sought at this time and surgical exploration was planned; however, this was refused by the family and the patient was taken to our hospital next day.\n\nAt the time of presentation in our hospital, the patient was vitally stable, pale-looking with a nasogastric tube placed in the right nostril. Her abdomen was soft, distended with centrally placed umbilicus and gut sounded sluggish on auscultation. Otherwise, she had no visceromegaly. The patient’s drain attached to the nasogastric tube collected 850ml greenish aspirates over 8 hours. Visual assessment of the aspirated fluids showed particles of ingested medicine. Basic laboratory workups, including complete blood count, electrolytes, and creatinine, were performed (Table 1). X-ray of the abdomen done at our hospital showed a deeply placed nasogastric tube with distended bowel loops (Figure 1 and Figure 2). The nasogastric tube was removed and the patient was kept on intravenous ceftriaxone (1 gram twice daily) and metronidazole (250mg every 8 hours for 3 days). Surgical consultation was reviewed and after 6 hours she was allowed liquids and foods orally, initially for liquids then after 8 hours solid food was given.\n\nThe patient passed stool and had no issues of vomiting, so she was discharged on the second day of admission after dietary counseling. X-ray abdomen showed normal gaseous shadows (Figure 3). Scheduled follow up after 10 days, showed that the patient was in good condition.\n\n\nDiscussion\n\nMechanical obstruction caused by pharmacobezoars accounts for 4% of all patients with intestinal obstruction, often subacute obstruction3. Mechanisms involved in formation of pharmacobezoars include dysmotility secondary to an anatomical defect or anticholinergic effects of the drug, a massive quantity of drug intake, or its hydrophobic nature4. Ingestion of medicine in huge quantities may be secondary to mental retardation, psychiatric disorders, or suicidal intentions. There is limited published data of pediatric pharmacobezoar internationally in this regard with no documented evidence in the local population in Pakistan.\n\nImaging modality used in diagnosis of pharmacobezoars includes plain radiographs and ultrasonography, and more advanced techniques, such as CT scan. Endoscopy may be considered as a diagnostic tool as well as a therapeutic option for an impacted bezoar. Visual assessment and biopsy of fragmented bezoar tissue are also helpful in diagnosis.\n\nTreatment options vary from a non-surgical approach, including conservative management, to laparoscopic approach or open laparotomy in rare circumstances. Non-surgical options include dissolution of bezoar, fragmentation, gastric lavage, or endoscopic removal, which is considered as the first line in management5. Irrigation of normal saline along with the withdrawal of the causative medicine has been effective in some cases6. One study has showed that the dissolution of pharmacobezoars was performed with cellulose and diet coke along with multiple medications1.\n\nOur patient had a history of intake of herbal medicine composed of tamarind, guava, mint, and other spices. There was an unintentional massive intake of tablets within a short time, leading to its concretion in the small intestine. She was diagnosed with X-ray of the abdomen and managed with normal saline irrigation and abstinence of the culprit medicine as we had a lack of availability of endoscopy for pediatrics in our city.\n\nIf left untreated, bezoar may lead to perforation, peritonitis, weight loss, ulcer, anorexia, and constipation7. Endoscopic removal of bezoar may lead to aspiration, particularly in the case of fragmentation. Timely management is key to avoid these complications. Risk factors attributing to the development of bezoars, such as gastrointestinal motility disorders, drug interactions and psychiatric illnesses, should be addressed appropriately to avoid recurrent bezoars.\n\n\nConclusion\n\nBezoars account for a sporadic number of patients with mechanical intestinal obstruction. History of pica should be evaluated in patients with bezoars. Proper dietary counseling, adequate chewing, and psychotherapy may prevent this condition.\n\n\nConsent\n\nWritten informed consent to publish the case report along with images was obtained from the father of the patient.\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\nEng K, Kay M: Gastrointestinal Bezoars: History and Current Treatment Paradigms. Gastroenterol Hepatol (N Y). 2012; 8(11): 776–778 ISSN 1554-7914; 1554-7914. PubMed Abstract \| Free Full Text\n\nSimpson SE: Pharmacobezoars Described and Demystified. Clin Toxicol (Phila). 2011; 49(2): 72–89 ISSN 1556-9519; 1556-3650. PubMed Abstract \| Publisher Full Text\n\nGandhi S, Arora E, Bhandarwar A, et al.: When a Cure Becomes the Pathology: Mechanical Bowel Obstruction due to Herbal Pharmacobezoar. A Case Report with Review of Literature. Clin J Gastroenterol. 2018; 11(5): 396–400 ISSN 1865-7265; 1865-7265. PubMed Abstract \| Publisher Full Text\n\nGori F, Cirronis M, Ieri A, et al.: Importance of Abdomen CT Scan and Gastroscopic Pharmacobezoar Removal Following Massive Acute Drug Overdose. Clinical Toxicology. 2015; 53(4). Reference Source\n\nCastle SL, Zmora O, Papillon S, et al.: Management of Complicated Gastric Bezoars in Children and Adolescents. Isr Med Assoc J. 2015; 17(9): 541–544 ISSN 1565-1088. PubMed Abstract\n\nPortuguez-Malavasi A, Aranda JV: Antacid Bezoar in a Newborn. Pediatrics. 1979; 63(4): 679–680 ISSN 0031-4005; 0031-4005. PubMed Abstract\n\nHon KL, Cheng J, Chow CM, et al.: Complications of Bezoar in Children: What is New? Case Rep Pediatr. 2013; 2013: 523569 ISSN 2090-6803. PubMed Abstract \| Free Full Text" }	[ { "id": "72257", "date": "09 Oct 2020", "name": "Roberto De Giorgio", "expertise": [ "Reviewer Expertise Gastrointestinal motility and functional bowel 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\nVery interesting, well detailed case.\nI suggest the Authors to improve the quality of the images presented in this case report.\n\nPlease note that Fig. 1 does not match with the text (no evidence of the N-G tube...where is it ?).\n\nExplain why a CT scan of the abdomen was not performed.\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": "73868", "date": "30 Nov 2020", "name": "Ajay Bhandarwar", "expertise": [ "Reviewer Expertise Minimal Access surgery in GI", "Endocrine and HPB surgeries." ], "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\nHistory and progression are detailed, however, the need for ayurvedk]ic tablet ingestion has not been mentioned in the first place. Indigestion associated 30 tablet intake seems unlikely.\n\nDoes the patient have any other previous Psychiatric illness history or Family history of the same? This is needed for reading practitioners to understand the association of Bezoars with psychiatric illness even at a young age.\n\nThe productive finding is that conservative Management can be approached in such cases, however, it should not be generalised treatment. Hence pre-op scoring systems can be employed to give a trial of conservative management in the future.\n\nManagement seems to be a basic conservative line of treatment, with no new findings. But reaffirmation of known treatment modality. Image of Nasogastric tube aspirate of 800+cc would have been more impactful.\n\nOverall, for inexperienced practitioners, it is a good read to understand the importance of a conservative trial and immediate stabilisation management steps. However, the above-mentioned points might make it more worthwhile a read.\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1037
https://f1000research.com/articles/10-817/v2	01 Nov 21	{ "type": "Research Article", "title": "The impact of vitamin D supplementation on peripheral neuropathy in a sample of Egyptian prediabetic individuals", "authors": [ "Mohamed Reda Halawa", "Iman Zaky Ahmed", "Nahla Fawzy Abouelezz", "Nagwa Roushdy Mohamed", "Naira Hany Abdelaziz Khalil", "Laila Mahmoud Ali Hendawy", "Mohamed Reda Halawa", "Iman Zaky Ahmed", "Nahla Fawzy Abouelezz", "Nagwa Roushdy Mohamed", "Laila Mahmoud Ali Hendawy" ], "abstract": "Background: Vitamin D deficiency is seen more frequently in diabetic patients with distal symmetrical polyneuropathy. Unfortunately, there is a shortage of data concerning prediabetic individuals with peripheral neuropathy (PN). Therefore, we aimed to study the association of vitamin D deficiency with PN severity and to determine the effect of vitamin D supplementation on PN in prediabetics. Methods: A case-control study was conducted consisting of 89 prediabetic individuals with PN and a control group of prediabetics without PN, recruited from the outpatient department of the National Institute of Diabetes and Endocrinology, Cairo, Egypt. All patients were screened for PN using clinical examination and Douleur Neuropathique 4 diagnostic questionnaire (DN4). Group A (with PN) was assessed for neuropathic severity using the Short-Form McGill Pain Questionnaire (SF-MPQ). In addition, 25-hydroxyvitamin D, ionized calcium, phosphorus, parathyroid hormone (PTH), glycated hemoglobin (HbA1c), fasting blood glucose (FBG), 2-hour post 75g glucose (2h-PPBG) and lipid profile were measured for both groups. Prediabetic patients with PN were given vitamin D3 200.000 IU IM monthly for three months. After three months, clinical assessment, DN4, SF-MPQ and all laboratory measures were repeated. Results: Vitamin D was not associated with the severity of PN patients. However, supplementation of vitamin D resulted in a highly significant improvement in glycemic parameters , p≤0.001. Interestingly, neuropathy score and severity before vitamin D supplementation were (6.4±1.6 and 28.3±7.2) and after became (2.5±0.9 and 17±6.3, p≤0.001).\nConclusion: Correction of vitamin D deficiency in prediabetics with PN as well as hypovitaminosis D, improves glycemic parameters, PN score and severity.", "keywords": [ "prediabetes", "vitamin D", "peripheral neuropathy", "neuropathic score" ], "content": "\n\nAfter being made aware of issues with this article, the F1000Research Editorial Team worked closely with the authors’ institution, who completed a rigorous investigation of the concerns raised. Investigation was carried out by a multidisciplinary internal auditing committee formed of endocrinology and public health individuals across the Faculty of Medicine - Ain Shams University. The committee reviewed the study datasets and analyses and their findings identified various inaccuracies that require correction. In line with our article policies around permanency of content, in order to maintain the integrity and completeness of the scholarly record, F1000Research allows authors to publish revised versions, and any errors that become apparent during peer review or later can be corrected through the publication of new versions. We have worked with the article authors towards correction of the manuscript and publication of a new version. Corrections and changes relative to the previous version are clearly summarized in the ‘Amendments’ section at the start of the new version.\n\n\nIntroduction\n\nDiabetes mellitus (DM), a significant world health problem, is a metabolic disease, which occurs due to a defect in insulin release and or insulin resistance1. Globally, the prevalence of type 2 diabetes (T2DM) is high and rising across all regions2.\n\nThere is a higher frequency of idiopathic polyneuropathy, small fiber neuropathy and painful sensory neuropathy among prediabetics. These findings suggest an involvement of the small unmyelinated nerve fibers that carry pain, temperature, and regulate autonomic function during prediabetes, before the development of diabetes3.\n\nVitamin D, which is a fat-soluble hormone, has multiple physiological roles, which extends far beyond calcium metabolism4. Vitamin D deficiency is a worldwide health problem, patients with prediabetes, T2DM, gestational diabetes and obesity represent a high-risk group5.\n\nRecently, a lot of studies have been done to assess the association between vitamin D level and the diabetic peripheral neuropathy in patients with diabetes mellitus and to study the effect of vitamin D on painful neuropathy, but there is a lack of data concerning prediabetic individuals1.\n\nThe aim of this work was to determine the association of vitamin D deficiency with peripheral neuropathy severity and evaluate the effect of vitamin D supplementation on peripheral neuropathy in prediabetics with hypovitaminosis D.\n\n\nMethods\n\nAn analytic case-control followed by an interventional one arm clinical trial design (quasi experiment) were performed to fulfill the preset objectives. The case-control study included 89 prediabetic patients with peripheral neuropathy (Group A) and 89 prediabetics without peripheral neuropathy (Group B).\n\nStudy Participants and Case Definition: Prediabetic individuals were diagnosed, according to the American Diabetes Association 2019, with impaired fasting (100–125 mg/dl) and/or impaired glucose tolerance (140–199 mg/dl), and/or glycated haemoglobin (5.7–6.4%)6 (they were all ages between 18–60 years old). Participants were recruited from the National Institute of Diabetes and Endocrinology (NIDE), Cairo, Egypt, in the period from September 2018 to March 2019 after proven informed written consent.\n\nEthical Considerations: A written informed consent was obtained from all patients. All data were made confidential and an ethical approval on study conduction was obtained from the Local Research Ethical Committee (REC) of the Faculty of Medicine, Ain Shams University. FWA 000017858.\n\nAll participants were subjected to full medical history including smoking habits, alcohol consumption, drug history, thorough clinical examination including blood pressure, weight, height and BMI.\n\nAll participants were screened for peripheral neuropathy by 10 g monofilament for assessing the loss of protective sensation, tuning fork (vibration sense testing using a 128-Hz tuning fork), ankle reflex, pinprick (for perception of pain) and Douleur Neuropathic 4 diagnostic questionnaire (DN4)‎7 that assesses symptoms reflecting pain in the form of burning, painful, cold, electric shocks, tingling, pins and needles. If the patients score is ≥4 the patient likely suffers from neuropathic pain. Patients found to have peripheral neuropathy were given the Short-Form McGill Pain Questionnaire (SF-MPQ)8 that assesses the severity of pain; an increase in the score indicates increasing severity.\n\nLaboratory studies included: ionized calcium, phosphorous, PTH, and 25(OH)vitamin D, FBG, 2h-PPBG, glycated hemoglobin and total lipid profile (total cholesterol, low density lipoprotein (LDL) and triglycerides)\n\nParticipants were first instructed to fast for eight hours (overnight fasting), 10 ml of venous blood were then collected by venipuncture without tourniquet. 2 ml of the collected blood were taken in an EDTA containing tube for the assay of the glycated hemoglobin and it was stored at 4°C to be carried out within one week. 2 ml were taken in a fluoride containing tube and then separated by centrifugation and the sample was used for measurement of FBG, serum Ca, phosphorus, PTH and 25(OH)vitamin D. 2 ml sample were collected two hours after 75 g oral glucose load for the measurement of the 2h-PPBG. On a separate day, 2 ml of venous blood were collected by venipuncture (after an overnight 12 hour fast), the sample was collected in a fluoride containing tube and then separated by centrifugation and used for measurement of total lipid profile (total cholesterol, low density lipoprotein (LDL), triglycerides (TG)) by enzyme colorimetric assay. Total cholesterol level was measured by Quantitative Enzymatic-Colorimetric assay (Catalogue Number: 1010/ manufacturer: Stanbio-Laboratory, Inc., USA/ Boerne, Texas/ 1/2018). Triglyceride level was measured by Quantitative Enzymatic-Colorimetric assay (Stanbio LiquiColor Triglycerides/ Catalog Number: 2100/ manufacturer: Stanbio-Laboratory, Inc., USA/ Boerne, Texas, USA/ 03/2018). LDL cholesterol can be determined as the difference between total cholesterol and the cholesterol content of the supernatant (HDL and VLDL) after precipitation of LDL fraction by polyvinyl sulphate in the presence of polyethylene-glycol monomethyl ether. Calculation LDL= Cholesterol- (HDL+ Triglyceride/5). HDL level was measured by Quantitative Enzymatic-Colorimetric assay (Stanbio HDL cholesterol/ Catalog Number: 0599/ manufacturer: Stanbio-Laboratory, Inc., USA/ Boerne, Texas, USA/ 02/2018). Serum 25-hydroxyvitamin D level was measured by an ELISA kit, which is a solid phase enzyme-linked immunosorbent assay (ELISA, Catalogue Number: 10501, Chemux Bioscience, Inc., Hayward, CA/ 10/2018). Parathormone level was measured by an ELISA kit with a normal range of 10–55 pg/ml (ELISA, Catalogue Number: KAP1481, DIAsource ImmunoAssays S.A, Nivelle, Belgium/ 2/2018). Glycated hemoglobin was measured by quantitative colorimetric determination of glycated haemoglobin in whole blood (Catalog Number: 0350/ manufacturer: Stanbio-Laboratory, Inc., Boerne, Texas, USA/ 06/2018). Fasting blood glucose, 2h-PPBG were measured by Stanbio Glucose LiquiColor (Oxidase) (Catalog Number: 1070, manufacturer: Stanbio-Laboratory, Inc., USA, Boerne, Texas, USA/ 04/2018). Vitamin D status was assessed according to Hovsepian et al., and classified as Sufficient if higher than 30ng/ml, Insufficient if between 20–29 ng/ml and Deficient if less than 20 ng/ml9.\n\nPatients with renal impairment, hypo or hyperthyroidism, patients on vitamin D supplementation or antiepileptic or any medication affecting calcium and vitamin D level, pregnant or breast-feeding females were excluded from the study.\n\nBased on the results of previous studies, the majority of patients with and without peripheral neuropathy were found to be either insufficient or deficient as regard their level of vitamin D. The current analytical study has also demonstrated that all prediabetics with and without peripheral neuropathy were also either insufficient or deficient as regard their level of vitamin D.\n\nInterventional Study: Patients of group A were invited to participate in a quasi-experiment by administration of therapeutic dose of vitamin D. None of the 89 patients participating in the case-control study refused to be enrolled in the interventional study. 200.000 IU of Vitamin D (cholecalciferol) were intramuscularly administered every month for three successive months to all patient with peripheral neuropathy.\n\nClinical assessments were repeated in the last visit after three months to assess the improvement in peripheral neuropathy in those patients. Retesting is advised after three months, as suppression of parathyroid hormone after supplementation with cholecalciferol takes at least three months and the response differs between individuals. So, most guidelines recommend repeat testing after three months10.\n\nAll laboratory tests were conducted at the beginning of the study and after three months of vitamin D supplementation.\n\nCollected data were analyzed using SPSS (version 17, 2012, IBM Corporation, USA) (An open-access alternative that can perform an equivalent function is the R Stats package). The continuous quantitative variables included Age (Years), BMI (kg/m2), Systolic BP (mmHg), Diastolic BP (mmHg), HbA1c (%), 2h-PPBG (mg/dl), T. cholesterol (mg/dl), LDL (mg/dl), HDL (mg/dl), TG (mg/dl), 25 (OH) Vit D (ng/ml), Ionized Ca (mg/dl), Phosphorus (mg/dl), PTH (pg/ml) and they were described as mean and standard deviation. Student’s T-test was used to compare two independent groups (group A and group B) for quantitative data. Continuous variables before and after vitamin D intake were assessed using paired t-test. Regarding categorical/qualitative data, we measured vitamin D status (Sufficient, Insufficient, Deficient) and they were presented as numbers and percentages and the Chi-Square test was used to compare two independent groups (group A and group B) with qualitative data. Additional qualitative data that were measured were clinical examination for peripheral neuropathy using ankle reflex, tuning fork (vibration) and 10 g monofilament and they were presented as numbers and percentages and McNemar and McNemar Bowker tests were used to compared group A before and after vitamin D supplementation.\n\n\nResults\n\nComparison between the two studied groups regarding clinical and laboratory characteristics is shown in Table 1. 20.2% of patients in group A (With PN) reported vitamin D insufficiency (N=18), while deficiency was evident in 79.8% (N=71) and none reported normal level of vitamin D. While group B (Without PN): 9 (10.1%) were insufficient, 80 (89.9%) were deficient and none were sufficient (Table 2); with a non-significant difference in the mean level of vitamin D level between the two groups (13.957 ± 6.3603 ng/ml (group A) vs 14.594 ± 3.9318 ng/mL (group B) (P>0.05) (Table 1).\n\nBMI= body mass index, BP= blood pressure, HbA1c = glycated haemoglobin, 2h-PPBG = 2 hour post 75g glucose, T.cholesterol = total cholesterol, LDL = low density lipoprotein, , HDL = high density lipoprotein, TG = serum triglycerides, 25 (OH) Vit D= 25 hydroxy Vitamin D, Ionized Ca= ionized calcium, PTH = serum parathyroid hormone.\n\nGroup A= Prediabetic patients with peripheral neuropathy\n\nGroup B= Prediabetic patients without peripheral neuropathy\n\nVitamin D level showed a negative but statistically insignificant correlation with the severity of peripheral neuropathy (assessed by SF-MPQ) (r= 0.032, p= 0.766) and pain score (assessed by DN4 questionnaire) (r= 0.052, p= 0.629)\n\nThere was a highly significant improvement in vitamin D level in group (A) after intramuscular injection of vitamin D, from which 42 (47%) prediabetic patients became sufficient, 38 (42.7%) became insufficient and only 9 (10.1%) remained deficient (P≤0.001). There was a highly significant improvement of glycemic profile as shown in (Table 3).\n\nHbA1c = glycated haemoglobin, FBG= fasting blood glucose, 2h-75g glucose=2-hour post 75g glucose\n\nA statistically significant improvement in neuropathic pain severity was observed by the SF-MPQ (28.3±7.2 before and 17±6.3 after vitamin D supplementation, P≤0.001). There was also a statistically significant reduction in the DN4 questionnaire score from 6.39 ±1.64 to 2.5 ± 0.9, ≤0.001 (≥4 denote neuropathic pain) with an improvement of neuropathic pain in 82% of patients (73 out of 89) (P≤0.001) (Figure 1).\n\nWe found a highly significant improvement in vibration sense by tuning fork and protective sense measured by the 10 g monofilament test (P≤0.001), while there was no improvement regarding ankle reflex (P>0.05) (Table 4).\n\nMNB=McNemar Bowker test between patients with peripheral neuropathy before and after vitamin D injection\n\nPaired t-test in both Short McGill and DN4 <0.001\n\n\nDiscussion\n\nDiabetic peripheral neuropathy in recently diagnosed diabetic patients may reach about 8% and more than 50% in patients with long-standing diabetes11. Recently, the American Diabetes Association stated that there is no strong evidence that supports the lifestyle management or efficacy of glycemic control in the treatment of neuropathic pain, which means that pharmaceutical interventions such as pregabalin, duloxetine, or tapentadol are the only way of treatment12. Accordingly, we aimed to demonstrate the association of vitamin D status with peripheral neuropathy and determine the effect of vitamin D supplementation on painful neuropathy in prediabetics.\n\nEven with our sunny country, none of our patients (0%) had sufficient vitamin D level.\n\nKuchay et al., 2015 in their study found that prediabetes patients were 54.3% vitamin D deficient, 21.3% were insufficient and only 24.4% were sufficient despite abundant sunshine in India13.\n\nKuchay et al., (2015) demonstrated an association between vitamin D status and prevalence of diabetes, with low prevalence in people with high vitamin D status and a belief that a serum 25(OH) vitamin D level of 15 ng/mL or less may be a threshold at which vitamin D deficiency confers negative effect on insulin sensitivity13. This was confirmed when nearly 50% of patients with prediabetes had serum 25(OH) vitamin D levels below 15 ng/mL13. On the contrary, Rolim et al., (2016) found the association between HbA1c and 25(OH) vitamin D controversial and glycemic control was not associated with vitamin D level14. Luo et al., (2009) stated that there was no impact of hypovitaminosis D on metabolic syndrome status and HbA1c15.\n\nThe association between vitamin D status and prevalence of diabetes can be explained through the effect of vitamin D on pancreatic β‐cell function and plasma calcium. Vitamin D deficiency decreases serum calcium, which regulates insulin synthesis and release16.\n\nOn the other hand, administration of vitamin D causes increase in serum calcium, decrease in circulating free fatty acid levels, increase in insulin release and improvement in glucose levels17.\n\nShehab et al., (2012) study on 210 diabetic patients, from which 87 had peripheral neuropathy, first found that vitamin D deficiency was significantly associated with diabetic peripheral neuropathy18. In agreement with Shillo et al., (2019) who reported that serum vitamin D levels were lower in patients with painful DPN than in those with painless DPN, and pain scores were negatively correlated with serum vitamin D levels19.\n\nOn the contrary, Basit et al., (2016) acknowledged that there was no significant correlation between 25 (OH) vitamin D status with either total McGill pain location, McGill pain score, DN4 or positive symptoms20 and this was inconsistent with the present study. Studies by Usluogullari et al., (2015) also found no difference in the prevalence of vitamin D deficiency between diabetic peripheral neuropathy patients and controls21.\n\nHowever, Shehab et al., (2012) study confirmed that vitamin D was the only independent risk factor for diabetic peripheral neuropathy18. While in China, He et al., (2017) declared that deficiency of vitamin D is an independent risk factor for diabetic peripheral neuropathy and can be considered a potential biomarker for peripheral neuropathy in diabetic Chinese patients22.\n\nOn the other hand, Alkhatatbeh et al., (2019) showed that the only significant predictor for neuropathic pain was female gender, while vitamin D level, BMI, age, FBG, duration of T2DM, DBP and SBP were not23. The divergence in the results of previous studies may be due to the use of different methods to assess neuropathy and because the studies were directed on different populations.\n\nInjection of vitamin D 200.000 IU intramuscular every month for three successive months is in accordance with the guidelines for vitamin D supplementation and treatment of deficiency in Central Europe individuals with proved vitamin D deficiency which require higher doses of vitamin D than doses recommended for the general population. The therapeutic dose in severe deficiency should be 1.000–10.000 IU/day (~50.000 IU/week), depending on the patient’s body weight and age. The duration of the treatment varies from 1–3 months, depending on the degree of vitamin D deficiency24. Our patients showed significant improvement and reduction in neuropathy severity score and also showed clinical improvement by monofilament and tuning fork. This is in line with Bell (2012) who found great improvement in neuropathic symptoms after supplementation with 50.000 IU of vitamin D2 every week in a case report of a patient suffering from diabetic peripheral neuropathy. The patient had been refractory to different types of treatment like tricyclic's, gabapentin, oxycodone and pregabalin25. As well, Shehab et al., (2015) in their study applied vitamin D replacement therapy as a single intramuscular vitamin D dose of 300.000 IU and this application significantly enhanced the DN4 questionnaire scores of the patients with diabetic neuropathy26. Correspondingly, Lee and Chen (2008) showed that oral cholecalciferol resulted in an approximate 50% reduction in painful neuropathic symptoms and a significant reduction in SF-MPQ score from 32.1 to 19.4; however, this study had neither a placebo group nor was randomized, leaving it open to considerable bias27.\n\nPossible explanation of previous studies was demonstrated in vitro by Fukuoka et al., (2001) and in vivo by Riaz et al., (1999) who considered vitamin D as a neurotrophic substance, which modulates neuronal growth and differentiation, and neuromuscular functions28,29. Its exact role in diabetic neuropathic pain is uncertain; insufficiency of vitamin D may increase damage of diabetic nerve and may affect the function of nociceptors leading to pain at a higher threshold of serum 25 (OH) vitamin D concentration higher than that in the non-diabetic individuals27.\n\nTherefore, the results of previous studies corroborate our findings that vitamin D supplementation improves peripheral neuropathy and can be used as a safe treatment for peripheral neuropathy in prediabetic patients with hypovitaminosis D.\n\nOpposing previous results, a study by Alam et al., (2016) reported no significant decrease in neuropathic pain scores after vitamin D administration30. This study was based on all or none values instead of assessing the quantity of pain score, which may have led to a failure of observing a reduction in pain scoring.\n\nGlycemic parameters of our patients showed significant improvement after the administration of 200.000 IU of vitamin D every four weeks for 12 weeks, which was the same result found by Kuchay et al., (2015) who revealed that correcting vitamin D deficiency in people with prediabetes significantly reduces FBG, two hours plasma glucose and A1C levels in 12 months13. However, contrary to our findings, He et al., (2018) proclaimed in their meta-analysis that vitamin D supplementation did not improve fasting glucose levels or insulin resistance, nor did it prevent T2DM in non-diabetics31. Furthermore, Moreira-Lucas et al., (2017) confirmed that vitamin D supplementation did not improve fasting or post challenge measures of insulin sensitivity, β-cell function or HbA1c32.\n\nAmong the limitations of the study were a small sample size compared to previous studies. Our study is the first to discuss the effect of vitamin D supplementation on peripheral neuropathy in prediabetic individuals whereas other studies have discussed the effect on diabetic patients. Finding prediabetic participants with peripheral neuropathy to include in the study was challenging.\n\n\nConclusion\n\nThis study found that vitamin D supplementation in prediabetics with peripheral neuropathy and hypovitaminosis D improves neuropathy in those patients as assessed by McGill and DN4 scores, as well as glycemic parameter namely HbA1c, FBG and 2h-PPG.\n\n\nData availability\n\nFigshare: Underlying data for ‘The impact of vitamin D supplementation on peripheral neuropathy in a sample of Egyptian prediabetic individuals’, https://doi.org/10.6084/m9.figshare.1683185833\n\nThis project contains the following underlying data:\n\nData file 1: prediabetic patients without peripheral neuropathy and their descriptive and laboratory data.\n\nData file 2: prediabetic with peripheral neuropathy and their descriptive, laboratory data, McGill Pain Questionnaire, clinical examination for neuropathy before vitamin D supplementation.\n\nData file 3: prediabetic with peripheral neuropathy and their descriptive and laboratory data, McGill Pain Questionnaire, clinical examination for neuropathy after Vitamin D supplementation.\n\n\nConsent statement\n\nWritten informed consent was obtained from all individual participants included in our study.", "appendix": "References\n\nQu GB, Wang LL, Tang X, et al.: The association between vitamin D level and diabetic peripheral neuropathy in patients with type 2 diabetes mellitus: An update systematic review and meta-analysis. J Clin Transl Endocrinol. 2017; 9: 25–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Diabetes Federation: IDF diabetes atlas 9th edition. 2019. Reference Source\n\nNebuchennykh M, Løseth S, Jorde R, et al.: Idiopathic polyneuropathy and impaired glucose metabolism in a Norwegian patient series. Eur J Neurol. 2008; 15(8): 810–816. PubMed Abstract \| Publisher Full Text\n\nHolick MF: Vitamin D deficiency. N Engl J Med. 2007; 357(3): 266–281. PubMed Abstract \| Publisher Full Text\n\nMuñoz-Garach A, García-Fontana B, Muñoz-Torres M: Vitamin D Status, Calcium Intake and Risk of Developing Type 2 Diabetes: An Unresolved Issue. Nutrients. 2019; 11(3): 642. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Diabetes Association: Criteria for testing for diabetes or prediabetes in asymptomatic adults. American Diabetes Association. 2019; 42(Supp): 1–17.\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\nHawker GA, Mian S, Kendzerska T, et al.: Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SF-MPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP). Arthritis Care Res (Hoboken). 2011; 63 Suppl 11: S240–52. PubMed Abstract \| Publisher Full Text\n\nHovsepian S, Amini M, Aminorroaya A, et al.: Prevalence of vitamin D deficiency among adult population of Isfahan City, Iran. J Health Popul Nutr. 2011; 29(2): 149–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlendenning P: Measuring vitamin D. Aust Prescr. 2015; 38(1): 12–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOraby MI, Srie MA, Abdelshafy S, et al.: Diabetic peripheral neuropathy: the potential role of vitamin D deficiency. Egypt J Neurol Psychiatr Neurosurg. 2019; 55: 10. Publisher Full Text\n\nAmerican Diabetes Association: 11. Microvascular complications and foot care: Standards of Medical Care in Diabetes-2020. Diabetes Care. 2020; 43(Suppl 1): 135–151. PubMed Abstract \| Publisher Full Text\n\nKuchay MS, Laway BA, Bashir MI, et al.: Effect of vitamin D supplementation on glycemic parameters and progression of prediabetes to diabetes: a 1-year, open-label randomized study. Indian J Endocrinol Metab. 2015; 19(3): 387–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRolim MC, Santos BM, Conceição G, et al.: Relationship between vitamin D status, glycemic control and cardiovascular risk factors in Brazilians with type 2 diabetes mellitus. Diabetol Metab Syndr. 2016; 8: 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo C, Wong J, Brown M, et al.: Hypovitaminosis D in Chinese type 2 diabetes: lack of impact on clinical metabolic status and biomarkers of cellular inflammation. Diab Vasc Dis Res. 2009; 6(3): 194–9. PubMed Abstract \| Publisher Full Text\n\nPalomer X, González-Clemente JM, Blanco-Vaca F, et al.: Role of vitamin D in the pathogenesis of type 2 diabetes mellitus. Diabetes Obes Metab. 2008; 10(3): 185–197. PubMed Abstract \| Publisher Full Text\n\nAlvarez JA, Ashraf A: Role of vitamin D in insulin secretion and insulin sensitivity for glucose homeostasis. Int J Endocrinol. 2010; 2010: 351385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShehab D, Al-Jarallah K, Mojiminiyi OA, et al.: Does Vitamin D deficiency play a role in peripheral neuropathy in Type 2 diabetes? Diabet Med. 2012; 29(1): 43–49. PubMed Abstract \| Publisher Full Text\n\nShillo P, Selvarajah PD, Greig M, et al.: Reduced vitamin D levels in painful diabetic peripheral neuropathy. Diabet Med. 2019; 36(1): 44–51. PubMed Abstract \| Publisher Full Text\n\nBasit A, Basit KA, Fawwad A, et al.: Vitamin D for the treatment of painful diabetic neuropathy. BMJ Open Diabetes Res Care. 2016; 4(1): e000148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUsluogullari CA, Balkan F, Caner S, et al.: The relationship between microvascular complications and vitamin D deficiency in type 2 diabetes mellitus. BMC Endocr Disord. 2015; 15: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe R, Hu Y, Zeng H, et al.: Vitamin D deficiency increases the risk of peripheral neuropathy in Chinese patients with type 2 diabetes. Diabetes Metab Res Rev. 2017; 33(2). PubMed Abstract \| Publisher Full Text\n\nAlkhatatbeh M, Abdul-Razzak KK: Neuropathic pain is not associated with serum vitamin D but is associated with female gender in patients with type 2 diabetes mellitus. BMJ Open Diabetes Res Care. 2019; 7(1): e000690. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPłudowski P, Karczmarewicz E, Bayer M, et al.: Practical guidelines for the supplementation of vitamin D and the treatment of deficits in Central Europe - recommended vitamin D intakes in the general population and groups at risk of vitamin D deficiency. Endokrynol Pol. 2013; 64(4): 319–27. PubMed Abstract \| Publisher Full Text\n\nBell DS: Reversal of the symptoms of diabetic neuropathy through correction of vitamin D deficiency in a type 1 diabetic patient. Case Rep Endocrinol. 2012; 2012: 165056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShehab D, Al-Jarallah K, Abdella N, et al.: Prospective evaluation of the effect of short-term oral vitamin d supplementation on peripheral neuropathy in type 2 diabetes mellitus. Med Princ Pract. 2015; 24(3): 250–256. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee P, Chen R: Vitamin D as an analgesic for patients with type 2 diabetes and neuropathic pain. Arch Intern Med. 2008; 168(7): 771–772. PubMed Abstract \| Publisher Full Text\n\nFukuoka M, Sakurai K, Ohta T, et al.: Tacalcitol, an active vitamin D3, induces nerve growth factor production in human epidermal keratinocytes. Skin Pharmacol Appl Skin Physiol. 2001; 14(4): 226–33. PubMed Abstract \| Publisher Full Text\n\nRiaz S, Malcangio M, Miller M, et al.: A vitamin D(3) derivative (CB1093) induces nerve growth factor and prevents neurotrophic deficits in streptozotocin-diabetic rats. Diabetologia. 1999; 42(11): 1308–1313. PubMed Abstract \| Publisher Full Text\n\nAlam U, Arul-Devah V, Javed S, et al.: Vitamin D and diabetic complications: true or false prophet? Diabetes Ther. 2016; 7(1): 11–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe S, Yu S, Zhou Z, et al.: Effect of vitamin D supplementation on fasting plasma glucose, insulin resistance and prevention of type 2 diabetes mellitus in non-diabetics: A systematic review and meta-analysis. Biomed Rep. 2018; 8(5): 475–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreira-Lucas TS, Duncan AM, Rabasa-Lhoret R, et al.: Effect of vitamin D supplementation on oral glucose tolerance in individuals with low vitamin D status and increased risk for developing type 2 diabetes (EVIDENCE): A double-blind, randomized, placebo-controlled clinical trial. Diabetes Obes Metab. 2017; 19(1): 133–41. PubMed Abstract \| Publisher Full Text\n\nHalawa MR, Ahmed IZ, Abouelezz NF, et al.: The impact of vitamin D supplementation on peripheral neuropathy in a sample of Egyptian prediabetic individuals. Figshare. Dataset, 2021. http://www.doi.org/10.6084/m9.figshare.16831858" }	[ { "id": "101602", "date": "24 Jan 2022", "name": "Zaynab Alourfi", "expertise": [ "Reviewer Expertise Vitamin D disorders", "osteoporosis", "hypothyroidism", "diabetes" ], "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 discusses a common health problem facing diabetologists and internal medicine professionals.\nThe authors have properly introduced the aim of the study in the introduction, especially the fourth paragraph. For instance, a lot of papers (Xiaohua G et al., 20211 and Shehab D et al., 20122) talked about Vit D deficiency in relation to diabetic neuropathy, and in clinical practice a lot of Vit D prescriptions are without clear evidence, but above that, no data about prediabetes, so focusing on this point is great.\n\nThe methodology was clear after the latest revision and helps to reproduce results by other researchers.\n\nI prefer that the 75 g glucose tolerance test be referred to with “75g-OGTT” rather than “2h-PPBG” in the laboratory studies and other places.\n\nRegarding statistical analysis, the authors described statistical tests properly but to give an accurate criticism, the reviewer should be more specialized in statistics. Data files are uploaded completely and allow who wants to use them in reproducing results.\n\nResults were well described and organized; authors started with sample characteristics and organized them in Table 1 properly, then moved to Vit D results and followed up with results after replacement, then described neuropathic pain scores and also organized it in tables.\n\nThe discussion was very good, citing the literature and comparing results to other papers was well established, and I really appreciate the authors' comment about how hard it is to find patients with such inclusion criteria given my expertise. The authors declared that one limitation was collecting patients compatible with the inclusion criteria, it's really challenging to find a prediabetic patient accept, confirming the diagnosis by fasting and doing OGTT test then assessment of neuropathic pain, so this effort from the authors is really appreciated in my opinion.\n\nThe conclusion was short but I think it is enough to support the results since it is mentioning the primary outcome for this paper which is that replacement of Vit D in neuropathic prediabetes patients is useful.\nFinally, I find this version accepted and well written.\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": "121333", "date": "15 Feb 2022", "name": "Ghada M. El Sagheer", "expertise": [ "Reviewer Expertise Endocrine 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\nThanks to the authors for their choice of this subject of study which appears to be interesting and not frequently discussed before. It’s a really well-organized study, well-constructed title, discussing the effect of vitamin D supplementation on prediabetic individuals which is the first study so far discussing this specific point\n\nThe introduction was clear and updated, showing that microvascular complications like peripheral neuropathy can occur before the development of diabetes (Nebuchennykh M. et al., 20081) and highlighting the association between peripheral neuropathy and vitamin D (Qu GB et al., 20172).\nThe methodology was perfectly planned, well written, and well-arranged so that the reader can understand the steps of the study and how the authors managed to find prediabetic individuals with peripheral neuropathy. Also, the number of patients is very suitable to the study design, helping to get more valid data on the effect of vitamin D supplementation on peripheral neuropathy as previous studies were lacking evidence due to either small sample size or defect in follow-up.\nThe statistics are well prepared, the sample size is quite enough for the study and the data is analyzed by SPSS. The statistical analysis and tests are properly described and appropriate for the study, used correctly which helped to get the results that are interpreted in a clear way. The tables and figures are well presented and have a crucial role in the study and writing of the manuscript and helped to finalize the results of the study. Finally, data files are uploaded which will help produce more results further on it, but it would be much better to present the raw data in a simplified way.\nThe results were nicely presented; the authors first presented that vitamin D deficiency was highly prevalent in the studied group. Second, the authors compared clinical and laboratory data between the two studied groups. Third, they presented the effect of vitamin D supplementation over the arm with the peripheral neuropathy and clarified the benefit on the vitamin D levels and neuropathy score and severity and clinically by peripheral neuropathy indicators (ankle reflex, tuning fork vibration, and 10 g monofilament).\nThe discussion was organized and updated, citing each part of the study with other papers, discussing with cons, and clarifying the cause of each result.\nThe conclusion was short, to the point, summarizing all the aims in the study design.\nDespite this, I would be thankful if the authors could clarify the following points:\nThe main results are not sufficiently presented in the abstract (e.g. the mean and SD of the vitamin D level in the study groups, the mean of HbA1c results, and the other glucose parameters, etc).\n\nIn the methods section, I need to know whether the pre-diabetic individuals received any management, e.g. lifestyle modifications, exercise, metformin, to clarify that neuropathy improvement is not related to other factors.\n\nWhat are the definitions of vitamin D deficiency, insufficiency, and sufficiency the authors used, and which reference?\n\nIn the results sections, the mean level of vitamin D of the studied groups was not written in any part of the results or tables.\nFinally, I find this study accepted, well-organized, and well-written.\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": [] } ]	2	https://f1000research.com/articles/10-817
https://f1000research.com/articles/10-261/v1	31 Mar 21	{ "type": "Brief Report", "title": "Modifications to the delivery of NHS face-to-face general practice consultations during the COVID-19 pandemic in England", "authors": [ "Lorna J. Duncan", "Kelly F.D. Cheng", "Kelly F.D. Cheng" ], "abstract": "Background: To minimise transmission of SARS-CoV-2, the virus causing COVID-19, delivery of general practice consultations has been modified to enable the separation of diagnosed or suspected COVID-19 patients from others. Remote triage and consultations are currently the default model, with adapted face-to-face contact used when clinically necessary. This study aimed to identify the modified face-to-face delivery models used across England, and evidence for their effectiveness. Methods: In June 2020, a national survey was sent by email to the 135 Clinical Commissioning Groups (CCGs) in England to identify local organisation of face-to-face general practice consultations since March 2020. An email was sent to Public Health England (PHE) requesting information about COVID-19 outbreaks or clusters linked to general practice. Results: All CCGs responded. Separation of COVID-19 patients from others was achieved using combinations of the following models:\nzoned surgeries (used in 47% of CCGs), where COVID-19 and other patients are separated within their own practice; ‘hot’ or ‘cold’ hubs (used in 90% of CCGs), separate sites where COVID-19 or other patients registered at one of several collaborating practices are seen; ‘hot’ and ‘cold’ home visits (used in 70% of CCGs).\nOne of seven model combinations was used across each CCG, with some flexibility according to changing need shown through hub availability. Concomitant PHE data showed less than 2% of COVID-19 outbreaks or clusters in England were linked to general practice. Conclusions: Varied, flexible ways of delivering face-to-face general practice consultations were identified. While COVID-19 outbreaks or clusters linked to general practice constituted a small proportion of totals, their investigation, together with evaluations of the modified delivery models in terms of management of COVID-19 and other conditions and impacts on staff and patients, may aid future management of the pandemic and identify aspects of adapted practice of benefit beyond this.", "keywords": [ "COVID-19", "SARS-CoV-2", "coronavirus", "general practice", "primary care", "face-to-face consultation", "delivery model", "transmission" ], "content": "Introduction\n\nWhile more than 80% of patients with COVID-19 may not require hospitalisation,1 many will seek treatment in general practice. In order to minimise transmission of the causative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during general practice (GP) consultations, NHS England’s Standard Operating Procedure was revised in March 2020 to a remote triage and consultation default, with adapted models for face-to-face contact when clinically necessary.2 The use of telephone, video and online consultations in English general practice has been studied elsewhere.3 In this paper we report on the delivery of face-to-face general practice consultations during the pandemic, largely focussed on the spring and summer of 2020.\n\nWhen, after remote triage, a face-to-face general practice consultation is considered necessary, the requirement to separate those patients with suspected or diagnosed COVID-19 [‘COVID-19’ patients] from others is clear. Protection must be afforded to all on-site and NHS guidance suggests two possible ways to manage patients, premises and workforce for optimal infection prevention and control (IPC), alongside dedicated home visiting services for those shielding:2\n\n(i) Zoned practices: In this model, patient cohorts are separated within their own practices. Designated areas e.g., ‘red’ and ‘green’ zones, may be used to manage COVID-19 and other patients, respectively. Careful management is needed to minimise cross-contamination between groups, including separate walkways and consultation rooms, and staff allocated to one zone only. Zoning may therefore be impractical in some surgeries.\n\n(ii) Hot and cold hubs: A primary care hub may be designated as either ‘hot’ or ‘cold’, to treat COVID-19 or other patients respectively. It is available to patients registered at one of several collaborating practices. Where hot hubs are sited separately to non-COVID services, IPC procedures may be more straightforward than in zoned practices.2,4\n\n(iii) Dedicated home visiting: Home visiting services, modified to minimise cross-contamination, are necessary for those patients unable to access other face-to-face services, or where such provision is otherwise considered appropriate during the pandemic. Staff work exclusively with COVID-19 or other patients, and the number of activities undertaken during visits should be maximised to limit the requirement for additional face-to-face consultations. This service may be organised collaboratively, such as across Primary Care Networks, or by individual practices.\n\nNHS guidance indicates that decisions regarding model use should be determined locally, in agreement with the relevant Clinical Commissioning Group (CCG) responsible for planning and commissioning NHS health care services in the area. It also recognises that flexible models may be required as patient demand and workforce capacity fluctuates throughout the pandemic.4\n\nThis study aimed to identify the ways in which delivery of NHS face-to-face general practice has been re-organised across England to meet IPC standards during the pandemic, together with any evidence regarding the effectiveness of these measures.\n\n\nMethods\n\nA cross-sectional survey of the 135 CCGs in England was conducted to identify how face-to-face general practice consultations were being delivered across the country.\n\nSurvey design\n\nSurvey questions were devised by the study team. They concerned models of face-to-face consultations in use and the patient populations each were available to; prior use of the hub model; and planned evaluations. Questions were pre-tested with a researcher experienced in survey design, two CCGs and one provider of primary healthcare. Minor changes to wording were made for clarity. The final questionnaire is available as Extended data.13\n\nData collection\n\nQuestions were sent by email to all CCGs in June 2020 under the Freedom of Information (FOI) Act 2000. This legislation enables public access to recorded information held by public authorities in England. Responsibility for cleansing data lies with the authorities responding to FOI requests5 and research ethics approval was not required.\n\nIndividual CCGs were identified on the NHS England website and their specific FOI procedures were followed. FOI regulations mandate a response timeframe of 20 working days. Where, rarely, replies were not received within this time, follow-up emails were sent, and telephone calls made if necessary. All responses were collated in an Excel spreadsheet for analysis.\n\nData analysis\n\nData was extracted for each survey question, with any queries regarding response interpretation discussed between the authors. Email and telephone communication with CCGs was also used occasionally for clarification or updates. Numeric and narrative analyses were undertaken for responses to each question. Different face-to-face delivery models were identified, and each CCG was assigned to a model combination of best fit for nationwide comparison.\n\nSpecific data on clusters or outbreaks of acute respiratory infections with at least one linked case testing positive for SARS-CoV-2 are recorded for hospitals and care homes in the weekly COVID-19 surveillance reports, published by Public Health England (PHE).6,7 [‘Clusters’ are cases linked to a setting through surveillance testing; ‘outbreaks’ are reported directly from settings]. To obtain similar information regarding general practice, an email was sent to PHE in December 2020, under the FOI Act 2000. The questions asked are available as Extended data.14\n\n\nResults\n\nResponses\n\nReplies were received from all CCGs, 99% in June and July 2020, with the final response received on 2nd October.\n\nResponse interpretation\n\nTerminology used in responses varied – ‘hot sites’, ‘hot clinics’ and ‘resilience hubs’ could be used to refer to the same service for example. Colour-coding, e.g., ‘green’ and ‘amber’ sites, could also be used differently, and service provision for non-COVID patients was not always clear. As a consequence the distinction between hot hubs, cold hubs and zoned practices was not always immediately evident. Complete response sets, including any additional documentation provided on model pathways and usage data, were therefore used to interpret and categorise all face-to-face consultation types according to the models in this paper.\n\nThe following interpretation of the data is the authors’ own and has not been approved or otherwise by the CCGs.\n\nAdapted delivery models\n\nGeneral practice face-to-face delivery has been modified in each CCG using combinations of the three models previously described:\n\n(i) Zoned practices (model 1 in Figure 1), available to the entire patient populations served, were reported by 63 (47%) of CCGs. All but 12 of these also used hubs at the time of reporting. Most commonly, two closed ‘red’ and ‘green’ areas with different entrances and exits were used. Rarely, cohorts could also be separated temporally, with COVID-19 patients alone seen at certain times. The latter model was described in updated NHS England guidance (version 2, dated 5th April 2020) for surgeries where provision of separate spaces for different cohorts was not possible.2\n\n(ii) ‘Hot’ or ‘cold’ hubs (models 2 and 3 in Figure 1), were reported by 90% of CCGs. All of these had hot hubs, with 17% also using cold hubs. Hubs were generally available to all of the combined patient populations served. Occasionally however, cold hubs had more specific uses - a ‘super-green’ hub for patients requiring additional shielding for example and a ‘purple’ hub for routine treatments including vaccinations and maternity checks. Hub reach extended from several practices to entire CCGs. They were sited in re-purposed buildings (surgeries for example, or hubs that usually offered extended GP access), a racecourse, temporary structures (e.g., portacabins and marquees) or provided as drive-through facilities. Most CCGs indicated they had used hubs prior to the pandemic, mainly for the provision of extended hours GP access for patients.\n\nThe use of ‘co-located’ hubs was indicated in 21 CCGs, whereby hot hubs were sited adjacent to cold hubs (n=4) or cold practices (n=17).\n\n(iii) ‘Hot’ and/or ‘cold’ home visiting services, were specified by 70% of CCGs - 15% for hot appointments only, 10% for cold and 45% for both cohorts. These usually served patients unable to access other face-to-face services but were occasionally the main form of COVID-19 service provision. Delivery could be provided by practices, collaborative networks or CCG acute visiting services, and in some cases operated out of hubs. A small number of CCGs offered home monitoring of COVID-19 patients, via pulse oximetry for example, as an additional service.\n\nThe different means of delivering face-to-face services were compared for each CCG. Overall approaches taken were found to fit one of seven model combinations, albeit with some distinctions, notably the different usage of home visits and co-located hubs.\n\nThe seven model combinations are illustrated in Figure 1, and their distribution among CCGs shown in Figure 2. The diverse spread of models and size of CCGs seen in Figure 2 reflect the varied geography and population across England. A degree of cross-CCG working was evident in replies, and a small number of CCGs had access to hubs across neighbouring boundaries.\n\nAuthors’ interpretations of CCG responses according to the following definitions:\n\n• Zoned practice: co-location of hot and cold services on a single site, serving own practice list\n\n• Hot or cold hub: site of multi-practice working for COVID-19 or other patients respectively\n\nModel combinations:\n\n1: zoned practices (+/- home visits)\n\n2: hot hubs + cold practices (+/- home visits)\n\n3: hot hubs, cold hubs + cold practices (+/- home visits)\n\n4: hot home visits + cold practices\n\n5: hot hubs, cold hubs + zoned practices (+/- home visits)\n\n6: hot hubs, cold hubs, zoned practices + cold practices (+/- home visits)\n\n7: zoned practices, cold practices + hot home visits.\n\nAuthors’ interpretations of CCG responses according to the following definitions:\n\n• Zoned practice: co-location of hot and cold services on a single site, serving own practice list\n\n• Hot or cold hub: site of multi-practice working for COVID-19 or other patients respectively\n\nN.B. 15 CCGs did not describe face-to-face consultations for ‘cold’ patients. 14 of these were assigned to model combination 2 as hot hubs were described which were not co-located with cold services; and 1 was assigned to model combination 5.\n\nThe face-to-face delivery data we have presented was correct between March 2020 and the date of response [June/July (n=134) and October (n=1) 2020].\n\nEvaluations and flexible models\n\n87% of CCGs reported on-going, complete or intended reviews, generally of hub and/or telephone triage use, although one CCG was considering the potential of its drive-through model for influenza vaccinations, and others were intending to consider staff or patient perspectives. In total 23 CCGs reported using reviews to facilitate dynamic models, with hubs either available but as yet unused (n=3) or having been used initially and then stood down (n=20). [Assignment to model combinations 1-7 was based on provision at the time of CCG reporting.] More recent communication (Oct 12th-16th 2020) with three such CCGs using the zoned practice model (#1) confirmed that, despite COVID-19 incidence requiring local lockdowns at that time,6,7 GP escalation plans had not yet been necessary although hub sites remained available. Some other CCGs also indicated lower use of COVID-19 face-to-face consultations than anticipated.\n\n17 CCGs provided data on face-to-face contact across 21 hot hubs. While representing only a small proportion of total hubs, wide variations in usage were seen, with average weekly consultation numbers ranging from 2 to 79 per hot hub (March to July 2020).\n\nPHE data received detailed regional outbreaks or clusters of acute respiratory infections with possible/confirmed COVID-19 cases linked to GP surgeries. Table 1 shows this data as monthly totals from March to December 2020, alongside numbers of English face-to-face GP consultations obtained from NHS Digital.8 In the March-August timeframe covered by model usage in this study, 25 outbreaks or clusters with possible/confirmed COVID-19 cases were reported in England in the context of over 104 million face-to-face GP appointments. This represented less than 2% of all COVID-19 outbreaks or clusters in England [calculated using data in COVID-19 surveillance reports].6,7 Numbers increased in October and November, alongside rising face-to-face appointment levels, but continued to represent a small proportion of totals.6,7 Final figures may in fact be lower after investigation of unconfirmed cases by local PHE Health Protection Teams.\n\n* Monthly data calculated from weekly totals given in Public Health England response to FOI request. [March: 02/03/2020 to 29/03/2020; April: 30/03/2020 to 26/04/2020; May: 27/04/2020 to 31/05/2020; June: 01/06/2020 to 28/06/2020; July: 29/06/2020 to 26/07/2020; Aug: 27/07/2020 to 30/08/2020; Sept: 31/08/2020 to 27/09/2020; Oct: 28/09/2020 to 01/11/2020; Nov: 02/11/2020 to 29/11/2020]. Some 'possible' COVID-19 cases reported may be excluded after investigation by local PHE Health Protection Teams, and final numbers may therefore be lower.\n\n** Face-to-face general practice appointment data obtained from NHS Digital.8\n\n\nDiscussion\n\nAll CCGs reported the use of zoned practices, hubs and/or home visits in varying combinations for face-to-face GP consultations. Factors influencing model selection included COVID-19 appointment demand, presence of pre-established collaborative networks and GP preferences to provide continuity of care. On-going model assessments enabled responsiveness to changing demand, mainly through altered hub availability, but also by changed provision of home monitoring for COVID-19 patients.\n\nCCG mergers on 1st April 2020, which decreased total numbers from 191 to 135, may also have impacted model patterning within CCGs. While the ‘hot hubs + cold practices’ model combination #2 was the most frequently used nationwide, larger CCGs tended to report ‘mixed’ models, giving model combination #5 the greatest overall coverage (Figure 2). Patterning within these mixed model combinations (#5-#7) could not always be identified, but in one CCG five areas of zoning and a sixth using a hot hub were identified, and in some others more diffuse patterning was noted.\n\nThe distinction between the zoned practice and hub models used is not as clear as indicated in our Introduction. The co-location of hot hubs with cold services means that the requirement for strict management between hot and cold areas is as important here as in the zoned practice model. Indeed, several CCGs using co-located hubs and some using zoned practices specified the use of separate entrances and exits, with some also indicating separate car parking facilities. By contrast, other zoned practices shared more similarities with distinctly sited hubs, where red and green areas were split between main and branch surgeries for example, or additional structures such as portacabins were used on site, to separate patient cohorts registered within one practice. Thus, it is not necessarily the case that the hub model provides clearer separation and thereby simpler IPC adherence than zoned practices, as indicated in the guidelines.2,4\n\nUsage of home visits also varied. In at least two CCGs this was the main or only form of COVID-19 face-to-face consultation. In 30% of CCGs however, the use of specific hot and/or cold home visits was not reported. Home monitoring, via pulse oximetry for example, was an additional service indicated by a small number of CCGs at the time of reporting.\n\nWhile the risk of transmission can be minimised in face-to-face consultations, it cannot be eliminated, particularly with the significant pre-symptomatic and asymptomatic transmission associated with COVID-19.9 PHE data shows a total of 247 possible or confirmed COVID-19 outbreaks or clusters in English GP surgeries over the first nine months of the pandemic, in the context of more than 216 million face-to-face appointments. While these numbers are a small proportion of total (all source) outbreaks in the UK, investigation of any confirmed outbreak may guide future measures to reduce transmission.\n\nThe variability in possible/confirmed outbreaks or clusters in general practice over time, as well as by region (shown in Table 1), may partly be accounted for by changing COVID-19 incidence rates, but also by other factors including differences in testing rates, population risk factors, and the numbers and means of delivery of face-to-face consultations taking place locally.\n\nIn addition to minimising SARS-CoV-2 transmission, the success of IPC delivery models used during the pandemic will be measured in terms of the management of COVID-19 and other conditions, and the perspectives of patients and staff. Several patient surveys have now been undertaken.10–12 The authors have also reported on a survey of the public, investigating experiences and understanding of the changes to general practice.15 These patient findings, taken together with staff perspectives3 and quantitative evaluations should provide helpful insights into the effective use of the adapted models.\n\nThere are several limitations to this study. As already described, terms used to describe novel concepts (e.g., hot/cold hubs, zoning) varied and could not readily be checked. This may, for a small number of CCGs, have led to misinterpretation of responses and consequent misassignment to model combinations. Studies at Primary Care Network or practice level would likely reveal more granular detail, particularly with respect to the ‘mixed model’ combinations identified. In terms of the PHE FOI request, data on outbreaks/clusters from ‘GP surgeries’ was provided, and it is unclear whether this includes hubs and all home visiting services. Finally, the service delivery details provided by CCGs were mainly received in summer 2020 and, as has been identified, these are dynamic models and may since have been modified.\n\n\nConclusions\n\nThis study provides an overview of nationwide IPC adaptation of face-to-face GP consultations in the spring and summer of 2020, with varied and dynamic models implemented to suit different and changing local conditions. Numbers of COVID-19 outbreaks linked to English GP surgeries indicate their relative success in minimising transmission. Shared learning from evaluation of these outbreaks across the different models may be instructive however for future management of SARS-CoV-2 transmission within general practice, where numbers of face-to-face consultations will need to increase again in future. Broader evaluation of the changes is also needed, including analysis of their impact on the management of non-COVID conditions, as well as on staff and patients. Taken together, this will identify beneficial elements of the rapidly enforced adaptations to inform practice both during this pandemic and beyond.", "appendix": "Acknowledgements\n\nWe would like to thank each CCG, as well as Public Health England for responding to our requests for information.\n\n\nData availability\n\nThe Re-use of Public Sector Information Regulations (RPSI) 2005 and copyright requirements have been invoked as imposing requirements around certain types of further use of survey data provided by some Clinical Commissioning Groups (CCGs). This may also apply to data received from other CCGs and Public Health England (PHE) and it is therefore not possible to share this data under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe data may be available from individual CCGs and PHE on request, with reference to the authors and this publication. Alternatively, Freedom of Information requests similar to those made by the authors may be used. Full details of these are provided in the Extended data and Methods section.\n\nFigshare: Survey sent to Clinical Commissioning Groups in England, https://doi.org/10.6084/m9.figshare.14156741.v113\n\nFigshare: Questions sent to Public Health England, https://doi.org/10.6084/m9.figshare.14156762.v114\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nVerity R, Okell LC, Dorigatti I, et al.: Estimates of the severity of coronavirus disease 2019: a model-based analysis. Lancet Infect Dis. 2020; 20: 669–677. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNHS_England: Guidance and standard operating procedures. General practice in the context of coronavirus (COVID-19). 2020 [Accessed 31 Oct 2020]. Reference Source\n\nMurphy M, Scott LJ, Salisbury C, et al.: Implementation of remote consulting in UK primary care following the COVID-19 pandemic: a mixed-methods longitudinal study. Br J Gen Pract. 2021; 71: e166–e177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNHS_England_and_NHS_Improvement: GP Preparedness update letter.2020 [Accessed 25 Feb 2021]. Reference Source\n\nSavage A, Hyde R: Using freedom of information requests to facilitate research. Int J Soc Res Method. 2014; 17: 303–317. Publisher Full Text\n\nPublic_Health_England: National COVID-19 surveillance reports.2020 [Accessed 31 Oct 2020]. Reference Source\n\nPublic_Health_England: National flu and COVID-19 surveillance reports.2020 [Accessed 25 Feb 2021]. Reference Source\n\nNHS_Digital: Appointments in General Practice.2020 [Accessed 25 Feb 2021]. Reference Source\n\nEuropean_Centre_for_Disease_Prevention_and_Control: Transmission of COVID-19.2020 [Accessed 25 Feb 2021]. Reference Source\n\nPublic_Health_England: Wider Impacts of COVID-19 on Health (WICH) monitoring tool.2020 [Accessed 3 Sept 2020]. Reference Source\n\nHealthwatch_Swindon: PPG chairs’ quick survey of Swindon GP practices.2020 [Accessed 31 Oct 2020]. Reference Source\n\nNational_Voices: What we need now.2020 [Accessed 25 Feb 2021]. Reference Source\n\nDuncan L: Survey sent to Clinical Commissioning Groups in England. figshare. Journal Contribution. 2021. Publisher Full Text\n\nDuncan L: Questions sent to Public Health England. figshare. Journal Contribution. 2021. Publisher Full Text\n\nDuncan LJ, Cheng KFD: Public perception of NHS general practice during the first six months of the COVID-19 pandemic in England. F1000Research. 2021. Publisher Full Text" }	[ { "id": "83182", "date": "04 May 2021", "name": "Helen Atherton", "expertise": [ "Reviewer Expertise Access to general practice" ], "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 useful and clear report summarising the modifications made in general practice during the height of the pandemic.\nThere are a few key things that would strengthen the manuscript particularly in terms of message.\nThe abstract background says the study looked at 'evidence for their effectiveness' which is an overstatement as this is very much a descriptive study. It would be more accurate to remove that.\n\nAt present the paper is written as though we are still 'in' this stage of the pandemic. With vaccinations and the benefit of time changes are happening. It would be useful to couch the study as a description of service delivery in a time of crisis. Hot hubs are not going to be used in the same way now rates are extremely low. Your report is a really useful for any future lockdowns/rise in rates and as a learning tool and you don't couch it in those terms at present.\n\nI completely appreciate why you used survey methodology but it isn't ideal as you later highlight. Using FOI requests is such a good idea but does make this a slightly different approach, and probably worth discussing pros/cons in the discussion for the benefit of other researchers. I was interested as to whether it is worth it for the data you get.\n\nThe data analysis section is very thin and would benefit from some extra detail.\n\nThe PHE element is probably the weakest bit, the link between the models and the outbreaks is tenuous as you don't have any other data around what happened there. Where you describe the 25 outbreaks it would be useful to have more context in the text - how many cases, which models, where. If this is not available I think this needs to be highlighted.\n\nThere are several limitations to be added to the discussion - for example the bit about the CCG mergers, which is important.\n\nThe section in the discussion about transmission is useful context but comes a bit late in terms of understanding that bit of the work.\n\nThe section on evaluation doesn't add anything at this point. Could you say instead what the practical application of your findings might be?\n\nOverall the manuscript lacks a bit of context, which is fine if it is just a descriptive exercise, but at the moment it isn't sure if it is something more than that. If it is a useful descriptive tool that is fine, but the conclusion probably needs to be toned down. In particular the line 'indicate their relative success in minimising transmission' - you just don't have the data to say that.\n\nFigure is really good and the succinct messaging was appreciated.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7058", "date": "20 Aug 2021", "name": "Lorna Duncan", "role": "Author Response", "response": "Dear Dr Atherton, Many thanks for taking the time to review this paper. We have used your detailed and helpful comments to improve our manuscript. Our specific responses to each of your points are listed below. Key points to strengthen the manuscript: 1. The abstract background says the study looked at 'evidence for their effectiveness' which is an overstatement as this is very much a descriptive study. It would be more accurate to remove that. We have removed this sentence further to your feedback. Please see also our response to your point 5 below. 2. At present the paper is written as though we are still 'in' this stage of the pandemic. With vaccinations and the benefit of time changes are happening. It would be useful to couch the study as a description of service delivery in a time of crisis. Hot hubs are not going to be used in the same way now rates are extremely low. Your report is a really useful for any future lockdowns/rise in rates and as a learning tool and you don't couch it in those terms at present. We have now ensured the entire manuscript is written from a historic perspective, looking back to the first wave of the pandemic in spring and early summer 2020. We wrote this report during the second wave as incidence fluctuated, and we had difficulty deciding how to contextualise it. We are grateful for your perspective and agree that this revision has given a much clearer context for the findings. 3. I completely appreciate why you used survey methodology but it isn't ideal as you later highlight. Using FOI requests is such a good idea but does make this a slightly different approach, and probably worth discussing pros/cons in the discussion for the benefit of other researchers. I was interested as to whether it is worth it for the data you get. We have now included further discussion of the pros and cons of the use of both surveys and FOI requests in the new ‘strengths and limitations’ section. We were somewhat limited in the design of our study as COVID-19 restrictions required it to be completed remotely, and because it was initially intended as a 6-week project. Despite the limitations however, we feel this survey using FOI requests yielded useful information, either for case site selection and further investigation, or for retrospective analysis for initial management of any future epidemics, or future lockdowns as you suggest. Although it was not possible to include a comparative table of the CCG data received, we hope that the additional information now included in our summary analysis table (as Underlying data) supports this and is found useful. Please see also our response to your point 8 below. 4. The data analysis section is very thin and would benefit from some extra detail. We have now incorporated more detail in our methodology section and have also added a summary version of our analysis spreadsheet as Underlying data. It is unfortunate that we are unable to include copies of the CCG and PHE responses under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0) used in this article. However, on considering your comments, we realised it may be helpful to offer permissible detail from which figures 1 and 2 were derived by adding a version of our analysis spreadsheet with the raw data removed. Of course this includes only an interpretation of the data received. 5. The PHE element is probably the weakest bit, the link between the models and the outbreaks is tenuous as you don't have any other data around what happened there. Where you describe the 25 outbreaks it would be useful to have more context in the text - how many cases, which models, where. If this is not available I think this needs to be highlighted. We agree that the data we have concerning general practice-linked COVID-19 outbreaks is limited and we are grateful to you for highlighting the possible inference of a direct link between the models used and outbreaks from the text. This was not our intention and we have now given a fuller account of the reasoning for requesting this data and for including it in our report. We have also separated this work out in each section of the report. To the best of our knowledge, this data has not otherwise been made publicly available and we feel it offers useful context around the scale of outbreaks in general practice. We requested further information both from PHE and NHS England & NHS Improvement and have now mentioned this in our methodology section and reported the finding that transmission in staff was more common in general practice than that involving patients in one region. PHE was not able to provide more detail on specific outbreaks due to the potential for deductive disclosure however, and the table we have included in our report provides the most detailed data made available to us. 6. There are several limitations to be added to the discussion - for example the bit about the CCG mergers, which is important. We have now expanded on our limitations section, to include discussion around the CCG mergers and several other items. Please see our response to item 8 below for more information. 7. The section in the discussion about transmission is useful context but comes a bit late in terms of understanding that bit of the work. We agree and thank you for highlighting this. We have now moved this section to the introduction and expanded it. 8. The section on evaluation doesn't add anything at this point. Could you say instead what the practical application of your findings might be? [Combined response for items 3, 6 and 8]. We have replaced both the ‘evaluation’ and ‘limitations’ sections with a fuller ‘strengths and limitations’ section. This includes further discussion of the survey methodology, using FOI requests at CCG level, the CCG mergers and possible applications of our study. We agree that this is likely to be far more useful than the two previously included sections – many thanks. 9. Overall the manuscript lacks a bit of context, which is fine if it is just a descriptive exercise, but at the moment it isn't sure if it is something more than that. If it is a useful descriptive tool that is fine, but the conclusion probably needs to be toned down. In particular the line 'indicate their relative success in minimising transmission' - you just don't have the data to say that. This report is intended to offer a description of the face-to-face services offered in general practice in the initial phases of the pandemic. The information we included concerning outbreaks in general practice perhaps confused this although we do feel it adds some useful context to our data. We have now removed the line you mention and agree that our conclusion now more clearly represents our study. 10. Figure is really good and the succinct messaging was appreciated. Many thanks, we are pleased you found this helpful! Thank you very much also for each of your comments which we feel have helped us to improve this report." } ] } ]	1	https://f1000research.com/articles/10-261
https://f1000research.com/articles/10-279/v1	08 Apr 21	{ "type": "Brief Report", "title": "Public perception of NHS general practice during the first six months of the COVID-19 pandemic in England", "authors": [ "Lorna J. Duncan", "Kelly F.D. Cheng", "Kelly F.D. Cheng" ], "abstract": "Background: In March 2020, the delivery of NHS general practice consultations was rapidly modified to mitigate against the spread of COVID-19. Remote triage and consultations became the default, with adapted models for face-to-face contact if clinically required. This study aimed to gain insight into public perception of these adaptations. Methods: Two online surveys were developed, and conducted between August and September 2020. Survey A, open to anyone receiving the link to it, considered respondents’ experiences of healthcare contacts since March 2020, and their understanding of the adapted delivery. Survey B, open to survey A respondents only, then considered how healthcare communication had been received and individual preferences for this. Survey participation was voluntary. Results: The views and experiences of 150 members of the public were obtained. 105 had considered contacting general practice, although half avoided this or delayed doing so for longer than usual. While some patients did so ‘to help the NHS’, others experienced reduced access for a variety of reasons including COVID-19 safety concerns. Some however reported benefitting from remote consultation availability and regular texts/emails from their practice. 68% (102/150) of respondents were unaware that patients with COVID-19 were seen separately from other patients during general practice appointments. 27% of those in survey B who had avoided or delayed contact said they would have felt more comfortable contacting general practice had they known about this. Conclusions: Experience and use of the adapted general practice models varied. Some patients felt their access to healthcare was reduced, often due to technological requirements. For some who found attending face-to-face appointments difficult however, remote contact was advantageous. Most patients surveyed were unaware of the COVID-19 control measures in place during face-to-face general practice consultations. Assessment of adapted delivery model accessibility and clearer public messaging about the changes may help reduce inequalities.", "keywords": [ "COVID-19", "general practice", "coronavirus", "SARS-CoV-2 transmission", "delivery model", "face-to-face consultation", "patient communication", "patient experience" ], "content": "Introduction\n\nIn March 2020, with the onset of the COVID-19 pandemic, the delivery of general practice consultations changed rapidly and extensively throughout England. National Health Service (NHS) standard operating procedure was adapted to ensure the physical separation of patients with suspected or diagnosed COVID-19 (‘COVID-19 patients’) from others, to minimise cross-infection.1 Remote triage and consultation became the default, with face-to-face contact only used when clinically necessary.1 As a result, the proportion of face-to-face general practice consultations dropped from 80% before March 2020, to 47% in April 2020. While this rose gradually after the end of the first national lockdown in July, it remained considerably below pre-pandemic levels at 56% in the most recently available (January 2021) data.2\n\nWhen face-to-face consultations were necessary, they required reorganisation to comply with Infection Prevention and Control (IPC) guidance.1 Our June 2020 study reports on the adapted models used to deliver NHS face-to-face general practice consultations in England.3 While several nuances to these models exist, the two most typical are shown in Figure 1. In model A, COVID-19 patients are seen at a ‘hot’ hub - a site shared between several locally collaborating practices. All other patients are seen at ‘cold’ GP practices. In model B meanwhile, COVID-19 and other patients are seen at their own practice, but in two separate ‘zones’. These are carefully managed to minimise cross-contamination, with staff working in one zone only, and separate entrances and exits.\n\nVariations of these models may be used, as well as designated ‘COVID-19’ and ‘other’ home visiting services.\n\nIn addition to the reduced proportion of face-to-face consultations, the number of all-mode general practice consultations (including telephone, video/online and face-to-face appointments as well as home visits) also dropped by around 30% in April 2020.2 Among many possible reasons for this are the change to total triage prior to arranging consultations, and public response to the adapted consultation models themselves.\n\nThe aims of this study were:\n\nto explore public experiences and perceptions of general practice in the first 6 months of the pandemic (March-Sept 2020);\n\nto understand public awareness of the changes to general practice and the ways in which information had been received about this.\n\n\nMethods\n\nTwo online surveys, A and B, were conducted sequentially to identify the public’s experience and perceptions of general practice in England from March to September 2020.\n\nSurvey A considered:\n\n• respondents' contacts with primary care for any symptoms since March 2020, their experience and satisfaction with this;\n\n• respondents' awareness of the separation of COVID-19 patients from others during general practice face-to-face consultations.\n\nSurvey B then considered:\n\n• how respondents who knew that COVID-19 patients were separated from others during face-to-face general practice consultations had received this knowledge;\n\n• COVID-19 information sources used and preferred by respondents.\n\nSurvey questions were developed by the study team and made available using JISC online surveys. They were pre-tested on five people (two experienced in survey design), and minor changes to wording were made for clarity. The final questionnaires and flyer giving password access to survey A are available as Extended data.21-23\n\nParticipation in both surveys was voluntary and anonymity was assured – completion of a survey indicated consent to participate. Ethical approval was not required due to the low risk nature of the surveys. Survey A was open to anybody receiving details of it, including the password. Survey B was open to survey A respondents who agreed to help further.\n\nSurvey A details were distributed by the Patient and Public Involvement and Engagement team (Centre for Academic Primary Care, University of Bristol) to their contacts list via email, attaching our flyer. A newsflash was also placed in People in Health West of England’s newsletter. Details were further distributed by Dr L. Farbus and others at NHS England and NHS Improvement, and by South Gloucestershire Council. Survey B was distributed from the survey website to email addresses supplied by survey A respondents.\n\nSurvey A was open August 4th—September 9th 2020; survey B between August 19th and September 14th 2020.\n\nStatistical analyses (counts and percentages) of closed questions were provided within the JISC online survey analysis tool. Free-text responses were analysed both numerically (grouped in relation to the items raised) and narratively by the team. Quotations in the results section represent key themes.\n\nAdditional reporting of methodology, following guidance for online surveys,4 is available as Extended data.24\n\n\nResults\n\nA total of 150 people completed survey A. Figure 2 shows their locations in relation to Clinical Commissioning Groups (CCGs), organisations responsible for planning and commissioning most hospital and community NHS services in England. Our respondents lived within the boundaries of 12 CCGs, labelled A-L on the map. 91% of respondents lived in South-West England (CCGs A-E), a region of relatively low COVID-19 incidence to date.5 71% were from NHS Bristol, North Somerset and South Gloucestershire CCG. 15 respondents were healthcare professionals, with three working in general practice. Closed responses to survey A questions are available as Underlying data.20\n\n5 respondent locations unknown.\n\nDecision to contact general practice or NHS 111\n\nIn total, 70% (105/150) of survey A respondents reported having considered contacting general practice or NHS 111 (a national telephone helpline and website) since March 2020; 10 thought they may have had COVID-19. Figure 3 shows the healthcare interactions of all respondents between March and September. It can be seen that twelve symptomatic respondents did not seek advice; a further 41 reported delaying doing so for longer than usual (data not shown). Excepting four people who managed their own symptoms, these two groups represented 47% of symptomatic respondents. The most common reason for this, given by 39% of these respondents, was a desire to ‘help the NHS’. Other factors included access issues (anticipated or experienced), lack of face-to-face consultations or feeling uncomfortable with telephone consultations, and fear of contracting COVID-19.\n\nNHS 111 is a national telephone helpline and website for patients.\n\nBy contrast, 11% of symptomatic respondents reported contacting general practice more quickly than usual, mainly due to symptom severity or anxiety. In two cases however, patients required prompt advice to establish whether they should self-isolate, according to COVID-19 regulations.\n\nContacts made\n\nIrrespective of time taken to seek help, a total of 93 people - 89% of those who were symptomatic - did so. While two people used occupational health or online searches only, the remaining 91 used NHS healthcare, as detailed in Figure 3. The vast majority - 78 people - contacted their GP surgery, 37 of these using only this method. 33 respondents used the NHS 111 service (15 by telephone, 18 accessing it online), but only 5 used it alone. Several other sources (identified in Figure 3) were also used by 30 people, but most used these alongside general practice or NHS 111 contacts.\n\nSatisfaction with contacts\n\nAs Figure 3 illustrates, 78% (71/91) of respondents who contacted general practice and/or NHS 111 felt they received the help they needed. This included all 25 who had face-to-face appointments, despite one-fifth having had initial concerns about attending related to COVID-19 safety. 20 respondents however were dissatisfied, with reasons involving the inability to make successful contact, or unsatisfactory outcomes where contact was made, due mainly to the unavailability of appointments and dissatisfaction with remote appointments.\n\nUnderstanding of adapted delivery models\n\nThe evident reluctance to seek help by half of the patients in this survey was explored by investigating all 150 respondents’ understanding of the changed general practice delivery models. A large proportion (68%) reported not knowing whether COVID-19 patients were separated from others in face-to-face consultations. Similar proportions were seen in each sub-group in Figure 3. Further confusion is indicated by the fact that 25% of those who had had symptoms were unaware that face-to-face general practice consultations could happen during lockdown.\n\nSurvey B was distributed to 71 survey A respondents who indicated their willingness to help further, and 56 of these (79%) completed it. Their characteristics were checked using responses to the first survey and this sub-group was found to be similar in terms of location (96% lived in South-West England compared to 91% in survey A) and occurrence of symptoms (73% compared to 70%). However, awareness of the separation of COVID-19 patients from others was somewhat higher in these respondents, at 39% compared to 32% in survey A respondents.\n\nCommunication regarding COVID-19 and changes to general practice service delivery\n\nIn total, 28% of respondents who knew about the measures used to control COVID-19 during face-to-face consultations had working links to general practice. Others were informed by their practice, had seen visible evidence on-site such as a marquee or signage, or learnt through news reports or by word-of-mouth. Among those who were unaware, 27% indicated they would have felt reassured to contact general practice had they known that the patient groups were being kept apart.\n\nTable 1 shows a sample of the perspectives of our respondents concerning their communication with general practice. Half (A-M) describe experiences of consultations or messaging about the changed delivery, the remainder (N-Z) indicate their preferences and suggestions for this. Examples have been selected to highlight themes evident in the surveys, rather than to represent, for example, satisfaction with this communication. As might be expected, these may be experienced positively or negatively, dependent upon individual circumstances:\n\nQuotations were selected to demonstrate themes across responses rather than to represent levels of satisfaction.\n\nAccess to healthcare\n\nWhile respondents A,F,W and Y benefitted from the use of digital technology and remote consultations, respondents B,J,M,Q and R saw potential barriers to accessing healthcare in this way. Busy telephone lines and unclear answerphone messages were also common issues (E,L,U).\n\nPerson-centred care\n\nSome patients preferred the modified forms of delivery, finding them more convenient (A,F,Y); for others, choice (H,Q,R), privacy (H,Z), dignity (Z) and continuity of care (K) could be compromised.\n\nMessaging\n\nSome respondents reported receiving sufficient, regular or timely communication from their practice (F,W) and 27 identified or described the use of hubs or zoned practices locally to them (data not shown). For others however, confusion arising from unclear, out-of-date or insufficient messaging was evident (C,D,G,I,L,P,T,X) and this could cause anxiety (C,I). Suggestions and preferences for explaining the relevant changes included the use of social media (O), graphics and posters (N,U), local broadcasting and newspaper coverage (V), and sending letters to patients (S). It was apparent that clear, relevant information from respondents’ practices and then other local sources was preferred. National sources of information were seen as less useful.\n\n\nDiscussion\n\nOf the 105 respondents with symptoms in our surveys, half reported not seeking advice or delaying doing so, most commonly to reduce demand on healthcare services and to a lesser extent due to fear of COVID-19, concurring with national and international findings.6–8 These reasons, together with those of perceived or actual access issues, and differing preferences for the altered consultation modes were also shared with an NHS survey of 6614 patients in South-West England [personal communication, Dr L. Farbus, NHS England and NHS Improvement, 22nd September 2020].\n\nDespite promotion of the NHS 111 service during the pandemic, only 36% of respondents either called 111 or accessed the NHS 111 website, with a mere 5% using this service alone. This may partly be due to the misunderstanding by some that only those with COVID-19 symptoms were to use the service. However, it was also clear that respondents wanted local, relevant communication, preferably from their own practices. Indeed 84% of people contacted their own practice directly, with 40% using no other method.\n\nSatisfaction levels among respondents who sought advice in general practice remained high at 78%, similar to pre-pandemic levels.9 Among those receiving face-to-face consultations, satisfaction was 100%. 20 respondents were dissatisfied however. Half of these were unable to make contact, while others were unhappy with the outcomes of contacts made, typically related to the availability and modes of consultations.\n\nOur surveys indicate that the changes to service delivery have decreased equity of access. While some respondents benefitted from video and telephone consultation availability, for example where it could be hard to fit face-to-face appointments around caring or work responsibilities, others experienced reduced access due to lack of relevant information, fear, loss of choice, logistic and/or technological barriers. The necessary speed of change has undoubtedly impacted all parties and limited co-production of the new models with patients and staff. It is of interest however that a small number of individuals successfully self-managed conditions they would previously have brought to general practice, including self-monitoring of blood pressure and treatment of corns.\n\nWhile some people were well-informed about the changes to face-to-face consultations, public awareness was generally low, and some respondents indicated that better understanding would have reassured them to seek healthcare advice. Some ambiguity in messaging was apparent, with both the understanding that COVID-19 patients were not being seen in general practice and, contrastingly, that patients without COVID-19 were not being seen, indicated in our survey. Email communication in January 2021 with a small number of respondents suggested that both the avoidance of general practice and reasons for this were still present, with some people remaining unable to make contact and/or having received minimal communication from their practice. Conversely, regular communication was reported by some respondents. This may be contributing to the different messages coming from the public, media and general practice concerning the availability of general practice appointments.10,11\n\nThe differences identified in both the communication received and its comprehension are perhaps unsurprising, given that national focus has been on secondary care of people with COVID-19,12 focus in general practice has necessarily been on adapting delivery and providing safer care,13 and that patients have been faced with volumes of information from multiple sources throughout the pandemic.14–16 It is clear though, that this has impacted on patient experience of general practice, causing confusion and increased anxiety in some, while delivering improved access for others. NHS guidance indicates the importance of informing the public of changes, and the need for accessible patient communication has also been identified.1,10,15,17,19 Evidence of regular communication by individual GP practices with their patients is available (https://www.facebook.com/Alvanleyfamilypractice/; https://youtu.be/kEXOSl0cIaA)18 and it is likely that the COVID-19 vaccination campaign has also re-established connection with some patients. Clear, current and specific messaging detailing the local measures in place to keep people safe, will empower others.\n\nThis online survey of 150 people was largely local to South-West England, an area of relatively low COVID-19 incidence to date. While our findings concerning the use of general practice during the pandemic reflect those obtained in other regional and national surveys, studies of populations in regions with different demographics and including those without internet access, may identify additional themes and establish whether our outcomes concerning knowledge of adapted general practice delivery are representative nationally.\n\n\nConclusions\n\n150 survey respondents have provided insights into the experience and communication of general practice between March and September 2020. While the adapted models of delivery were preferred by some patients, they were inaccessible to others. Possible reasons for general practice avoidance were also indicated, including a significant lack of awareness of the measures taken to optimise safety during face-to-face consultations. Evaluation of all delivery models, incorporating perspectives from both staff and patients, as well as the checking of current messaging, should help to ensure that all patients are able to access general practice.", "appendix": "Acknowledgements\n\nWe would like to thank everybody who completed our surveys, and those who helped to distribute them, including the Patient and Public Involvement and Engagement team, Centre for Academic Primary Care, University of Bristol; People in Health West of England; Dr L. Farbus and others at NHS England and NHS Improvement; and South Gloucestershire Council.\n\n\nData availability\n\nFigshare: Survey A responses, https://doi.org/10.6084/m9.figshare.14269967.v120\n\nFree text responses in surveys A and B cannot be made openly accessible as it is not possible to successfully anonymise each of these responses and the data cannot be shared outside of the research team. Either respondents to the surveys, their friends or family, healthcare sites and staff may be identifiable. Some anonymised responses reflective of overall responses are available in Table 1.\n\nFigshare: Survey A questions, https://doi.org/10.6084/m9.figshare.14269469.v121\n\nFigshare: Survey A flyer, https://doi.org/10.6084/m9.figshare.14269475.v122\n\nFigshare: Survey B questions, https://doi.org/10.6084/m9.figshare.14269478.v123\n\nFigshare: Reporting of the design, conduct and analysis of surveys A and B, https://doi.org/10.6084/m9.figshare.14269454.v124\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nNHS_England: Guidance and standard operating procedures. General practice in the context of coronavirus (COVID-19).2020 [Accessed 18 March 2021]. Reference Source\n\nNHS_Digital: Appointments in General Practice.2020 [Accessed 18 March 2021]. Reference Source\n\nDuncan LJ, Cheng KFD: Modifications to the delivery of NHS face-to-face general practice consultations during the COVID-19 pandemic in England. F1000Res. 2021. Publisher Full Text\n\nEysenbach G: Improving the Quality of Web Surveys: The Checklist for ReportingResults of Internet E-Surveys (CHERRIES). J Med Internet Res. 2004; 3: e34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDepartment_of_Health_and_Social_Care_and_Public_Health_England: Coronavirus (COVID-19) in the UK.2020 [Accessed 18 March 2021]. Reference Source\n\nPublic_Health_England: Wider Impacts of COVID-19 on Health (WICH) monitoring tool.2020 [Accessed 21 Feb 2021]. Reference Source\n\nThe_Health_Foundation: Use of primary care during the COVID-19 pandemic.2020 [Accessed 1 March 2021]. Reference Source\n\nHuston P, Campbell J, Russell G, et al.: COVID-19 and primary care in six countries. BJGP Open. 2020; 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMayor S: Patient satisfaction with GPs remains high despite current pressures. Bmj. 2019; 366(14687). PubMed Abstract \| Publisher Full Text\n\nHealthwatch_Swindon: PPG chairs’ quick survey of Swindon GP practices.2020 [Accessed 12 Feb 2021]. Reference Source\n\nLind S: NHSE asked to issue ‘correction’ to claims about face-to-face GP consultations. Pulse. 2020. Reference Source\n\nPark SE, J, Berlin A, et al.: Strengthening the UK primary care response to covid-19. Bmj. 2020; 370: m3691. PubMed Abstract \| Publisher Full Text\n\nKhan N, Jones D, Grice A, et al.: A brave new world: the new normal for general practice after the COVID-19 pandemic. BJGP Open. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRawaf S, Allen LN, Stigler FL, et al.: Lessons on the COVID-19 pandemic, for and by primary care professionals worldwide. Eur J Gen Pract. 2020; 26: 129–133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational_Voices: What we need now.2020 [Accessed 18 March 2021]. Reference Source\n\nWorld_Health_Organisation: Managing the COVID-19 infodemic: Promoting healthy behaviours and mitigating the harm from misinformation and disinformation.2020 [Accessed 18 March 2021]. Reference Source\n\nPublic_Health_England: Beyond the data: Understanding the impact of COVID-19 on BAME groups.2020 [Accessed 18 March 2021]. Reference Source\n\nStanley S: Patient instructions for HOT site. BJGP 2020; COVID-19 Clinical Solutions [Accessed 18 March 2021]. Reference Source\n\nNHS_England_and_NHS_Improvement: Access to general practice communications toolkit.2020 [Accessed 12 Feb 2021]. Reference Source\n\nDuncan L: A. Survey A responses. figshare. Journal contribution. 2021. Publisher Full Text\n\nDuncan L: 2. Survey A questions. figshare. Journal contribution. 2021. Publisher Full Text\n\nDuncan L: 3. Survey A flyer. figshare. Journal contribution. 2021. Publisher Full Text\n\nDuncan L: 4. Survey B questions. figshare. Journal contribution. 2021. Publisher Full Text\n\nDuncan L: 1. Reporting of the design, conduct and analysis of surveys A and B. figshare. Journal contribution. 2021. Publisher Full Text" }	[ { "id": "84461", "date": "06 May 2021", "name": "Joanne Parsons", "expertise": [ "Reviewer Expertise Primary care", "vaccination adherence", "remote consultations", "digital healthcare interventions." ], "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 articles provides an examination of the perceptions of general practice patients of adaptations made to appointments as a result of the COVID-19 pandemic. Participants completed online surveys to explore use and perceptions of delivery and healthcare communication.\n\nThis is a very timely and relevant article, and is interesting in providing understanding about patients' experience and perceptions of this area. Background and rationale for the need for the project is clear, and methodology and findings are reported clearly and logically, making it an interesting and accessible paper to read.\n\nI have read the paper in detail, and there are a few minor areas that some clarification on would improve the paper for readers.\n\nIntroduction:\nParagraph 1; you mention the percentage of face-to-face consultations before and during COVID-19. It would be interesting to also include the percentages of consultations that were delivered using other (remote) methods during the time that face-to-face consultations were reduced.\n\nMethods:\nWhen you detail what Survey A considered, you state 'respondents' contacts with primary care for any symptoms' - it would be helpful to state if this refers to COVID-19 symptoms only, or any symptoms of any health condition. Just to ensure readers are clear.\n\nWhen you explain that five people pre-tested the survey questions (two were experienced in survey design), who were the other three? Was there input from lay/PPI representatives?\n\nResults:\nIt would be interesting for readers to have a brief summary of the participants that responded to each of the surveys. Before you discuss the location of respondents, could you include the mean age, and percentage of respondents that were male/female?\n\nUnderstanding of adapted delivery models:\nThere are a couple of occasions in the results section where you refer to participants not being aware that COVID-19 patients were 'separated from others in face-to-face consultations.' This wording is slightly unclear and perhaps rewording to make it clear that practices have different processes in place to ensure that patients with COVID-19 are seen in a different location/different part of the practice to other patients (you discuss it clearly in the introduction section).\n\nYou refer to respondents having 'working links to general practice' (p7), could you please provide some examples of such links?\n\nDiscussion:\nWhen you cite the personal communication from Dr L. Farbus - should this be a numerical reference in line with the other references?\n\nThe only other comment I have, is that you do not include any strengths or implications in the Discussion. This research is really interesting, and likely has real-world implications for ensuring that clear messages are provided to patients about when and how it is appropriate to contact the practice, and the availability of appointments during a situation such as COVID-19. It would be useful for readers to understand about how the findings of this research can improve patient experience.\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": [ { "c_id": "7366", "date": "01 Nov 2021", "name": "Lorna Duncan", "role": "Author Response", "response": "Dear Dr Parsons, Many thanks for reviewing our paper and for your specific comments which we found very helpful. We have now addressed each of your points to improve our manuscript as indicated below: 1. Paragraph 1; you mention the percentage of face-to-face consultations before and during COVID-19. It would be interesting to also include the percentages of consultations that were delivered using other (remote) methods during the time that face-to-face consultations were reduced. Thank you, we have now included the full breakdown of consultations in a new table, and have expanded on this in the Introduction text. We agree this has added useful perspective and we have also taken the opportunity to update the figures to include those most recently available (August 2021). 2. When you detail what Survey A considered, you state 'respondents' contacts with primary care for any symptoms' - it would be helpful to state if this refers to COVID-19 symptoms only, or any symptoms of any health condition. Just to ensure readers are clear. Thank you for highlighting this, we have now indicated in the Methods section that contacts were for any symptoms, both COVID-19 and/ or those related to other conditions. 3. When you explain that five people pre-tested the survey questions (two were experienced in survey design), who were the other three? Was there input from lay/PPI representatives? We have now expanded this sentence to clarify that the remaining three people were lay representatives. 4. It would be interesting for readers to have a brief summary of the participants that responded to each of the surveys. Before you discuss the location of respondents, could you include the mean age, and percentage of respondents that were male/female? While the only required data from our respondents was their location (at town or similar level) and we are unable to give statistics on age and gender, it is possible to say from the information given by some, that the known ages of our participants ranged from 20’s to 70’s, and both men and women were included. We have now included this detail in our Results section, and have also indicated that people with both chronic and acute conditions were included, as well as those who had not had symptoms requiring them to contact general practice. While our survey size limited the generalisability of our findings and we focused rather on gathering the breadth of perceptions of our respondents, we feel that, this supplemental information has offered useful context in addition to that available in the quotations in Table 2. Many thanks for your suggestion. 5. Understanding of adapted delivery models: There are a couple of occasions in the results section where you refer to participants not being aware that COVID-19 patients were 'separated from others in face-to-face consultations.' This wording is slightly unclear and perhaps rewording to make it clear that practices have different processes in place to ensure that patients with COVID-19 are seen in a different location/different part of the practice to other patients (you discuss it clearly in the introduction section). Thank you for your suggestion, we have now clarified this throughout accordingly. 6. You refer to respondents having 'working links to general practice' (p7), could you please provide some examples of such links? We have now included elaboration that these people with worked in general practice or were members of patient participation groups or made deliveries to general practice. 7. When you cite the personal communication from Dr L. Farbus - should this be a numerical reference in line with the other references? We appreciate your comment and agree that this is sometimes the case. Here, we have complied with the style required for personal communication by f1000research. 8. The only other comment I have, is that you do not include any strengths or implications in the Discussion. This research is really interesting, and likely has real-world implications for ensuring that clear messages are provided to patients about when and how it is appropriate to contact the practice, and the availability of appointments during a situation such as COVID-19. It would be useful for readers to understand about how the findings of this research can improve patient experience. Many thanks for highlighting this and for your interest in our findings. We have now replaced the ‘limitations’ section with a fuller ‘strengths and limitations’ section. In this, we now highlight the importance of considering the communication of changed practices, including the checking of access to, and understanding of, this communication, for change to be implemented successfully, and of maximum benefit for both patients and health care professionals. Thank you for the points you have raised, each has been really useful in helping us to improve this report." } ] }, { "id": "93309", "date": "07 Sep 2021", "name": "Karen McBride-Henry", "expertise": [ "Reviewer Expertise Primary health care", "mixed methods research", "peoples experiences of health care and health care access" ], "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 article; it was interesting to read and is timely given the current COVID-19 pandemic. Research that seeks to understand how people experience accessing health care is essential to deliver care effectively. The results have some similarities to findings from research that we have conducted here in New Zealand.\nI offer the following comments that will help to refine the article further.\nThe introduction literature was limited, citing only the authors’ research. I would encourage the authors to broaden out the introduction to include additional research.\n\nInformation provided on the study methods was limited. For example, how were the survey respondents identified? How was the survey distributed? Although this information is provided in the “Checklist for reporting results of internet surveys” for this study on Figshare, additional information related to the study methods should be in the article. No information on the analysis of the open text answers is available; this information needs to be presented in the article.\n\nA significant issue with the research was the lack of ethical approval for the study. Who deemed the survey as ‘low risk’? If it was the authors, this raises concerns. The lack of detail around ethical practices embedded in the study is a weakness. For example, how was anonymity assured? What were the procedures embedded in this study that kept respondents ‘safe’?\n\nThe figures helped explain the survey results.\n\nReferring to respondents by alphabet letters was somewhat confusing given the limited information about the method of analysis. It is not clear whether the alphabet letters referred to respondents from survey A or B. This information should be noted in Table 1, as it indicates potential bias in the survey respondents.\n\nNew findings are presented in the Discussion, in the ‘Communication of changes’ section; these should be removed.\n\nIt is also reported that the researchers were in email contact with the respondents; while this is not necessarily unusual, it raises additional concerns about how respondent information was managed given the lack of ethical approval or ethical information related to this research.\n\nThe study authors used descriptive statistics only, which given the small sample size, is appropriate but impacts the generalisability of the results; this needs to be more clearly articulated in the limitation section.\nThank you again for the opportunity to review this research.\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? No\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? Yes", "responses": [ { "c_id": "7371", "date": "01 Nov 2021", "name": "Lorna Duncan", "role": "Author Response", "response": "Dear Professor McBride-Henry, Many thanks for taking the time to review our paper and for your helpful comments. We have used these to improve our manuscript as indicated below in our responses to each of your points: 1. The introduction literature was limited, citing only the authors’ research. I would encourage the authors to broaden out the introduction to include additional research. Many thanks for highlighting this, we agree and have now expanded our introduction. We prepared the manuscript as a brief report and, as you mention, linked it only to our related publication identifying the pandemic-related modifications made to English face-to-face GP consultations. We can see that the additions we have now made, incorporating both national and international consideration of general practice adaptations and clarification of the purpose of our evaluation, have strengthened the paper. A table has also been added which we hope more clearly demonstrates the changes to general practice consultation modes described in the text. We were interested to read your findings around patient experience and preferences for telehealth in general practice during the March-May 2020 lockdown in New Zealand. We noted the similarities in several of the themes identified in both our studies and have included your publication, as well as further relevant references, to provide greater perspective in our Discussion. 2. Information provided on the study methods was limited. For example, how were the survey respondents identified? How was the survey distributed? Although this information is provided in the “Checklist for reporting results of internet surveys” for this study on Figshare, additional information related to the study methods should be in the article. No information on the analysis of the open text answers is available; this information needs to be presented in the article. We have now expanded on the Survey design, Data collection and Data analysis sections in the Methods, to address your points. We have incorporated data from the “Checklist for reporting results of internet surveys” on Figshare and supplemented this with additional information. As mentioned in point 1 above, we had prepared this manuscript as a ‘brief report’, referring to the checklist as supplemental information, but we agree with you that these additions have improved clarity in the report. 3. A significant issue with the research was the lack of ethical approval for the study. Who deemed the survey as ‘low risk’? If it was the authors, this raises concerns. The lack of detail around ethical practices embedded in the study is a weakness. For example, how was anonymity assured? What were the procedures embedded in this study that kept respondents ‘safe’? We agree that we had not indicated fully in the text the ways in which we acted to minimise harm to our respondents, although we had given some detail in the “Checklist for reporting results of internet surveys” on Figshare. We have now incorporated this detail into our Methodology section and added further explanation around the ethical considerations, including issues of anonymity and safety of respondents. The comment that the surveys were deemed ‘low risk’ was added by the editorial team during processing for publication. We should have requested that this be removed and have now done so - we are grateful to you for highlighting this. The work was undertaken as an evaluation of general practice delivery and we hope it is now clear in our expanded methodology section that we have considered the safety of our respondents throughout and taken appropriate steps to ensure security of their data and to preserve their anonymity. In fact, the first half of their postcode (which indicates their town or similar area) was the only identifiable element required from any respondent. Where respondents volunteered further detail we ensured this was treated and stored confidentially according to the methodology indicated. We also provided our study email address to which people could write additionally if they wanted to. A small number did so and we replied as the study team. Although the potential for harm for respondents in recollecting negative experiences was possible, this was minimised in our survey by not requesting specific details of these, focusing largely on service delivery and its communication, rather than on respondents’ health conditions or on the detail of any consultations they may have had. In addition, we feel that the brief nature of the surveys, with no consequence incurred by the respondents for non-completion (this was not in any way linked to their healthcare and the distributors of survey A had no knowledge of whether or not they had completed it), enabled us to maximise safety for each respondent. Quotations were also selected for our report to encompass our respondents’ reflections overall while maintaining their individual anonymity. 4. The figures helped explain the survey results. Thank you, we are pleased they are helpful. 5. Referring to respondents by alphabet letters was somewhat confusing given the limited information about the method of analysis. It is not clear whether the alphabet letters referred to respondents from survey A or B. This information should be noted in Table 1, as it indicates potential bias in the survey respondents. Many thanks for highlighting possible confusion in our table. We have now included an explanation both in the table and within the text that the letters linked to each example are identifiers given purely to enable reference to them within the text. Our aim was not to suggest any hierarchy in our ‘coding’ system, so the alphabet was used rather than numbers. The quotations detailed in Table 2 indicate the breadth of experiences, views and ideas expressed by our participants, but not the frequency with which these ideas were expressed. They may have been indicated by one or several participants in our sample population, but have been included to represent possible factors for consideration in future consideration and planning of general practice delivery. We have now indicated which survey the quotations came from - most were from survey B, as this was where questions concerning respondents’ preferences and suggestions for communication were asked (none of the included quotes in the right-hand column were derived from survey A). In the left-hand column 4 of the 13 responses shown (D,J,K,M) were given in survey A, and this is not unexpected as both surveys considered respondents’ experience of general practice and all open text responses were scanned for examples for inclusion in this table. Our selection was made to include as many ideas as possible, with the views of all 150 participants being equally important. We have also clarified this within the manuscript - in the Data analysis [‘Communication methods used and preferred’] section in the Methods, and in the Communication regarding COVID-19 and changes to general practice service delivery section in the Results. 6. New findings are presented in the Discussion, in the ‘Communication of changes’ section; these should be removed. Thank you, we have now moved this item to the end of the Results section. 7. It is also reported that the researchers were in email contact with the respondents; while this is not necessarily unusual, it raises additional concerns about how respondent information was managed given the lack of ethical approval or ethical information related to this research. We have now clarified the limited nature of this email contact, as well as the management of all respondent information, in the Data collection section in the Methods. 8. The study authors used descriptive statistics only, which given the small sample size, is appropriate but impacts the generalisability of the results; this needs to be more clearly articulated in the limitation section. We agree and have now clarified this in the appropriate section, which has been reorganised into a ‘strengths and limitations’ section in the Discussion. Many thanks for each of the points you have raised. In addressing each, we feel this has very much helped us to refine our report." } ] } ]	1	https://f1000research.com/articles/10-279
https://f1000research.com/articles/10-1092/v1	27 Oct 21	{ "type": "Research Article", "title": "Myofunctional training with negative airway pressure for obstructive sleep apnea: a prospective non-randomized cohort feasibility study", "authors": [ "Suzanne Karan", "William A. Voter", "Denham Ward", "William A. Voter", "Denham Ward" ], "abstract": "Background: Orofacial myofunctional therapy (OMT), for obstructive sleep apnea (OSA) is emerging with recognition of specific phenotypes. Since many OSA sufferers are already familiar with positive airway pressure machines, we designed an OMT protocol involving the modification of such a machine to deliver negative airway pressure. We hypothesized that a three-month trial of using this protocol while awake would reduce the signs and symptoms of OSA. Methods: Fifteen adults with OSA enrolled for 30-minute sessions/three days a week for a three-month trial of “upper airway muscle physical therapy” while awake. Overnight sleep studies were performed before and after the intervention to determine each subject’s apnea hypopnea index (AHI), the primary outcome measure. Negative airway pressure application was determined by estimation of each subject’s one-repetition maximum (an exercise physiology measure of strength capacity). Exercise sessions consisted of subjects breathing against this pressure for a series of repetitions, and sets. Results: While the mean post study AHI was slightly improved (-4.3 ± 12.0 [ -10.9, 2.3], mean ± standard deviation, 95% confidence interval), it was not statistically significant. The results were similar when the AHI was divided by the sleep state (REM vs. non-REM). The nadir saturation was also essentially unchanged. The eight subjects with mild or moderate OSA (AHI < 30) showed similar results. Conclusions: Though there was no significant reduction in AHI in this small cohort, the methods elucidate a new daytime use for a machine with which many OSA sufferers are familiar. Trial registration: ClinicalTrials.gov NCT02109731 (registered on April 10th 2015)", "keywords": [ "Sleep apnea", "therapy", "airway pressure" ], "content": "Abbreviations\n\nAHI: apnea hypopnea index\n\nBMI: body mass index\n\nNAP: negative airway pressure\n\nOSA: Obstructive Sleep Apnea\n\nOMT: orofacial myofunctional therapy\n\nPAP: positive airway pressure (could be continuous or auto-titrated)\n\n1RM: one-repetition maximum\n\nREM: rapid eye movement\n\n\nIntroduction\n\nObstructive sleep apnea (OSA) is caused by upper airway collapse during sleep resulting in a disruption of regular periodic breathing. The phenotypes of OSA pathology are variable and a particular patient can have the disorder because of anatomical and/or non-anatomical reasons.1 Continuous or auto-titrated positive airway pressure (PAP) is the current standard treatment; however, other therapies have emerged as alternatives. Mandibular advancement devices (or oral appliances) are currently recommended as a second line therapy2; though the ability to properly track compliance (and thus efficacy) is a challenge. Surgical interventions have targeted anatomical or morphological problems in those unresponsive to standard therapies. However, the evidence supporting its widespread application is lacking.3\n\nWe have been studying the collapsibility of the upper airway during sedation-induced sleep by provoking its occurrence through the use of negative airway pressure (NAP).4,5 People who are awake can breathe against this NAP (it feels like a vacuum on the back of the throat) down to negative pressures that would readily collapse the airway during sleep or sedation. Awake and healthy OSA patients have been able to tolerate breathing with the application of up to -40 cm of water.6 The ability to tolerate these pressures is the result of increased airway muscle tone (both tonic and reflex phasic activation of the airway dilator muscles).7\n\nSubjects in our laboratory have commonly described their breathing against this vacuum as a “workout” on their airway. This idea sparked our curiosity to test the ability of NAP application as a therapeutic intervention, exercising the oropharynx, increasing muscle tone and thus decreasing upper airway collapsibility during sleep. Over the last decade, orofacial myofunctional therapy (OMT) has been applied to treat this disorder.8 Both as a stand-alone therapy and in conjunction with PAP, OMT has been shown to reduce the apnea hypopnea index (AHI), reduce snoring, and improve quality of sleep and life.9 While other OMT protocols involve methods that require specific training,8,10,11 we considered that OSA sufferers already familiar with PAP would more easily adapt to our protocol which involves the use of a similar device. The key to our proposed therapy is the use of NAP when awake so that the increased reflex phasic drive to the muscles will result in muscle conditioning. Thus, we developed a regimen similar to OMT, with a primary hypothesis that AHI measured via attended polysomnography after treatment intervention would be %50 reduced compared to baseline AHI.\n\n\nMethods\n\nThis study was approved by the University of Rochester Research Subject Review Board (RSRB#24149) and conducted between December 2008 and January 2012. At the time the study was approved, it was not standard practice or mandatory to publicly register trials. This trial’s information was first submitted in 2012, posted in 2014, and results posted in 2015 in ClinicalTrials.gov (NCT02109731) after the study was completed, but in line with our understanding of what was evolving as a research standard at that time.\n\nAll subjects provided written informed consent to participate in this non-randomized, non-blinded feasibility study. The estimated sample size for a one sample comparison of means of AHI utilizing the s.d. of the delta AHI between two measurements was 11 subjects to have a 90% power to detect a 50% difference or a reduction in the AHI of 20 at alpha = 0.05. Taking into account possible dropout of subjects over time, we planned to enroll 15 subjects in the study. For the primary analysis, differences between baseline and follow-up were analyzed using two sample t-tests.\n\nTwenty subjects (>18 years of age) were recruited from local sleep medicine laboratories via flyers or mailing from their treating sleep physicians. Once consented, subjects were additionally screened for inclusion and exclusion criteria and 15 recruited for the study. All subjects had a diagnosis of OSA (apnea hypopnea index, AHI >10) but were not currently using their previously prescribed PAP. Each participant received a targeted history and physical exam to assess for stability of medical conditions, including having a normal electrocardiogram and a negative urine drug test. Measurements of body mass index (BMI), neck circumference, and an airway exam were collected, and the Epworth sleepiness scale survey was administered.\n\nExclusion criteria included: major upper airway morphologic abnormality (such as profound micrognathia), history of airway surgery, regular use of central nervous system depressants or alcohol (e.g., > 14 alcoholic drinks per week or >2 per day), morbid obesity (BMI > 40 kg/m2), or unstable medical or psychiatric illness. Participants were also excluded if they were undergoing or planning to undergo an intervention for weight loss, or were pregnant or lactating.\n\nParticipants were initially evaluated by the University of Rochester Strong Sleep Disorders Center with an overnight polysomnographic study incorporating 16 electrophysiologic signals: 2-channel electro-oculogram, 8-channel electroencephalogram (Fz, C3, CZ, C4, Pz, Oz, T3, and T4), bipolar mentalis electromyogram, 2 lead electrocardiogram, nasal/oral airflow thermocouple, two respiratory effort sensors, a pulse oximeter, and a channel representing A1/A2. The resulting data were analyzed in 30-second epochs by an independent and certified sleep scorer (LL; see Acknowledgements).\n\nThe NAP myofunctional therapy protocol was designed to minimize the development of muscle hypertrophy and optimize strength and/or endurance.12 In muscle conditioning therapy, one-repetition maximum (1RM) is the unit designated to describe a maximum weight or resistance that a muscle group can move. For this study, the 1RM was defined as the maximum negative pressure the participant could comfortably tolerate for one breath, not less than −25 cm H2O. As is conventional in exercise physiology, ideal strength training involves targeting each subject’s 1RM repetitiously for 1-5 breaths per set and repeating this set 4-7 times with rests of 1-2 minutes between sets.\n\nTraining visits\n\nThe initial visit to the University of Rochester outpatient clinical research center was used to familiarize subjects with the apparatus, which appears similar to a typical PAP machine. Subjects were acclimated to both the mask fit (over the mouth and nose) and the feeling of breathing against a negative pressure. Airflow was measured with a pneumotachograph (Hans Rudolph, Shawnee, KS) inserted into the mask, which was calibrated with a rotameter. Mask pressure was measured by connecting a port to a pressure transducer (Validyne, Northridge, CA) calibrated with a water manometer.\n\nSubjects were instructed to breathe through the nose with a closed mouth while sitting in order to minimize any tendency for the upper airway to collapse. At the first visit, the participants were exposed to a variety of negative pressures in an increasing-decreasing manner (from −5 cm H2O for five breaths and down) and also in random sequence in order to determine each subject’s 1RM.\n\nExperiment\n\nThereafter, the subjects would schedule three observed sessions per week for three months in our clinical research center. As described above, each session entailed the subject breathing NAP at the predefined 1RM for five breaths per set (reps) and repeating this set 4-7 times with rests of 1-2 minutes between sets. Constant observation by study personnel allowed for verification of wakefulness (eyes open) and breathing with the mouth closed during the entire procedure. Each session lasted approximately 30 minutes. After the first and second month, the 1RM was re-evaluated and adjusted, as appropriate. For the following month, the new 1RM of NAP was used for the training exercises. Each subject’s BMI and neck circumference were re-measured after the first and second months.\n\nParticipants completed a post-intervention overnight sleep study within a week of finishing the prescribed course of airway physical therapy. Data from this study were collected and analyzed in the same manner as the initial overnight sleep study.\n\nParticipants were also instructed to maintain nightly sleep diaries. This included recording and rating aspects of sleep quality and sleep-related quality of life on a 1-5 scale (1 = never; 5 = frequently) upon waking, and recording bedtimes, time out of bed, time required to fall asleep, number of awakenings during the night, time awake after falling asleep, time awake before alarm or intended wake time, and time out of bed at night.\n\nSince this was a pilot study without a control group, the primary outcome was a difference in AHI (as measured by overnight attended polysomnography) between the initial sleep study and the final study in each subject. Specifically, the study was sized to detect a 50% reduction in AHI or an AHI reduction of more than 10. Other secondary outcomes include nadir saturation, non-REM AHI, and protocol compliance. A pre-planned subgroup analysis of the subjects with a mild to moderate AHI (AHI < 30) was performed.\n\nFor the primary analysis, the differences between baseline and post-intervention data were analyzed using paired Student’s t-tests. Symptoms scores were analyzed nonparametrically.\n\nData are reported as mean ± standard deviation and 95% confidence intervals. p < 0.05 was considered significant. All statistical analyses were performed using STATA/IC 13.1, (Stata Corp LP, College Station, TX).\n\n\nResults\n\nFifteen subjects completed the study and Table 1 provides their demographics, which demonstrates no significant changes in BMI, weight or neck circumference after the training session (see Figure 1 for CONSORT flow chart). The initial airway training pressures ranged from -8 to -18 cm H2O and all airway training pressures became more negative except for one subject who stayed at -18 cm H2O (Figure 2), which was the most extreme pressure utilized for the study.\n\nBMI = body mass index. Data is mean ± standard deviation.\n\nThe left panel shows the change in the AHI from before and after the training for all subjects. The right panel shows the change in the initial to the final training pressure.\n\nWhile the mean post study AHI was slightly improved (−4.3 ± 12.0 [−10.9, 2.3]), it was not statistically significant (Table 2). Six subjects actually increased their AHI, only one subject decreased to below 10 and this was the subject with the lowest pre-study AHI of 10.1 (Figure 2). The results were similar when the AHI was categorized by the sleep state (REM vs. non-REM). The nadir saturation was also essentially unchanged. The eight subjects with mild or moderate OSA (AHI < 30) showed similar results.\n\nAHI – apnea hypopnea index. The AHI was calculated during rapid eye movement (REM) and non-REM sleep. Data given as mean ± stand deviation, except for difference which is mean and 95% confidence interval.\n\nOf the 15 subjects, only 10 completed their sleep diaries. There were no differences noted in sleep quality or feeling refreshed from the intervention.\n\n\nDiscussion\n\nThis study demonstrates the novel use of a modified standard PAP machine to provide NAP as a form of quantifiable myofunctional therapy for OSAS. In this small cohort, the intervention did not cause significant changes in AHI. The small size of this pilot study makes it difficult to definitively exclude a clinically helpful effect, but we can exclude a clinically significant increase in the AHI, thus indicating its safety. However, we believe that sharing the methodology and results may provide clues for further study.\n\nThough PAP therapy remains the gold standard approach for effective treatment of OSA,13 compliance remains a challenge.14 OMT has emerged as a promising alternative or adjunct to standard OSA therapy.8 In a recent review of the 11 studies published to date, de Felicio et al. provide an analysis of comparative methodology and ideas for further study.15 In most OMT studies, the intervention exercises target a variety of oral and facial muscles including the tongue, palate, pharynx, mouth, and cheeks. In our study, the NAP therapy was hypothetically the reverse of PAP and may have exercised similar structures. However, we do not know whether NAP targets specific muscles whose tone (or lack thereof) contributes to OSA. With increasing attention being paid to OSA phenotypes, it has been suggested that most would benefit from anatomic intervention.1 In addition, standardized classification of location, directionality and severity of pharyngeal collapse as determined during drug-induced sedation endoscopy16 could be explored as a method to target appropriate candidates who might benefit from myofunctional therapy, as it has been used to evaluate success with other OSA interventions such as surgery and electrical stimulation.\n\nWe considered that prior familiarity with the PAP machine but failure to adhere to nighttime usage would be a reasonable approach for recruitment of subjects for our study, which included daytime and more limited use of the machine. In hindsight, non-use of PAP may have actually been an inappropriate criterion for inclusion as this may have predicted a phenotype á priori which would not benefit from our approach of myofunctional therapy.1 Most PAP machines can now be interrogated to ensure patient compliance. Future investigations may benefit from recruiting PAP users and assessing devices for sleep-related pressure changes over the course of an exercise intervention during wakefulness.\n\nAs was done in our study, OMT is typically utilized during wakefulness as a method to decrease muscle hypotonia during sleep. Theoretically, the frequent repetitions at low levels of resistance may target Type 1 (slow-resistant to fatigue) muscles, such as those found in the tongue.17 These muscles and Type 2A (fast-intermediate fatigue) muscles have been stimulated electrically.18,19 and trained11,20 In patients with OSA there are even more type 2A fibers than in normal controls, which could contribute to the increase fatigability.21\n\nOur protocol lasted for three months, a time period that is frequently used in muscle training.15 Unlike other protocols, we chose to have subjects observed in a laboratory and reasoned that visiting three times per week would not be too onerous. Many of the training protocols describe daily exercises, sometimes three8,22 to five times23 per day. It is possible that our training regimen was not sufficient to see an effect.\n\nWe asked our subjects to maintain a mouth closed position while performing exercises, mostly as a means to standardize their positions. It has also been shown that mouth breathing exacerbates upper airway obstruction with advocates for mouth closure training to promote airway patency maintenance during sleep.24,25\n\nDespite the limitations of our study, there were no reported adverse events. In contrast, participants shared that they appreciated doing this therapy, and could envision doing it at home were it to become available. We have been contacted by former subjects and a few interested citizens who have located this study on ClinicalTrials.gov to inquire about inclusion in ongoing investigations.\n\n\nConclusions\n\nIn conclusion, subjects with OSAS were able to tolerate a three-month exercise protocol using the novel application of NAP as orofacial myofunctional therapy. Though was no significant reduction in AHI in this small cohort, the methods elucidate a new daytime use for a machine with which many OSAS sufferers are familiar. Future studies utilizing this protocol may target current PAP users or those whose drug-induced sedation endoscopies indicate this approach as an alternative therapy.\n\n\nData availability\n\nFigshare: AirwayPT Data Original 100121.csv, https://doi.org/10.6084/m9.figshare.16727488.v1.26\n\nFigshare: pt_sleep_diaries_042412.xls, https://doi.org/10.6084/m9.figshare.16798138.v1.27\n\nFigshare: CONSORT checklist for ‘Myofunctional training with negative airway pressure for obstructive sleep apnea: a prospective non-randomized cohort feasibility study’, https://doi.org/10.6084/m9.figshare.16818352.v2.28\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledegments\n\nThe authors thank Dr. Lynn Liu for help in reading the sleep studies, Dr. Michael Perlis and Michael Leonard for input into the study design, and Dr. Elia Rackovsky for assistance in equipment setup for the experiment.\n\n\nReferences\n\nEckert DJ, White DP, Jordan AS, et al.: Defining phenotypic causes of obstructive sleep apnea. Identification of novel therapeutic targets. Am J Respir Crit Care Med. 2013; 188: 996–1004. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrzepizur W, Cistulli PA, Glos M, et al.: Health Outcomes of Continuous Positive Airway Pressure versus Mandibular Advancement Device for the Treatment of Severe Obstructive Sleep Apnea: an Individual Participant Data Meta-analysis. Sleep. 2021, 44. PubMed Abstract \| Publisher Full Text\n\nHolty JE, Guilleminault C: Surgical options for the treatment of obstructive sleep apnea. Med Clin North Am. 2010; 94: 479–515. Publisher Full Text\n\nNorton JR, Ward DS, Karan S, et al.: Differences between midazolam and propofol sedation on upper airway collapsibility using dynamic negative airway pressure. Anesthesiology. 2006; 104: 1155–1164. PubMed Abstract \| Publisher Full Text\n\nLitman RS, Hayes JL, Basco MG, et al.: Use of dynamic negative airway pressure (DNAP) to assess sedative-induced upper airway obstruction. Anesthesiology. 2002; 96: 342–345. PubMed Abstract \| Publisher Full Text\n\nSuratt PM, Wilhoit SC, Cooper K: Induction of airway collapse with subatmospheric pressure in awake patients with sleep apnea. J Appl Physiol Respir Environ Exerc Physiol. 1984; 57: 140–146. PubMed Abstract \| Publisher Full Text\n\nWheatley JR, Mezzanotte WS, Tangel DJ, et al.: Influence of sleep on genioglossus muscle activation by negative pressure in normal men. Am Rev Respir Dis. 1993; 148: 597–605. Publisher Full Text\n\nGuimaraes KC, Drager LF, Genta PR, et al.: Effects of oropharyngeal exercises on patients with moderate obstructive sleep apnea syndrome. Am J Respir Crit Care Med. 2009; 179: 962–966. PubMed Abstract \| Publisher Full Text\n\nHsu B, Emperumal CP, Grbach VX, et al.: Effects of respiratory muscle therapy on obstructive sleep apnea: a systematic review and meta-analysis. J Clin Sleep Med. 2020; 16: 785–801. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeumannova K, Hobzova M, Sova M, et al.: Pulmonary rehabilitation and oropharyngeal exercises as an adjunct therapy in obstructive sleep apnea: a randomized controlled trial. Sleep Med. 2018; 52: 92–97. PubMed Abstract \| Publisher Full Text\n\nPuhan MA, Suarez A, Lo Cascio C, et al.: Didgeridoo playing as alternative treatment for obstructive sleep apnoea syndrome: randomised controlled trial. BMJ. 2006; 332: 266–270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBird SP, Tarpenning KM, Marino FE: Designing resistance training programmes to enhance muscular fitness: a review of the acute programme variables. Sports Med. 2005; 35: 841–851. PubMed Abstract \| Publisher Full Text\n\nMediano O, Romero-Peralta S, Resano P, et al.: Obstructive Sleep Apnea: Emerging Treatments Targeting the Genioglossus Muscle. J Clin Med. 2019; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeaver TE, Grunstein RR: Adherence to continuous positive airway pressure therapy: the challenge to effective treatment. Proc Am Thorac Soc. 2008; 5: 173–178. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Felicio CM , da Silva Dias FV , Trawitzki LVV: Obstructive sleep apnea: focus on myofunctional therapy. Nat Sci Sleep. 2018; 10: 271–286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKezirian EJ: Nonresponders to pharyngeal surgery for obstructive sleep apnea: insights from drug-induced sleep endoscopy. Laryngoscope. 2011; 121: 1320–1326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClark HM: Neuromuscular treatments for speech and swallowing: a tutorial. Am J Speech Lang Pathol. 2003; 12: 400–415. PubMed Abstract \| Publisher Full Text\n\nMann EA, Burnett T, Cornell S, et al.: The effect of neuromuscular stimulation of the genioglossus on the hypopharyngeal airway. Laryngoscope. 2002; 112: 351–356. PubMed Abstract \| Publisher Full Text\n\nSmith PL, Eisele DW, Podszus T, et al.: Electrical stimulation of upper airway musculature. Sleep. 1996; 19: S284–287PubMed Abstract\n\nLequeux T, Chantrain G, Bonnand M, et al.: Physiotherapy in obstructive sleep apnea syndrome: preliminary results. Eur Arch Otorhinolaryngol. 2005; 262: 501–503. PubMed Abstract \| Publisher Full Text\n\nSeries F, Cote C, Simoneau JA, et al.: Upper airway collapsibility, and contractile and metabolic characteristics of musculus uvulae. FASEB J. 1996; 10: 897–904. PubMed Abstract \| Publisher Full Text\n\nDiaferia G, Santos-Silva R, Truksinas E, et al.: Myofunctional therapy improves adherence to continuous positive airway pressure treatment. Sleep Breath. 2017; 21: 387–395. PubMed Abstract \| Publisher Full Text\n\nVerma RK, Johnson JJ, Goyal M, et al.: Oropharyngeal exercises in the treatment of obstructive sleep apnoea: our experience. Sleep Breath. 2016; 20: 1193–1201. PubMed Abstract \| Publisher Full Text\n\nCatlin G: Shut Your Mouth and Save Your Life. Paternoster ROW:N. Truebner & Co.;Fourth ed.1870.\n\nSuzuki H, Watanabe A, Akihiro Y, et al.: Pilot study to assess the potential of oral myofunctional therapy for improving respiration during sleep. J Prosthodont Res. 2013; 57: 195–199. PubMed Abstract \| Publisher Full Text\n\nKaran S: AirwayPT Data Original 100121.csv. figshare. Dataset. 2021. Publisher Full Text\n\nKaran S: pt_sleep_diaries_042412.xls. figshare. Dataset. 2021. Publisher Full Text\n\nKaran S: CONSORT checklist. figshare. J. Contribution. 2021. Publisher Full Text" }	[ { "id": "98530", "date": "09 Nov 2021", "name": "Carlos O'Connor-Reina", "expertise": [ "Reviewer Expertise I am expertise in sleep medicine and myofunctional therapy" ], "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 manuscript based on a therapy called Nap therapy where patients breaths against like a vacuum cleaner with negative pressure. They try to evaluate if this activity can increase the tone of the upper airway muscles and improve OSA patients. Authors considered that breathing against a pipe with negative pressure is an oropharyngeal exercise. This kind of exercise has not developed any improvement in sleep apnea in any worldwide manuscript. Exercises reported by Guimarães KC, et al. Those reported by O'Connor Reina et al, have been proven to be effective, but not this kind of activity. We believe there is great issue in the design of this trial as there was no scientific basis to support this. There is no scientific evidence about this therapy to be considered as oropharyngeal exercises.\nWe believe there is a big misunderstanding by the authors of the concept of orofacial myofunctional therapy. We think the title should be changed to Upper Airway Physical Therapy for the Treatment of Obstructive Sleep Apnea as It was indicated in the clinical trial. On the other hand authors considered this therapy as a potential use when the results did not reflect this point. On the other hand, authors speak about drug induced sleep endoscopy as a target for myofunctional therapy but they do not explain what patients are suitable for this therapy.\nThe study was finished in 2015 and since then, there was no other reports about NAP therapy. Due this fact, we recommend the authors to rewrite this paper and explain the use of this therapy in the treatment of OSA.\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? No\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?\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? No", "responses": [] }, { "id": "142395", "date": "18 Jul 2022", "name": "Maria R Bonsignore", "expertise": [ "Reviewer Expertise Respiratory sleep 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\nIn this paper, Karan and coworkers report the results of a small study testing the effects of orofacial myofunctional therapy (OMT) in OSA patients. The rationale was to train upper airway muscles to negative pressure application during wakefulness for 3 months. The protocol was conducted in the laboratory 3 times a week, and the intensity of training was established based on the maximum negative pressure the subject could tolerate for 1 breath (1RM). Negative airway pressure (NAP) was applied for 5 breaths, and each set was repeated 4-7 times with 1-2 min rest between sets. The pressure was adjusted each month. 15 subjects were studied, all of them with OSA and prescription of CPAP but not using it. The study did not show any significant change in the expected outcome, i.e. 50% reduction in AHI or reduction of AHI below 10), although the training pressure decreased during the study in all subjects.\n\nThe study is interesting, but the result were negative, raising more questions than answers, especially on the value of this specific training procedure. The study was done between 2008 and 2012, and the possibility to train upper airway muscle has been explored in a more structured way in recent years, and with different devices, some of them using muscle stimulation. I agree with the comments of the previous reviewer and can only add that a more accurate endotyping might show a subgroup of patients especially responsive to this or other treatments aimed at upper airway muscle training. Currently, the treatment discussed in this study cannot be recommended.\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?\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/10-1092
https://f1000research.com/articles/10-1089/v1	27 Oct 21	{ "type": "Research Article", "title": "Production and characteristics of sailfin catfish (Pterygoplichthys pardalis) protein hydrolysate", "authors": [ "Asep Awaludin Prihanto", "Rahmi Nurdiani", "Lina Widya Sari", "Rahmi Nurdiani", "Lina Widya Sari" ], "abstract": "Background: The sailfin catfish (Pterygoplichthys pardalis) is a freshwater fish from the Loricariidae family, and is considered an invasive species in Indonesia. The fish is usually neglected and discarded. Its protein hydrolysate is the product of the breakdown of proteins into amino acids through the hydrolysis of acids, bases, or enzymes. Therefore, this study aims to determine the hydrolysate characteristics of sailfin catfish (Pterygoplichthys pardalis) proteins, produced with different pH and hydrolysis durations. Methods: The hydrolysis was carried out with variable pH (control, 5, 7, 9) and hydrolysis durations (12 and 24 hours) in three replicates. Results: The hydrolysis duration, pH, and interaction of both variables had significant effects (p<0.05) on the parameters of yield, antioxidant activity, degree of hydrolysis, protein levels, and ash content. Similarly, the moisture content, hydrolysis duration, and pH were significantly affected (p<0.05), while their interactions were not (p>0.05). The pH treatment had a significant effect on fat content (p<0.05), while the hydrolysis length and the interaction between the two had no significant effect (p>0.05). Based on this study, the best resulting production of fish protein hydrolysate (FPH) from sailfin catfish were a for a combination of pH 9 and a hydrolysis duration of 24 hours. The characteristics of FPH produced were as follows: a yield of 57.39%, antioxidant activity 63.99%, degree of hydrolysis 40.67%, water content 7.28%, ash content 7.63%, fat content 5.10%, protein content 34.51%, molecular weight 6.14 -118.17 KDa, total essential amino acids 49.30%, and nonessential amino acids 50.71%. Conclusions: Two factors affected FPH characteristics in sailfin catfish (Pterygoplichthys pardalis). The best processing conditions to obtain optimal characteristics for FPH were a yield of 57.39%, antioxidant levels of 63.99%, a DH of 40.67%, a moisture content of 7.28%, ash content of 7.63%, fat content of 5.10%, and protein content of 34.51%.", "keywords": [ "endogenous", "enzymes", "fish protein hydrolysate", "Sapu-sapu." ], "content": "Introduction\n\nThe sailfin catfish (Pterygoplichthys pardalis) is a freshwater fish from the Loricariidae family and native to the Americas (Susanto, 2014). In Indonesia, sailfin catfish are present in various locations with high levels of pollution (Qoyyimah et al., 2016), and have a negative impact because they prey on other species. The fish can dominate the water and become a predator or competitor of domestic fish (Hadiaty, 2011). Sailfin catfish is considered as an under exploited fish (Guo et al., 2019). Despite being discarded as bycatch, the nutritional content of sailfin catfish is relatively high, especially the protein, fat, and carbohydrates contents (Panase et al., 2018). Hence, the use of proteins in less valuable fish species can increase the functional value of the product (Hsu, 2010). The production of fish protein hydrolysate (FPH) is considered the most important source of protein and bioactive peptides (Chalamaiah et al., 2012).\n\nFPH is a product of the decomposition of fish proteins into smaller fragments of peptides that contain between two and 20 amino acids through hydrolysis by enzymes, acids, or bases (Latifah, 2013; Chalamaiah et al., 2012). FPH is produced mainly through protease activity controlled by additional enzymes or endogenous protease enzymes from raw materials (Swanepoel and Goosen, 2018).\n\nPrevious studies on the production of FPH using endogenous enzymes have been carried out on Alaska pollack (Je et al., 2005), ornate threadfin bream (Nalinanon et al., 2011), barramundi fish, silver warehou fish, salmon (Nurdiani et al., 2015), and parrotfish (Chlorurus sordidus) (Prihanto et al., 2019). During the production process, precise hydrolysis conditions such as temperature, pH, and hydrolysis duration need to be considered to achieve optimal results (Meldstad, 2015). Therefore, this study aims to determine the optimum conditions for the production of FPH from the sailfin catfish (Pterygoplichthys pardalis) protein and characterize its product.\n\n\nMethods\n\nSailfin catfish (Pterygoplichthys pardalis); methanol; 2,2-diphenyl-1–picrylhydrazyl, alcohol (DPPH); ascorbic acid; TCA 20%; H2SO4; NaOH; methyl orange; borax; HCL 0.01 N; petroleum ether; tris base; HCl 6 N; dH2Os, acrylamide; N'N'-bis-methylene-acrylamide; glycine; SDS; APS; TEMED; glacial acetic acid and Coomasie Brilliant Blue G-250 (All chemicals and reagents were analytical grade and purchased from Merck, Germany).\n\nAbout 5 kg of Sailfin catfish (Pterygoplichthys pardalis) were collected from the river in Blitar Regency, East Java, Indonesia. The sailfin catfish were washed with water to remove dirt and brought to the laboratory in a cool box at a temperature of 4°C.\n\nA preliminary study was conducted to determine the best comparison of sample and Aqua Dest (w/v) for the production of FPH. At this stage, a whole sailfin catfish (Pterygoplichthys pardalis) was chopped and smoothed using a Philips food processor (model HR7627, 650 W), and the chopped fish (20 grams) was added to Aqua Dest distilled water with a ratio of sample to Aqua Dest as follows 1:0, 1:1, 1:2, and:3 (w/v). The hydrolysis process was carried out for 18 hours using a shaker at 150 rpm and a temperature of 30 ± 2oC. Furthermore, the FPH was centrifuged at 3000 rpm for 30 minutes and the ratio with the highest yield was used for the subsequent investigation.\n\nThe main study was carried out to determine the characteristics of sailfin catfish FPH, and the investigated factors were variations in pH and duration of hydrolysis. The pH levels used were 5, 7, 9, and 6.4 (control) with 12 and 24 hours of hydrolysis, while in the sample comparison, 1:2 (w/v) Aqua Dest was used. Meanwhile, the hydrolysis process was carried out following the same procedure as the preliminary study, and the supernatant obtained was dried using a spray drying method (Prihanto et al., 2019).\n\nThe FPH yield was calculated from the percentage of the number of hydrolysate products obtained against the volume of initial raw materials as of the hydrolysis process. The yield was calculated using the following formula:\n\nThe inhibitory effect of DPPH free radicals was determined according to the method by Donkor et al. (2012), with a slight adjustment on the wavelength used. The inhibitory effect of DPPH free radicals was determined according to the method by Donkor et al. (2012), with slight modifications on wavelength, where they originally used 515 nm. Briefly, a dissolved protein fraction (100 μL) was added to the 3900 μL solution of DPPH 0.075 mM in methanol. In a dark room, an aliquot was homogenized and incubated for 30 minutes at room temperature (28 ± 2 °C). A UV-Vis spectrophotometer (Genesys 6, Thermofisher Scientific, USA) was used to measure the absorbance of the aliquot at a wavelength of 517 nm. The antioxidant activity was based on the equation below:\n\nA = Blank absorbance\n\nB = Sample absorbance\n\nThe degree of dissolved protein hydrolysis in the production of fish protein hydrolysate was calculated according to the method by Hoyle and Merritt (1994). The dissolved protein fraction (2 mL) was added to 2 mL of 20% TCA and the solution was centrifuged at a speed of 5000 rpm for 10 minutes at room temperature. Furthermore, the supernatant was taken to determine the nitrogen content after adding a 20% TCA solvent using the Kjeldahl method (AOAC, 2005). The degree of hydrolysis (DH) was defined by the following equation:\n\nThe analysis of FPH proximal was carried out using standard methods from AOAC (2005). Protein levels were determined using the Kjeldahl method, while fat content was determined using the Soxhlet method. Furthermore, the water content analysis was conducted using the drying method, in an oven at a temperature of 105°C for 2 hours. The ash levels were also determined using the drying method in the furnace at 550oC, for at least five hours until the stable weight was achieved.\n\nAn SDS-PAGE analysis was carried out based on the Laemli (1970) method. A sample of 15 μL was mixed with a 15 μL sample buffer and heated at 100°C for 15 minutes. The SDS-PAGE analysis used a 12% gel-separating concentrate and a 4% gel stacking, and the running process was started at a constant current of 20 mA 100 V for three hours. Meanwhile, staining was carried out by soaking the gel in a staining solution which contained one gram of Coomasie Brilliant Blue G-250 (Merck, Catalogue No. 1154440025), 450 mL of methanol, 100 mL of glacial acetic acid, and 450 mL of Aqua Dest. The molecular weight calculations were analyzed by comparing the bands appearing on the samples with the standard markers.\n\nThe amino acid content was analyzed based on Boogers et al. (2008), using the ultra-performance liquid chromatography (UPLC) method. Moreover, the sample liquid (0.50 mL) was added to 2.0 mL of AABA's standard internal solution of 10 mM, diluted with HCl 0.1 N, and homogenized. The solution was filtered using a filter membrane of 0.22 μm, and 10 μL of the solution were taken and placed in a vial. The aliquot was added to 70 μL AccQ-fluor borate and vortexed. Similarly, approximately 20 μL of the reagent fluor A was added and vortexed. The solution was allowed to stand for a minute and further incubated for 10 minutes at a temperature of 55°C, and the sample was later injected into the UPLC system.\n\nThe experiment was optimized using the response surface method (RSM), which was presented in a contour plot using the Minitab 18 software tool (Minitab Pty Ltd., Sydney, NSW, Australia). Apart from optimization, all the data obtained were analyzed with the one-way variant analysis, followed by a post-hoc test. Furthermore, all data were analyzed in triplicate, and the results are presented as the mean ± SD.\n\n\nResults\n\nThe proximate content of sailfin catfish (Pterygoplichthys pardalis) is shown in Table 1. Based on the results, the sailfin catfish protein content was 30.42%, while the fat content was 5.24%.\n\n* This study.\n\n** Munandar and Eurika (2016).\n\nAfter the centrifugation process, the sample formed five layers of different fractions. These layers included the oil, the light lipoprotein, the dissolved protein (liquid/soluble protein), the fine particles, and the rough particles at the bottom of the cuvette, as shown in Figure 1. The different sample and Aqua Dest ratios affected FPH production yields and antioxidants (P < 0.05). Meanwhile, the highest yield and antioxidants were obtained for a sample and Aqua Dest ratio of 1:2 (w/v) (Figure 2), with a soluble protein yield of 78.25 ± 1.75% and antioxidant levels of 46.70 ± 0.30%. These samples with an Aqua Dest ratio of 1:2 (w/v) were used to manufacture FPH in the advanced study and analyzed to determine the characteristics of FPH.\n\nYield\n\nThe hydrolysis duration and pH had a significant effect on the yield of FPH, which ranged from 20.29 ± 0.27% to 57.39 ± 0.17% (Table 2). The highest yield value (57.39 ± 0.17%) was obtained at pH 9 and after a 24-hour hydrolysis duration, while the lowest value was obtained in control treatment after a 12-hour hydrolysis duration, with a value of 20.29 ± 0.27%.\n\nAntioxidant activity\n\nThe results showed that the antioxidant values of FPH ranged from 11.56 ± 1.59% to 63.99 ± 0.62% (Table 2). Furthermore, there was a significant difference (P<0.05) in antioxidant activity with variations in pH and duration of hydrolysis. The highest antioxidant activity of 63.99 ± 0.62% was obtained at pH 9 with a 24-hour hydrolysis, while the lowest value (of 11.56 ± 1.59%) was obtained for the control treatment with a 12-hour hydrolysis.\n\nDegree of hydrolysis (DH)\n\nThe duration of hydrolysis and pH significantly affected the DH (P < 0.05). Based on the results, the DH of the fish protein hydrolysis samples ranged from 22.08 ± 1.77% to 40.67 ± 1.19%. The highest DH (40.67 ± 1.19%) was obtained at pH 9 after a 24-hour hydrolysis duration, while the lowest value was obtained for pH 5 after 12 hours (22.08 ± 1.77%).\n\nProximate analysis\n\nBased on the approximate FPH content as shown in Table 2, the hydrolysis duration, pH, and interaction between both factors gave significantly different protein content results (P < 0.05), which ranged from 8.51 ± 1.34% to 34.51 ± 1.56%. Furthermore, the highest protein content (34.51 ± 1.56%) was obtained at pH 9 for a 24-hour hydrolysis duration.\n\nDifferent pH treatments gave significantly different results (P < 0.05), while the duration of hydrolysis and the interaction of both factors had no significant impact (P > 0.05) on fat levels. Meanwhile, fat content ranged from 4.37 ± 0.22% to 5.30 ± 0.13%. The fat content in the study decreased compared to the content of the raw materials of sailfin fish due to the separation of fat and lipoproteins during centrifugation, while some lipids still existed in the hydrolysate.\n\nThe results showed that the moisture content of the FPH ranged from 7.28 ± 1.06% to 13.84 ± 0.36%. Additionally, hydrolysis duration and pH variations gave significantly different results (P < 0.05), while the interaction of these treatments had no significant effect (P > 0.05) on moisture content. Furthermore, the moisture content decreased compared to that of the raw materials of the Sailfin fish by 59.14%.\n\nAll treatments had significantly different ash levels (P < 0.05), with values ranging from 3.75 ± 0.33% to 7.63 ± 0.19%. In addition, there was a significant increase in ash levels with pH treatments of 5 and 9.\n\nMolecular weight\n\nThe molecular weight characteristics of FPH from different treatments were visualized using the SDS-PAGE method (Figure 3). Moreover, the 12-hour hydrolysis formed 11-protein bands with molecular weights ranging from 7.23-123.03 kDa, while the 24-hour hydrolysis formed a 13-protein band with a molecular weight range of 6.14-118.17 kDa.\n\nDetermining the optimum process\n\nThe response surface methodology (RSM) method is a common method for analyzing process optimization. Therefore, to achieve the best characteristics of FPH (in terms of yield, antioxidants, DH, moisture content, ash content, fat content, and protein content), two factors were optimized, namely pH and hydrolysis duration. The white area on the contour plot indicated the optimum condition for (Pterygoplichthys pardalis) hydrolysis process (Figure 4). It shows that the optimum pH for hydrolysis ranged from pH of 8.8 to 9 and hydrolysis duration for 22.8 – 24 hours. Therefore, based on our experimental treatment, the best Sailfin catfish FPH processing was at pH 9 for 24-hour hydrolysis. The treatment that still yielded acceptable results based on FPH quality standard is shown from the formation of white areas on the contour plot (Figure 4). The treatment received by the response ranges from a pH of 8.8 to 9 and a hydrolysis duration of 22.8 – 24 hours. It results that the best FPH processing was for a pH 9 and a 24-hour hydrolysis.\n\nAmino acid composition\n\nThe amino acid content analysis was carried out only on the FPH produced using the optimal treatments. The amino acids detected in the yielded FPH is shown in Table 3. The result showed that eight essential and seven non-essential amino acids were detected. The amino acids such as lysine, leucine, glutamic acid, aspartic acid, and glycine were dominant in the FPH samples from sailfin catfish.\n\n* This study.\n\n Foh et al. (2011).\n\n* International Quality Ingredients (2019).\n\n\nDiscussion\n\nThe protein and fat values obtained were higher than in a previous study by Munandar and Eurika (2016) which showed that the protein levels and fat content were 19.71% and 1.73%. According to Tunjungsari (2007), the chemical composition of fish meat varies depending on the species, age level, habitat, and feeding locations.\n\nThe yield obtained in this study was lower than the hydrolyzed protein yield of salmon waste protein of 65.17%, obtained without added enzymes, for a pH 2.5 and at room temperature, and for a hydrolysis duration of 18 hours (Nurdiani et al., 2015). According to Jamil et al. (2016), differences in yields from FPH are due to variations in species, parts of the fish used, types of enzymes used, and the applied hydrolysis conditions.\n\nThe antioxidant activity of FPH from sailfin catfish was previously processed without the addition of enzymes, and for pH 7, at a temperature of 55oC, and a hydrolysis duration of six hours (Baehaki et al.,2015). Data on the antioxidant activity of snakehead (Channa striata) FPH were significantly higher compared to those of sailfin catfish. The FPH was processed using the addition of protease enzyme at pH 7 for 90 min at 55°C, which gave an antioxidant activity of 20.7% (Baehaki et al., 2020). The antioxidant peptides from fish proteins are considered safe and healthy due to their low molecular weight, easy absorption, low cost, and high activity (Sarmadi and Ismail, 2010).\n\nThe degree of hydrolysis measured in this study was lower than that of the yellowfin fish’s (Limanda aspera) waste protein hydrolysis samples with the addition of mackerel digestive tract enzymes, at pH 10, a temperature of 50°C, and a 3-hour hydrolysis duration, by 67% (Jun et al., 2004); it was also lower than that in catfish (Pangasius pangasius) FPH obtained without the addition of enzymes, at pH 7, a 55°C temperature, and a 6-hour hydrolysis, by 63.21% (Baehaki et al., 2015). According to Hasnaliza et al. (2010), the increase in DH is due to an increase in dissolved peptides and amino acids in TCA, which was terminated during hydrolysis.\n\nThe protein levels obtained were similar to the hydrolysate protein of catfish (Pangasius pangasius) without enzymes, at pH 7, 55 °C, and hydrolysis duration of 6 hours, with a value of 20.86% (Baehaki et al., 2015). The protein level in FPH was higher than that in raw material due to the dissolution of proteins during hydrolysis, and the separation of solids and non-protein substances during centrifugation (Chalamaiah et al., 2010). According to Bautista (1999), the hydrolysis duration affects the protein levels of FPH. The protein will increase by approximately 3% when the hydrolysis length is extended from 60 to 120 minutes.\n\nIn this study, the fat content produced was lower than the hydrolysate fat content of the salmon waste protein obtained without adding enzymes, at pH 2.5, and a hydrolysis duration of 18 hours, which was 18.90% (Nurdiani et al., 2015; Idowu et al., 2018). The process reduces the moisture of the product. This is due to the use of high-temperature spray drying methods for the processing of FPH, which removes most of the water content. According to Riansyah et al. (2013), the ability of the material to release water from its surface is more remarkable with increased drying air temperature used, and a longer drying process to produce a lower moisture content. This shows that the water content of the FPH produced was lower than that of the spray-dried FPH from Nile tilapia and Tambelo, which yielded water contents of 9.06 ± 0.09% and 10.16 ± 0.17%, respectively (Anwar and Rosmiati, 2013; Annisa et al., 2017). Mixing acidic and alkaline compounds in protein hydrolysate solutions leads to the formation of salt compounds, causing the ash levels to increase in protein hydrolysates (Wijayanti et al., 2015). In addition, the ash content in the hydrolysate was also influenced by the release of minerals during hydrolysis, especially from bones (Ido et al., 2018).\n\nThe longer hydrolysis time was more effective in breaking protein bonds into peptides and amino acids with low molecular weight. This hydrolysis process caused structural changes, where proteins slowly degraded into smaller peptide units (Rawdkuen et al., 2018). The low molecular weight of FPH also support its potential for application as a functional food product.\n\nOptimal hydrolysis depends on the reaction conditions, such as the type of endogenous protease. Moreover, the raw material has specific optimum conditions for its endogenous enzymes. For example, the endogenous enzymes of the digestive tract of snapper and mackerel function best at pH 9 and temperatures of 45°C and 55°C, respectively (Singh and Benjakul, 2018). Meanwhile, parrotfish have showed better results when processed at pH 8–9 and after reaction times of 21.5–24 h (Prihanto et al., 2020).\n\nVariations in the composition of the amino acids produced were related to differences in prevailing hydrolysis conditions such as enzyme type, pH, and hydrolysis temperature (Klompong et al., 2009) and the total essential amino acid contents were 49.30%. This result is higher than that of total essential amino acid in Nile tilapia (Oreochromis niloticus) and commercial hemp seed protein isolate (HPI), which were previously found to be 40.77% and 40.6%, respectively (Foh et al., 2011; International Quality Ingredients, 2019). Compared to other amino acids, glutamate showed the highest proportion with 12.35%. This result is in line with the FPH of Nile tilapia, which had the highest glutamic acid content of 21.03% (Foh et al., 2011). According to Chalamaiah et al. (2012), most reported FPH to have the highest content of amino acids of the types of aspartic acid and glutamate.\n\n\nConclusion\n\nThe FPH characteristics of the sailfin catfish (Pterygoplichthys pardalis) were affected by two factors, which were pH and hydrolysis duration. Based on the results, the best processing conditions to obtain optimal characteristics for FPH were a yield of 57.39%, antioxidant levels of 63.99%, a DH of 40.67%, a moisture content of 7.28%, ash content of 7.63%, fat content of 5.10%, and protein content of 34.51%. Furthermore, the treatments ranged from a pH of 8.8 – 9 and a hydrolysis length of 22.8 – 24 hours, which led to total essential and non-essential amino acid levels of 49.30% and 50.71%, respectively.\n\n\nData availability\n\nFigshare: Raw data for Sailfin Catfish (Pterygoplichthys pardalis) Fish Protein Hydrolysate.\n\nhttps://doi.org/10.6084/m9.figshare.16566360 (Prihanto et al., 2021)\n\nThis project contains the following underlying data:\n\n- Proximate of Sailfin Catfish (Pterygoplichthys pardalis)\n\n- Yield, antioxidant, and proximate of the Sailfin Catfish (Pterygoplichthys pardalis) Fish Protein Hydrolysate\n\n- Amino acid of the best treatment\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 ministry of Research, Technology, and Higher Education, Republic of Indonesia.\n\n\nReferences\n\nAnnisa S, Darmanto YS, Amalia U: Influence of differences in fish species on fish protein hydrolysate with the addition of the papain enzyme. 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Publisher Full Text\n\nPrihanto AA, Nurdiani R, Bagus AD: Production and characteristics of fish protein hydrolysate from parrotfish (Chlorurus sordidus) head. PeerJ. 2019; 7: e8297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrihanto AA, Nurdiani R, Sari LW: Raw Data of Sailfin Catfish (Pterygoplichthys pardalis) Fish Protein Hydrolysate. figshare. Dataset. 2021. Publisher Full Text\n\nQoyyimah FD, Elfidabari D, Fahmi MR: Identification of Sailfin fish (Loricariidae) based on the character of abdominal patterns in the waters of Ciliwung. J. Biol. 2016; 20(1): 40–43.\n\nRawdkuen S, Rodzi N, Pinijsuwan S: Characterization of sacha inchi protein hydrolysate produced by crude papain and Calotropis proteases.2018; 98: 18–24.\n\nRiansyah A, Supriadi A, Nopianti R: Effect of temperature and drying time differences on the characteristics of siamese salted fish (Trichogaster pectoralis) using an oven. Fishtech. 2013; 2(1): 53–68.\n\nSarmadi BH, Ismail A: Antioxidative peptides from food proteins: A review. Peptides. 2010; 31(10): 1949–1956. PubMed Abstract \| Publisher Full Text\n\nSingh A, Benjakul S: Proteolysis and its control using protease inhibitors in fish and fish products. Compr Rev Food Sci Food Saf. 2018; 17: 496–509. PubMed Abstract \| Publisher Full Text\n\nSusanto DA: Pleco, exotic ornamental Sailfin. Jakarta.2014.\n\nSwanepoel JC, Goosen NJ: Evaluation of fish hydrolysate in juvenil African catfish (Clarias gariepinus) diets. Aquaculture. 2018; 496: 262–269. Publisher Full Text\n\nTunjungsari RM: Utilization of Sailfin fish (Hyposarcus pardalis) in the manufacture of fish chips. Thesis.Bogor: IPB;2007.\n\nWijayanti I, Romadhon, Rianingsih L: Effect of the concentration of the enzyme papain on proximal levels and the yield value of protein hydrolysate bandeng fish (Chanos chanos Forskkal). PENA akuatika. 2015; 12(1): 13–23." }	[ { "id": "128074", "date": "08 Apr 2022", "name": "Celia Garcia-Sifuentes", "expertise": [ "Reviewer Expertise Fish by-products" ], "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\nAlthough the manuscript presents interesting information; there are multiple grammatical and drafting mistakes that make it very hard to understand the context and its relevance. In general, the manuscript lacks a fluent and practical reading and needs to be edited to improve the writing and structure.\nThe title of the manuscript “Production and characteristics of sailfin catfish (Pterygoplichthys pardalis) protein hydrolysate [version 1; peer review: awaiting peer review]” is in my opinion misleading since the small-scale lab methods applied can hardly be called “production”, therefore, I suggest improving the manuscript title.\n\nDescribe the significance level and the post-hoc test used for the data analysis\n\nFor the fish hydrolysis process, described if an enzyme was used for the FPH production. The FPH process is not clear. Were autolytic enzymes used?\n\nYield calculation is not clear, what does the number of hydrolysates mean?\n\nFigures and tables seem to be incomplete, example, table 2. Write a footnote, different letters in the same row indicate significant differences. Figure 3, what is A1B1, A2B2, etc.\n\nFigure 3: The protein/peptides patterns are not clear, maybe because of the separating gel and running conditions of the analysis.\n\nSDS PAGE Analysis. I suggest including the following information on the method. A) µg of protein loaded per well? B) MW of the standard? C) Software used for the band's analysis?\n\nSome newer references should be included for the discussion to compare with other authors.\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? 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 source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "137246", "date": "02 Sep 2022", "name": "Nilesh Nirmal", "expertise": [ "Reviewer Expertise Waste utilization", "bioactive compounds", "bioactivities" ], "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\nPrihanto et al. have studied the production and catherization of sailfin catfish protein hydrolysate. The research topic is important and could influence the nutraceutical market.\n\nHowever, the authors failed to present well the structure and sound understanding of the outcome. In fact, the whole manuscript need to be re-written with scientific and technical merits and explanations.\n\nSecondly the English language is very poor and lacks consistency. The author can do much better writing and comparing the results. Also the labels of each section need to improve.\nThe present manuscript lacks thorough discussion and presentation.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1089
https://f1000research.com/articles/10-829/v1	19 Aug 21	{ "type": "Case Report", "title": "Case Report: Cyclophosphamide in COVID-19 – when an absolute contraindication is an absolute necessity", "authors": [ "Kamila Bołtuć", "Ada Bielejewska", "Alejandro Coloma-Millar", "Robert Dziugieł", "Arkadiusz Bociek", "Agnieszka Perkowska-Ptasińska", "Andrzej Jaroszyński", "Kamila Bołtuć", "Ada Bielejewska", "Alejandro Coloma-Millar", "Robert Dziugieł", "Arkadiusz Bociek", "Agnieszka Perkowska-Ptasińska" ], "abstract": "Background: Despite many studies on COVID-19, our knowledge of it remains incomplete. In some cases, treating SARS-CoV-2 infection concomitant with other diseases can be particularly challenging, as finding an appropriate treatment may involve some risks. Case presentation: A 34-year-old SARS-CoV-2 positive patient admitted due to fever, dyspnoea, haemoptysis and pneumonia, developed alveolar haemorrhage and acute kidney injury. Due to his severe state, abnormalities in laboratory tests and rapidly progressing loss of kidney function, kidney biopsy, as well as antibody panel were carried out, in which perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) were found with a high titer (>200; N: <1:20). The results of kidney biopsy, combined with clinical manifestation and laboratory findings prompted the diagnosis of rapidly progressing glomerulonephritis (RPGN) in the course of p-ANCA vasculitis. Initial treatment consisted of heamodialyses, remdesivir, plasmaphereses, intravenous immunoglobulins, antibiotics, corticosteroids and fraxiparine. Once the haemorrhage had subsided, kidney function had been partially retrieved and heamodialyses had no longer been necessary, cyclophosphamide treatment was initiated, despite being contraindicated in COVID-19 according to its summary of product characteristics. Immunotherapy is still continued. The patient has already received a total of 2.4g of cyclophosphamide (4 cycles of 600mg each every three weeks). Pulmonary and radiological regression, as well as improvement of renal parameters have been achieved.\n\nConclusions: We suspect that cyclophosphamide, the drug of choice in p-ANCA vasculitis, could be a potential factor providing regression of the radiological changes in the lungs and it could have prevented the patient from developing acute respiratory distress syndrome. COVID-19 diagnosis should not exclude searching for other diseases which can have a similar course. When treating a patient in a life-threatening condition, a departure from trying to find the perfect timing of cyclophosphamide delivery should be considered, as delaying it could cause potentially greater harm.", "keywords": [ "COVID-19", "vasculitis", "alveolar haemorrhage", "p-ANCA", "cyclophosphamide" ], "content": "List of abbreviations\n\nAKI: acute kidney injury\n\nANA: antinuclear antibodies\n\nanti-GBM: anti-glomerular basement membrane antibodies\n\nARDS: acute respiratory distress syndrome\n\nBP: blood pressure\n\nc-ANCA: cytoplasmic anti-neutrophil cytoplasmic antibodies\n\nCRP: C-reactive protein\n\nCT: chest computed tomography\n\nIL-6: interleukin 6\n\nIVIG: intravenous immunoglobulins\n\np-ANCA: perinuclear anti-neutrophil cytoplasmic antibodies\n\nRPGN: rapidly progressing glomerulonephritis\n\n\nIntroduction\n\nMore than a year has passed since COVID-19 emerged as a global pandemic. Despite many studies, our knowledge of this disease remains incomplete, especially in SARS-CoV-2 infection with concomitant diseases. Thus, many clinicians face difficulties with finding an appropriate treatment for some cases.\n\n\nCase report\n\nA 34-year-old SARS-CoV-2-positive Caucasian male with a history of borderline hypertension, hypothyroidism and transient arthralgia 2 years prior (without any further investigation), was admitted to a pulmonology ward in November 2020 due to fever (up to 38.5°C), dyspnoea, haemoptysis and developing bilateral pneumonia. During hospitalization the patient developed alveolar haemorrhage followed by acute kidney injury (AKI), for which he was transferred to a Nephrology Clinic on November 21, 2020. On admission he was afebrile and presented with mild haemoptysis and conjunctivitis, oxygen saturation 86-94% (oxygen flow 10-15 l/min), blood pressure (BP) 120/70 mmHg, numerous erythrocytes in urine sediment and proteinuria of 0.47 g/l. His condition was severe and deteriorating.\n\nOwing to the anamnesis of transient arthralgia and rapid deterioration of the kidney function, antibody panel was carried out in which anti-glomerular basement membrane antibodies (anti-GBM), antinuclear antibodies (ANA), as well as cytoplasmic anti-neutrophil cytoplasmic antibodies (c-ANCA) were negative, whereas perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA) were found with a high titer (>200; N: <1:20), suggesting p-ANCA vasculitis complicating COVID-19. Laboratory tests revealed AKI requiring haemodialysis. Anaemia was particularly noticeable. Additionally, leukocytosis, hypoproteinemia, dysproteinemia, elevated D-dimers, C-reactive protein (CRP), interleukin 6 (IL-6), creatinine and urea levels, proteinuria and haematuria were present (Table 1). The parameters of the blood coagulation system were within normal range. Chest computed tomography (CT) demonstrated changes suggesting both viral pneumonia and alveolar haemorrhage (Figure 1, scans A-C). Kidney biopsy performed on the first day of hospitalisation in the Nephrology Clinic and examined with a light microscope revealed crescents in segmental tuft necrosis in two, cellular crescent in one and fibrocellular crescents in three out of nine glomeruli. Glomerular lesions were accompanied by mild interstitial inflammation and acute tubular necrosis. There was no interstitial fibrosis nor tubular atrophy. Immunofluorescence did not reveal any deposits of immunoglobulins nor complement components (Figure 2). The biopsy result, combined with clinical manifestation, prompted the diagnosis of rapidly progressing glomerulonephritis (RPGN) in the course of p-ANCA vasculitis.\n\nScans A-C - CT performed on admission - scans show diffuse ground-glass opacities and septal interlobular thickening in the right lung, as well as paving-stone findings which could represent alveolar haemorrhage. Scans D-F - CT performed 2 months later - visible radiological regression.\n\nScan A - segmental necrosis and cellular crescent, Masson stain. Scan B - Glomeruli with fibrocellular crescents, PAS stain.\n\nIn spite of haemodialysis, the patient was treated with remdesivir (0.1 g). Parallelly to remdesivir therapy, in order to stop the immunological process causing the bleeding, the patient underwent four plasmaphereses with both fresh frozen plasma and albumin as replacement fluids and citrate as anticoagulant. On account of anaemia, the patient was given four units of packed red blood cells. On the fifth and sixth day of hospitalization, a total of 40 g of intravenous immunoglobulins (IVIG) were ordered to inhibit the underlying immunological process responsible for alveolar bleeding in the course of p-ANCA vasculitis. Moreover, owing to the alveolar haemorrhage and probable bacterial superinfection, clarithromycin and piperacillin with tazobactam were ordered. Simultaneously, corticosteroid treatment was started when the results of p-ANCA antibodies were received (1.0 g of intravenous methylprednisolone for 4 consecutive days, followed by 40 mg of oral prednisone). Initially, due to alveolar bleeding, fraxiparine treatment could not be initiated, despite increasing values of D-dimers (2378.0 ug/l vs 4053.0 ug/l 3 days later; N: 0.0-500.0). It was not until after the bleeding had stopped, that it was prescribed (2× 0.6 ml). Once the haemorrhage had subsided, kidney function had been partially retrieved and heamodialyses had no longer been necessary, cyclophosphamide treatment was initiated on the eighth day of hospitalization, despite being contraindicated in COVID-19 according to its summary of product characteristics.\n\nCurrently, immunotherapy is continued. To date, the patient has received a total of 2.4 g (four cycles of 600 mg each, 3 weeks apart) of cyclophosphamide. The therapy is well-tolerated and a pulmonary clinical and radiological remission has been achieved (Figure 1, scans D-F). Additionally, renal parameters have improved with creatinine level at 1.1 mg/dl and stable proteinuria of 1.8 g/24 h.\n\n\nDiscussion\n\nWhile there are cases of continuing immunosuppressive therapy during COVID-19, no data related to the onset of this treatment while undergoing the acute phase of SARS-CoV-2 infection and the follow-up has been reported yet. To our best knowledge, this is the first report of introducing the cyclophosphamide treatment during the acute phase of COVID-19 with the follow-up.\n\nOur case presents a critically ill patient whose state was deteriorating despite the standard treatment. Even though SARS-CoV-2 has been known to induce AKI even in 22.2% of hospitalized patients,1 as well as to cause pneumorrhagia,2 the coincidence of renal and pulmonary symptoms prompted us to search for another reason for the patient’s state other than merely COVID-19. Confirmed p-ANCA vasculitis was an indication to introduce immunosuppressive treatment. However, an active SARS-CoV-2 infection is considered an absolute contraindication for this therapy. Even so, the treatment was still initiated, since the patient’s life was in danger and it was the last resort. We decided to try to stabilize the patient and wait till the estimated time of the viral replication phase end to introduce cyclophosphamide.3\n\nDespite the fact that COVID-19 could have been the reason for pneumorrhagia and such severe state of the patient, systemic vasculitis seemed to be the more likely cause. Thus, the limitation of vasculitis effects and induction of immunosuppression was essential. To this end, plasmapheresis and high doses of IVIG were ordered. It is worth noting that IVIG and plasmapheresis could limit a SARS-CoV-2 infection and increase the tolerance to immunosuppressive treatment4 which was twice as beneficial for our patient. The patient also received remdesivir to inhibit the virus multiplication and invasion. It should be noted that starting immunosuppression in the acute phase of the disease was very risky. Surprisingly, not only did cyclophosphamide not harm the patient, but also the whole applied treatment improved the patient’s state remarkably. CT performed 32 days after the initial CT (after three cycles of cyclophosphamide) showed total regression of ground-glass opacities and septal interlobular thickening and paving-stone findings (Figure 1, scans D-F), even though radiological changes in the lungs in COVID-19 can persist for a long time after recovering from the disease.5\n\nIt seems that our case may indirectly be in line with the hypothesis about the positive influence of immunosuppressive treatment in COVID-19.6 Although IVIG and plasmapheresis therapy was not meaningless, this treatment alone would not lead to remission. We suspect that it was in fact cyclophosphamide, the drug of choice in p-ANCA vasculitis, that could be a potential factor providing regression of the radiological changes in the lungs and it could have prevented the patient from developing acute respiratory distress syndrome (ARDS). It could have happened by the drug’s immunomodulatory function - the second phase of COVID-19 is strongly linked to immunological response, as innate immunity mediated damage of pneumocytes and capillary leak, brought by activation of cytotoxic and effector T-cells, take place in it. The immunomodulatory function of cyclophosphamide led to the elimination of inflammatory damages’ origin. We point out that the onset of treatment happened during the second phase of COVID-19. Having regard to the high potential of replication of SARS CoV-2, immunosuppressive treatment in the replication phase could be tragic.\n\n\nConclusion\n\nCOVID-19 diagnosis should not exclude searching for other diseases which can have a similar course. When treating a patient in a life-threatening condition, a departure from trying to find the perfect timing of cyclophosphamide delivery should be considered, as delaying it could cause potentially greater harm.\n\n\nDeclarations\n\nConsent for publication – Written informed consent for publication was obtained from the patient.\n\nData availability - All data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\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 - J Am Med Assoc. 2020; 323: 2052–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLöffler C, Mahrhold J, Fogarassy P, et al.: Two Immunocompromised Patients With Diffuse Alveolar Hemorrhage as a Complication of Severe Coronavirus Disease 2019. Chest. 2020; 158: e215–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffin DO, Brennan-Rieder D, Ngo B, et al.: The Importance of Understanding the Stages of COVID-19 in Treatment and Trials. Aids Rev. 2021; 23. PubMed Abstract \| Publisher Full Text\n\nPourahmad R, Moazzami B, Rezaei N: Efficacy of Plasmapheresis and Immunoglobulin Replacement Therapy (IVIG) on Patients with COVID-19. SNCompr Clin Med. 2020; 2: 1407–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMyall KJ, Mukherjee B, Castanheira AM, et al.: Persistent Post-COVID-19 Inflammatory Interstitial Lung Disease: An Observational Study of Corticosteroid Treatment. Ann Am Thorac Soc. 2021; 0: AnnalsATS.202008-1002OC. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRevannasiddaiah S, Kumar Devadas S, Palassery R, et al.: A potential role for cyclophosphamide in the mitigation of acute respiratory distress syndrome among patients with SARS-CoV-2. Med Hypotheses. 2020; 144: 109850. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "92236", "date": "03 Sep 2021", "name": "Anna Clementi", "expertise": [ "Reviewer Expertise Nephrology" ], "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 is very well described. Both the patient's clinical history and progression are presented in a detailed way, as well as diagnostic tests and outcomes. This is a case of p-ANCA (perinuclear anti-neutrophil cytoplasmic antibodies) vasculitis complicating COVID-19 in a 34-year-old Caucasian male with a history of borderline hypertension, hypothyroidism, and transient arthralgia 2 years prior admission.\nThe patient was admitted to the hospital because of fever, dyspnoea, haemoptysis, and bilateral pneumonia. Hospitalization was then complicated by acute kidney injury and alveolar haemorrhage. Due to his clinical history and progression, an antibody panel was carried out and a high titer of p-ANCA was found, suggesting another disease in addition to COVID-19. Chest computed tomography (CT) demonstrated the presence of both viral pneumonia and alveolar haemorrhage. A kidney biopsy revealed a rapidly progressing glomerulonephritis in the course of p-ANCA vasculitis. The patient was treated with remdesivir, antibiotics, corticosteroids, IV immunoglobulins, and hemodialysis, and plasmaphereses were performed as well. Also, cyclophosphamide treatment was initiated, despite being contraindicated in the case of COVID-19.\nFirst of all, this case report highlights the importance of considering other diseases which can have a similar course to COVID-19. Furthermore, the choice of each treatment is reported in a reasonable and clear way. In particular, the introduction of immunosuppressive treatment with cyclophosphamide may have improved the patient’s clinical condition by modifying the immune response to SARS-CoV-2.\nI think this is a very interesting case report which can be very useful for clinicians, especially during this period of pandemic.\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": [ { "c_id": "7269", "date": "04 Oct 2021", "name": "Kamila Bołtuć", "role": "Author Response", "response": "Thank you so much for this detailed review. We tried to highlight the possibility of the COVID-19 concomitance with various diseases. Moreover, based on this case we wanted to demonstrate the complexity of treatment in COVID-19 patients. We are glad that our case report met your expectations and due to sufficient detail may be useful for other clinicians." } ] }, { "id": "93992", "date": "10 Sep 2021", "name": "Krzysztof J. Filipiak", "expertise": [ "Reviewer Expertise 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\nA very interesting case report describing the possibility of cyclophosphamide use in COVID-19. Immunosuppressive treatment in COVID-19 is a question of discussion. Cyclophosphamide, the drug of choice in p-ANCA vasculitis, could be a potential factor providing regression of the radiological changes in the lungs and it could have prevented the patient from developing acute respiratory distress syndrome (ARDS). The case is well illustrated and well written. As the immunomodulatory function of cyclophosphamide led to the elimination of inflammatory damages’ origin, broader studies are needed.\n\nOne thing requiring change: there is no such drug as \"fraxiparine\" - do the authors mean nadroparin?\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": [ { "c_id": "7270", "date": "04 Oct 2021", "name": "Kamila Bołtuć", "role": "Author Response", "response": "Thank you so much for your review. In this particular case, we wanted to highlight the complexity of treatment in COVID-19 patients and present the difficulties associated with it. We are glad that this case report met your expectations and may be useful for other clinical practitioners. Additionally, thank you for finding an error with the drug. It should be Nadroparin and it will be changed." } ] } ]	1	https://f1000research.com/articles/10-829
https://f1000research.com/articles/10-1083/v1	25 Oct 21	{ "type": "Research Article", "title": "Investigating the impact of university image on international students’ participation behavior: An empirical study", "authors": [ "Shamima Raihan Manzoor", "Chinnasamy Agamudai Nambi Malarvizhi", "Abdullah Al-Mahmud", "Junainah Mahdee", "Abdullah Al-Mahmud", "Junainah Mahdee" ], "abstract": "Background: The competition among higher education institutions (HEIs) around the world coupled with the rising mobility of international postgraduate students has created challenges for universities to maintain their competitive advantage. There is continuous emphasis from higher education administrators and governments on internationalization and creating global prominence in this sector. As an emerging hub in southeast Asia, Malaysia is no exception to this. Realizing this need for internationalization, this study has attempted to analyze the impact of university image as a higher-order formative construct on international students' participation behavior as a multidimensional construct. Methods: The sample of this study comprised 150 international students from public and private HEIs in Malaysia. The study focused on this group of students as their participation in university life and face-to-face interactions with university personnel have been significantly affected due to the restrictions on global travel during the COVID-19 pandemic. Purposive sampling was employed to select the respondents. A structured questionnaire based on a seven-point Likert scale was used to collect the responses. The hypothesized relationships of this cross-sectional study were examined using partial least square-based structured equation modeling (PLS-SEM). Results: The results showed that all the four hypotheses developed in relation to the dimensions of participation behavior were supported. This research outcome has evidenced that university image can influence international students’ participation behavior such as information seeking, and information sharing behavior that can be perceived as positive behavioral outcomes towards their respective HEIs. Conclusions: Since the present study was conducted in one country, future studies may reproduce this in other southeast Asian countries. While cross-validation to local students lies outside the scope of this study, this empirically tested behavior model offers practical implications for universities, particularly in this uncertain period where HEIs are going through tremendous challenges to uphold their strong rapport with international students.", "keywords": [ "Higher Education", "International Students", "Students’ Participation Behavior", "University Image" ], "content": "Introduction\n\nThe rising number of international students can have strong social effects such as creating diverse communities as well as an economic effect through educational investment in the form of paying their tuition fees, thus contributing to the host country and the home country’s wellbeing (Kumi-Yeboah & James, 2014). International students predominantly come from developing nations, pursue higher education with internationally recognized accreditations and seek lucrative jobs upon returning to their home countries (Ahrari et al., 2019). However, HEIs are currently under immense pressure from the decline of student enrolment and funding grants (Lee et al., 2018) as a result of the ongoing pandemic. Middle-class students in particular are likely to be affected most by the post-COVID-19 higher education situation, with a severe revenue crisis and uncertain impacts upon HEIs (Altbach & de Wit, 2020). In the last decade, the enormous investments from South Korea, Singapore, Taiwan and Hong Kong in higher education projects to create “the best education hub in Asia” (Munusamy & Hashim, 2019, p. 2293) demonstrate the significant competition approaching Malaysia, a country that entices successful international students. To stabilize economies during and after the effects of COVID-19, a strict tightening of the future budget allocations to HEIs is very likely in Malaysia and elsewhere, which will make the universities more reliant on international student intake.\n\nHowever, the usual mobility of Asian international students is towards Organization for Economic Co-operation and Development (OECD) countries (OECD, 2014). This trend undoubtedly drives Malaysia, as Munusamy & Hashim (2019) state, towards rethinking and rebuilding their HEIs’ identity in terms of their image, reputation and ranking on a global platform. The concern is also a result of scholars’ prediction that in the post-COVID world, 20% of institutions may face closure (Altbach & de Wit, 2020). In this context, Manzoor et al. (2020) have defined university image as, “the result of an aggregate process using students’ mental perceptions of their own reality, based on the evaluation of various university attributes through the expression of their feelings, ideas, beliefs, impressions, and real-life experiences at the university” (p.2). As such, high-image universities with stable income streams might have better chances to survive due to their strong identity compared to their lower-performing counterparts.\n\nWilliams & Leahy (2020) point out in the U21 report that Malaysia is still ranked only 31st out of 50 countries in terms of connectivity despite its substantial investment in the higher education sector. Therefore, Munusamy & Hashim (2019) have expressed the necessity of taking extensive initiatives and forming strategies to make Malaysia’s approach towards internationalization more successful in establishing itself as the new global education hub. The trend also creates a challenge for the country to establish and sustain itself as an education hub in Asia and compete with other emerging hub nations such as South Korea, Singapore, Taiwan, and Hong Kong.\n\nThe meta-analysis of Lafuente-Ruiz-de-Sabando, Zorrilla and Forcada (2018) reveals that university image is not only multi-dimensional but also that it needs to be viewed as a higher-order formative construct (HOFC). According to Hair et al. (2017), higher-order structures contain two layers of concrete constructs that capture separate attributes. Referring to Manzoor et al. (2020), the university image model in this study is considered as a higher-order construct containing three lower-order constructs (LOCs). These LOCs such as ‘External communication and values’, ‘National and international awareness and facilities’, and ‘Economic values’ possess formative relationships with university image as a higher-order construct thus termed as a HOFC.\n\nPast researchers have viewed students as a direct and vital source of funding and survival for HEIs (Tamer, 2016). Manzoor & Al-Mahmud (2021) have acknowledged the importance of students’ participation considering their roles as active participants for service improvement and creating competitive effectiveness for HEIs, particularly during uncertain times such as the COVID-19 pandemic. On the other hand, Frasquet-Deltoro, Alarcón-del-Amo and Lorenzo-Romero (2019) have emphasized the need for further research related to customer participation behavior in the service setting (such as that of students in the university context).\n\nHence, there is a serious need for a student-centered relational model that integrates the concept of ‘students as coproducers’ that can lead to international students’ prosocial behavior such as participation behavior (Elsharnouby, 2015). The importance and relevance of customer participation in service settings are endorsed by a lot of evidence (Eisingerich et al., 2013). Realizing this need in the current situation, this study explores how the university image as a HOFC influences students’ participation behavior. To address this central question, the current study analyzes the impact of the university image model developed by Manzoor, Ho and Al-Mahmud (2020) and its impact on international students' participation behavior as a multidimensional concept based on Yi & Gong (2013).\n\n\nLiterature review and hypotheses\n\nAhrari, Krauss, Suandi, Abdullah, Sahimi, Olutokunbo and Dahalan (2019) have mentioned that international students are vital to universities and the host and source countries in today’s neoliberal environment of global competition. Various researchers have considered students, and particularly university students, as the primary customers because they consciously choose and buy the services of their desired university. This modified view underscores viewing students “not only as customers who seek personalized services and high-quality outcomes but also as active players in shaping the university experience” (Tamer, 2016, p.680). The service firms’ capability for better quality service depends on a key step in the co-creation process, which is customer participation (Yi & Gong, 2013).\n\nFirstly, this study is based on the social identity theory (SIT) (Boroş, 2008). According to this theory, organizational identification is “a form of social identification, whereby a person comes to view him- or herself as a member of a particular social entity - the organization” (p.2). Customers, who identify with the organization, have prosocial behavior (such as participation behavior) toward the organization and other stakeholders (Gruen et al., 2000). The meta-analysis done by Santini et al. (2017) reveals that one of the important constructs, which is image, is associated with the identity of HEIs. Customers, who identify with the organization, have prosocial behavior (such as participation behavior) toward the organization and other stakeholders (Gruen et al., 2000).\n\nVarious researchers (e.g. Azoury et al., 2014; Duarte et al., 2010; Wilkins & Huisman, 2013) have defined university image using various dimensions and as a first-order construct. However, Manzoor et al. (2020) have defined university image as a higher-order formative construct (HOFC) consisting of three dimensions: ‘external communication and values’, ‘national and international awareness and facilities’ and ‘economic value’. This means that a university’s image is formed by its national and international name and brand, academic practices and facilities, and psychological environment. The large influence of rising digital media has allowed “constant and active information flow from the inside of an organization to the outside” (Lee et al., 2018, p. 311). Therefore, as well as the course image, there is a clear connection between communications and a university’s image (Duarte et al., 2010). Azoury, Daou and Khoury’s (2014) research on university image shows that ‘cost to quality ratio’ is one of the cognitive factors of image that has the utmost positive impact. According to these researchers, students tend to evaluate if the tuition fees they pay truly complement the quality of their education and if the university support facilities are QS World University Rankings (2021) worth their value for money.\n\nIn this study, social exchange theory has also been used to analyze the role of students' participation behavior as an exchange and expected outcome due to the impact of the positive image of HEIs. According to Blau (1964), social exchange theory postulates that the relationship between an individual and his or her partner is an exchange. The researchers in this study postulate participation behavior as an exchange in the higher education context.\n\nAccording to Mursid & Wu (2021), customer participation is significantly influenced by the image of the institutional services. Moreover, Cheng & Xue (2014, June) have also argued that, through a strong company image, service firms can influence customers’ participation behavior. Also, recent study findings by Manzoor et al. (2020) illustrate university image as a higher-order construct that influences prosocial behavior such as satisfaction and extra-role behavior.\n\nTherefore, we propose the following main hypothesis based on the above literature review:\n\nH1: University image (as HOFC) positively related to students’ participation behavior.\n\nAs suggested earlier, based on Yi & Gong’s (2013) ideas, the present study conceptualizes student participation behavior as a multidimensional concept that has four broad dimensions: information seeking, information sharing, personal interaction, and responsible behavior. In university settings, students seek information in various ways, such as asking other persons (e.g. students or employees). Likewise, information sharing in service settings includes providing employees with essential information that enables them to perform their roles better. Personal interaction between customers [or students] and service providers [or a university] is crucial for successful service delivery (Ennew & Binks, 1999). Customers must demonstrate responsible behavior accepting their duties (Deltoro et al., 2019) as a part of participation behavior (Yi & Gong, 2013). Accordingly, we postulate the H1 expansion and conceptual model (Figure 1) as follows:\n\nH1(a): University image as a HOFC is positively related to students’ information seeking.\n\nH1(b): University image as a HOFC is positively related to students’ information sharing.\n\nH1(c): University image as a HOFC is positively related to students’ personal interaction.\n\nH1 (d): University image as a HOFC is positively related to students’ responsible behavior.\n\nThe above conceptual model illustrates the relations of university image as a HOFC and its relationship with students’ participation behavior as a multidimensional construct consisting of elements such as information seeking, information sharing, personal interaction, and responsible behavior.\n\n\nMethods\n\nEthical approval was obtained for this project from the Multimedia University Research Ethics Committee (REC) (approval Number: EA0272021). The questionnaire was accompanied by a cover letter stating the purpose of the study and confirming confidentiality of participant data. Each respondent was given adequate time to go through the cover letter prior to filling in their survey questionnaire. Participants who wanted to proceed with the survey could then advance to the next page by clicking a button. This study therefore ensured that the participants intended to continue with the survey voluntarily after reading the cover letter.\n\nData were collected from questionnaires conducted at three public and three private universities in Malaysia. International students were chosen due to their multiplicity in terms of university experience, background, and cultural diversity which could offer deeper insights for this study’s findings. The responses from international students in selected Malaysian HEIs were used as data for analysis. However, in a quantitative study such as this, non-responses are likely to persist, despite the survey design (Dillman, 1991). To reduce non-responses in this study, the questionnaire was pretested to ensure that respondents could clearly understand each question, thus increasing their response rate.\n\nThis cross-sectional quantitative study used a structured questionnaire for data collection. These were conducted both face-to-face and online from November 2018 to February 2019. Three fellow research students and several Student Representative Council (SRC) members in the respective universities helped to facilitate the survey distribution. The face-to-face questionnaire copies were distributed by fellow research students with the help of SRC members and the online copies were disseminated through the Google Forms link. Reminder emails were sent through SRCs at two-week intervals as suggested by Toepoel & Schonlau (2017) to increase the response rate for this study.\n\nThe questionnaire included a one-page front cover informing the respondents about the purpose of the study and assuring them about data confidentiality and anonymous use. A seven-point Likert scale ranging from “1-totally disagree” to “7-totally agree” was used to collect the responses. The items for the three lower-order constructs to measure the university image were adapted from Manzoor et al. (2020). Thus, ‘external communication and values’ was measured using five items, ‘economic value’ using two items, and ‘national and international awareness and facilities’ using eight items. The items used to measure four dimensions of students' participation behavior were adapted from Yi & Gong (2013). ‘Information seeking’ comprised three items, ‘information sharing’ four items, ‘personal interaction’ five items, and ‘responsible behavior’ four items. To guarantee the content validity and relevance of the method, the questionnaire was pre-tested beforehand by two academics with several years of experience in the HE sector and three international students with over a year of study experience in Malaysia. The pre-testing was conducted with these experts in their respective university settings. No major feedback was reported regarding the clarity of the questionnaires.\n\nHenceforth, a miniaturized walk-through pilot study (n=35) was conducted among the randomly selected international students who were enrolled in either public or private universities in Malaysia. After the pilot respondents provided positive feedback about the overall structure and presentation of the questionnaire, the final questionnaire was distributed among the actual survey participants.\n\nThe final questionnaire consisted of three sections. The first section comprised demographic information such as nationality, gender, program of study, and marital status. The second section comprised 15 items measuring the university image. The last section consisted of 16 items measuring the dimensions of participation behavior. A copy of the questionnaire can be found here (Manzoor, 2021).\n\nPurposive sampling was used in this study, as non-probability sampling is commonly employed for sampling from international students in the context of Malaysia (Singh & Jack, 2018). The sample of this study comprised 150 existing undergraduate and postgraduate international students of both public and private HEIs in Malaysia with a response rate of 60%. The HEIs were selected for this study due to their higher number of international students and their competitive positions in the QS World University Rankings, 2021 report. Out of the 158 responses, 150 were maintained and the remaining eight responses were excluded due to a ‘straightlining’ issue (Kim et al., 2019). The sample size was acceptable for structural equation modeling (Hair et al., 2010). Moreover, the missing value is less than 15%, thus not creating any issue for this study (Sarstedt et al., 2014). For each answer to the Likert scale questions, participants were removed if their responses scored a standard deviation of <0.3. The accompanying dataset can be found here (Manzoor, 2021).\n\nSPSS (Statistical Package for the Social Sciences) Version 23 was used to organize and refine the data for further analysis. To control the common method bias (CMB), this study used the unrotated factor analysis known as Harman’s Single Factor test following Lowry & Gaskin’s (2014) study to ensure the single-source data is not an issue for this study. This study also used Cronbach’s Alpha coefficient to estimate the internal consistency of the measures based on the threshold value suggested by Nunnally (1978).\n\nThis study then employed SmartPLS 3.2.9 (also available as a 30-days downloadable trial version) for model estimations and analyzing the structural paths to illustrate the relationships between the university image (HOFC) with participation behavior dimensions and the predictive value to interpret such image as perceived by the international students. According to Hair et al.’s (2019), PLS-SEM is adopted when there is a concern for testing a theoretical framework from a prediction aspect and one is required to use latent variable scores to pursue further analyses.\n\nThe evaluation of PLS-SEM results starts with analyzing the measurement models and then the structural model. Following Hair et al. (2017), convergent validity for PLS-SEM has been examined from the measurement by evaluating their indicator reliability and average variance extracted (AVE). As a part of assessing the indicator reliability, the composite reliability was used where the items are weighted based on the construct indicators’ individual loadings. Subsequently, AVE was assessed as it represents the commonality of the constructs (grand mean value of the squared loadings of the indicators). To analyze the discriminant validity, this study used heterotrait-monotrait (HTMT) ratio proposed by Henseler et al. (2015).\n\nFollowing Hair et al.’s (2017) recommendation, a two-stage approach was used to assess the structural model validity for the university image as the higher-order formative construct (HOFC). Variance inflation factor (VIF) and bootstrapped outer weights were used to assess the validity of the university image as HOFC. While assessing the HOFC measurement model for collinearity issues, the outer variance inflation factor values were derived using the latent variable scores (LVS) (Hair et al., 2010). Structural model relationships (i.e. the path coefficients that represent the hypothesized relationships among the constructs) are obtained in this study by means of bootstrapping and assessing the t-value as suggested by Hair et al. (2017), as coefficients ultimately depend on their standard error. Subsequently, the R2 value has been reported as an indicator of the model’s explanatory power as it measures the variance of the endogenous constructs (Rigdon, 2012) such as the participation behavior dimensions in this study. Finally, combining the aspects of out-of-sample prediction and in-sample explanatory power as input, the blindfolding procedure was applied to derive the Q2 value to evaluate the predictive accuracy of the PLS path model analyzed in this study.\n\n\nResults\n\nData were checked for common method bias (CMB) that used Harman's Single Factor test as a statistical fix (Podsakoff et al., 2012) as this study is based on cross-sectional data. No CMB issues were found as 48.98% of the variance is explained by just one factor (Podsakoff et al., 2003). The Cronbach’s alpha values in this study are greater than 0.7, thus indicating the stability of the scale (Nunnally, 1978).\n\nLooking into the demographic profile of this study, the nationalities of the respondents broadly represented the international student population in Malaysia, with 35.1% from Bangladesh, 20.2% from Pakistan, 15.8% from Yemen, 12.6% from India, 8% from Indonesia, and 8.3% from other countries. The samples consisted of 70.5% male respondents and 29.5% female respondents. 55% of the students were postgraduate students and the rest (45%) were undergraduate students. The majority of the respondents (65%) were single and 35% of the respondents were married.\n\nFor the measurement of model validity, all the constructs reached the satisfactory level of average variance extracted (see Hair et al., 2017) with a value greater than 0.5 and composite reliability (CR) >0.8, thus ensuring the convergent validity. For discriminant validity, the values of the Heterotrait-Monotrait ratio (HTMT) also met the essential threshold of HTMT.85 (see Henseler et al., 2015), thus establishing discriminant validity for the constructs in this study.\n\nBy following Manzoor et al. (2020), university image was considered a higher-order construct in this study which also comprises three lower-order constructs, namely ‘external communication and values’, ‘national and international awareness and facilities’, and ‘economic value’ (p. 29). According to Hair et al. (2017), a two-stage approach is used “to assess the structural model validity for the higher-order formative construct” (p.23). In order to evaluate the validity of university image as a HOFC, this study used variance inflation factor and bootstrapped outer weights. The outer variance inflation factor (VIF) values for all indicators using the latent variable score (LVS) are less than 5 and the t-values for all the other indicators’ outer weights are significant (t-values>1.96) (see Hair et al., 2010 for the requirements) (Table 1).\n\nNote: Bootstrap confidence intervals for 5% probability of error (alpha = 0.05), p<0.05, *p<0.001\n\nThe structural model validity was evaluated using a bootstrapping analysis to assess the direct effects of H1(a)-H1(d). The bootstrapping procedure is administered based on 5000 samples. The t-values of the bootstrapped results of above 1.645 at a 5% level of significance (one-tailed) show that all four hypotheses are supported (Table 2).\n\nThe hypotheses results show that there is a positive relationship between ‘university image’ and ‘information seeking’ (H1a: t-value=3.735, p<0.05); ‘university image’ and ‘information sharing’ (H1b: t-value=5.252, p<0.01); ‘university image’ and ‘personal interaction’ (H1c: t-value=2.554.735, p<0.03); ‘university image’ and ‘responsible behavior’ (H1d: t-value=6.121, p<0.01). The R2 values for the four dimensions of participative behavior are 0.26, considered substantial based on the threshold recommended by Cohen (2013). The Q2 values are obtained from the blindfolding procedure to examine the model’s predictive relevance. In a structural model, Q2 values larger than zero indicate that “exogenous constructs have predictive relevance for endogenous constructs” (Hair et al., 2017, p.37). The results of this study revealed that all Q2 values range from 0.001 to 0.398 and are more than 0, thus indicating that the model’s predictive relevance is satisfactory.\n\n\nDiscussion\n\nThe aim of this study was to analyze the university image as a higher-order formative construct and its impact on international students’ participation behavior in HEIs in Malaysia. The results have revealed that there are positive associations between university image and the dimensions of participation behavior such as information seeking, information sharing, personal interaction, and responsible behavior. This study’s outcomes complement the findings of Mursid & Wu (2021) that institutional image positively influences the customers’ participation (such as that of HEIs on student participation). Thus, HEIs having a sound image can ultimately encourage international students to interact openly and enhance the latter’s information exchange and spontaneous participation behaviors with their personnel or staff.\n\nThis study has revalidated the implications of social exchange theory, particularly in the higher education context. The relationship-oriented model used in this study addresses the importance of focusing on university image as a higher-order formative construct consisting of three lower-order constructs (i.e., ‘external communication and values’, ‘national and international awareness and facilities’ and ‘economic value’) enhancing a strong identity based on the underlying assumptions of social identity theory for the HEIs. This higher-order image model can then act as a relationship enhancement catalyst and encourage students in participation behavior that is defined by Yi & Gong (2013) as multidimensional. This indicates that the higher education practitioners need to develop their regional and global image through analyzing its multidimensional nature beyond one factor for the sustainability of other factors as well such as their ranking, facilities, and communications with various stakeholders.\n\nFurthermore, the results of this study show that HEIs’ image can positively influence information seeking, information sharing, personal interaction, and responsible behavior as parts of students’ participation behavior. The study thus confirms the positive relationship between university image and participation behavior dimensions which is compatible with the views of Cheng & Xue (2014, June). In line with the fundamentals of social exchange theory, students' participation behavior is the outcome of their prosocial behavior derived as a reciprocal exchange between the customer and an organization.\n\nTherefore, higher education administrators can create a flexible platform (both online and offline) to facilitate international students’ participation behavior in university service provision and equip them with knowledge of the university’s services, something that provides students with a ‘sense of belonging’. This ultimately helps the HEIs to nurture a relationship with their students even in cases of uncertainty such as during and post-pandemic situations (Manzoor et al., 2020).\n\n\nConclusion\n\nBy considering the competitive situation of HEIs around the world, this study has investigated the impact of university image on the participation behavior of international students in Malaysia. In this regard, the study has taken ‘image’ as a higher-order construct on the multidimensional participation behavior of students. International students have been concentrated particularly due to the imposition of travel restrictions worldwide due to the COVID-19 pandemic that has largely affected their mobility and interactions with university personnel. The study has confirmed the positive associations between university image and the multiple dimensions of participation behavior such as information seeking, information sharing, personal interaction, and responsible behavior. Future studies can assess this model of university image and students’ participation behavior among local students to perform the multigroup analysis to assess if any variability exists in their behavior. Moreover, the relationship-based behavioral model explored in this study could be tested with a larger sample size incorporating private colleges and community colleges to enhance the generalizability of the research findings. The outcome of this research has evidenced that university image can encourage students’ participation behavior such as information seeking, information sharing, and responsible behavior, which are perceived as positive behavioral outcomes towards their respective HEIs. This empirically tested behavioral model in the present study offers practical implications for universities particularly in this current period of uncertainty following the COVID-19 pandemic where HEIs are facing tremendous challenges to uphold their strong rapport with international students.\n\n\nData availability\n\nMendeley: University image and participation behavior data. https://data.mendeley.com/datasets/fn2kdnbv4j/2 (Manzoor, 2021)\n\nThis project contains the following underlying data:\n\n- Main study with full data entry 04011920.csv\n\n- University image and participation behavior latest.csv\n\nMendeley: University image and participation behavior data. https://data.mendeley.com/datasets/fn2kdnbv4j/2 (Manzoor, 2021)\n\nThis project contains the following extended data:\n\n- Questionnaire\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nThis paper is an outcome of the shared concept and contribution of the four authors. The first author formulated the idea, conducted the data curation, completed the analysis and the writing; the second author helped with data collection, validation, and supervision; the third author edited and formatted the manuscript, and the fourth author was involved in visualization and networking.\n\n\nReferences\n\nAhrari S, Krauss SE, Suandi T, et al.: A stranger in a strange land: Experiences of adjustment among international postgraduate students in Malaysia. Issues in Educational Research. 2019; 29(3): 611–632. Reference Source\n\nAltbach PG, de Wit H: COVID-19: The internationalization revolution that isn’t. University World News. 2020; 14. Reference Source\n\nAzoury N, Daou L, Khoury CE: University image and its relationship to student satisfaction-case of the Middle Eastern private business schools. International strategic management review. 2014; 2(1): 1–8. Publisher Full Text\n\nBlau P: Power and exchange in social life. USA: Routledge. 1964.\n\nBoroş S: Organizational identification: Theoretical and empirical analyses of competing conceptualizations. Cognition, Brain, Behavior. 2008; 12(1): 1–27. Reference Source\n\nCheng P, Xue W: Corporate image, customer participation and service quality: From social identity theory perspective. In 2014 11th International Conference on Service Systems and Service Management (ICSSSM). IEEE. 2014; 1–5. Publisher Full Text\n\nCohen J: Statistical power analysis for the behavioral sciences. Academic Press. 2013. Reference Source\n\nDeltoro FM, Amo AM, Romero LC: Antecedents and consequences of virtual customer co-creation behaviors. Internet Res. 2019; 29(1): 218–244. Publisher Full Text\n\nDillman DA: The design and administration of mail surveys. Annu Rev Sociol. 1991; 17(1): 225–249. Publisher Full Text\n\nDuarte PO, Alves HB, Raposo MB: Understanding university image: a structural equation model approach. 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Publisher Full Text\n\nGruen TW, Summers J, Acito F: Relationship marketing activities, commitment, and membership behaviors in professional associations. J Mark. 2000; 64(3): 34–49. Publisher Full Text\n\nHair JF Jr, Black WC, Babin BJ: Multivariate Data Analysis: A Global Perspective. (7th ed.). New Jersey: Pearson Education Inc, 2010. Reference Source\n\nHair JF Jr, Hult GTM, Ringle CM, et al.: A primer on partial least squares structural equation modeling (PLS-SEM). Sage publications, 2017. Reference Source\n\nHair JF, Sarstedt M, Ringle CM: Rethinking some of the rethinking of partial least squares. Eur J Mark. 2019; 53(4): 566–584. Publisher Full Text\n\nHenseler J, Ringle CM, Sarstedt M: A new criterion for assessing discriminant validity in variance-based structural equation modeling. J Acad Market Sci. 2015; 43(1): 115–135. Publisher Full Text\n\nKim Y, Dykema J, Stevenson J, et al.: Straightlining: overview of measurement, comparison of indicators, and effects in mail-web mixed-mode surveys. Soc Sci Comput Rev. 2019; 37(2): 214–233. Publisher Full Text\n\nKumi–Yeboah A, James W: Transformative learning experiences of international graduate students from Asian countries. Journal of Transformative Education. 2014; 12(1): 25–53. Publisher Full Text\n\nLafuente-Ruiz-de-Sabando A, Zorrilla P, Forcada J: A review of higher education image and reputation literature: Knowledge gaps and a research agenda. European research on management and business economics. 2018; 24(1): 8–16. Publisher Full Text\n\nLee Y, Park H, Cameron GT: Strategic communication in U.S. higher education: Testing congruity effects of university identity and image among parents of prospective students. International Journal of Strategic Communication. 2018; 12(3): 308–327. Publisher Full Text\n\nLowry PB, Gaskin J: Partial least squares (PLS) structural equation modeling (SEM) for building and testing behavioral causal theory: When to choose it and how to use it. IEEE Trans Prof Commun. 2014; 57(2): 123–146. Publisher Full Text\n\nManzoor SR, Al Mahmud A: Redefining a Value Co-creation Behaviour Model for Student Satisfaction in HEIs. International Journal of Services and Operations Management. 2021. Publisher Full Text\n\nManzoor SR, Ho JSY, Al Mahmud A: Revisiting the 'university image model' for higher education institutions' sustainability. Journal of Marketing for Higher Education. 2020; 1–20. Publisher Full Text\n\nManzoor S: University image and participation behavior data combined. Mendeley Data, V2, 2021. http://www.doi.org/10.17632/fn2kdnbv4j.2\n\nMunusamy MM, Hashim A: Internationalization of higher education in Malaysia: insights from higher education administrators. AEI Insights: An International Journal of Asia-Europe Relations. 2019; 5(1): 21–39. Reference Source\n\nMursid A, Wu CHJ: Customer participation, value co-creation and customer loyalty: evidence from Umrah travel agencies in Indonesia. Journal of Islamic Marketing. 2021. Publisher Full Text\n\nNunnally JC: Psychometric theory. New York: McGraw-Hills Book Company, 1978.\n\nOECD: Indicator C4: Who studies abroad and where? In Education at a Glance 2014: OECD Indicators, OECD Publishing, Paris. 2014. Publisher Full Text\n\nPodsakoff PM, MacKenzie SB, Lee JY, et al.: Common method biases in behavioral research: a critical review of the literature and recommended remedies. J Appl Psycho. 2003; 88(5): 879–903. PubMed Abstract \| Publisher Full Text\n\nPodsakoff PM, MacKenzie SB, Podsakoff NP: Sources of method bias in social science research and recommendations on how to control it. Annu Rev Psychol. 2012; 63: 539–569. PubMed Abstract \| Publisher Full Text\n\nQS World University Ranking: (2021, January/February). 2021. Reference Source\n\nRigdon EE: Rethinking partial least squares path modeling: In praise of simple methods. Long Range Planning. 2012; 45(5–6): 341–358. Publisher Full Text\n\nSantini FDO, Ladeira WJ, Sampaio CH, et al.: Student satisfaction in higher education: A meta-analytic study. Journal of Marketing for Higher Education. 2017; 27(1): 1–18. Publisher Full Text\n\nSarstedt M, Ringle CM, Henseler J, et al.: On the emancipation of PLS-SEM: A commentary on Rigdon (2012). Long Range Planning. 2014; 47(3): 154–160. Publisher Full Text\n\nSingh JKN, Jack G: The benefits of overseas study for international postgraduate students in Malaysia. Higher Education. 2018; 75(4): 607–624. Publisher Full Text\n\nTamer H: Participation behavior among international students. The International Journal of Educational Management. 2016; 30(5): 679–697.\n\nToepoel V, Schonlau M: Dealing with nonresponse: Strategies to increase participation and methods for post survey adjustments. Mathematical Population Studies. 2017; 24(2): 79–83. Publisher Full Text\n\nWilkins S, Huisman J: Student evaluation of university image attractiveness and its impact on student attachment to international branch campuses. J Stud Int Educ. 2013; 17(5): 607–623. Publisher Full Text\n\nWilliams R, Leahy A: U21 Ranking of National Higher Educational Systems 2020. Melbourne: University of Melbourne. 2020.\n\nYi Y, Gong T: Customer value co-creation behavior: Scale development and validation. J Bus Res. 2013; 66(9): 1279–1284. Publisher Full Text" }	[ { "id": "129135", "date": "26 Apr 2022", "name": "Purificación Alcaide-Pulido", "expertise": [ "Reviewer Expertise Higher Education" ], "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\nCurrent literature:\nPlease cite the original research on the development of the university image model by Alcaide-Pulido, Alves & Gutierrez-Villar, 20171.\n\n\"Past researchers have viewed students as a direct and vital source of funding and survival for HEIs\", and \"Hence, there is a serious need for a student-centered relational model that integrates the concept of ‘students as coproducers’ that can lead to international students’ prosocial behavior such as participation behavior\" - Please cite recent research:\n\nThis article by Alcaide-Pulido, O’Sullivan, & Chapleo, 20212 addresses the links between the importance of students and a relational model in the context of HEIs. It addresses an important topic, especially given ongoing management on this and other studies and research throughout the topic. There is an important reference to students as a vital source of survival for HEIs. The findings highlight areas of commonality as well as differences between the different cultures and nationalities examined as part of the study. They show areas that brand managers from each country should concentrate on, making recommendations that could help to shape the marketing direction of universities in each of the nations included in the research.\n\nIn addition, the role of students satisfaction and student trust related to a relational model should be explained by Abdelmaaboud, Peña & Mahrous, 20213. The results also confirm the presence of the significant indirect effect of student-university identification on students’ advocacy intentions via student satisfaction and student trust.\n\nThe role of brand experience and brand effect should be connected to Farhat, Mokhtar & Salleh, 20214. The findings showed the critical rule of brand interactivity towards brand engagement behavior. The study suggests leveraging the power of brand experience and brand interactivity to drive brand engagement behavior through the critical role of brand effect in HEIs.\n\nStudy design:\nAbstract and conclusion indicate: \"The study focused on this group of students as their participation in university life and face-to-face interactions with university personnel have been significantly affected due to the restrictions on global travel during the COVID-19 pandemic.\" However, the study design presents the data collection: \"This cross-sectional quantitative study used...were conducted both face-to-face and online from November 2018 to February 2019\", it is not a pandemic period. It is a mistake.\n\nStatistical analysis: In my opinion, it is necessary for the figure resultant of the model with factors and charges after applying the analysis. It is important to detect the weights in the model analysis SmartPLS.\n\nRecommendation of a larger sample size in future research and real period of uncertainty due to the COVID-19 pandemic.\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? 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": [] }, { "id": "136419", "date": "16 May 2022", "name": "Rosalind Latiner Raby", "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\nThis manuscript is relevant to the field of Comparative and International Education because it highlights a critical issue facing universities - that of image, status, and student orientation. The authors point out that the aim of this study was to analyze the university image as a higher-order formative construct and its impact on international students' participation behavior in HEIs in Malaysia. The article is firmly organized within empirical research standards. However, there are revisions that I suggest for the introduction, methodology, and discussion.\nThe manuscript does acknowledge a range of literature and uses that literature to help support the authors’ thesis that attrition is complex. The authors use very current literature and literature representing the voices of Malaysian researchers. A solid description of the theoretical framework is also provided. However, I would suggest making that a sub-heading.\nIn terms of methodology, there is a solid discussion on why methodological choices were made. However, the choice to access international students in all the universities in Malaysia is not explained in the introduction. It is also curious why so few international students were included in the sample given that all the universities were assessed. This needs to be further explained. The use of international students also seems contradictory to the literature that talks about domestic student orientations. These need to be better explained in the introduction that addresses a) why international students make a good sample; b) why all universities were chosen; c) why both public and private were chosen; and d) how many international students are in Malaysia and how that relates to the sample size. This then needs to be summarized at the beginning of the methods section.\nI am not convinced that a hypothesis model is needed. The research is not really testing anything, which is a criterion for using the hypothesis. I would suggest using research questions instead. That also fits better with your discussion section as you can then dive deep into the responses given.\nOverall, this is an interesting article and brings out some issues for future discussion. I do believe that this article has the potential for being a strong article with minor revisions.\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? No\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/10-1083
https://f1000research.com/articles/9-1299/v1	05 Nov 20	{ "type": "Research Article", "title": "Common genetic signatures of Alzheimer’s disease in Down Syndrome", "authors": [ "Ayati Sharma", "Alisha Chunduri", "Asha Gopu", "Christine Shatrowsky", "Wim E. Crusio", "Anna Delprato", "Ayati Sharma", "Alisha Chunduri", "Asha Gopu", "Christine Shatrowsky", "Wim E. Crusio" ], "abstract": "Background: People with Down Syndrome (DS) are born with an extra copy of Chromosome (Chr) 21 and many of these individuals develop Alzheimer’s Disease (AD) when they age. This is due at least in part to the extra copy of the APP gene located on Chr 21. By 40 years, most people with DS have amyloid plaques which disrupt brain cell function and increase their risk for AD. About half of the people with DS develop AD and the associated dementia around 50 to 60 years of age, which is about the age at which the hereditary form of AD, early onset AD, manifests. In the absence of Chr 21 trisomy, duplication of APP alone is a cause of early onset Alzheimer’s disease, making it likely that having three copies of APP is important in the development of AD and in DS. In individuals with both DS and AD, early behavior and cognition-related symptoms may include a reduction in social behavior, decreased enthusiasm, diminished ability to pay attention, sadness, fearfulness or anxiety, irritability, uncooperativeness or aggression, seizures that begin in adulthood, and changes in coordination and walking. Methods: We investigate the relationship between AD and DS through integrative analysis of genesets derived from a MeSH query of AD and DS associated beta amyloid peptides, Chr 21, GWAS identified AD risk factor genes, and differentially expressed genes in DS individuals. Results: Unique and shared aspects of each geneset were evaluated based on functional enrichment analysis, transcription factor profile and network analyses. Genes that may be important to both disorders: ACSM1, APBA2, APLP1, BACE2, BCL2L, COL18A1, DYRK1A, IK, KLK6, METTL2B, MTOR, NFE2L2, NFKB1, PRSS1, QTRT1, RCAN1, RUNX1, SAP18 SOD1, SYNJ1, S100B. Conclusions: Our findings indicate that oxidative stress, apoptosis, and inflammation/immune system processes likely underlie the pathogenesis of AD and DS.", "keywords": [ "Alzheimer’s Disease", "Down Syndrome", "Behavior", "Memory", "Learning" ], "content": "Introduction\n\nAmyloids are peptide or protein aggregates that form from the misfolding of normally soluble proteins, which then stick together due to their chemical properties and accumulate in extracellular compartments and organs1. Amyloids form fibrous structures and plaques that are highly insoluble, resistant to degradation, and are involved in several diseases such as Alzheimer’s disease (AD), Down syndrome (DS), spongiform encephalopathies, and type II diabetes2,3. The amyloid plaques associated with AD are formed from peptides derived from the mis-processing of APP, a protein that typically resides around nerve cells2. The toxic peptide fragments are called beta amyloids. In AD, the amyloid plaques deposit in brain tissue, destroy neuronal connectivity, disrupt signaling at synapses, and eventually result in nerve cell death, tissue loss, and a reduction in brain mass2. Smaller aggregates of beta-amyloid and not the plaques themselves trigger the immune system and inflammatory processes4.\n\nEarly onset AD that runs in families is linked to the APP and PSEN1/PSEN2 genes5. A mutation in one of these three genes may cause AD to develop early whereas the more general form of the disease, late onset, is typically linked to the APOE gene6. PSEN1/PSEN2 are transmembrane proteins that are the catalytic subunit of gamma secretase, the enzyme responsible for cleaving APP. Mutations in the PSEN genes may result in the abnormal cleaving and processing of APP to smaller toxic beta amyloid fragments which aggregate and accumulate7,8.\n\nPeople with Down Syndrome (DS) are born with an extra copy of chromosome 21 (Chr 21) and many of these individuals develop AD as they age9. This is due at least in part to the extra copy of the APP gene located on Chr 21. By the age of 40, most people with DS have amyloid plaques which disrupt brain cell function and increase their risk for AD3. About half of the people with DS develop AD and the associated dementia around 50 to 60 years of age, which is about the age at which the hereditary form of AD, early onset AD, manifests10. Duplication of APP alone, in the absence of Chr 21 trisomy, is another cause of early onset AD11,12 making it likely that having three copies of APP is important in the development of AD in DS.\n\nIn both early and late onset AD the clinical symptoms include dementia, memory decline, and the inability to retain recent information or store new memories13. As the disease progresses, AD individuals may exhibit problems with language, reasoning, decision making, executive function, mood swings, aggressive behavior, and apathy. Late stage symptoms of AD may result in seizures, hypertonia myoclonus, incontinence, and mutism14. Death commonly occurs from general inanition, malnutrition, and pneumonia.\n\nMemory loss and forgetfulness, which is typical in AD individuals, is less pronounced in people with both DS and AD. This may in part be a floor-effect due to the memory deficit already present in DS individuals15–17. Studies report an impairment in verbal short-term memory (example: serial order of a list of words) relative to visuo-spatial memory (manual selection in serial order of locations) and deficits in explicit long-term memory18. Also in individuals with DS there is evidence of hippocampal dysfunction and deficits in prefrontal systems as compared with mental age-matched controls19. In DS individuals, the hippocampal volume is reduced prior to the onset of dementia, and these reductions were found to relate to memory mainly due to the loss of neurons and neuronal volume as a result of neurofibrillary tangle formation20.\n\nIn this study we investigate the relationship between AD and DS through integrative geneset analysis of genes derived from peptides associated with amyloid plaques found in AD and DS individuals, Chr 21 genes, AD risk factor genes, and differentially expressed genes (DEX) identified through a transcriptome analysis of DS individuals for both the dorsal frontal cortex (DFC) and cerebellar cortex (CBC).\n\n\nMethods\n\nAll genesets used in this study are presented in Extended data Workbook 121. The Chr 21 geneset was obtained from NCBI Gene. A total of 250 unique gene IDs were obtained at the time of manuscript preparation (September 1st, 2020). The AD-DS geneset, consisting of 292 genes, was obtained from GeneWeaver using “Alzheimer’s Down Syndrome” as the search term. The geneset was originally generated via gene2mesh v.1.1.1 (updated: 2019-01-07) from Medical Subject Headings (MESH Terms) GS236695 • [MeSH] Amyloid beta-Peptides: D016229. The AD risk factor geneset is comprised of 279 genes, many of which were identified and/or confirmed through a large scale GWAS of 71,880 clinically diagnosed AD and AD-by-proxy cases and 383,378 controls22.\n\nThe DEX genesets for the DFC (842) and CBC (570) were obtained from The Down Syndrome Developmental Brain Transcriptome database. Human Brain Transcriptome, Department of Neurobiology Yale University School of Medicine which is a publicly accessible database containing searchable differential gene expression information of transcriptome data in developing and adult DS versus control human brains. The data was generated from 15 sets of a DS and a matched control brain each. The specimens ranged from embryonic development to adulthood23.\n\nCommon genes among the AD-DS, Chr 21, DEX DFC, DEX CBC and AD risk factor genesets were assessed using venn diagram analysis (http://www.interactivenn.net/) and visualized with the UpSet Library in RStudio, R Version 4.0.2.\n\nKeyword categories were used to evaluate the genesets. The keyword categories were chosen based on the major phenotypes associated with AD and DS. The terms used were: aging, Alzheimer's disease, amyloid; apoptosis, behavior cholesterol, circadian, cognition; Down Syndrome, face, fibril, immune, inflammation, insulin, learning, leptin, memory, muscle, myelin; obesity, sleep, speech, and tau.\n\nGene ontology characterization of the genesets was performed in both DAVID and the Gene Ontology database for Biological Process (BP). The Benjamini corrected P-value was used to determine enrichment significance. Functional information based on GO annotations for the genes associated with a keyword search term related to AD and DS were identified and noted.\n\nGene Ontology pathway enrichment was used to further characterize the AD-DS and Chr21 genesets in order to obtain a broader overview of collective gene function. The Benjamini-corrected P-value was used to determine significance.\n\nThe APP protein-protein interaction network was built in STRING (version 11.0), based on experimentally validated interactions. The combined scores for the interactions are computed by combining the probabilities from the different evidence channels and corrected for the probability of randomly observing an interaction. First and 2nd shell interactions are included in the network. The network was exported from STRING and analyzed in Cytoscape (version 3.7). Network bottlenecks and clusters were identified with Cytoscape plugins CytoHubba (version 0.1) and MCODE (version 1.6.1), respectively.\n\n\nResults\n\nThe number of common genes among all of the 5 genesets (AD-DS, Chr 21, AD risk factors, DEX DFC, and DEX CBC) along with the gene names and Gene Ontology classifiers are shown in Figure 1 and Extended data Workbook 224. The AD-DS, Chr 21 and AD risk factor genesets overlap by eight genes: APP, BACE2, COL18A1, DYRK1A, RCAN1, SOD1, SYNJ1, and S100B (Figure 1). BACE2 encodes an integral membrane glycoprotein that cleaves the APP protein into amyloid-β, a critical step in the cause of AD and DS. COL18A1 encodes the alpha chain of type XVIII collagen. It is associated with vascular deposits and senile plaques in AD brains25. The DYRK1A gene product can phosphorylate APP and alter the protein’s stability and the formation of amyloid-β26,27. Increased RCAN1 expression is associated with neuronal death and Tau hyperphosphorylation, as well as neurofibrillary tangle formation in DS and AD individuals28.\n\nUpSet plot showing geneset overlap highlighting gene content similarity between the AD-DS, Chr21, AD risk factors DEX DFC, and DEX CBC genes.\n\nSOD1 is the only gene present in all of the genesets. SOD1 is associated with apoptosis and oxidative stress29. The extra copy of SOD on Chr 21 results in increased gene expression and increased production of H2O2 which is believed to underlie many of the DS-related pathologies29. SOD1 is also associated with neurodegeneration in amyotrophic lateral sclerosis and AD30,31. SYNJ1 encodes a lipid phosphatase that is involved in autophagosomal/endosomal trafficking and synaptic vesicle recycling. Its dysfunction has been linked to several neurodegenerative diseases, including AD and DS32. S100β belongs to a family of cytokines that are strongly associated with activity underlying AD related pathologies such as APP processing, protein inclusion formation, and Tau post-translational modifications. S100β is also linked to DS. S100β levels are increased in neuronal progenitor cells of DS patients33 and in human induced pluripotent stem cells derived from DS patients34. Two additional genes, KLK6 and BCL2L, are shared among the AD-DS, AD risk factors, DEX DFC and DEX CBC genesets. KLK6 has been proposed as a biomarker for AD35. BCL2L is located on the outer mitochondrial membrane and is a negative regulator of apoptosis36.\n\nEach of the genesets were evaluated for association with AD and DS related phenotypes (Figure 2 and Extended data, Workbook 337). The keyword categories shared among all genesets are muscle, immune, insulin, glucose, behavior, oxidation and heart. The AD-DS geneset has a high frequency of genes associated with most of the keyword categories. The largest represented categories are: AD, muscle, inflammation/immune system, insulin, amyloid, behavior, aging, learning/memory, circadian processes and face/facial features. There were no genes directly associated with DS. For the Chr 21 geneset, unlike the AD-DS geneset, there were very few genes associated with the keyword categories. The highest frequency categories are immune, muscle, aging, behavior and insulin. Three genes are connected to AD (NDUFV3, APP and BACE2) and one with DS (DSCAM).\n\nIdentification of genes associated with terms relating to AD and DS based on gene ontology term classification. AD-DS genes: blue, Chr 21 genes: orange, DEX DFC genes: green, DEX CBC genes: gray, X-axis, keyword categories; Y-axis, frequency of occurrence in the geneset.\n\nThe enriched keyword categories for the DEX DFC are very similar to the results obtained for the AD-DS geneset: muscle, inflammation/immune system, insulin, aging, face/facial features, behavior, AD, and learning/memory. There are 13 genes directly associated with AD (NDUFS2, APAF1, BACE1, CACNA1F, COX5B, COX6A2, GRIN1, GRIN2A, LPL, PLD3, PSEN1, RYR3, UQCRC1) and one gene associated with DS (DSCR9). For the DEX CBC geneset the most representative categories are again similar to the AD-DS geneset as well as the DEX DFC geneset: muscle, immune/inflammation, insulin, behavior, face/facial features, aging and amyloid. There are four genes directly linked to AD (ATP5H, APOE, APAF1, RYR3). There are no genes directly associated with DS.\n\nGiven that behavioral phenotypes are highly shared between AD and DS, the specific types of behaviors identified from the keyword enrichment were evaluated more in depth. The AD-DS geneset has a large number of behavior related genes and genes related to learning and memory: (Behavior 33, Learning 26, Memory 21). This observation is based on the GO results obtained for three random genesets of the same size: Behavior 7,2,1; Learning 0,1,1; Memory: 0,0,1. The behavior gene categories are diverse and include fear, locomotion, eating and feeding, addiction related (nicotine, cocaine, ethanol), social, and others such as circadian, mating, and response to pain. The learning categories include visual learning, associative learning, and also olfactory, motor, and nonassociative learning. The memory related categories are short-term and long-term memory, and in one instance, susceptibility to memory impairment (Figure 3).\n\nComparison of frequencies of behavior related genes in the AD-DS, Chr 21, DEX DFC, and DEX CBC genesets. X-axis, geneset; Y-axis, frequency of occurrence (percentage of total genes) in the geneset.\n\nMany of the significant BP enrichment classifiers for the AD-DS geneset are associated with cell death (P=3.01E-83,) apoptosis (P=1.30E-70) and inflammation/immune system (P= 1.65E-36). For the Chr21 geneset, the significant BP enriched terms are linked to keratin (keratinization, P=1.04E-37), skin (skin development (P=2.83E-29) and epithelium processes (P=3.19E-15) as well as tissue (P= 3.56E-14), organ (P=3.40E-09) and anatomical structure development (P=8.66E-09). The significant pathways associated with the AD-DS geneset are related to neurodegenerative disorders (AD P=3.1E-23, Parkinson’s disease (P=1.39E-04) and Huntington’s disease P=1.36E-07) as well as many signaling pathways linked to insulin (P=1.86E-09) and inflammation (Jak/Stat P=9.49E-04), Toll receptor (P=4.04E-10), Interferon-gamma signaling (P=8.90E-06). There were no significant pathways associated with Chr 21. All pathways are listed in Extended data Workbook 438.\n\nThe transcription profiles of the AD-DS and Chr 21 genesets were evaluated here and compared with the DEX genesets which were previously evaluated by Olmos-Serrano et al.23 (Extended data, Workbook 539). There are 64 transcription factors present in the AD-DS geneset. Several of these are directly associated with AD (GSK3B, IL1B, MAPK3/8/10/14, WNT1, WNT3A, KAT5 NOTCH1 and TNF), Tau (GSK3B and CLU) and amyloid (CD36, NLRP3, CLU, FOXO3, PARP1, PRNP). Of these, many are related to mitochondria processes (AKT1, CLU, GSK3B, HIF1A, MAPK3,8,10,14, MTOR, NFKB1, PPARGC1A, PARP1, PRNP, PRKCA, SIRT1, STAT3, SREBF2, UBB) and also inflammation, oxidative stress, and aging (TP53, STAT1/3, NFKB1, HIF1A, and NEF2L2).\n\nFor the Chr 21 geneset, 18 transcription factors were identified. RUNX1 which is associated with ossification40 and nervous system development41 observed comparable expression in a study comparing AD and DS brains. Gene variants of RUNX1 are associated with both AD and DS42. The OLIG1/OLIG2 transcription factors regulate oligodendroglial differentiation and myelination and neuron fate commitment43. In DS, due to the gene triplication, OLIG1/OLIG2 causes alterations in brain development44. OLIG2 is associated with the psychotic symptoms of AD and also schizophrenia45. Of the Chr 21 transcription factors, only one is associated with mitochondria—GABPA— which is involved in the activation of cytochrome oxidase expression and nuclear control of mitochondrial function46.\n\nThere is one common transcription factor between the AD-DS and DEX-DFC genesets: NFE2L2 (also known as NRF2), which is associated with the oxidative stress response with aging, spatial learning, memory, and neuro-inflammmation via regulation of antioxidant response elements47,48. NFE2L2/NRF2 regulates BACE1, the rate-limiting enzyme for amyloid-β peptide (Aβ) generation. NRF2 activation decreases production of BACE1 and BACE1 antisense RNA (BACE1-AS) transcripts and Aβ production and ameliorates cognitive deficits in animal models of AD49. Depletion of NFE2L2/NRF2 increases BACE1 and BACE1-AS expression and Aβ production and worsens cognitive deficits50.\n\nThere are two transcription factors common between the AD-DS and DEX-CBC genesets. MTOR has been identified as a key target for therapeutic intervention in AD because of its regulation of several key signaling pathways: phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt), glycogen synthase kinase 3 [GSK-3], AMP-activated protein kinase (AMPK), and insulin/insulin-like growth factor 1 (IGF-1)51. Both upstream and downstream components of mTOR signaling are associated with AD progression and pathogenesis. MTOR inhibits autophagic processes and contributes to amyloid β-peptide generation and/or clearance52. MTOR activation also contributes to aberrant hyperphosphorylated tau53. The other common TF is NFKB1 which is a key regulator of innate immunity and strongly associated with the inflammatory response involving cytokines and chemokines54. NFKB1 is also linked to aging and AD55,56.\n\nAn APP protein-protein interaction network was created to identify genes from the genesets evaluated in this study that are connected to APP through 1st and 2nd shell interactions. A total of 362 proteins make up the network (Extended data Workbook 657).\n\nThe APP protein interaction network overlaps by 48 genes with the AD-DS geneset, 41 with the AD risk factor geneset, 21 with the DEX DFC, 12 with the DEX CBC geneset and four with the Chr 21 geneset. The shared genes are highlighted in the network to visualize and forecast additional genes that are potentially involved in APP signaling and that are relevant to both AD and DS (Figure 4A). The top proteins that bridge (bottlenecks) the different sections of the network and that may signify information flow are: APP, ENSG00000259680 (a novel protein coding gene with similarity to immunoglobulin heavy chain variable region.), SHC1, DLG4, STUB1, KLC1, GFA1, CENPJ, and GNO1.\n\n(A) Geneset overlap between 1st and 2nd shell interactions and the AD-DS, Chr 21, DEX DFC, and DEX CBC genesets. AD-DS genes unique: red; Chr 21 genes unique: gray; DEX DFC genes unique: purple; CBC genes unique: orange; AD risk factors (RF) and AD-DS genes shared: green; DEX DFC genes shared with RF & AD-DS genes: green oval; CBC and DFC shared genes: dark blue V; CBC genes shared with RF: green triangle; APP: yellow rectangle. (B) Interaction network gene clusters. Cluster 1: red – COP subunits, signalosome complex, development; Ubiquitin, Cluster 2: yellow – Tubulin, microtubules, motors, intracellular transport; Cluster 3: green – apoptosis, insulin signaling, ubiquitin, VEGFR growth factor signaling; Cluster 4: blue – Ubiqitin, autophagy; and Cluster 5: black – APP processing (PSEN, gamma secretase complex). (C) Distribution frequency for interaction score.\n\nThe APP network contains six major clusters (Figure 4B). Cluster 1: COP subunits, signalosome complex, development, ubiquitin; Cluster 2: TUBULIN, microtubules, motors, intracellular transport; Cluster 3: apoptosis, insulin signaling, ubiquitin, VEGFR growth factor signaling; Cluster 4: UBIQUITIN, autophagy; Cluster 5: APP processing (PSEN, gamma secretase complex); and Cluster 6: TUBULIN, microtubules.\n\nThe AD risk factor genes, Chr 21, and AD-DS genes are mostly dispersed throughout the network but a couple of areas in the network contain several connected AD risk factor genes. Predicted genes of interest based on their connectivity to these areas are METTL2B (tRNA methylation), IK (immune response), SAP18 (RNA splicing), QTRT1 (tRNA modification), APLP1 (Prion pathway), PRSS1( proteolysis, extracellular matrix digestion), ACSM1 (lipid metabolism), APBA2 (binds beta amyloid, synaptic transmission, and nervous system development). The validity of all of the interaction scores range from 0.4–1.00 and, for the most part, are uniformly distributed with 695 of the interactions falling in the low to mid-range of 0.4 and 0.7 and 617 falling in the mid to high-range of 0.7 and 1.0 (Figure 4C).\n\n\nConclusion\n\nGenesets associated with AD, DS, and Chr 21 were evaluated to identify genes, transcription factors, and pathways that may shed light on the relationship between AD and DS. Genes common to multiple genesets are either directly involved in APP processing or in TAU post translational modification. Many of the genes associated with the amyloid plaques in AD and DS function in learning and memory. A network analysis of APP protein-protein interactions was used to analyze the topology and connectivity of the genesets and, based on interactions with common AD-DS genes and AD risk factor genes, provide the foundation to predict potential genes of interest. Genes that connect the network and represent information flow as well as regions of high interconnectivity are also of interest for follow up studies. Given the central role of APP related processes in the pathology of AD and DS, all of the proteins in the APP interaction network are either potential risk factors for AD or may contribute to disease progression in both AD and DS. Taken together, our findings indicate that oxidative stress, cell death/apoptosis, and inflammation/immune system processes likely underlie the pathogenesis of both AD and DS.\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 Workbook 1. Genesets: AD-DS, Chr 21, AD risk factors, DEX DFC and CBC, https://doi.org/10.6084/m9.figshare.13106693.v121.\n\nFigshare: Extended Data Workbook 2. Common Genes: Gene overlap between the AD-DS, Chr 21, AD risk factors, DEX DFC and CBC genesets, https://doi.org/10.6084/m9.figshare.13106741.v124.\n\nFigshare: Extended Data Workbook 3. Keyword Gene Enrichment: Enrichment of the AD-DS, Chr 21, AD risk factors, DEX DFC and CBC genesets, https://doi.org/10.6084/m9.figshare.13106750.v137.\n\nFigshare: Extended Data Workbook 4. GO Terms and Pathways: Gene Ontology Biological Process terms and pathways associated with the AD-DS and Chr 21 genesets, https://doi.org/10.6084/m9.figshare.13106762.v138.\n\nFigshare: Extended Data Workbook 5. Transcription Factors: TFs present in the AD-DS, Chr 21 genesets, https://doi.org/10.6084/m9.figshare.13106774.v139.\n\nFigshare: Extended Data Workbook 6. APP Network File: APP protein-protein interaction network, https://doi.org/10.6084/m9.figshare.13106777.v157.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nRambaran RN, Serpell LC: Amyloid fibrils: abnormal protein assembly. Prion. 2008; 2(3): 112–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGouras GK, Olsson TT, Hansson O: β-Amyloid peptides and amyloid plaques in Alzheimer’s disease. Neurotherapeutics. 2015; 12(1): 3–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnnus T, Wilson LR, Hong YT, et al.: The pattern of amyloid accumulation in the brains of adults with Down syndrome. Alzheimers Dement. 2016; 12(5): 538–45. 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PubMed Abstract \| Publisher Full Text\n\nJones SV, Kounatidis I: Nuclear Factor-Kappa B and Alzheimer Disease, Unifying Genetic and Environmental Risk Factors from Cell to Humans. Front Immunol. 2017; 8: 1805. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelprato A: Extended Data Workbook 6. APP Network File: APP protein-protein interaction network. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13106777.v1" }	[ { "id": "74335", "date": "24 Nov 2020", "name": "Daniel W. Nixon", "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\nThis is a well-written paper that adds useful information to the literature. The connection between Down syndrome and Alzheimer’s disease is clinically well established and is currently not preventable, leading to increased difficulty in Down syndrome management. Genetic examination as in this paper should stimulate further molecular research in Down syndrome including the genetic influences on obesity, common in Down syndrome and a risk factor in Alzheimer's disease. I suggest further research by this group in adipokines such as leptin and adiponectin as well as interactions between Wnt, BACE, Notch, BCL and DYRK. This could lead to better understanding of the relationships between Down syndrome, Alzheimer’s disease, leukemia and solid tumors. Down syndrome is associated with an increased risk of leukemia and a decreased risk for solid tumors while Alzheimer’s disease is associated with a decreased risk for various solid tumors.\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": "7322", "date": "25 Oct 2021", "name": "Anna Delprato", "role": "Author Response", "response": "Reviewer 1: This is a well-written paper that adds useful information to the literature. The connection between Down syndrome and Alzheimer’s disease is clinically well established and is currently not preventable, leading to increased difficulty in Down syndrome management. Genetic examination as in this paper should stimulate further molecular research in Down syndrome including the genetic influences on obesity, common in Down syndrome and a risk factor in Alzheimer's disease. I suggest further research by this group in adipokines such as leptin and adiponectin as well as interactions between Wnt, BACE, Notch, BCL and DYRK. This could lead to better understanding of the relationships between Down syndrome, Alzheimer’s disease, leukemia and solid tumors. Down syndrome is associated with an increased risk of leukemia and a decreased risk for solid tumors while Alzheimer’s disease is associated with a decreased risk for various solid tumors. Response: Thank you for the encouraging review and the information pertaining to cancer and solid tumor formation in the context of AD and DS. We will keep this in mind for future studies." } ] }, { "id": "76772", "date": "08 Feb 2021", "name": "William C. Mobley", "expertise": [ "Reviewer Expertise Studies on the pathogenesis of AD in DS." ], "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\nSharma and colleagues have examined data for genetic contributions to the emergence of Alzheimer's disease (AD) in those with Down syndrome (DS). Their methods include the creation and/or mining of various genesets, including one for AD and DS derived from a MeSH query, as well as genesets representing Chromosome 21, AD risk factors from GWAS data, and differentially expressed genes in the DS cortex and cerebellum. Overlaps between genesets were examined to identify genes of interest, including tissues and biological processes, functional analyses, behavior and transcription as well an APP protein-protein interaction network. Overall, the findings confirm some expected associations and point to possible novel associations that may prove useful to those exploring the biology of AD in DS (AD-DS).\nHaving stated the positives, additional comments may prove useful to enhance the appreciation of the work. First, it should be clarified that APP is expressed in neurons wherein it is processed to Aβ peptides, some of which are found in plaques. It is not true that Aβ peptides are due to misprocessing of APP but instead that one of its products the Aβ42 peptide is preferentially deposited in plaques. An increase in the relative levels of Aβ42 to shorter Aβ species is cited as playing a role in pathogenesis, as well it might, but Aβ42 is nevertheless a normal product of APP. Note, however, also, that Aβ42 and other Aβ species are present in toxic oligomeric complexes that are now viewed as more significant for pathogenesis than are the amyloid plaques. A second comment concerns the section examining transcriptional profiles wherein a listing of differentially regulated genes is given. While the corresponding RNA levels corresponding to these genes differ in AD-DS versus controls only some of the genes listed encode transcription factors. I suspect the authors meant to convey that the products of these genes may impact transcription.\nIn the end, this paper will be important to the extent that it drives the testing of specific hypotheses. The large amount of data that comes from such analyses may not readily suggest follow-on studies. Given the extensive work invested here, the authors might choose to highlight a small subset of findings and predict relationships that, if further supported, would more incisively speak to the biology of AD in DS.\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": "7323", "date": "25 Oct 2021", "name": "Anna Delprato", "role": "Author Response", "response": "Reviewer 2: Sharma and colleagues have examined data for genetic contributions to the emergence of Alzheimer's disease (AD) in those with Down syndrome (DS). Their methods include the creation and/or mining of various genesets, including one for AD and DS derived from a MeSH query, as well as genesets representing Chromosome 21, AD risk factors from GWAS data, and differentially expressed genes in the DS cortex and cerebellum. Overlaps between genesets were examined to identify genes of interest, including tissues and biological processes, functional analyses, behavior and transcription as well an APP protein-protein interaction network. Overall, the findings confirm some expected associations and point to possible novel associations that may prove useful to those exploring the biology of AD in DS (AD-DS). Having stated the positives, additional comments may prove useful to enhance the appreciation of the work. First, it should be clarified that APP is expressed in neurons wherein it is processed to Aβ peptides, some of which are found in plaques. It is not true that Aβ peptides are due to misprocessing of APP but instead that one of its products the Aβ42 peptide is preferentially deposited in plaques. An increase in the relative levels of Aβ42 to shorter Aβ species is cited as playing a role in pathogenesis, as well it might, but Aβ42 is nevertheless a normal product of APP. Note, however, also, that Aβ42 and other Aβ species are present in toxic oligomeric complexes that are now viewed as more significant for pathogenesis than are the amyloid plaques. Response: We thank the reviewer for the clarification concerning the processing of Aβ peptides. We have included this information in the introduction of the revised manuscript. A second comment concerns the section examining transcriptional profiles wherein a listing of differentially regulated genes is given. While the corresponding RNA levels corresponding to these genes differ in AD-DS versus controls, only some of the genes listed encode transcription factors. I suspect the authors meant to convey that the products of these genes may impact transcription. Response: We have clarified this point in the revised manuscript. In the end, this paper will be important to the extent that it drives the testing of specific hypotheses. The large amount of data that comes from such analyses may not readily suggest follow-on studies. Given the extensive work invested here, the authors might choose to highlight a small subset of findings and predict relationships that, if further supported, would more incisively speak to the biology of AD in DS. Response: There is a subset of findings highlighted in the Results section of the abstract. We have added more context to emphasize the significance of these findings." } ] }, { "id": "77580", "date": "15 Feb 2021", "name": "Ann-Charlotte Granholm", "expertise": [ "Reviewer Expertise Down syndrome and Alzheimer's disease." ], "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 excellent genetic study of comparisons between AD and DS-related AD by Delprato and collaborators. Overall, this is an interesting and timely publication. The GeneSet overlay in Figure 1 is an excellent demonstration of the findings. There are only a few comments to be made and these are listed below.\nIn the abstract, last sentence, the authors state that “Our findings indicate that oxidative stress, apoptosis, and inflammation/immune system processes likely underlie the pathogenesis of AD and DS”. However, these contributors to AD in DS have already been known for quite some time, albeit maybe not at the genetic level. It would be better to reformulate this sentence to something like this: “Our findings confirm that oxidative stress, apoptosis, and inflammation/immune system processes likely contribute to AD in DS – these processes have been investigated in animal models and post mortem human tissues previously but not at the human genetic level”. Or something like that.\n\nIntroduction, first sentence: “stick together” should be replaced with “aggregate”.\n\nFirst paragraph of Introduction: “a protein that typically resides around nerve cells” should be replaced with “an integral membrane protein that is concentrated in synapses of neurons”. Introduction first paragraph: “Smaller aggregates of beta-amyloid and not the plaques themselves…” This could be more specific. For example “Oligomeric forms of the beta-amyloid, and not the beta-pleated sheets in plaques” or something like that.\n\nSecond paragraph of Introduction: “About half of the people with DS develop AD and the associated dementia around 50 to 60 years of age”. It is actually thought by most people now that 90% or more of those with DS developed AD – this should be referenced and corrected.\n\nThroughout the text, “AD individuals” or “DS individuals” should be replaced with “people or individuals with AD or DS” – those who have either condition do not wish to be defined by their disease. This is very important.\n\nGenerally, people refer to DS-related AD as DS-AD and not AD-DS, since the DS condition came first.\n\nLast sentence of Conclusions: it would not hurt to follow this statement with some literature citations that, indeed, confirm that these same processes have been proposed by others using both post mortem human tissue and animal models for DS. This would place these findings in the light of previous literature and acknowledge that these processes have been proposed by others and are here confirmed by gene expression 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? 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": "7324", "date": "25 Oct 2021", "name": "Anna Delprato", "role": "Author Response", "response": "Reviewer 3: This is an excellent genetic study of comparisons between AD and DS-related AD by Delprato and collaborators. Overall, this is an interesting and timely publication. The GeneSet overlay in Figure 1 is an excellent demonstration of the findings. There are only a few comments to be made and these are listed below. In the abstract, last sentence, the authors state that “Our findings indicate that oxidative stress, apoptosis, and inflammation/immune system processes likely underlie the pathogenesis of AD and DS”. However, these contributors to AD in DS have already been known for quite some time, albeit maybe not at the genetic level. It would be better to reformulate this sentence to something like this: “Our findings confirm that oxidative stress, apoptosis, and inflammation/immune system processes likely contribute to AD in DS – these processes have been investigated in animal models and post mortem human tissues previously but not at the human genetic level”. Or something like that. Response: Done. Introduction, first sentence: “stick together” should be replaced with “aggregate”. Response: Done. First paragraph of Introduction: “a protein that typically resides around nerve cells” should be replaced with “an integral membrane protein that is concentrated in synapses of neurons”. Response: Done. Introduction first paragraph: “Smaller aggregates of beta-amyloid and not the plaques themselves…” This could be more specific. For example “Oligomeric forms of the beta-amyloid, and not the beta-pleated sheets in plaques” or something like that. Response: Done. Second paragraph of Introduction: “About half of the people with DS develop AD and the associated dementia around 50 to 60 years of age”. It is actually thought by most people now that 90% or more of those with DS developed AD –this should be referenced and corrected. Response: We have added this information to the introduction. Throughout the text, “AD individuals” or “DS individuals” should be replaced with “people or individuals with AD or DS” – those who have either condition do not wish to be defined by their disease. This is very important. Response: Thank you for the correction. We have edited the text accordingly. Generally, people refer to DS-related AD as DS-AD and not AD-DS, since the DS condition came first. Response: To the best of our knowledge each instance of the “AD-DS” designation refers to the datasets and figures generated in the study and not the condition of AD in people with DS. Last sentence of Conclusions: it would not hurt to follow this statement with some literature citations that, indeed, confirm that these same processes have been proposed by others using both post mortem human tissue and animal models for DS. This would place these findings in the light of previous literature and acknowledge that these processes have been proposed by others and are here confirmed by gene expression studies. Response: The Conclusion section has been modified and references have been added." } ] }, { "id": "78528", "date": "09 Mar 2021", "name": "Joseph H. Lee", "expertise": [ "Reviewer Expertise Human genetics", "Genetic epidemiology", "Alzheimer’s disease", "Down syndrome", "Autosomal dominant forms of Alzheimer’s disease." ], "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 by Delprato, Sharma, and colleagues examined genetic and transcriptomic factors that may explain Alzheimer’s disease in Down syndrome by putting together five genesets: genes on chromosome 21; genes identified from the search term “Alzheimer’s Down syndrome” using GeneWeaver; genes identified from a large-scale genome-wide association study (GWAS); genes identified from the differential gene expression study of the dorsal frontal cortex (DFC) and cerebellar cortex (CBC) using the Down Syndrome Developmental Brain Transcriptome database. Subsequently, they performed a series of bioinformatic analyses to generate associated genes and concluded that oxidative stress, apoptosis, and inflammation/immune system processes were important biological processes in AD in DS. This paper is timely as an increasing number of genomic and transcriptomic studies are being carried out to better understand the biology of AD in adults with DS and the non-DS population. However, there are a few points that need to be clarified:\nThe title, “Common genetic signatures of Alzheimer’s disease in Down Syndrome,” suggests that this paper deals with genes that are likely to be involved in the neurodegenerative disease process in adults with Down syndrome. However, it is stated that the differential gene expression geneset was obtained from the Down Syndrome Developmental Brain Transcriptome Database, which included 15 sets of DS brains and matched control brains, where some of the brain specimens in the database were in embryonic stages. It is unclear how well these DS brain samples from embryonic stages would represent the neurodegenerative processes associated with AD in DS. It would be informative to repeat the analysis after excluding tissues that were from embryonic stages, if the sample size is sufficient.\n\nIn the Introduction (page 3, lines 4-8), it is stated that “Amyloids form fibrous structures and plaques … are involved in several diseases, such as Alzheimer’s disease (AD), Down Syndrome (DS), ….” Most would agree that adults with DS have a high risk of developing AD. However, it is unclear whether the formation of excess amyloids is biologically involved in the Down Syndrome itself, not just AD in DS. It should be clarified whether the wide range of DS phenotypes shown in Figure 2 can be attributed to the amyloid form fibrous structure and plaques.\n\nComparisons of the genes identified from AD-DS vs. DEX DFC and AD-DS vs. DEX CBC were interesting. It would be equally interesting to present DEX DFC vs. DEX CBC comparisons as they may represent genes involved in cognitive vs. motor processes.\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": [ { "c_id": "7325", "date": "25 Oct 2021", "name": "Anna Delprato", "role": "Author Response", "response": "Reviewer 4: APPROVED WITH RESERVATIONS This paper by Delprato, Sharma, and colleagues examined genetic and transcriptomic factors that may explain Alzheimer’s disease in Down syndrome by putting together five genesets: genes on chromosome 21; genes identified from the search term “Alzheimer’s Down syndrome” using GeneWeaver; genes identified from a large-scale genome-wide association study (GWAS); genes identified from the differential gene expression study of the dorsal frontal cortex (DFC) and cerebellar cortex (CBC) using the Down Syndrome Developmental Brain Transcriptome database. Subsequently, they performed a series of bioinformatic analyses to generate associated genes and concluded that oxidative stress, apoptosis, and inflammation/immune system processes were important biological processes in AD in DS. This paper is timely as an increasing number of genomic and transcriptomic studies are being carried out to better understand the biology of AD in adults with DS and the non-DS population. However, there are a few points that need to be clarified: The title, “Common genetic signatures of Alzheimer’s disease in Down Syndrome,” suggests that this paper deals with genes that are likely to be involved in the neurodegenerative disease process in adults with Down syndrome. However, it is stated that the differential gene expression geneset was obtained from the Down Syndrome Developmental Brain Transcriptome Database, which included 15 sets of DS brains and matched control brains, where some of the brain specimens in the database were in embryonic stages. It is unclear how well these DS brain samples from embryonic stages would represent the neurodegenerative processes associated with AD in DS. It would be informative to repeat the analysis after excluding tissues that were from embryonic stages, if the sample size is sufficient. Response: The aim of the study was not to investigate and compare the neurodegeneration process in DS and AD. Our aim was to analyze gene expression patterns associated with both disorders in the context of genes located on Chr 21 because individuals with DS have a third copy of this chromosome which contains the APP gene as well as other relevant genes. In the Introduction (page 3, lines 4-8), it is stated that “Amyloids form fibrous structures and plaques … are involved in several diseases, such as Alzheimer’s disease (AD), Down Syndrome (DS), ….” Most would agree that adults with DS have a high risk of developing AD. However, it is unclear whether the formation of excess amyloids is biologically involved in Down Syndrome itself, not just AD in DS. It should be clarified whether the wide range of DS phenotypes shown in Figure 2 can be attributed to the amyloid form fibrous structure and plaques. Response: We have addressed this point in the introduction by stressing that APP may account for some of the DS associated phenotypes but is most likely not solely responsible for the wide range of phenotypes associated with DS.. APP may account for some of the phenotypes but given the widespread alteration throughout the DS genome, an extra copy of APP is most likely not solely responsible for the multiple and varied phenotypes associated with DS. Comparisons of the genes identified from AD-DS vs. DEX DFC and AD-DS vs. DEX CBC were interesting. It would be equally interesting to present DEX DFC vs. DEX CBC comparisons as they may represent genes involved in cognitive vs. motor processes. Response: The DFC and CBC comparison of differentially expressed genes was performed in the original study (Olmos-Serranoon et al. 2016) on which the DS developmental transcriptome database was generated. These results are reported in this Table S5 Summarized here. The DFC Gene ontology enrichment analysis indicated potential dysregulated biological categories including transmission of nerve impulse (Benjamini-Hochberg-adjusted (BHA) p=2.9×10-8), synaptic transmission (BHA p=1.4×10-5), cell morphogenesis (BHA p=1.4×10-5), cell-cell signaling (BHA p=6.7×10-5), and axon ensheathment (BHA p=4.8×10-2) (Table S5). For the CBC, enriched terms for DNA, RNA processing as well as endocytosis and vesicle transport and budding. Also of interest, for the DFC the number of differentially expressed (DEX) genes increased in an age-dependent manner, with significantly more DEX genes emerging over postnatal development and adulthood (periods 8-14) (Figure 1D, and Table S4) whereas for the CBC the number of DEX genes remained constant." } ] } ]	1	https://f1000research.com/articles/9-1299
https://f1000research.com/articles/10-1081/v1	25 Oct 21	{ "type": "Review", "title": "A brief review of simulated Kalman Filter Algorithm – variants and applications", "authors": [ "Nor Hidayati Abdul Aziz", "Zuwairie Ibrahim", "Nor Azlina Ab. Aziz", "Mohd Saberi Mohamad", "Muhammad Razlan Kamaruzaman", "Mohd Saberi Mohamad", "Muhammad Razlan Kamaruzaman" ], "abstract": "Simulated Kalman Filter (SKF) solves optimization problems by finding the estimate of the optimum solution. As a multi-agent algorithm, every agent in the population acts as a Kalman filter by using a standard Kalman filter framework, which includes a simulated measurement process and a best-so-far solution as a reference. This paper presented an overview of the research progress in SKF from the day it was introduced until the present day, discussing the progress, improvements, modifications, and applications of SKF. The fundamental and standard algorithm were first introduced. Then, the work on the algorithm improvements was surveyed. Finally, the remaining unresolved problems and some directions of SKF research were discussed. We reviewed 57 SKF papers. 16 of them on fundamental improvements, 9 on extension of the algorithm to discrete problems and 25 on their applications. Researchers have worked on ideas to improve exploration capability to prevent premature convergence by trying prediction operators, opposition-based learning, and different iteration strategies. There were also attempts to hybridize SKF with other famous algorithms such as Particle Swarm Optimization (PSO), Gravitational Search Algorithm (GSA), and Sine Cosine Algorithm (SCA) to improve its performance. Lastly, a single-agent variant of SKF and a multi-objective SKF were introduced. SKF algorithms and its variants have been implemented in at least nine areas of applications: drill path optimization, airport gate allocation problem (AGAP), assembly sequence planning (ASP), system identification, feature selection, image template matching, controller tuning, wireless sensor network, and engineering design problem. The literature reviewed solely depended on the keyword search that contained the terms simulated Kalman filter from December 2015 to the present date. This is the first review paper on SKF. It is hoped that this survey would be beneficial for the researchers of this area and attracting interest towards the algorithm.", "keywords": [ "SKF", "review", "population-based", "single-agent", "continuous problems", "discrete problems", "single objective optimization", "multiobjective optimization" ], "content": "Introduction\n\nOptimization in terms of time and complexity is often needed in this technological era. Optimization helps in cost minimization and profit maximization. The complexity of the traditional exact optimization methods led to enormous amount of computational work which is not practical. Therefore, approximate methods especially metaheuristics are becoming popular. These general-purpose algorithms have lower complexity and offer good solutions.\n\nStudies of well-known Kalman Filter1 have contributed to the development of many algorithms in estimation as well as in other fields, such as optimization. In optimization, two optimizers have been developed based on Kalman filter. They are Heuristic Kalman Algorithm (HKA)2 and Simulated Kalman Filter (SKF).3 This paper emphasizes the fundamental advancements and applications of SKF, which originally proposed by Ibrahim et al. in 2015. The intention of this paper is to gather and briefly review all the papers related to SKF for the beneficial of students and researchers who would like to venture in this field.\n\n\nSimulated Kalman Filter (SKF) algorithm\n\nThe SKF, which is also an estimation-based metaheuristic algorithm4 was first introduced as a solution to unimodal optimization problems.3 A year later, it was tested on various optimization problems and found to be a promising optimizer.5\n\nIn principle, SKF tries to solve an optimization problem by estimating the optimum solution. By taking the inspiration from the Kalman filter algorithm, each agent in the SKF will go through the same three-step process available in the Kalman filter that consist of prediction, measurement and estimation. The SKF uses the same prediction and measurement equation as in the Kalman filter. Moreover, since the measurement in Kalman filter estimation comes from sensors, the measurement is simulated in the optimization algorithm, thus the algorithm is named Simulated Kalman Filter (SKF).\n\nFigure 1 shows the flowchart of the SKF algorithm. The optimization process starts with the initialization of solutions, followed by the fitness evaluation of each solution, and then the generation of new solutions for the next iteration. This process will stop when the stopping criteria is met. To further understand how the SKF works, a tutorial published in 2019 can be referred.6 In addition, further studies of the SKF algorithm revealed that the P, Q and R parameters can be replaced with random numbers ∈01 without affecting the performance of the algorithm.7-9\n\n\nThe SKF improvements\n\nThe exploration in the SKF algorithm solely depends on the measurement equation. The sine operator in the measurement equation gives a 50-50 chance for exploration to occur in the measurement phase. In the estimation phase, exploitation is set to take place. The predicted value is updated by a multiplication of two small numbers (the Kalman gain and the distance between the predicted and measurement value). This may cause the SKF algorithm to prematurely converge.\n\nIn 2018, Ibrahim et al. introduced Opposition-Based Learning (OBL) concept in SKF to overcome the problem of premature convergent. In SKF with oppositional-learning prediction operator (SKF-OPO), the opposition population is generated around the best-so-far solution identified in the prediction phase.10 If the opposite prediction has a better fitness value than the original prediction (which is the previous estimated solution), the opposite prediction will be used as the predicted solution. This method proves that a proper prediction operator will help SKF to escape from premature convergence. Further experiments were conducted to observe the impact of opposition population generation in SKF-OPO with the introduction of jumping rate.11 This jumping rate is later compared to a random number between 0-1. If the random number is smaller than the jumping rate, then the opposition population will be generated at prediction phase. Results show that the higher the jumping rate, the better the performance of the algorithm. Another variation of OBL implementation in SKF was introduced in 2019 by Mohd Azmi et al. known as Current Optimum Opposition-Based Learning SKF (COOB-SKF). In this algorithm, the formation of the opposite population uses the best-so-far solution as the center between the estimate population and the opposition estimate population.12\n\nThe original population-based SKF algorithm uses the synchronous update mechanism, in which all agents have to go through all optimization steps before the best-so-far solution is updated. However, in 2018, Ab. Aziz et al. found that when the update strategy of SKF is made individual-oriented, the results are better.\n\nThe researchers later explore the possibility to use both mechanisms in SKF. There are three variants of SKF adaptive switching algorithms: the fitness-based adaptive switching synchronous-asynchronous SKF (ASSA-SKF), fitness-evaluated adaptive switching SKF with randomness (ASSKFR), and diversity-based adaptive switching synchronous-asynchronous SKF (DASSA-SKF). In ASSA-SKF, the SKF algorithm starts with synchronous update and later changes its update mechanism when the fitness is found to be static for a number of fitness evaluations.13 In ASSKFR, the switching happens when the switching counter is greater than a random number which is chosen every time a switching occurs.14 One may choose to start with synchronous update or with asynchronous update. Lastly in DASSA-SKF, instead of using fitness as the switching indicator, the decision to switch depends on the diversity of the population.15 Every time the diversity of the population is not changing for a certain number of iterations, then the iteration strategy will switch from synchronous update to asynchronous update and vice versa. All findings have shown that the algorithms benefit when there is a greater number of switching happening, thus encouraging more explorations.\n\nHybridization between algorithms can be used to improve an algorithm’s performance. SKF has been subjected to hybridization with three algorithms: the Particle Swarm Optimization (PSO),16 the Gravitational Search Algorithm (GSA),17 and the Sine Cosine Algorithm (SCA).18\n\nMuhammad et al. have proposed four ways to show how GSA19 and PSO20 can be hybridized with SKF during its prediction step due to the absence of prediction operator in SKF.\n\n\n\n• Model 1: GSA/PSO as prediction operator\n\n• Model 2: GSA/PSO as prediction operator when a better solution is found\n\n• Model 3: GSA/PSO as prediction operator with jumping rate\n\n• Model 4: GSA/PSO as prediction operator with jumping rate and when a better solution is found\n\nIn another paper, although the fourth model has a better performance compared to the original SKF algorithm, this method is not the best hybrid method for SKF-GSA and SKF-PSO.21\n\nA fairly recent SKF hybrid is the hybridization between SKF with SCA in 2018.18 The SCA algorithm is formulated using the mathematical sine and cosine term. In the hybrid version of SKF and SCA, namely the Kalman Filter-based Sine Cosine Algorithm (KFSCA), the prediction and estimation phases of SKF are implemented in SCA. Instead of using the simulated measurement equation of SKF during the measurement phase, SCA equations are used to update the individual agent’s position. Five unimodal benchmark functions are used to compare the performance of the hybrid KFSCA algorithm with the original SKF and SCA algorithm. The statistical results showed that the KFSCA performed significantly better than the original SKF and SCA algorithms, and had a higher convergence rate compared to the original SKF algorithm.\n\nHigh convergence rate is a signature for all Kalman filter-based algorithms. While most researchers tried many ways to introduce more explorations to prevent the SKF algorithm from prematurely converging due to its fast convergence speed, Mat Jusof et al. opined that a faster convergence speed was favored especially in solving unimodal problems.22 In his paper, Simulated Kalman Filter with Improved Accuracy, a new exponential-based SKF named SKFIA was introduced. This SKFIA algorithm uses modified equations in the estimation phase where exponential term is introduced in the calculation of the Kalman gain and the corresponding error covariance. Instead of using the suggestion constant for measurement noise, an exponential term is added in the calculation of the measurement noise and is made dependent on the predicted error covariance. This is to enable a large step size at the beginning of the search and smaller size towards the end. Performance evaluation comparing the SKFIA with the original SKF using the first four benchmark functions of CEC2014 (3 unimodal and 1 multimodal function) shows SKFIA is able to find better results compared to the original SKF.\n\n\nSKF extensions\n\nBesides being subjected to various modifications to improve its performance, SKF also has been modified to extend its usage. Three main extension of SKF are SKF algorithms for discrete problems, single solution version of SKF algorithm, and SKF for multi-objective problems.\n\nThe first extension of SKF algorithm is to make it available to solve discrete problems. There are three approaches proposed by Md. Yusof et al. in solving combinatorial optimization problems using SKF, giving birth to three new modified algorithms: the binary SKF (BSKF), angle modulated SKF (AMSKF) and distance evaluated SKF (DESKF).23\n\nRight after the introduction of SKF, Md. Yusof et al. published Binary SKF (BSKF) algorithm to enable SKF to operate in binary search space BSKF.24 This is followed by the publication of DESKF and AMSKF a year later.25,26 Another variant of the DESKF is the local optimum DESKF algorithm27 and the DESKF with state encoding.28 All these discrete SKF algorithms are meant to solve binary problems. The state-encoded DESKF (SEDESKF) was introduced in 2018 to solve combinatorial optimization problems that were not in binary search space.\n\nIn the analysis on the number of agents towards the performance of the SKF optimizer, the results showed that the SKF algorithm performed its best at a surprisingly large population of 700 agents.29 Due to this reason, another variant of the SKF algorithm was introduced in 2018 which was a single agent version of the SKF algorithm.30 In order to use the same equations as the SKF algorithm, a prediction operator needs to be introduced during the prediction phase. Thus, a simple local adaptive neighborhood method centered around the best-found solution so far is used to predict the location of the optimum solution. However, the introduction of this new adaptive prediction operator introduces another parameter. The ssSKF algorithm with adaptive coefficient value of 5 is proven to be significantly better than the original SKF of 100 agents. Another tutorial published in 2019 can be referred to understand the difference between the population-based and the single-solution algorithm.31 The parameter tuning analysis published last year revealed that ssSKF performed best when the adaptive coefficient was set at 10.32\n\nThe last but quite significant extension of SKF is the introduction of multi-objective SKF (MOSKF).33 Research on multi-objective algorithm is gaining attention worldwide because it deals with problems with two or more objectives that might be conflicting to one another. The SKF is transformed into MOSKF by applying the non-dominated sorting approach. Each agent is associated with cost function value and diversity spacing parameter. Besides that, the mutation and crossover operators are also adopted in MOSKF. Only a limited number of agents undergo mutations and crossovers to reduce the complexity of the algorithm while at the same time promote randomness. The experimental results on three multi-objective benchmark functions showed MOSKF performed better than the Non-dominated Sorting Genetic Algorithm II. However, this MOSKF has no real-world application or has not been subjected to solve more complex problems yet.\n\n\nSKF applications\n\nApplications of SKF algorithm and its variants can be categorized into nine different area of applications:\n\n\n\nA. Drill path optimization\n\nB. Airport gate allocation problem (AGAP)\n\nC. Assembly sequence planning (ASP)\n\nD. System identification\n\nE. Feature selection\n\nF. Image template matching\n\nG. Controller tuning\n\nH. Wireless sensor network\n\nI. Engineering design problem\n\nTable 1 shows the summary of applications of SKF algorithm and its variants in the 9 fields mentioned above.\n\nSKF: simulated Kalman filter, SKF+OBL: SKF – opposition-based learning, SSSKF: single solution SKF, BSKF: binary SKF, AMSKF: angle modulated SKF, DESKF: distance evaluated SKF.\n\n\nConclusions\n\nIn this paper, we provided a brief review of SKF, its improvements, extensions and applications. It does not include any comparison study between SKF and other algorithms. Since the introduction of SKF at the end of 2015, more than 57 papers have been published. The majority of them focused on methods to improve the SKF performance and their applications in various fields due to its simple implementation and fast convergence behavior. However, additional works might need to be done to test the algorithms performance to solve more complex applications and large-scale problems, especially for multi-objective optimization. Another interesting area to explore is on improving the exploration of the single agent version of SKF since no single work has been carried out yet for the ssSKF algorithm.\n\n\nData availability\n\nNo data associated with this paper.\n\n\nAuthor contributions\n\nNHAA & NAAA: wrote most of the manuscript.\n\nZI & MSM: revised the manuscript.\n\nMRK: compiled all SKF related papers.", "appendix": "Acknowledgements\n\nThe authors would like to acknowledge all parties directly or indirectly involved in the preparation of this review paper.\n\n\nReferences\n\nKalman RE: A new approach to linear filtering and prediction problems. J. Fluids Eng. Trans. ASME. 1960; 82(1): 35–45. Publisher Full Text\n\nToscano R, Lyonnet P: Heuristic kalman algorithm for solving optimization problems. IEEE Trans. Syst. 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Publisher Full Text\n\nAb Aziz NA, Aziz ZI, Aziz NHA, et al.: A fitness-based adaptive synchronous-asynchronous switching in simulated kalman filter optimizer. 2019 Int Conf Computer Information Sci, ICCIS 2019. May 2019: 1–5. Publisher Full Text\n\nAb Aziz NA, Rahman TA, Aziz NHA: Fitness-evaluated Adaptive Switching Simulated Kalman Filter Algorithm with Randomness. Mekatronika. 2019; 1(2): 45–65. Publisher Full Text\n\nAb Aziz NA, et al.: A Diversity-Based Adaptive Synchronous-Asynchronous Switching Simulated Kalman Filter Optimizer. 2019; 632(July).\n\nMuhammad B, et al.: A new hybrid simulated Kalman filter and particle swarm optimization for continuous numerical optimization problems. ARPN J. Eng. Appl. Sci. 2015; 10(22): 17171–17176.\n\nMuhammad B, Ibrahim Z, Mat Jusof MF, et al.: A Hybrid Simulated Kalman Filter - Gravitational Search Algorithm (SKF-GSA). Proc. Int. Conf. Artif. Life Robot. 2017; 22(1): 707–710. Publisher Full Text\n\nMat Jusof MF, et al.: A Kalman-Filter-Based Sine-Cosine Algorithm. 2018 IEEE Int Conf Automatic Control Intelligent Systems (I2CACIS 2018). 2018: 137–141.\n\nMuhammad B, et al.: Four Different Methods to Hybrid Simulated Kalman Filter (SKF) with Gravitational Search Algorithm (GSA). National Conf Postgraduate Res. 2016. 2016: 854–864.\n\nMuhammad B, et al.: Four Different Methods to Hybrid Simulated Kalman Filter (SKF) with Particle Swarm Optimization (PSO). National Conf Postgraduate Res. 2016. 2016: 843–853.\n\nMuhammad B, et al.: Performance Evaluation of Hybrid SKF Algorithms: Hybrid SKF-PSO and Hybrid SKF-GSA. Natl. Conf. Postgrad. Res. 2016; 2016: 865–874.\n\nMat Jusof MF, et al.: Simulated Kalman Filter Algorithm with Improved Accuracy. 2019; vol. 538. . Springer Singapore.\n\nMd Yusof Z, et al.: Three Approaches to Solve Combinatorial Optimization Problems using Simulated Kalman Filter. National Conf Postgraduate Res 2016. 2016: 951–960.\n\nMd Yusof Z, Ibrahim I, Satiman SN, et al.: BSKF: Simulated kalman filter. Proc - AIMS 2015, 3rd Int Conf Artificial Intelligence, Modelling Simulation. 2015: 77–81. Publisher Full Text\n\nMd Yusof Z, et al.: Distance evaluated simulated Kalman filter algorithm for combinatorial optimization problems. ARPN J. Eng. Appl. Sci. 2016; 11(7): 4911–4916.Reference Source\n\nMd Yusof Z, et al.: Angle modulated simulated kalman filter algorithm for combinatorial optimization problems. ARPN J. Eng. Appl. Sci. 2016; 11(7): 4854–4859.\n\nMd Yusof Z, et al.: Local Optimum Distance Evaluated Simulated Kalman Filter for Combinatorial Optimization Problems. National Conf Postgraduate Res 2016, Universiti Malaysia Pahang. 2016: 892–901.\n\nMd Yusof Z, et al.: Distance evaluated simulated kalman filter with state encoding for combinatorial optimization problems. Int. J. Eng. Technol. 2018; 7(4): 22–29. Publisher Full Text\n\nAbdul Aziz NH, et al.: An analysis on the number of agents towards the performance of the simulated kalman filter optimizer. Proc. - Int. Conf. Intell. Syst. Model. Simulation, ISMS. 2018; (2018-May): 16, 21. Publisher Full Text\n\nAbdul Aziz NH, Ibrahim Z, Ab Aziz NA, et al.: Single-solution Simulated Kalman Filter algorithm for global optimisation problems. Sadhana - Acad. Proc. Eng. Sci. 2018; 43(7): 1–15. Publisher Full Text\n\nAbdul Aziz NH, et al.: A Tutorial on Single-solution Simulated Kalman Filter. Mekatronika. 2019; 1(2): 33–44. Publisher Full Text\n\nAbdul Aziz NH, et al.: Parameter tuning in the single-solution simulated Kalman filter optimizer. Lecture Notes in Mechanical Engineering. Springer;2020, pp. 48–56.\n\nAzwan A, Razak A, Jusof MFM, et al.: A multiobjective simulated Kalman filter optimization algorithm. Proc 4th IEEE Int Conf Applied System Innovation 2018, ICASI 2018. 2018: 23–26. Publisher Full Text\n\nAziz NHA, Ab Aziz NA, Ibrahim Z, et al.: A Kalman Filter approach to PCB drill path optimization problem. Proc - 2016 IEEE Conf Systems, Process and Control, ICSPC 2016. 2017: 33–36. Publisher Full Text\n\nAziz NHA, Ibrahim Z, Aziz NAA, et al.: Single-solution simulated kalman filter algorithm for routing in printed circuit board drilling process. Lecture Notes in Mechanical Engineering. 2018: 649–655.\n\nMd Yusof Z, et al.: Solving Airport Gate Allocation Problem using Simulated Kalman Filter. Int Conf Knowledge Transfer. 2015; (December): 1–3.\n\nMohd Azmi KZ, et al.: Solving airport gate allocation problem using angle modulated simulated Kalman filter. National Conf Postgraduate Res 2016. 2016: 875–885.\n\nMustapa A, Yusof ZM, Adam A, et al.: Solving Assembly Sequence Planning using Angle Modulated Simulated Kalman Filter. IOP Conf. Ser. Mater. Sci. Eng. 2018; vol. 319, no. 1, pp. 0–6. Publisher Full Text\n\nMustapa A, Ibrahim Z, Md Yusof Z, et al.: Solving Assembly Sequence Planning using Distance Evaluated Simulated Kalman Filter. J. Intell. Manuf. Mechatronics. 2019; 319(02): 012044–012047. Publisher Full Text\n\nMohd Azmi KZ, Ibrahim Z, Pebrianti D, et al.: Simultaneous computation of model order and parameter estimation for ARX model based on single Swarm and Multi Swarm simulated Kalman filter. J. Telecommun. Electron. Comput. Eng. 2017; 9(1–3): 151–155.\n\nMuhammad B, Mohd Azmi KZ, Ibrahim Z, et al.: Simultaneous computation of model order and parameter estimation for system identification based on opposition-based simulated Kalman Filter. 2018 SICE International Symposium on COntrol Systems (SICE ISCS). 2018: 105–112.\n\nAhmad Zamri N, Bhuvaneswari T, Aziz NAA, et al.: Feature selection using simulated Kalman filter (SKF) for prediction of body fat percentage. ACM Int Conf Proc Series. 2018: 23–27. Publisher Full Text\n\nMuhammad B, et al.: Feature selection using binary simulated kalman filter for peak classification of EEG signals. Proc - Int Conf Intelligent Systems, Modelling Simulation, ISMS. 2018: 1–6. Publisher Full Text\n\nAdam A, Ibrahim Z, Mokhtar N, et al.: Feature selection using angle modulated simulated Kalman filter for peak classification of EEG signals. Springerplus. 2016; 5(1580): 1–24. Publisher Full Text\n\nAdam A, Muhammad B: Distance evaluated simulated kalman filter algorithm for peak classification of EEG signals. Int. J. Simul. Syst. Sci. Technol. 2018; 19(5): 6.1–6.7. Publisher Full Text\n\nAnn NQ, Pebrianti D, Ibrahim Z, et al.: Illumination-invariant image matching based on Simulated Kalman Filter (SKF) algorithm. J. Telecommun. Electron. Comput. Eng. 2018; 10(1–3): 31–36.\n\nAnn NQ, et al.: SKF-based image template matching for distance measurement by using stereo vision. Springer Singapore;2018.\n\nAnn NQ, Pebrianti D, Ibrahim Z, et al.: Image template matching based on Simulated Kalman Filter (SKF) algorithm. J. Telecommun. Electron. Comput. Eng. 2018; 10(2–7): 37–41.\n\nMuhammad B, et al.: An application of simulated Kalman filter optimization algorithm for parameter tuning in proportional-integral-derivative controllers for automatic voltage regulator system. SICE ISCS 2018-2018 SICE International Symposium on Control Systems. 2018: 113–120. Publisher Full Text\n\nMat Jusof MF, Nasir ANK, Ahmad MA, et al.: An exponential based simulated Kalman filter algorithm for data-driven PID tuning in liquid slosh controller. Proc 4th IEEE Int Conf Applied System Innovation 2018, ICASI 2018. 2018: 984–987. Publisher Full Text\n\nRamli MS, Sian SM, Salim MN, et al.: A Fictitious Reference Iterative Tuning Method for Buck Converter-Powered DC Motor Control System. Lecture Notes in Electrical Engineering. 2019: 47–58.\n\nTaha Z, et al.: The control of an upper extremity exoskeleton for stroke rehabilitation by means of a hybrid active force control. Springer International Publishing;2019; vol. 751. .\n\nAb Aziz NA, Ibrahim Z, Aziz NHA, et al.: Simulated kalman filter optimization algorithm for maximization of wireless sensor networks coverage.May 2019. Publisher Full Text\n\nLazarus K, Noordin NH, Mat Jusof MF, et al.: Adaptive Beamforming Algorithm based on a Simulated Kalman Filter. Int. J. Simul. Syst. Sci. Technol. 2016; 18(4): 10.1–10.5. Publisher Full Text\n\nLazarus K, Noordin NH, Mohd Azmi KZ, et al.: Adaptive Beamforming Algorithm based on Generalized Opposition-based Simulated Kalman Filter. National Conf Postgraduate Res 2016, Universiti Malaysia Pahang. 2016: 1–9.\n\nLazarus K, et al.: An opposition-based simulated kalman filter algorithm for adaptive beamforming. Proc. 2017 IEEE Int. Conf. Appl. Syst. Innov. Appl. Syst. Innov. Mod. Technol. ICASI 2017. 2017; 4: 91–94. Publisher Full Text\n\nLazarus K, Noordin NH, Ibrahim Z: An Enhanced Simulated Kalman Filter Algorithm and its Application in Adaptive Beamforming. RFM 2018-2018 IEEE International RF and Microwave Conference, Proceedings. 2018: 321–324. Publisher Full Text\n\nFauzi H, Batool U: A Three-bar Truss Design using Single-solution Simulated Kalman Filter Optimizer. Mekatronika. 2019; 1(2): 98–102. Publisher Full Text\n\nAzzam MA, Batool U, Fauzi H: Design of an Helical Spring using Single-solution Simulated Kalman Filter Optimizer. Mekatronika. 2019; 1(2): 93–97. Publisher Full Text" }	[ { "id": "120972", "date": "21 Feb 2022", "name": "Roberta Veloso Garcia", "expertise": [ "Reviewer Expertise Specialist in optimal estimation methods", "with emphasis on Kalman filters applied to aerospace 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 purpose of the article is to present an overview of the progress of SKF research from the day it was introduced to the present day, discussing SKF progress, improvements, modifications and applications. The importance of the proposal is due to the researchers' work on ideas to improve SKF's exploitability to avoid premature convergence of the algorithm, trying prediction operators, opposition-based learning and different iteration strategies. There have also been attempts to hybridize SKF with other algorithms to improve their performance. A SKF single-agent variant and a multi-purpose SKF were also introduced. SKF algorithms and their variants have been implemented in at least nine application areas, all cited in this article.\nSome comments and suggestions are pointed out below, in order to resolve doubts and contribute to the excellence of the work:\nThe subject reviewed is relevant, since studies that approach optimal estimation have been the object of interest of several researchers in different areas. In addition, having an overview of how such studies have been advancing provides a more robust parameter for researchers to dedicate themselves to areas that have not yet been explored.\n\nIn Figure 1, several fundamental terms and indices for the KF appear. I suggest defining them in the text.\n\nIn “The SKF Improvement”, the so-called premature convergence is cited in several studies. In this case, the authors must define what this convergence means, in addition to explaining why it is not appropriate, since the convergence of filters is always something desirable.\n\nOn page 5, 1st paragraph, I suggest that the authors detail better why and in what aspects the 4th model is better than the hybrid methods (SKF-GSA and SKF-PSO).\n\nIn table 1, check the writing “Modi-fied SKF” or “Modified SKF”.\n\nIn “SKF Applications”, the authors were too succinct. I suggest, if possible, detailing why the algorithms were chosen for the cited applications. Note that the Modi-fied SKF algorithm is in only one application and SKF is in practically all applications. What is this fact due to?\n\nThe conclusions were superficial, given the importance of the work. Even though it is a review article, it is necessary to explore in greater detail the improvements obtained and under which applications they are most relevant in relation to SKF. The authors cite that of the more than 57 published articles, most focus on methods to improve SKF performance. However, in table 1, it is noted that in terms of applications, SKF is still the most discussed. Is there any explanation, since the purpose of improving the performance of algorithms is to have better results in applications?\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? Partly", "responses": [] }, { "id": "126077", "date": "21 Apr 2022", "name": "Komeil Nosrati", "expertise": [ "Reviewer Expertise Kalman Filtering", "Estimation", "Fractional Calculus and Descriptor Systems" ], "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 my opinion, the methods in 57 papers will be compared together. Their advantages and disadvantages need to be listed in a table. Some simulation and example can make the topic clearer and help the readers to make a difference between the presented algorithms. I am also wondering if there is any work related to the nonlinear SKF. I think the literature review has not been covered completely.\nAlso, the structure and organization of the paper needs to be modified. In case, the abstract is too long, and the introduction is too short.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1081
https://f1000research.com/articles/10-1079/v1	25 Oct 21	{ "type": "Research Article", "title": "Neural matrix factorization++ based recommendation system", "authors": [ "Kyle Ong", "Kok-Why Ng", "Su-Cheng Haw", "Kyle Ong", "Su-Cheng Haw" ], "abstract": "In recent years, Recommender System (RS) research work has covered a wide variety of Artificial Intelligence techniques, ranging from traditional Matrix Factorization (MF) to complex Deep Neural Networks (DNN). Traditional Collaborative Filtering (CF) recommendation methods such as MF, have limited learning capabilities as it only considers the linear combination between user and item vectors. For learning non-linear relationships, methods like Neural Collaborative Filtering (NCF) incorporate DNN into CF methods. Though, CF methods still suffer from cold start and data sparsity. This paper proposes an improved hybrid-based RS, namely Neural Matrix Factorization++ (NeuMF++), for effectively learning user and item features to improve recommendation accuracy and alleviate cold start and data sparsity. NeuMF++ is proposed by incorporating effective latent representation into NeuMF via Stacked Denoising Autoencoders (SDAE). NeuMF++ can also be seen as the fusion of GMF++ and MLP++. NeuMF is an NCF framework which associates with GMF (Generalized Matrix Factorization) and MLP (Multilayer Perceptrons). NeuMF achieves state-of-the-art results due to the integration of GMF linearity and MLP non-linearity. Concurrently, incorporating latent representations has shown tremendous improvement in GMF and MLP, which result in GMF++ and MLP++. Latent representation obtained through the SDAEs’ latent space allows NeuMF++ to effectively learn user and item features, significantly enhancing its learning capability. However, sharing feature extractions among GMF++ and MLP++ in NeuMF++ might hinder its performance. Hence, allowing GMF++ and MLP++ to learn separate features provides more flexibility and greatly improves its performance. Experiments performed on a real-world dataset have demonstrated that NeuMF++ achieves an outstanding result of a test root-mean-square error of 0.8681. In future work, we can extend NeuMF++ by introducing other auxiliary information like text or images. Different neural network building blocks can also be integrated into NeuMF++ to form a more robust recommendation model.", "keywords": [ "Recommender System", "Matrix Factorization", "Collaborative Filtering", "Deep Neural Networks", "Neural Collaborative Filtering." ], "content": "Introduction\n\nCollaborative Filtering (CF) based Recommender System (RS) typically suggests items based on user-item interactions. Users’ interests are predicted based on analyzing other users’ tastes and preferences in the system. Matrix Factorization (MF),1 popularized by the Netflix price,2 has emerged as a powerful CF recommendation tool. However, its simple interaction function, which is the inner product, has hindered its performance. Not to mention that CF methods also suffer from cold start and data sparsity.\n\nMuch effort has been devoted to improving MF’s accuracy throughout the years, but one approach that has caught much attention is deep learning (DL). DL has drastically improved MF’s accuracy by exploiting deep neural networks (DNN). Eventually, many researchers have also suggested incorporating side information into CF methods. This subsequently forms a hybrid-based (HB) method that solves CF’s cold start and data sparsity.3\n\nIn this paper, we proposed a novel hybrid-based RS named Neural Matrix Factorization ++ (NeuMF++). NeuMF++ is an improved version of NeuMF that incorporates an effective latent representation of side information via Stacked Denoising Autoencoders (SDAEs). In the original work, NeuMF has achieved outstanding results. It is surprising to see that not much prior work has been done to enhance NeuMF. In NeuMF++, SDAEs extract high-level representations from side information and later incorporate them as latent feature vectors. Incorporating user-item features in the learning process enhances its learning capabilities and improves its recommendation performance. Experiments on a real-world dataset have demonstrated the effectiveness of side information in NeuMF++, yielding state-of-the-art results.\n\nThe rest of the paper is organized as follows. Section 2 discusses the related work. Section 3 introduces our proposed framework, NeuMF++, in detail. Section 4 discusses the result. Finally, section 5 summarizes the paper and briefly introduces our future work.\n\n\nRelated work\n\nThere are different DL models ranging from standard Multilayer Perceptrons (MLP) to Convolutional Neural Network (CNN). DL models like MLP are utilized to add the non-linear transformation to existing linear techniques and interpret them as neural extensions.4,5 NCF frameworks,2 which include Generalized MF (GMF), MLP and NeuMF, make use of DNN into traditional MF to further enhance its recommendation performance and quality. The differences between the three models are their interaction functions. GMF uses a linear kernel by taking user and item latent vectors and multiplying them element by element (element-wise product). In contrast, MLP uses a non-linear kernel by concatenating user and item latent vectors and then fully connects to an MLP. Lastly, NeuMF integrates the linearity of GMF and non-linearity MLP by combining both of their outputs with a single-layer MLP.\n\nAnother popular DL model is the Autoencoder (AE). AE is a powerful tool for dimensionality reduction and can be considered a strict generalization of Principal Component Analysis. It aims to reconstruct the input data as output. Many popular MF techniques can be thought of as a form of dimensionality reduction.3 Therefore, AE can be adapted for this task as well, such as AutoRec.6 Subsequently,7 further enhances AutoRec by training it much deeper, which aids the network to generalize better8 proposed Collaborative Denoising Autoencoder, which utilized a Denoising Autoencoder (DAE) to perform CF tasks. Noises are added intentionally to the rating input and reconstructing the original rating input as the output. This allows the network to be more noise-resistant and helps it to learn more stable features.\n\nMost studies only focus on ratings, but ratings alone are unable to reveal user-item relation fully. Additionally, most CF methods also suffer from cold start and data sparsity. Hence, several researchers suggested incorporating side information into the model, forming an HB method3,8 proposed a new HB method known as CF Network (CFN). Instead of only adding the side information into the first layer, the author injected that information into every layer except the output layer of the network.\n\nHowever, most AE-based CFs utilize side information as regularization in their models. However, due to the sparse nature of the rating matrix together with side information, the learned latent vectors might not be very effective. Therefore,9 introduced Collaborative Deep Learning (CDL), in which a DAE learns item features and is then utilized as an item latent vector for MF. Subsequently,10 proposed a marginalized DAE for CF (mDA-CF), an extension of CDL by adding user latent vectors learned by another AE. The key of mDA-CF is to extract user and item features from mDAs and combine them in a joint framework.\n\nEven though both CFL and mDA-CF utilize DNN to improve recommendation performance, their CF’s core is still a linear MF. Therefore,3 proposed two models 一 GMF++ and MLP++. GMF++/MLP++ enhances the GMF/MLP of the NCF frameworks by incorporating user and item latent vectors extracted from SDAEs into neural collaborative filtering.\n\n\nMethods\n\nThe real-world dataset was obtained from the GroupLens Research Project. The GroupLens Research Project is a research group in the Department of Computer Science and Engineering at the University of Minnesota. The Movielens-1M dataset from the GroupLens Research is available at: https://grouplens.org/datasets/movielens/1m/.\n\nEthical Approval Number: EA1572021\n\nEthical Approval Body: Research Ethic Committee 2021, Multimedia University\n\nFirst, we will present NeuMF++ as a general framework. Then, we will describe feature extraction and neural collaborative filtering in detail. Lastly, we will explain the learning and optimization of NeuMF++. Table 1 shows the frequent notations.\n\nIn this section, the proposed NeuMF++ is introduced in general. As illustrated in Figure 1, NeuMF++ is a hybrid model that bridges multiple SDAEs to a NeuMF. NeuMF++ contains two major components: feature extraction and neural collaborative filtering.\n\nIn feature extraction, each user and item features are assigned with 2 SDAEs for feature extraction. As discussed earlier, recommendation performance and accuracy can be improved by incorporating side information. NeuMF++ utilizes SDAEs to learn user-item features by minimizing the errors of the reconstructed and the original input features. Then, compressed high-level features can be extracted from the bottleneck layer, located in the middle-most layer. In neural collaborative filtering, NeuMF has been chosen as our framework due to its outstanding performance. As mentioned earlier, NeuMF combines the output of GMF and MLP interaction functions. Similarly, NeuMF++ combines the output of GMF++ and MLP++ interaction functions. First, user and item latent vectors can be formed by concatenating the user and item embeddings of GMF and MLP, with the learned user and item latent feature vectors extracted from the SDAEs. Then, the user and item latent vector will be fed to the respective GMF++ and MLP++ interaction function. Finally, the outputs obtained from GMF++ and MLP++ are concatenated and fed into a single-layer MLP 一 NeuMF layer to generate ratings.\n\nSDAE can be formed by stacking multiple DAEs on top of one another. Side information (features) is usually composed of the subject attributes like users’ age and occupation or item’s shape and size. In NeuMF++, SDAEs take user features X and item features V as input, encode them in a low-dimensional latent space, and then reconstruct X̂ and V̂ in the output space. At the same time, noises are added intentionally between layers during training.\n\nFor example, given a set of features X∈Rm×p the SDAE minimize the reconstruction error,\n\nwhere ω denotes as the model parameters, λω as the regularization term, and X̂ as the reconstruction of X∈Rm×p, where\n\nwhere ∇ denotes the noise function. During inference, the values of the bottleneck layer can be extracted as in Eq. (3).\n\nNeuMF++ can be seen as the combination of GMF++ and MLP++. The ++ acronym denotes that side information is appended to the model. At first, one-hot encoding is performed on user and item ID to obtain the user and item embeddings. Then, user and item latent feature vectors are extracted and concatenated with their respective embedding to form user and item latent vectors pu and qi, formulated as such\n\nAs discussed earlier, GMF++ and MLP++ use different computations and layers in their interaction function. GMF++ performs an element-wise product between pu and qi as shown in Eq. (6). In contrast, MLP++ utilizes a standard MLP by adding several hidden layers on the concatenated latent vectors, as shown in Eq. (7).\n\nFinally, the NeuMF layer, a single-layer MLP, is introduced to combine both GMF++ and MLP++ interaction output. Specifically, NeuMF++ integrates GMF++ and MLP++ with a single-layer MLP can be formulated in Eq. (8).\n\nFrom Eq. (8), we can see that GMF++ and MLP++ shared the same pu and qi which extracted from the same user and item SDAEs. This might limit the performance and learning capabilities of NeuMF++. For example, the hyperparameters and latent vector size between GMF++ and MLP++ might vary. Hence, we allow GMF++ and MLP++ to perform user-item feature extraction separately. This provides more flexibility to the NeuMF++. Hence, the final NeuMF++ algorithm can be written as,\n\nNeuMF++ objective function consists of user-item feature reconstruction error in feature extraction and prediction error in neural collaborative filtering. The loss function of user and item SDAE can be seen in Eq. (1). Since NeuMF++ is a rating prediction model, its output ruî range between 0NWhere N is the maximum rating number. Hence, the loss function can be defined in Eq. (12),\n\nwhere θ denotes as the parameters of the models, λθ as the regularization term.\n\nTherefore, the general loss function for optimizing NeuMF++ is formulated in Eq. (13).\n\n\nResults\n\nThis paper uses the public MovieLens 1-M dataset.11 The dataset contains approximate 1 million ratings from 6040 unique users across 3706 unique movies, with 95.8% sparseness. Concurrently, we also use the side information provided by the dataset. The user side information consists of age, occupation and gender attributes, while the item consists of 18 different movie genres. All features are preprocessed and encoded as one-hot numeric arrays.\n\nThe evaluation index used in this paper is the root mean square error, RMSE, as shown in Eq. (14). RMSE is directly related to our loss function. The smaller the RMSE, the better the recommendation accuracy.\n\nWe compared our proposed NeuMF++ with related baseline models which include MF, GMF, MLP, NeuMF, GMF++ and MLP++.1-3\n\nAll the experiments were implemented using Pytorch, a deep learning framework built on top of the Python programming language. We utilized the Adam optimization method to optimize our model by setting the batch size of 1024, regularization term of 0.001 and learning rate of 0.001. Concurrently, we split the dataset into 70:30 ratios, where 70% of the dataset is used for training, while another 30% is used for testing. The hyperparameters used on the related baseline models are based on their respective publications.2,3\n\nAs mentioned previously, we used different hyperparameters on GMF++ and MLP++ for user-item feature extraction. We used 8 neurons on 1 hidden layer in GMF++ user-item SDAEs, and 16:8:16 neurons on 3 hidden layers in MLP++ user-item SDAEs. Hence, the latent vector dimensions for all SDAEs are 8. Each SDAE layer is also inputted with some Gaussian noises. In neural collaborative filtering, the embedding vector dimension, d chosen is 8. We used ReLU as GMF++ activation function, while SeLU as MLP++ activation function. Concurrently, MLP++ composed of [32,16,8] neurons in its interaction MLP layers. Finally, we set all the trade-off parameters α,β,γ,δ to 0.000001.\n\nIn Table 2, we can see that NeuMF++ has proved to outperform all the other baseline models with 0.7964 in train RMSE and 0.8681 in test RMSE. NeuMF++ has achieved a 1.37% improvement than its predecessor NeuMF and 2% improvement than traditional MF. As a result, NeuMF++ has demonstrated the effectiveness of employing DNN and side information for rating prediction.\n\nFigures 2 and 3 show that most models converged very fast, except for MF and GMF. This shows that models with DNN learn much faster than the models without DNN in this dataset. Also, MLP++ does not converge as much as MLP. Therefore, side information does not provide much effect on MLP.\n\nTo demonstrate the effectiveness of separate feature extraction and pre-trained weights for NeuMF++, we compared the performance on three versions of NeuMF++ as seen in Table 3. As expected, NeuMF++, with pre-trained weights and feature extraction separated among the GMF++ and MLP++ layers, achieve the best performance.\n\nConcurrently, we also observed that NeuMF++ with feature extraction shared among the GMF and MLP layers, over-fitted in the early iterations, as shown in Figure 4.\n\nAt first, we found out that NeuMF++ did not perform as well as NeuMF. Hence, inspired by the concept of a pre-training method from,2 we loaded and froze pre-trained GMF++ and MLP++ weights into NeuMF++. As a result, we noticed a 8.11% improvement, as shown in Table 3. This pre-training method updates weights within the NeuMF layer but not within the GMF++ and MLP++ layers. As a result, NeuMF++ with pre-trained weights performed much better as compared to NeuMF++ without pre-trained weights. This justified that the usefulness of the pre-training method for initializing NeuMF++.\n\n\nConclusion\n\nIn this paper, we proposed an HB recommendation model, namely NeuMF++, which is an enhanced version of NeuMF that incorporates effective latent representations of side information. Throughout the experiment, we found that incorporating side information to neural collaborative filtering can improve the recommendation performance and eliminate CF cold start and data sparsity.\n\nNeuMF++ is also not limited to categorical or numerical type information, and can be extended with other information types such as text or even images. For example, pre-trained word embedding models such as word2vec, ELMO or BERT, can transform textual information into input bags of words. Besides, CNN can also learn features from images and aid feature extraction or neural collaborative filtering.\n\nDL’s flexibility also allows different neural network building blocks to be integrated. This concept can also be applied to NeuMF++ to form a more robust recommendation model and further improve its recommendation precision.\n\n\nAuthor contributions\n\nOng, Ng and Haw conceived the presented idea. Ong carried out the experiment and wrote the manuscript. Ng and Haw supervised the project and provided critical feedback.\n\n\nData availability\n\nNone.", "appendix": "Acknowledgements\n\nWe thank the anonymous reviewers for their careful reading of our manuscript and their insightful comments and suggestions.\n\n\nReferences\n\nKoren Y, Bell R, Volinsky C: Matrix factorization techniques for recommender systems. Computer (Long. Beach. Calif). 2009; 42(8): 30–37.\n\nHe X, Liao L, Zhang H, et al.: Neural collaborative filtering. Proceedings of the 26th international conference on world wide web. 2017; pp. 173–182.\n\nLiu Y, Wang S, Khan MS, et al.: A novel deep hybrid recommender system based on auto-encoder with neural collaborative filtering. Big Data Min. Anal. 2018; 1(3): 211–221. Publisher Full Text\n\nOng K, Haw S-C, Ng K-W: Deep Learning Based-Recommendation System: An Overview on Models, Datasets, Evaluation Metrics, and Future Trends. Proceedings of the 2019 2nd International Conference on Computational Intelligence and Intelligent Systems. 2019; pp. 6–11.\n\nAng J-S, Ng K-W, Chua F-F: Modeling Time Series Data with Deep Learning: A Review, Analysis, Evaluation and Future Trend. 2020 8th International Conference on Information Technology and Multimedia (ICIMU). 2020; pp. 32–37.\n\nSedhain S, Menon AK, Sanner S, et al.: Autorec: Autoencoders meet collaborative filtering. Proceedings of the 24th international conference on World Wide Web. 2015; pp. 111–112.\n\nKuchaiev O, Ginsburg B: Training deep autoencoders for collaborative filtering. arXiv Prepr. arXiv1708.01715. 2017.\n\nWu Y, DuBois C, Zheng AX, et al.: Collaborative denoising auto-encoders for top-n recommender systems. Proceedings of the ninth ACM international conference on web search and data mining. 2016; pp. 153–162.\n\nWang H, Wang N, Yeung D-Y: Collaborative deep learning for recommender systems. Proceedings of the 21th ACM SIGKDD international conference on knowledge discovery and data mining. 2015; pp. 1235–1244.\n\nLi S, Kawale J, Fu Y: Deep collaborative filtering via marginalized denoising auto-encoder. Proceedings of the 24th ACM international on conference on information and knowledge management. 2015; pp. 811–820.\n\nHarper FM, Konstan JA: The movielens datasets: History and context. Acm Trans. Interact. Intell. Syst. 2015; 5(4): 1–19. Publisher Full Text ." }	[ { "id": "101096", "date": "15 Feb 2022", "name": "Dino Caesaron", "expertise": [ "Reviewer Expertise virtual/augmented reality", "human factors engineering" ], "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\nOverall, the work is fine, and is suitable for indexing. The experimental design has been meticulously planned. The proposed methodology is an improvement to the existing work. The experiment tested on 1 million movielens data. Hence, the proposed method is considered robust and stable. The only weakness is that perhaps it would be useful to test on data obtained from different domains.\nPlease include the benefit of this research's finding in the abstract as well as in the introduction sections, if possible.\nI recommend indexing this article with minor revisions or as is.\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": "211221", "date": "16 Oct 2023", "name": "Khanh Luong", "expertise": [ "Reviewer Expertise unsupervised learning", "dimensionality reduction", "multi-view/multi-modal learning" ], "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 a novel Hybrid recommendation model that leverages two distinct models: GMF (Generalized Matrix Factorization) and SDAE (Stacked Denoising Autoencoder). This hybrid model is composed of two fundamental components: feature extraction and collaborative filtering, where feature extraction step was done on both user and item data (using both GMF and SDAE).\nThe underlying concept behind this hybrid model is straightforward yet promising. The paper's structure and writing are well-organized and easily comprehensible.\nExperiment has been done and reported the RMSE on both training and testing data, however, it is crucial to measure the accuracy of the proposed hybrid model against benchmark models. Therefore, it is suggested that authors provide accuracy comparisons in their paper in order to strengthen its rigour and provide a more comprehensive picture of the hybrid proposed model.\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? Partly", "responses": [] }, { "id": "211206", "date": "27 Mar 2024", "name": "Zhigang Liu", "expertise": [ "Reviewer Expertise Latent factor analysis", "community detection" ], "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 proposes a hybrid recommender system based on neural matrix factorization and stacked denoising autoencoders. The authors claim that their model, called NeuMF++, can effectively learn user and item features from side information and improve the accuracy of rating prediction. The proposed method was evaluated on a real-world dataset and compare it with several baseline methods, showing that NeuMF++ achieves the best performance in terms of root mean square error. In general, this study is interesting and has some significance to recommender system community. However, considering its current form, I have some major concerns for the authors to address: (1) The paper lacks a clear motivation and contribution statement. The authors should explain why they choose to combine neural matrix factorization and stacked denoising autoencoders, and what are the main advantages and challenges of their approach. The authors should also highlight how their work differs from the existing literature and what are the novel aspects of their model. (2) The paper should provide a more comprehensive literature review that covers more related work on MF-based recommendation systems, and latent factor analysis, e.g., Professor Xin Luo’s work. The paper should also highlight the novelty and significance of the proposed method in relation to existing work. (3) The paper does not provide enough details and justification for the design choices and hyperparameters of the model. For example, the authors should explain why they use different activation functions, noise functions, and latent vector dimensions for GMF++ and MLP++. The authors should also report how they tune the trade-off parameters α, β, γ, δ and the regularization terms λω and λθ, and what are the effects of these parameters on the model performance. (4) The paper does not conduct a comprehensive evaluation of the model. The authors only use one very small dataset and one metric to assess their model, which limits the generalizability and robustness of their results. The authors should also perform ablation studies to analyze the contribution of each component of their model. (5) The paper does not discuss the limitations and future directions of their work. The authors should acknowledge the potential drawbacks and challenges of their model, such as scalability, interpretability, cold start, 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? 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": [] } ]	1	https://f1000research.com/articles/10-1079
https://f1000research.com/articles/10-841/v1	20 Aug 21	{ "type": "Brief Report", "title": "First two years of reimbursed enzyme replacement therapy in the treatment of Fabry disease in Poland", "authors": [ "Michał Nowicki", "Monika Komar", "Mariusz Kusztal", "Katarzyna Mizia-Stec", "Tomasz Liberek", "Jolanta Małyszko", "Katarzyna Muras-Szwedziak", "Krzysztof Pawlaczyk", "Piotr Podolec", "Jarosław Sławek", "Monika Komar", "Mariusz Kusztal", "Katarzyna Mizia-Stec", "Tomasz Liberek", "Jolanta Małyszko", "Katarzyna Muras-Szwedziak", "Krzysztof Pawlaczyk", "Piotr Podolec", "Jarosław Sławek" ], "abstract": "Fabry disease (FD) is an ultra-rare genetic lysosomal storage disease caused by pathologic gene variants resulting in insufficient expression of α-galactosidase A. This enzyme deficiency leads to accumulation of globotriaosylceramide and globotriaosylsphingosine in plasma and in different cells throughout the body, causing major cardiovascular, renal, and nervous system complications. Until 2018, reimbursed enzyme replacement therapy (ERT) for FD was available in all European Union countries except Poland.\n\nWe present the preliminary results of the first two years of reimbursed ERT in Poland. We obtained data from the seven largest academic centers in Katowice, Kraków, Wrocław, Poznań, Gdańsk, Warszawa, and Łódź. The questionnaire included the following data: number of patients treated, number of patients qualified for ERT, and patient characteristics.\n\nAll centers returned completed questionnaires that included data for a total of 71 patients (28 men and 43 women) as of June 2021. Thirty-five patients with the diagnosis of FD confirmed by genetic testing (22 men and 13 women) had already qualified for reimbursed ERT. Mean (SD) age at the commencement of the ERT program was 39.6 (15.5) years (range 18-79 years). Mean time from the first clinical symptoms reported by the patients to the FD diagnosis was 21.1 (8.9) years, and the mean time from the final diagnosis of FD to the beginning of ERT was 4.7 (4.6) years.\n\nFD is still underdiagnosed in Poland. To identify undiagnosed FD patients and to ensure that patients in Poland benefit fully from ERT, implementation of an effective nationwide screening strategy and close cooperation with a network of rare disease centers is advised.", "keywords": [ "Fabry disease", "enzyme replacement therapy", "ultra-rare disease", "α-galactosidase", "globotriaosylceramide", "globotriaosylsphingosine" ], "content": "Introduction\n\nFabry disease (FD) is an ultra-rare genetic lysosomal storage disease caused by pathologic gene variants resulting in insufficient expression of α-galactosidase A (GLA).1 This enzyme deficiency leads to accumulation of globotriaosylceramide (GL-3) and its deacylated form, globotriaosylsphingosine (lyso-GL-3), in plasma and in different cells throughout the body. Accumulation of GL-3 and lyso-GL-3 causes major organ damage which leads to multiorgan complications involving the central and peripheral nervous systems, kidney, and heart that decrease the quality of life and shorten lifespan.1 FD is largely underdiagnosed and diagnosis is most often established only after target organ damage has already occurred.\n\nThe introduction of enzyme replacement therapy (ERT) in 2001 revolutionized the treatment landscape of FD. Clinical studies have proven the efficacy of ERT in the treatment of FD. Specifically, it has been shown that ERT inhibits the progression of target organ damage and stabilizes or even improves organ function. Therefore, international standards and guidelines recommend ERT as the optimal treatment of FD, although for some patients with amenable mutations, an alternative oral chaperone therapy is also available.2,3\n\nThe available ERT treatment currently includes recombinant α-galactosidase A enzymes: agalsidase alfa (Replagal, marketed by Shire) and agalsidase beta (Fabrazyme, marketed by Sanofi Genzyme). In the EU, both agalsidase alfa and agalsidase beta have been approved and available for 20 years. Agalsidase alpha and beta are administered every two weeks by intravenous infusion at a dose of 0.2 mg/kg and 1.0 mg/kg, respectively.4,5 Although two ERT preparations are currently available and approved for the treatment of FD, there are ongoing debates as to what dose of agalsidase preparation may offer better target organ protection. Two recent national guidelines suggested that higher doses of the recombinant enzyme may result in better clinical outcomes, at least in males with a classic phenotype.6,7\n\nUntil 2018, reimbursed ERT for FD was available in all EU countries except Poland, where only a limited number of patients who participated in clinical trials or compassionate drug use programs received the treatment.8 To help patients with FD obtain access to the therapy, emphasize the challenges they face, and gain public attention, the Association of Families with Fabry Disease, with the help of medical professionals and parliament members, initiated several public campaigns such as “Where is Fabry,”9 “Who is Fabry,”10 and “Fabry Disease – a burning problem”.11 After an initial rejection of the application in 2014, the Polish Ministry of Health eventually included ERT for FD to the list of reimbursable drugs in 2019.\n\nInitially, one of the major challenges in the treatment of FD in Poland was a lack of guidelines for diagnosis and management of the disease.12 In 2020, an interdisciplinary group of Polish clinicians prepared a comprehensive position statement providing practical recommendations for physicians who treat patients with FD. The position statement was approved by the Boards of the Polish Cardiac Society, Polish Society of Inborn Errors of Metabolism, Polish Society of Internal Medicine, Polish Society of Nephrology, and Polish Society of Neurology.13\n\nThe introduction of the reimbursable ERT was a major step towards the improvement of the quality of life of Polish patients with FD. However, to date, the results of this treatment program in Poland have not been published. The choice of one of the two available recombinant drugs was the sole decision of the treating physician, but patients had to be centrally approved for the participation in the program by a group of rare disease experts.\n\n\nMethods\n\nIn 2021, two years after the introduction of reimbursed ERT therapy for FD in Poland, we designed a short survey to gather data on the FD patients currently treated in rare disease centers. The survey was distributed via e-mail to the Fabry disease attending physicians at seven largest academic centers in Katowice, Kraków, Wrocław, Poznań, Gdańsk, Warszawa, and Łódź. The centers were selected based on the number of patients with Fabry disease treated. The questionnaire included the following data: number of patients treated, number of patients qualified for ERT, and patient characteristics (gender, age, date of the qualification to the ERT program, age at the time of qualification, and time from the appearance of the first disease symptoms to the clinical diagnosis).\n\nData were analyzed using descriptive statistics and Statistica 13.1 PL (StatSoft Polska) software.\n\n\nEthics\n\nThe following study was non-interventional, questionnaire-based research, therefore, according to local regulations, Ethics Committee approval and patient informed consent were not required. The authors received permission to collect the data from all the centers involved and the patients' personal data were anonymized.\n\n\nResults\n\nAll centers returned completed questionnaires that included data for a total of 71 patients (28 men and 43 women) as of June 2021. Thirty-five patients with the diagnosis of FD confirmed by genetic testing (22 men and 13 women) had already qualified for reimbursed ERT. Mean (SD) age at the commencement of the ERT program was 39.6 (15.5) years (range 18-79 years). The mean time from the final diagnosis of FD to the beginning of ERT was 4.7 (4.6) years, although there was a substantial delay from the first clinical symptoms reported by the patients to the diagnosis - 21.1 (8.9) years. The centers with the largest number of patients with FD was Łódź, Cracow, and Wrocław. Detailed numbers of the patients diagnosed and receiving ERT reported by each center in Poland are provided in Table 1.\n\n\nDiscussion\n\nFD is still underdiagnosed in Poland since the reported disease prevalence and number of patients currently receiving the therapy is lower than in other EU countries. For example, in Germany, the estimated treated FD prevalence was 0.85 per 100,000 insured patients from 2010 to 2017,14 which when extrapolated to the Polish population, may suggest that there should be at least 300 patients with FD that may require specific treatment. The situation is improving since the survey showed that almost half (48%) of the Polish patients with FD are already on reimbursed ERT therapy. This reflects the important role that the program has already played, but much remains to be done to implement an effective nationwide screening strategy to identify undiagnosed FD patients and establish close cooperation with a network of rare disease centers to ensure that patients in Poland benefit fully from ERT.\n\n\nData availability\n\nZenodo: First two years of reimbursed enzyme replacement therapy in the treatment of Fabry's disease in Poland. https://doi.org/10.5281/zenodo.5163859.15\n\nThis project contains the following underlying data:\n\n- FD Polska Res Letter FINAL database.xlsx.\n\nZenodo: First two years of reimbursed enzyme replacement therapy in the treatment of Fabry's disease in Poland. https://doi.org/10.5281/zenodo.5163859.16\n\nThis project contains the following extended data:\n\n- Copy of survey (translated to English)\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 Proper Medical Writing and Sanofi Genzyme for their editorial support in preparation of this manuscript.\n\n\nReferences\n\nGermain DP: Fabry disease. Orphanet J Rare Dis. 2010; 5: 30.\n\nWanner C, Arad M, Baron R, et al.: European expert consensus statement on therapeutic goals in Fabry disease. Mol Genet Metab. 2018; 124: 189–203. PubMed Abstract \| Publisher Full Text\n\nOrtiz A, Germain DP, Desnick RJ, et al.: Fabry disease revisited: management and treatment recommendations for adult patients. Mol Gen Metabol. 2018; 123: 416–427. PubMed Abstract \| Publisher Full Text\n\nReplagal – Summary of product characteristics. Accessed June 28, 2021. https://www.ema.europa.eu/en/documents/product-information/replagal-epar-product-information_en.pdf\n\nFabrazyme – Summary of product characteristics. Accessed June 28, 2021. Reference Source\n\nAmsterdam UMC: Protocol Diagnosis, evaluation and treatment of Fabry disease in the Netherlands V.2020 Updated by: M. Langeveld, C.E. Hollak, S. Klein van Loon, S. van der Veen, M. el Sayed, E. Eskes\n\nSirrs S, Bichet DG, Iwanochko RM, et al.: Canadian Fabry Disease Treatment Guidelines 2019.Oct 2019; 4: 2019.\n\nSpada M, Baron R, Elliott PM, et al.: The effect of enzyme replacement therapy on clinical outcomes in paediatric patients with Fabry disease: a systematic literature review a European panel of experts. Mol Genet Metab. 2018 [Epub ahead of print]. PubMed Abstract \| Publisher Full Text\n\nWhere is Fabry social campaign video. Accessed on July 29, 2021. Reference Source\n\nWho is Fabry social campaign video. Accessed on July 29, 2021. Reference Source\n\nFabry Disease – a burning problem. Accessed on July 29, 2021. Reference Source\n\nBazan-Socha S, Kuczia P, Musiał J, et al.: Fabry disease in Poland. Pol Arch Intern Med. 2018; 128: 567–568. PubMed Abstract \| Publisher Full Text\n\nNowicki M, Bazan-Socha S, Błażejewska-Hyzorek B, et al.: Enzyme replacement therapy in Fabry disease in Poland: position statement. Pol Arch Intern Med. 2020; 130: 91–97. PubMed Abstract \| Publisher Full Text\n\nHilz M, DasMahapatra P, Fan Q, et al.: PRO100 Evaluation of treatment patterns of Fabry Disease utilizing medical claims analyses of a German sickness fund database. Value in Health. 2020; 23: 347. Publisher Full Text\n\nMichal N, Monika K, Mariusz K, et al.: First two years of reimbursed enzyme replacement therapy in the treatment of Fabry's disease in Poland [Data set]. Zenodo. 2021. Publisher Full Text\n\nNowicki M, Komar M, Kusztal M, et al.: First two years of reimbursed enzyme replacement therapy in the treatment of Fabry's disease in Poland.2021. Publisher Full Text" }	[ { "id": "92673", "date": "31 Aug 2021", "name": "Andrzej J. Jaroszynski", "expertise": [ "Reviewer Expertise Nephrology", "cardiology" ], "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 well-written manuscript that reports on the situation of FD patients in Poland two years after the introduction of the ERT reimbursement which enabled the treatment of FD patients in this country. The findings are interesting with some novel data presented regarding the relation between the FD diagnosis and the reimbursement of the ERT. However, the authors should address the following issues:\nHas there been an increase in the number of diagnoses of FD after the introduction of ERT reimbursement? The lack of reimbursement and hence the lack of therapy may be at least partly the reason why the number of FD diagnosed patients per million of the population is much lower in Poland than in other European countries;\n\nThe authors should also explain why in the eastern part of Poland patients with FD are not treated/diagnosed. I suggest that these two issues should be discussed in the discussion section.\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": "7319", "date": "22 Oct 2021", "name": "Michał Nowicki", "role": "Author Response", "response": "Dear Prof. Jaroszyński, We are very pleased to find out that our study met your interest. We are thankful to the Editor and Reviewers for their comments and suggestions, which undoubtedly improved the quality of our manuscript. We enclose a revised manuscript with introduced changes highlighted as track changes. We addressed all the comments as follows: Has there been an increase in the number of diagnoses of FD after the introduction of ERT reimbursement? The lack of reimbursement and hence the lack of therapy may be at least partly the reason why the number of FD diagnosed patients per million of the population is much lower in Poland than in other European countries; Author response: Thank you for raising this issue. Previous lack of ERT reimbursement indeed seems to be the main cause of the lower number of patients diagnosed with FD compared to other European countries. Unfortunately, we are unable to source the information regarding the number of patients diagnosed with FD before the reimbursement was introduced. We updated the Discussion section accordingly. The Authors should also explain why in the eastern part of Poland patients with FD are not treated/diagnosed. I suggest that these two issues should be discussed in the discussion section. Author response: Indeed, our study suggests some disproportions between eastern and western Poland. This might be due to national fund criteria for medical centers that can be contracted for ERT, with only highly specialized facilities being eligible, therefore, some patients need to move to another province to receive treatment. Also, eastern Poland is less populated than the western part. On the other hand, our study concerned only the seven biggest centers, selected based on the number of patients with FD treated. Therefore, in eastern Poland, there may be patients with FD diagnosed and treated, however, the study inclusion criteria did not capture them. We updated the Discussion section accordingly." } ] }, { "id": "92671", "date": "13 Sep 2021", "name": "Bojan Vujkovac", "expertise": [ "Reviewer Expertise Fabry Disease", "Chronic Kidney Disease" ], "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 presenting brief report by Dr. Nowicki and colleagues: \"First two years of reimbursed enzyme replacement therapy in the treatment of Fabry disease in Poland\" is a very interesting paper showing the importance of available treatment for Fabry disease (FD) patients. According to presenting data, FD patients are managed in the seven largest academic centers in the country. Similar to other countries, also in Poland there is a large time delay from the first clinical sign to the final diagnosis, therefore the authors are correctly pointing out the importance of raising awareness of FD as a rare disease.\nI have just a few minor suggestions for the authors:\nIntroduction\n(Line 9-10): I suggest emphasizing the importance of early treatment. Namely, disease-specific therapy is efficient only when started before irreversible changes develop. Due to that fact, it is also important to diagnose FD patients at an early age.\nResults:\nIf possible, it would be of great interest to also include in the Results data on how many families were affected. Namely according to Laney DA et al.1, family screening is very effective in diagnosing new patients as there were five family members diagnosed for every proband.\n\nTable 1: I would suggest renaming the first group of patients (\"N of treated patients with FD\" to \"N of diagnosed patients with FD)\", as it is duplicated and misleading. Also, check the numbers of treated patients with ERT - under Wroclaw numbers and sum are not correct. Check also the final sum of treated in the table and also in text.\nDiscussion:\n\nIn order to diagnose young patients (i.e. children) and females, the most effective way is family screening. I would suggest adding that fact to the discussion part and explain if it was done or not in Poland. It could be elaborated in a part where you mentioned \"effective nationwide screening strategy\", which is probably too vague expression and should be explained.\n\nExplain the main reasons or obstacles as to why there are still patients not receiving disease-specific treatment despite it being reimbursed.\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": [ { "c_id": "7320", "date": "22 Oct 2021", "name": "Michał Nowicki", "role": "Author Response", "response": "Dear Prof. Vujkovac, We are very pleased to find out that our study met your interest. We are thankful to the Editor and Reviewers for their comments and suggestions, which undoubtedly improved the quality of our manuscript. We enclose a revised manuscript with introduced changes highlighted as track changes. We addressed all the comments as follows: Introduction (Line 9-10): I suggest emphasizing the importance of early treatment. Namely, disease-specific therapy is efficient only when started before irreversible changes develop. Due to that fact, it is also important to diagnose FD patients at an early age. Author response: Thank you for raising this issue. We emphasized the need for early treatment introduction at the end of the first paragraph: “Early diagnosis and introduction of disease-specific treatment is essential to stop the disease progression at early stage and prevent from unreversible tissue and organ damage.”. Results: If possible, it would be of great interest to also include in the Results data on how many families were affected. Namely according to Laney DA et al.1, family screening is very effective in diagnosing new patients as there were five family members diagnosed for every proband. Author response: Unfortunately, we have no data on family connections of the investigated patients—this issue certainly needs to be explored in further research. We emphasized the need for family screening at the end of the Discussion section as follows: “The screening should particularly concern high-risk groups, i.e., young patients with cardio-vascular accidents and family members of patients already diagnosed with FD.”. We also suggested screening patients with cardiovascular accidents at an early age due to the fact that this is quite a common symptom of FD bringing patients to emergency department units. Table 1: I would suggest renaming the first group of patients (\"N of treated patients with FD\" to \"N of diagnosed patients with FD)\", as it is duplicated and misleading. Also, check the numbers of treated patients with ERT - under Wroclaw numbers and sum are not correct. Check also the final sum of treated in the table and also in text. Author response: Thank you for pointing this out, we have corrected and double-checked the numbers throughout the table and the manuscript. Discussion: In order to diagnose young patients (i.e. children) and females, the most effective way is family screening. I would suggest adding that fact to the discussion part and explain if it was done or not in Poland. It could be elaborated in a part where you mentioned \"effective nationwide screening strategy\", which is probably too vague expression and should be explained. Author response: We emphasized the need for family screening at the end of the Discussion section as follows: “The screening should particularly concern high-risk groups, i.e., young patients with cardio-vascular accidents and family members of patients already diagnosed with FD.” We also suggested screening patients with cardiovascular accidents at an early age due to the fact that this is quite a common symptom of FD bringing patients to emergency department units. Explain the main reasons or obstacles as to why there are still patients not receiving disease-specific treatment despite it being reimbursed. Author response: To our knowledge, the main reason for some patients not receiving disease-specific FD treatment is the reimbursement criteria, which in Poland are particularly strict. Patients qualify to ERT if they are 8 years or older, have FD confirmed in both genetic and enzyme activity testing (however, women with evident clinical symptoms can be qualified despite normal galactosidase activity), and if they present with at least one organ complication, attributed to FD in the differential diagnosis. Unfortunately, this excludes patients with asymptomatic FD, diagnosed during family screening." } ] } ]	1	https://f1000research.com/articles/10-841
https://f1000research.com/articles/10-228/v1	22 Mar 21	{ "type": "Clinical Practice Article", "title": "Case series of the first three severe COVID-19 patients treated with the secretome of hypoxia-mesenchymal stem cells in Indonesia", "authors": [ "Agung Putra", "Agus Widyatmoko", "Sugeng Ibrahim", "Fajar Amansyah", "Farid Amansyah", "Mukti Arja Berlian", "Retnaningsih Retnaningsih", "Zenitalia Pasongka", "Flora Eka Sari", "Basuki Rachmad", "Agus Widyatmoko", "Sugeng Ibrahim", "Fajar Amansyah", "Farid Amansyah", "Mukti Arja Berlian", "Retnaningsih Retnaningsih", "Zenitalia Pasongka", "Flora Eka Sari", "Basuki Rachmad" ], "abstract": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the outbreak of coronavirus disease 2019 (COVID-19), which has been rapidly spreading. Several guideline therapies have been proposed as a possible treatment for SARS-CoV-2, however, these therapies are not sufficient to treat a severe condition of SARS-CoV-2 infection characterised by the increase of D-dimer and C-reactive protein (CRP) levels, and patchy ground-glass opacities (GGOs). Secretome-mesenchymal stem cells (S-MSCs) produced by MSCs under hypoxia could excessively release several anti-inflammatory cytokines and growth factors to control the COVID-19 cytokine storm and accelerate lung injury improvement. This is the first study investigating the clinical outcomes of three severe COVID-19 patients admitted to the intensive care unit of three different hospitals in Indonesia treated with S-MSCs. The decrease of D-dimer and CRP level was reported for all patients treated with S-MSCs. This was in line with improvement of pulmonary radiology, blood gas level, and hematologic assessment. In conclusion, these cases suggest that S-MSCs could effectively control D-dimer, CRP level and GGOs of severe COVID-19 patients associated with recovered pulmonary function.", "keywords": [ "COVID-19", "secretome", "mesenchymal stem cells" ], "content": "Introduction\n\nSince December 2019, severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), responsible for the outbreak of coronavirus disease 2019 (COVID-19), has been rapidly spreading worldwide1. The number of infected persons has exceeded 87 million with 2 million deaths globally2. Several guideline therapies such as remdesivir and convalescent plasma have been proposed as possible treatment for SARS-CoV-2, however these treatments remain controversial. Moreover, these therapies were not effective to treat severe infection of SARS-CoV-2 due to these treatments potentially inducing the robust cytokine storm3,4. A previous study demonstrated that there is a correlation between disease severity and the release of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, IL-1B, IL-4, IFN-γ, IFN-γ-induced protein 10 (IP10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1a (MIP-1a), and granulocyte-colony stimulating factor (G-CSF)5. This finding was confirmed by the high plasma cytokines found in the most severe COVID-19 patients associated with extensive lung damage6,7. Therefore, finding an effective therapeutic option to hamper the devastating cytokine storm of COVID-19 and regenerate the damaged lung is crucial. Previous studies recently reported several benefits of mesenchymal stem cells (MSCs) under hypoxia condition to inhibit robust proinflammatory cytokines and repair extensive tissue damage by releasing several anti-inflammatory cytokines and growth factors8.\n\nThe use of hypoxia-MSCs (H-MSCs) could become an alternative solution to treat the severe cytokine storm of COVID-19. A previous study reported that hypoxia precondition treated on MSCs (H-MSCs) could enhance their survival to reach the damaged area9. However, blood clots appearing during the severe phase of COVID-19 could block the H-MSCs’ trajectory into the damaged area10. Other studies reported that H-MSCs could enhance the release of their active soluble molecules, known as secretome-MSCs (S-MSCs) such as IL-10, TGF beta, VEGF and PDGF, which are useful in hampering inflammation and improving tissue healing11. Therefore, isolating and concentrating the exact active soluble molecule of S-MSCs is a possible strategy to control the cytokine storm of COVID-19, and, in addition, to accelerate the damaged lung improvement.\n\nIn a recent study, we successfully isolate S-MSCs from their culture medium using tangential flow filtration (TFF) strategy with several molecular weight cut-off category12. In this Clinical Practice article, we report on three severe COVID-19 patients with several comorbidities who were treated with S-MSCs in three different hospitals in Indonesia. This is the first report to describe the complete monitoring of these three patients.\n\n\nEthical considerations\n\nEthical clearance for the use of S-MSCs in COVID-19 cases and the protocol for administration was obtained from the Health Research Ethics Committee of Bethesda Hospital, Yogyakarta, Indonesia (approval number, No.91/KEPK-RSB/VI/20). Written informed consent for treatment with S-MSCs was obtained from each patient prior to treatment. All patients were treated with standard treatments for severe condition of COVID-19 infection, in addition to novel S-MSCs therapy regarding. Each patient was treated with three, four and six doses of 1 mL S-MSCs every 12 h (with molecular weight cut-off combination of 10–50 kDa 50%, 50–100 kDa 25%, and 100–300 kDa 25%) via deltoid intramuscular injection, respectively. A different S-MSCs dose was utilized in the three patients due to the preliminary nature of this treatment.\n\n\nCase reports\n\nA 54-year-old Indonesian male with severe hypertension was diagnosed with COVID-19 on August 28, 2020 and admitted to Dr. Esnawan Antariksa Air Force Hospital, Jakarta, Indonesia intensive care unit (ICU) with cough and dyspnea (Table 1) and was treated with standard treatment (Table 2). The examination showed a temperature of 36°C, a 102/min pulse, a respiratory rate of 32/min, and a blood pressure of 200/100. Blood gas analysis showed decreased oxyhemoglobin saturation (SO2, 80.6%; normal reference: 95–100%), CO2 partial pressure (PCO2, 22.9 mmHg; normal reference: 38–42 mmHg), oxygen partial pressure (PO2, 37.6 mmHg; normal reference: 70–99 mmHg) and HCO3- (18.9 mmol/L; normal reference: 22–29 mmol/L). Laboratory studies showed increased white blood cells (WBC) count (17.2 × 109 /L; normal reference: 4–10 × 109 /L), monocyte count (9%; normal reference: 2–8%) and decreased lymphocyte count (15%; normal reference: 20–40%). On August 29, D-dimer was increased (1540 ng/mL; normal reference: 0–231 ng/mL) in line with the elevation of C-reactive protein (CRP, 61.7 mg/dL; normal reference: 0–8.1 mg/L) (Table 3). Chest X-ray showed bronchopneumonia with bilateral ground glass opacities (GGOs) and cardiomegaly condition (Figure 1).\n\npo., per os; i.v., i.v. injection; q12h, every 12 h; q24h, every 24 h.\n\n(A and B) Patient 1. (A) August 29 (a day post onset of illness (dpoi), showing bronchopneumonia with bilateral ground glass opacities (GGOs) and cardiomegaly; (B) September 10 (11 dpoi), showing the absorption of bilateral GGOs with no bronchopneumonia and cardiomegaly. (C and D) Patient 2. (C) November 19 (3 dpoi), showing cardiomegaly with lung edema, bilateral GGOs aorta elongation and aorta atherosclerosis; (D) November 22 (6 dpoi), showing improvement with minimum infiltrate on pulmonalis dextra and sinistra. (E and D) Patient 3. (C) December 23 (7 dpoi), showing worsened bilateral GGOs with cardiomegaly and aortic atherosclerosis; (F) December 28 (12 dpoi), showing decreased bilateral GGOs, cardiomegaly and aortic elongation.\n\nThe patient was treated with 1 mL S-MSCs three times every 12 h via deltoid intramuscular on August 30 and 31.\n\nOn September 4, SO2 was increased (99.6%) with increased PCO2 (36.2 mmHg), PO2 (198.7 mmHg), and HCO3- (24.7 mmol/L). Laboratory studies showed normal WBC count (7.4 × 109/L), monocyte count (5%) and lymphocyte count (20%). D-dimer and CRP level were decreased (1297 ng/mL and 2.33 mg/dL, respectively) (Table 3). The chest X-ray still showed a bronchopneumonia with cardiomegaly condition (Figure 1). On September 10, the patient was reported negative from COVID-19 infection. The chest X-ray showed improvement with no both bronchopneumonia and cardiomegaly observed. D-dimer was decreased (384 ng/mL), and CRP was normal (0.31 mg/dL). On 20 September, the patient has no cough and dyspnea. The examination showed that SO2 was 98.7%. He was discharged from ICU and mobilized into rehabilitation room for standard recovery of physical activity.\n\nA 53-year-old Indonesian male with type 2 diabetes mellitus was diagnosed with COVID-19 on November 16, 2020. He was admitted to Gatot Soebroto Army Hospital, Jakarta, Indonesia on November 17 with cough, dyspnea, chest pain and fatigue (Table 1). On November 19, he was admitted to the ICU due to worsened dyspnea and treated with standard treatment (Table 2). Chest X-ray revealed cardiomegaly with lung edema, bilateral GGOs, aorta elongation and aorta atherosclerosis (Figure 2). Blood gas analysis showed PCO2 (29.9 mmHg), PO2 (177.1 mmHg) and HCO3- (20.8 mmol/L), however SO2 was still normal (98%). On November 21, the SO2 was 90.6% (abnormal) with reduced PO2 (57.5 mmHg). Laboratory studies showed normal WBC count (6.02 × 109/L) with increased neutrophil count (81%, normal reference: 50–70%), monocyte count (9%) and decreased lymphocyte count (10%). D-dimer was abnormal (880 ng/mL). Blood chemistries revealed elevations in CRP (160 mg/L) and fasting plasma glucose (FPG, 398 mg/dL; normal reference: 70–140 mg/dL (Table 3).\n\nMSCs were incubated in 5% O2 hypoxia condition and S-MSCs was isolated from the culture medium using TFF technique12. S-MSCs contain soluble molecules, including IL-10, TGF-β, PDGF and VEGF. IL-10 and TGF-β inhibit NF-kB pathways activation of overactivated immune cells leading to cytokine storm inhibition, characterized by the decreased level of CRP and D-dimer. Under controlled inflammatory milieu, VEGF and PDGF promote the improvement of bilateral GGOs marked by pulmonary recovery.\n\nDue to the patient’s worsening condition, he was injected with 1 mL S-MSCs four times every 12 h via deltoid intramuscular injection on November 21 and 22. On the same day, 6 hours after injection, SO2 increased (98.7%) with increased PO2 (138.5 mmHg), pH (7.509) and HCO3- (29.7 mmol/L). On November 22, chest X-ray showed improvement with minimum infiltrate on pulmonalis dextra and sinistra (Figure 1). On November 23, oxygen saturation was normal (95.9%), with normal PCO2 (36.1 mmHg) and PO2 (71.5 mmHg). Laboratory studies showed increased neutrophil count (85%) with normal monocyte count (6%) and decreased lymphocyte count (9%). D-dimer was decreased (660 ng/mL). Blood chemistries revealed decreased fasting plasma glucose (277 mg/dL) and CRP (5.12 mg/dL (Table 3). On December 5, the patient has no cough, dyspnea, chest pain and fatigue. Examination showed that SO2 was 99.2%. He was discharged from ICU and mobilized into rehabilitation room for physical activity recovery. On December 28, laboratory studies showed normal neutrophil (57%) and lymphocyte count (27%) with increased monocyte count (11%). Chest X-ray showed normal cardiac physiology and no infiltrate or nodule in both pulmonalis.\n\nA 72-year-old Indonesian male with mild hypertension, liver failure, long-term sequelae of stroke and thalassemia minor was diagnosed with COVID-19 on December 16, 2020 and admitted to Bhayangkara Hospital, Makassar, Indonesia with abdominal pain, diarrhea, anosmia, cough and sore throat in the last three days (Table 1). The examination showed a temperature of 37.3°C, pulse of 82/min, respiratory rate of 24/min, blood pressure of 140/90 mmHg, and SO2 of 97%. Laboratory studies showed a decreased WBC count (3.1 × 109 /L), with normal neutrophil count (53.4%), lymphocyte count (26.8%) and increased monocyte count (18.8%). Chest X-ray showed bilateral GGOs. On December 23, the patient’s dyspnea worsened and he was admitted to the ICU and treated with standard treatment (Table 2). Blood gas analysis revealed that oxygen saturation was decreased (85%) with decreased PCO2 (30 mmHg), PO2 (49 mmHg) and HCO3- (19 mmol/L). Laboratory tests showed an increased neutrophil count (72.9%), monocyte count (14.9%) and decreased lymphocyte count (11.7%). D-dimer was increased (235 ng/mL) and blood chemistries revealed elevations in CRP (118 mg/L (Table 3). Chest X-ray showed worsened bilateral GGOs with cardiomegaly and aortic atherosclerosis (Figure 1).\n\nOn December 24–26, the patient was treated with 1 mL S-MSCs six times every 12 h via deltoid intramuscular injection in addition to other standard treatment (Table 2). The day after the first S-MSC injection, oxygen saturation increased (98%). The examination showed a temperature of 37°C, pulse of 90/min, respiratory rate of 28/min and blood pressure of 120/80 mmHg. On December 28, examination showed a temperature of 37°C, pulse of 80/min, respiratory rate of 24/min and blood pressure of 120/80 mmHg. Oxygen saturation was normal (98%). Laboratory studies showed normal neutrophil count (78.3%) monocyte count (7.2%) and lymphocyte count (25.8%). D-dimer was decreased (86.9 ng/mL). Blood chemistries revealed decreased CRP (8.5 mg/dL (Table 2). Chest X-ray showed decreased bilateral GGOs, cardiomegaly and aortic elongation (Figure 1). On December 30, the patient has no abdominal pain, diarrhea, anosmia, cough and sore throat. The examination showed that SO2 was 99%. He was discharged from ICU and mobilized into rehabilitation room for standard physical activity recovery. On January 6, the patient was negative for COVID-19.\n\n\nDiscussion\n\nSevere pneumonia COVID-19 is characterized by rapid viral infection, excessive inflammatory cell infiltration and robust cytokine storm associated with an increase of D-dimer and CRP levels, resulting in acute respiratory distress syndrome (ARDS)13. A previous study reported that the cytokine storm of COVID-19 was associated with an increase of several proinflammatory cytokines, including TNF-α, IL-1, IL-6, IL-17A and granulocyte macrophage colony-stimulating factor (GM-CSF) in the plasma. In line with this phenomenon, lymphocyte count in severe COVID-19 patients' peripheral blood was decreased14,15. Another study also revealed that lymphocyte count in severe COVID-19 patients was reduced due to overactivated immune cells, known as macrophage activated syndrome (MAS). The MAS potentially promotes the excessive cytokines storm characterized by the increase of CRP and D-dimer level leading to bilateral GGOs16. S-MSCs contain several anti-inflammatory cytokines, including IL-10 and TGF-β, to control the overactivated immunity and hamper the storm's excessive cytokines. In addition, S-MSCs also have several growth factors, such as VEGF and PDGF, that could accelerate lung injury improvement in COVID-19 patient8,11.\n\nThis is the first report that suggests the feasibility of S-MSC therapy of three severe COVID-19 patients in Indonesia. All patients showed bilateral GGOs on chest X-ray before treatment. The hematologic findings showed elevated D-dimer and CRP level in addition to lymphocytopenia and decrease of SO2 and PO2. These patients received several doses of S-MSCs (1 mL/dose) / 12 h with molecular weight cut-off combination of 10–50 kDa (50%), 50–100 kDa (25%) and 100–300 kDa (25%), which results in favorable outcomes. This study showed that between three and six doses of S-MSCs were well tolerated by the patients. The clinical symptoms were significantly improved with patchy GGO improvement, associated with the decrease of D-dimer and CRP level and increase of SO2 and PO2. From our observation, we would suggest that six doses of S-MSCs performed the most optimal treatment in the COVID-19 patients. These results suggest that the immune system's excessive inflammation and overactivation were alleviated by anti-inflammatory cytokines contained in S-MSCs, while the high level of growth factors in S-MSCs could also accelerate the improvement of GGOs. Based on our preliminary results, S-MSC therapy could be a promising and safe rescue option to treat severe COVID-19 patients.\n\nThe first key factor associated with effective S-MSC therapy is the controlled immune system overactivation resulting in the alleviated cytokines storm characterized by the decrease in CRP and D-dimer levels. In our observations, the level of CRP and D-dimer was decreased in all patients treated with S-MSCs. This data suggest that S-MSCs could effectively control the overactivated immune cells. A previous study reported that S-MSCs could control proinflammatory immune cells due to their anti-inflammatory cytokines, such as IL-10 and TGF-β11,17. IL-10 could hamper the inflammatory cells' activity by activating tyrosine kinase-2 and Janus tyrosine kinase 1 (JAK1) and inhibiting NF-kB pathways leading to the decreased expression of proinflammatory cytokines such as TNF-α, IL-1, IL-6 and IL-17A9,18. On the other hand, TGF-β could activate the regulatory subset of T lymphocyte (Treg) by initiating the FoxP3 expression, resulting in suppressing overactivated immune cells19 (Figure 2).\n\nAnother key factor associated with the efficacy of S-MSCs treatment is the acceleration of lung injury improvement characterized by the improvement pulmonary radiological results, decrease of CRP and D-dimer level resulting in the controlled inflammation leading to normal pulmonary function marked by normal SO2. In our patients, decreased CRP and D-dimer was associated with the improvement of patchy GGOs and normal SO2 in all patients treated with S-MSCs. These data suggest that along with the controlled immune overactivation, the growth factors contained in S-MSCs could rapidly improve lung regeneration post excessive cytokine storm exposure. A previous study revealed that S-MSCs could accelerate wound healing due to their growth factors, particularly VEGF and PDGF20. VEGF and PDGF could accelerate the repair of leaky pulmonary blood vessels and accelerate lung injury improvement through the MEK and Akt pathway resulting in the acceleration of angiogenesis and reduced pulmonary infiltrate characterized by decreased bilateral GGOs21,22 (Figure 2).\n\nIn our patients, no severe adverse effects were observed. However, one limitation of our report is that the dynamic changes of cytokine and growth factors during treatment were not investigated. Nevertheless, the preliminary result seem promising.\n\nIn conclusion, we report that S-MSC therapy shows a potential therapeutic effect and low risk in severe COVID-19 patient treatment. We observed that between three and six doses of S-MSCs, with weight cut-off combination of 10–50 kDa (50%), 50–100 kDa (25%) and 100–300 kDa (25%), rapidly controlled the excessive cytokine storm in our patients and improved lung injury. Six doses showed optimum outcome. The treatment time point and the clear clinical advantages of S-MSCs therapy need to be further investigated in randomized clinical studies.\n\n\nConsent\n\nWritten informed consent for publication of this case report, along with any associated images, was obtained from all three patients.\n\nWritten informed consent was also obtained from the patients to undergo treatment using the novel treatment.\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": "Acknowledgements\n\nWe would like to thank Gatot Soebroto Army Hospital, Dr. Esnawan Antariksa Air Force Hospital Jakarta and Bhayangkara Hospital, Makassar, Indonesia for supporting the medical data collection. We would also thank to Stem Cell and Cancer Research (SCCR) laboratory for supporting this study.\n\n\nReferences\n\nMunster VJ, Koopmans M, van Doremalen N, et al.: A Novel Coronavirus Emerging in China - Key Questions for Impact Assessment. N Engl J Med. 2020; 382(8): 692–694. PubMed Abstract \| Publisher Full Text\n\nJohns Hopkins University & Medicine: Coronavirus Resource Center. 2021; Accessed January 22,2021. Reference Source\n\nDubert M, Visseaux B, Isernia V, et al.: Case report study of the first five COVID-19 patients treated with remdesivir in France. Int J Infect Dis. 2020; 98: 290–293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgarwal A, Mukherjee A, Kumar G, et al.: Convalescent plasma in the management of moderate covid-19 in adults in India: open label phase II multicentre randomised controlled trial (PLACID Trial). BMJ. 2020; 371: m3939. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang J, Huang X, Han X, et al.: Endothelial progenitor cells promote viability and nerve regenerative ability of mesenchymal stem cells through PDGF-BB/PDGFR-β signaling. Aging (Albany NY). 2020; 12(1): 106–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhingra S, Sharma AK, Arora RC, et al.: IL-10 attenuates TNF-alpha-induced NF kappaB pathway activation and cardiomyocyte apoptosis. Cardiovasc Res. 2009; 82(1): 59–66. PubMed Abstract \| Publisher Full Text" }	[ { "id": "85411", "date": "05 Jul 2021", "name": "Rajesh Ramasamy", "expertise": [ "Reviewer Expertise Mesenchymal Stem Cell and Immunomodulation" ], "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 tested the usefulness of mesenchymal stem/stromal cells-derived filtered supernatant to treat severe ARS due to COVID-19. The outcomes of the intervention were measured through the parameter of CRP, D-Dimer, and improvement of patients' clinical conditions. The tested treatment indeed showing a range of positive outcomes on the patients. The following technical queries require clarifications:\n\nProvided the supplementary laboratory data on the characterisation of MSC.\n\nInclude the details of the hypoxic conditions that catered to the MSC.\n\nWhat would be the rationale for selecting various percentages and molecular weight for filtering the supernatant?\n\nAny profiling of cytokines or growth factors had been conducted prior to injecting the secretome into the patients?\n\nThe discussion should be made with achieved results rather than the anticipated immunosuppressive action of MSCs. There is no standardisation of MSC secretome. It could be possible that the exosome and microRNA in the secretome mediate the reparative function of MSCs through variable actions that include tissue repair.\n\nIs the background of the cases’ 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? Yes\n\nIs the conclusion balanced and justified on the basis of the findings? Partly", "responses": [ { "c_id": "6925", "date": "22 Jul 2021", "name": "Agung Putra", "role": "Author Response", "response": "Dear Reviewer Thank you for giving us the opportunity to submit a revised of our manuscripts “Case series of the first three severe COVID-19 patients treated with the secretome of hypoxia-mesenchymal stem cells in Indonesia”. We appreciate the time and effort that reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated most of the suggestions made by the reviewers. Those changes are highlighted within the manuscript and extended data. Please see below, in bold, for a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript file with tracked changes (yellow highlight). Provided supplementary laboratory data on the characterization of MSC. Author response: Thank you for your suggestion. We have added the suggestion content on extended data, figure S1. Include the details of the hypoxic conditions that catered to the MSCs. Author response: Thank you for this suggestion. We have added the details of hypoxic conditions on the discussion section that mark as the yellow highlight. What would be the rationale for selecting various percentages and molecular weight for filtering the supernatant? Author response: We selected the 10-50 and 50-100 kDa molecular weight filtering to obtain the desired cytokines and growth factors, such as IL-10, TGF-β, VEGF, and PDGF. We also use 25% S-MSCs with 100-300 kDA categories to obtain the miRNA and exosome. However, we do not analyze the exosome and miRNA contained in S-MSCs. Further studies need to be carried out. Any profiling of cytokines or growth factors had been conducted prior to injecting the secretome into the patients? Author response: We have evaluated the cytokine and growth factor profiles before injecting secretome MSCs into patients. We are confirming that the quality of the secretome MSCs administered is consistent on all three patients (See extended data, table S1 for more details). The discussion should be made with achieved results rather than the anticipated immunosuppressive action of MSCs. There is no standardisation of MSC secretome. It could be possible that the exosome and microRNA in the secretome mediate the reparative function of MSCs through variable actions that include tissue repair. Author response: Thank you for your suggestion. Accordingly, throughout the manuscript, we have revised and discussed the results in the discussion section such as: “The clinical symptoms were significantly improved with patchy GGO improvement, associated with the decrease of D-dimer and CRP level and increase of SO 2 and PO 2. From our observation, we would suggest that six doses of S-MSCs performed the most optimal treatment in the COVID-19 patients. These results suggest that the immune system's excessive inflammation and overactivation were alleviated by anti-inflammatory cytokines contained in S-MSCs, while the high level of growth factors in S-MSCs could also accelerate the improvement of GGOs.” “In our observations, the level of CRP and D-dimer was decreased in all patients treated with S-MSCs. This data suggest that S-MSCs could effectively control the overactivated immune cells. A previous study reported that S-MSCs could control proinflammatory immune cells due to their anti-inflammatory cytokines, such as IL-10 and TGF-β 11, 17.” We also agree with the reviewer’s assessment that there is no standardization of secretome MSCs. Secretome MSCs contain exosome and soluble molecule active. This is a potential limitation of the study because we have not standardized the secretome MSCs. Therefore, further studies of secretome MSCs standardization need to be carried out." } ] }, { "id": "87539", "date": "05 Jul 2021", "name": "Guido Moll", "expertise": [ "Reviewer Expertise Immunology", "Cardiovascular System", "COVID-19", "Stem Cell Therapy", "Mesenchymal Stromal Cells (MSCs)" ], "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 Case Study, Putra et al. report the experimental treatment of three patients suffering from severe COVID-19 with hypoxia-conditioned mesenchymal stromal cell (MSC)-derived secretome (S-MSC) prepared by tangential flow filtration (TFF), to prepare the highly concentrated/enriched MSC secretome for clinical application. In their introduction and discussion, the authors identify among others the COVID-19 cytokine storm and concomitant severe lung and tissue damage, as targets of MSC therapy, anticipating that MSC therapy may exert pleiotropic beneficial effects.\nThe Case Study was conducted at Bethesda Hospital in Yogyakarta, Indonesia (With Local Health Research Committee approval number No.91/KEPK-RSB/V1/20), with written informed consent for the treatment with S-MSCs obtained from each patient prior to treatment. All patients received standard treatment for severe COVID-19, with on-top adjunct treatment with S-MSCs. This is a Case Report of three patients, who were all treated in exploratory fashion with different doses of S-MSCs, and there is no control group or control patients reported in the study.\nThe S-MSC was prepared by TFF to enrich bioactive factors secreted by the MSCs into 1ml therapeutic doses. The S-MSCs were applied in each of the three patients six times every 12 hours via deltoid intramuscular injection. Molecular weight cut-off combination of 10-50 kDa 50%, 50-100 kDa 25%, and 100-300 kDa 25% were used. The production method of S-MSC is outlined in reference number 12: Haraszti et al. 1 Exosomes Produced from 3D-cultures of MSCs by Tangential Flow Filtration Show higher Yield and Improved Activity published in Molecular Therapy in 2018.\nThe authors report:\nIn Table 1) Clinical Characteristics of Patients Receiving S-MSC: Patient Sex, Age, Clinical Classification (Severe COVID-19), Principal Symptoms, Respective Comorbidities (e.g. Hypertension, T2D, Liver Failure, Stroke, Thalassemia);\n\nIn Table 2) Standard Treatment Received by the Three Patients: Drugs Administered (Antiviral, Antibiotic, Antifungal, and Corticosteroid Treatment) and Oxygen Support (Before / After S-MSC Therapy);\n\nIn Table 3) Comparison of Laboratory Parameters Before and After S-MSC Treatment (CRP, D-Dimer, Lymphocytes, SO2); and in Figure 1 and 2) Chest X-ray’s of the Three Patients and a Schematic Study Overview, respectively.\n\nGiven the standard of care treatment, that there was no control group, and that this was an open label Case Study, the key findings of this report are as follows (although any of these findings could also be a result of the standard treatment):\nCRP was strongly log-scale reduced after S-MSC treatment (from 62, 160, 118 to 0.4, 5.1, and 8.5),\n\nD-dimer was reduced around 2-fold on average (1540, 880, 235 to 384, 660, 87),\n\nLymphocyte counts normalized/2-fold increased on average (15, 10, 12 to 20, 27, 26),\n\nSO2 increased/improved (89.6, 90.6, 85 to 99.6, 95.9, 98) together with PO2, and 5) Chest x-rays mainly indicated that lung pathology and cardiomegaly improved (reduction of bilateral ground glass opacities and cardiomegaly).\n\nIn their introduction and discussion, the authors briefly outline the study hypothesis and why MSC / S-MSC treatment may be beneficial and relate this to the improved functional parameters in their Case Study, and these findings are in line with other clinical reports on MSC treatment of COVID-19. So it stands to reason that hypoxia conditioned S-MSC (concentrated soluble fraction of MSC media) is first of all safe, and second may be just as efficient as cellular MSCs in treating COVID-19. However, given the lack of a control group, and open-label treatment of these three patients the findings should be interpreted with caution. Interestingly, not only inflammation and lung parameters improved, but also D-dimer levels, as typically observed in the follow-up of successfully treated patients, see “Viscoelastic Testing Reveals Normalization of the Coagulation Profile 12 Weeks After Severe COVID-19” 2 published in Scientific Reports in 2021.\nThe authors recognized COVID-19 coagulopathy as a challenge for MSC therapy, as reported earlier in “MSC Therapies for COVID19: Importance of Patient Coagulopathy, Thromboprophylaxis, Cell Product Quality and Mode of Delivery for Treatment Safety and Efficacy” 3 published in Frontiers in Immunology 2020 and “Intravascular MSC Therapy Product Diversification: Time for New Clinical Guidelines” 4 published in Trends in Molecular Medicine 2019). Another crucial point is the appropriate timing and dosing and mode of application of MSC therapy in COVID-19, \"Mesenchymal Stromal Cell Therapy for Coronavirus Disease 2019: Which? When? And How Much?\" 5 published in Cytotherapy 2021.\nThe authors indicate their approach as a rescue treatment for patients progressed to severe COVID-19 stage (often presenting with pronounced coagulopathy). Here, the application of cellular secretome instead of the actual cellular MSC products may be somewhat superior/safer given the expression of varying levels of highly procoagulant Tissue Factor (TF/CD142) on MSCs (See PMID’s 32574263 3 and 30711482 4. In this context, another key advantage of this study may be that the S-MSC was applied via deltoid intramuscular injection.\nFuture studies should also put more emphasis on well-controlled dose escalation, pharmacokinetics and pharmacodynamics, as well as biodistribution of the product with tight monitoring of coagulation/complement markers and other safety parameters, adjunct with deeper monitoring of a broader panel of biomarkers of response (e.g. systemic and potentially tissue cytokine profiling in blood and BAL as well as longitudinal monitoring of immune cell subsets in the periphery and local tissue BAL). Another important point is the timing of the treatment relative to the diseases stage (e.g. newly diagnosed vs. progressed patients, identify optimal treatment window). Presumably, S-MSC is an allogeneic off-the-shelf product, thus aspects of alloimunization should be studied in more detailed (humoral and cellular alloimunization). Another important issue is the GMP-compliant production of S-MSC for use in human patients.\n\nIs the background of the cases’ 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "6926", "date": "22 Jul 2021", "name": "Agung Putra", "role": "Author Response", "response": "Dear Reviewer Thank you for giving us the opportunity to submit a revised of our manuscripts “Case series of the first three severe COVID-19 patients treated with the secretome of hypoxia-mesenchymal stem cells in Indonesia”. We appreciate the time and effort that reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We have incorporated most of the suggestions made by the reviewers. Those changes are highlighted within the manuscript and extended data. Please see below, in bold, for a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript file with tracked changes (yellow highlight). This is a Case Report of three patients, who were all treated in exploratory fashion with different doses of S-MSCs, and there are no control group or control patients reported in the study. Author response: Thank you for pointing this out. The reviewer is correct that we are reporting the findings of a case report study, and we do not utilize a control group to compare the relevance of the results. So, it stands to reason that hypoxia conditioned S-MSC (concentrated soluble fraction of MSC media) is first of all safe, Author response: We confirm the reviewer's statement if certainly what was emphasized at the outset was the safety of the use of S-MSCs. We believe that S-MSC is safe because it has undergone a sterile ultrafiltration process using a TFF strategy of up to 10 kDA in size, so contamination is not possible. Future studies of S-MSCs will lead the safety parameters. Second maybe just as efficient as cellular MSCs in treating COVID-19. Author Response: Regarding the efficacy of S-MSCs to treat the severe condition of COVID-19, we suggest that the S-MSCs is more efficient than cellular MSCs. This is due to the pulmonary intravascular coagulopathy (PIC) that present in severe COVID-19 patients could blockage the direction of cellular MSCs to homing to injured pulmonary. We use S-MSCs molecules under 300 kDA which makes it possible to get past the blockages due to PIC to modulate the inflammatory condition and enhance pulmonary regeneration. Interestingly, not only inflammation and lung parameters improved, but also D-dimer levels, as typically observed in the follow-up of successfully treated patients Author response: We suggest that the possibility of S-MSCs to improve D-dimer levels, besides lung parameters is the controlled immune system's excessive inflammation and overactivation and the improvement of the lung caused by anti-inflammatory cytokines and growth factors contained in S-MSCs which possible to pass the PIC blockages. Future studies should also put more emphasis on well-controlled dose-escalation, pharmacokinetics, and pharmacodynamics, as well as biodistribution of the product with tight monitoring of coagulation/complement markers and other safety parameters, adjunct with deeper monitoring of a broader panel of biomarkers of response (e.g. systemic and potentially tissue cytokine profiling in blood and BAL as well as longitudinal monitoring of immune cell subsets in the periphery and local tissue BAL). Another important point is the timing of the treatment relative to the disease's stage (e.g. newly diagnosed vs. progressed patients, identify optimal treatment window). Author response: We agree that this is a potential limitation of the study. In our future study, we will analyze the pharmacokinetics, pharmacodynamics, and biodistribution of the secretome. Furthermore, in the prospective study, we will also evaluate the parameter related to cytokine profiling in blood and BAL and monitoring of immune cell subsets in the periphery and local tissue BAL. We also agree that another potential limitation is no information about the disease stage of patients receiving secretome MSCs treatment. In the future study, it is planned to make several groups of patients who describe the different disease stages to determine the optimal treatment window. S-MSC is an allogeneic off-the-shelf product, thus aspects of alloimmunization should be studied in more detail (humoral and cellular alloimmunization). Another important issue is the GMP-compliant production of S-MSC for use in human patients Author response: Thank you for pointing this out. We agree that this is an important consideration, we have added the MSCs characterization in extended data, figure S1. Based on this data, the expression CD34, CD45, CD11b, CD19, and HLA-DR are represented as Lin <2%. It can be concluded that the possibility of an allergic reaction is quite limited. Although the potential of allergic reactions is limited, further studies need to be carried out." } ] } ]	1	https://f1000research.com/articles/10-228
https://f1000research.com/articles/10-1073/v1	22 Oct 21	{ "type": "Research Article", "title": "Optimal design for a psychosocial intervention on severely acute malnourished children in humanitarian settings: results of an expert survey", "authors": [ "Dieynaba S N'DIAYE", "Cécile Salpéteur", "Cécile Bizouerne", "Karine Le Roch", "Cécile Salpéteur", "Cécile Bizouerne", "Karine Le Roch" ], "abstract": "Background: Common psychosocial interventions focus on feeding, stimulation, emotional responsiveness and childcare and parenting. Inclusion of such a component in the outpatient management of severe acute malnutrition (SAM) children is recommended. However, clinical assessment of such interventions is tedious in humanitarian settings and modalities evidence on the impact of psychosocial interventions alongside nutrition protocol for SAM is scarce. This survey aimed to gather expert opinions on the optimal design of a combined psychosocial and nutrition intervention feasible in humanitarian settings. Methods: From March to May 2018, an online survey was emailed to international experts in nutrition and mental health and psychosocial support, mainly from academia and international non-governmental organisations (INGOs). It included multiple choices questions on the key components of an optimal combined intervention. Results: Of the 76 experts targeted, 20 responded. 11 (55%) belonged to INGOs, 2 (10%) to academia, and 4 (20%) to international organizations and donors. For most respondents, a combined intervention should be provided in weekly 45-minuites counselling sessions, provided individually (rather than in a group) and at home (rather than at a health center). None of the proposed ideal duration (two, four or six months) gained the majority of votes. Experts thought that 35% staff training should be in “Active listening for psychosocial support”, and 30% in “Early child development”, 25% in “Maternal depression” and 9% in “Anthropometric measurements”. They estimated that a combined intervention could improve SAM recovery rate by 10% (min-max: 0-19%) vs. the nutritional protocol alone. Qualitative results highlighted the importance of tailoring the intervention to the individual, the population and the settings; as well as considering feasibility and scalability at the design stage. Conclusion: These findings could guide further research on the impact of psychosocial interventions on SAM children’s health and development, and help designing innovative approaches to treat undernutrition.", "keywords": [ "Expert survey", "severe acute malnutrition", "psychosocial intervention", "children" ], "content": "Introduction\n\nSince child undernutrition is a major public health concern worldwide, the community management of acute malnutrition (CMAM) approach1–3 has constantly evolved to improve prevention and treatment of severe acute malnutrition (SAM) children treated in outpatient clinics and followed up by community health workers. While health information systems exist, robust evidence on the effectiveness of the CMAM approach in terms or children’s recovery, death and defaulter rates, coverage and quality of services, are sparse.4 Most of the reviews on SAM treatment effectiveness indicate the need for more high-quality research to improve the performance of such approaches across contexts.5–7\n\nSince 1999, the World Health Organization (WHO) has been recommending that, when feasible, the in-patient management of SAM children should include a psychosocial component.8 This recommendation was reinforced in 2013 by Ruel et al who suggested that a combined intervention could be cost-saving and enhance benefits on both nutrition and development outcomes.9 Since early childhood is being recognized as a critical time for growth, appropriate psychosocial interventions during this window of opportunity are highly relevant wherever counselling in early child development (ECD), hygiene promotion, breastfeeding and appropriate feeding practices could be embedded with health and nutrition objectives. However, a systematic review found that only two non-randomized control trials have formally tested the effectiveness of approach in SAM children development, and only one, implemented in a hospital Nutritional Rehabilitation Unit, included nutrition outcomes.10\n\nIndeed, several challenges are foreseen. First, implementation of the out-patient nutrition protocols has resulted in less contact time between the patient and the health care staff. This poses the question of the modality and duration of the psychosocial interventions. Secondly, the available psychosocial support is likely to be implemented in the health centers without additional staff or equipment, by staff without any additional training, which represent feasibility and economic obstacles. Thirdly, research shows that the psychosocial component might improve nutrition treatment programs but applicability of those interventions at a large scale even further precludes buy-in of stakeholders.\n\nIn order to facilitate the implementation of psychosocial interventions in areas with limited resources, this study aims to collect expert opinions and recommendations, from both nutrition and psychosocial fields, on the optimal modalities for designing a combined psychosocial intervention among SAM children in humanitarian settings.\n\n\nMethods\n\nAn online expert consultation was conducted. We selected experts from the authors’ contact lists with known expertise in the fields of mental health and psychosocial support (MHPSS), and nutrition working in academia, non-governmental organizations (NGOs), United Nations (UN) organizations, the World Bank, the Organisation for Economic Co-operation and Development (OECD), institutional donors, and foundations. We created a questionnaire on the modalities to tailor an intervention targeting children aged 6-23 months who had received a standard outpatient SAM treatment in humanitarian settings. It included 14 multiple answer questions in English on the characteristics of an optimal psychosocial intervention implemented within a SAM treatment program: frequency, duration and modalities of the counselling sessions, and component of the psychosocial workers training prior to implementation. Most multiple choices answers could be completed by comments and the last question encouraged open commentary and information sharing on the subject of the enquiry. The survey questions can be found in Table 1 and the Underlying Data. The questionnaire was e-mailed in March 2018 and once again (without any changes compared to its first launch) April 2018 and closed on May 2018 after several weeks without new responses.\n\nThe online survey software SurveyMonkey® (SurveyMonkey, Palo Alto, Calif., USA) was used for the questionnaire, the data collection and the quantitative analysis. Qualitative responses of the participants were de-identified and reported in their integrity without alteration and complete raw data of the participant responses can be found in the Underlying Data.11 A thematic content analysis was performed on the qualitative data provided by the open-ended comments, using word recurrence, and key-words-in-contexts techniques to identify and extrapolate themes and concepts. The textual analysis was also enriched by the analysis of technical and practical references cited by the authors. Due to the nature of the questions, it was considered that researchers characteristics (women researchers working in an NGO) were unlikely to influence the results. Nevertheless, researchers engaged in reflexivity notably by reporting raw qualitative responses without editing them. Triangulation was ensured by targeting participants with different backgrounds, fields of work, and institutions. Furthermore, the integration of the theoretical and practical references of the respondents was also an element of triangulation. Standards for Reporting Qualitative Research12 was followed to report our results.\n\nSince we followed the reference methodology for the processing of personal data in the context of research not involving the individual, for studies and evaluations in the field of health MR 0004 defined by the French National Commission on Informatics and Liberty, no ethical approval was needed. Procedures were carried out in accordance with the decree 2017-884: Article 2 du code de la santé publique R.1121-1.-I3°:“For the purposes of this title, research involving the human person is not research having a purpose of public interest research, study or evaluation in the field of health carried out exclusively from the exploitation of treatment of personal data mentioned in I of article 54 of the amended law n° 78-17 of 6 January 1978 relating to data processing, files and freedoms and which fall under the competence of the expert committee for research, studies and evaluations provided for in 2° of II of the same article.” (Translation by the authors.)\n\nAs it was an online-administered survey, participants were contacted via email, which included a brief summary of the project, contact details of the researchers implementing the survey for any future enquires and the link to the online questionnaire. The email included a statement that participants’ responses would only be used for research purpose. According to the European Data Protection Regulation (DPR), the consent of the person whose data is stored in a file is not required when the data is collected for a legitimate interest as stated in the General Data Protection Regulation, May 23, 2018, CHAPTER II - Principles, Article 6 - Legality of treatment. Following the European DRP, it was considered that respondents were consenting by completing the survey and a separate form was not necessary. The name of the respondent was not collected as part of the survey, but the respondent could state their name if they wanted to be mentioned in the acknowledgment section of the publication relating the result of the survey.\n\n\nResults\n\nResults of the expert consultation can be found in Table 1. Among the 76 international experts targeted, 20 responded to the online questionnaire that was available between March 2nd and May 26th (response rate = 26.3%). The background of respondents was nutrition (40%), public health (15%) and MHPSS (30%). They worked either in nutrition (65%) or MHPSS (30%) fields. 10% belonged to an academia, 55% to NGOs and 20% to various bodies (UN, World Bank and OECD, institutional donors, foundations).\n\nMost respondents believed that the counselling sessions should be taking place weekly, with a 45 minutes duration, provided individually rather than in a group and at the children’s home rather than at the outpatient nutrition treatment (OTP). None of the proposed ideal durations (two, four or six months) appeared more favourable. Indeed, 45% respondent chose the ‘Other’ option and the ‘four months’ option came second with 25%. The respondents thought that 35% of the time spent for the training of the health care workers should focus on ‘Active listening for psychosocial support’, 30% on ‘The development of the child from 0 to 6 years’, 25% on ‘Maternal depression’ and 9% on ‘How to take anthropometric measurements’.\n\nThe respondents estimated that SAM recovery would be improved by 10% with a psychosocial intervention in order to attain a child recovery rate of 70% (min: 55 % max: 79%), considering SAM definition as Z-score weight-for-height ≥ -2 and/or middle upper arm circumference ≥ 125 mm, within a standard outpatient nutrition treatment, without any hospital visits.\n\nAmong the 20 respondents, 15 gave qualitative statements to justify or complete their answers and commented the overall enquiry (see the Underlying Data).\n\nImportance of tailoring the intervention\n\nOn question 4 on the intensity duration of the intervention, longer treatment period above the maximum 6 months proposed by the authors were also recommended with a maximum at 18 months. For question 4, 5 and 6, 11 respondents stated that the design of the intervention should depend on the context. It was stated multiple times that the intervention should be tailored to the individual, the child’s age, his/her culture, his/her diagnosis, nutritional status, his/her caregiver, the population and the settings.\n\nRespondent 5 on question 4, 5 and 6: ‘Depend on the culture, people, primary vulnerabilities, the patient and his/her path as some of them need time to unfold themselves.’\n\nNo consensus on one-fits-all solution on the modalities of the intervention\n\nAcross question 5, 6 and 7, three respondents stated that the intervention modalities should not necessarily be fixed throughout the protocol. One respondent proposed that session be longer at first and then shorter, occurring weekly and then less frequently.\n\nRegarding question 7 on the delivery of the intervention in group rather than with the mother and baby only, respondents mentioned feasibility, scalability and cost-effectiveness in favour of group session, while the need for privacy, the mental state of the mother and the capacity of the health care worker to facilitate a group intervention were cited as potential reasons for individual sessions. However, three respondents proposed a combination of individual and group intervention.\n\nRegarding question 8 on the location of the psychosocial intervention, respondents heavily mentioned the home environment and atmosphere, the risk of inhibitions and the opportunity for the mother to have a safe space as important factors to decide for or against providing the intervention at home.\n\nTwo experts proposed that the intervention starts at the OTP, while other recommended to follow the will of the mother and to adapt to family context to have the best results.\n\nRespondent 20 on question 8 commented: ‘Individual sessions may be delivered at home or at the health centre. Mothers are more comfortable in home; however, in extended families, they feel inhibited.’\n\nChallenges\n\nThe open qualitative comments on question 11 mainly addressed potential challenges related to the field implementation of the psychosocial intervention or its impact assessment.\n\nRespondent 1 on question 11 emphasized that ‘The psychosocial intervention will do more than just offering a development component, it will provide a contact moment between the caregiver and project/health system. A clean evaluation of the value of this component would mean that a comparison group gets the same frequency of visits without the specific psychosocial intervention. Rather than assessing the impact of psychosocial interventions on recovery rate, it is important to assess the impact it has on development in the SAM subgroup. The potential impact is expected to be much larger than in a general child population.’\n\nThe experts highlighted the necessity to arbitrate between the desire to have a comprehensive and effective psychosocial care and the feasibility constraints of humanitarian settings. The needs for the psychosocial intervention to be extensive, tailored, intensive and managed by highly trained staff were balanced with more pragmatic considerations that suggest shorter sessions at the OTP, for a period matching that of the nutrition protocol, in order to ensure feasibility, cost-effectiveness, and scalability of the proposed combined intervention.\n\nOne respondent mentioned the risk of performance bias when comparing the two approaches. Four respondents discussed the difficulty to select an outcome that would best summarise the impact of the combined intervention on child health. They suggested that child growth, SAM relapse rate, ECD and neurodevelopment outcomes could also, or even better, encompass the impact of a combined psychosocial and nutrition intervention.\n\nRespondent 16 on question 11: ‘I think the plus value of a psycho-social intervention in SAM would more be on middle or long term (i.e. after the recovery, on child development, child health, etc.).’\n\nOn respondent proposed to add a training component on how to help caregivers stimulate their children through massage, play, talk, reading, telling stories, etc.., and mentioned education of WASH as a topic that could also be included. The other resources cited by experts for designing an optimal combined intervention included looking at the implementation science literature in the ECD field for recommendations on intensity of exposure to psychosocial activities, the use of WHO validated tools for mental health and development and the elaboration of an ‘intervention guidance’ building of existing resources for non-MHPSS staff like nutrition field workers.\n\n\nDiscussion\n\nThrough this expert consultation, the proposed model of a combined nutrition and psychosocial intervention that could be implemented in emergency settings includes a 45-minute weekly psychosocial session, delivered individually rather than in a group and at the child’s home or a combination of both.\n\nNo consensus on optimal duration of the overall intervention has emerged from the survey as it was mainly believed to be dependent of the individual and its context. For further considerations, our results should be put in regards with WHO recommendations of 15–30 minutes of psychosocial stimulation per day for inpatient SAM children13 where close and direct access to the children could enable session of this duration. As shown in few other studies, the feasibility of delivery of those interventions remains a challenge10,14 and this confirms the qualitative responses from our expert’s panel that also emphasized the need for optimal design features to be balanced with pragmatic considerations of SAM treatment.\n\nRegarding the trainings of health practitioners, the respondents recommended training in topics related to child development and active listening in order to perform this intervention optimally. The need to address capacity building and retention strategies in view of the current global shortage of skilled health workers has been recently highlighted as one of the top-ranked research questions in the ECD field.15 Indeed, high staff turnover and low level of qualification in psychosocial skills are common examples of the implementation challenges that could hamper the integration into the nutritional treatment of malnourished children.16 However, an introduction to child development could be proposed for various nutrition and health professionals and community health workers who would, among other benefits, facilitate the integration of psychosocial approach to the SAM outpatient treatment.15,17\n\nThis intervention, if optimally implemented, was thought by the expert panel to improve recovery rate by 10% compared to the nutrition treatment alone, but assessing the impact of a combined intervention and notably on the outcome that would best capture this impact remains a challenge. However, providing further evidence on this requires multidisciplinary teams and designing experimental studies sufficiently powered to detect an impact on each outcome deemed relevant.\n\nIn addition, the experts that we surveyed highlighted the need to tailor it to the context and its target population, which would allow building a tailored intervention with improved effectiveness, quality and sustainability.18 Indeed, understanding complex contextual factors and pathways happening in a given situation is crucial before implementation of new actions or modification of current ones.19 An extensive formative evaluation or a pilot phase involving relevant nutrition and MHPSS actors to assess current resources, barriers and capacities as well as the intervention acceptability by both staff and beneficiaries might be a way to: 1) ensure cultural and contextual sensitivity of the intervention modalities via qualitative assessments, 2) propose a combined intervention that really meets the target population’s needs and 3) identify, overcome or adapt to field constraints.20\n\nWe found that the expert responses highlight the importance of feasibility considerations. It includes practicality regarding resources, time, and commitment. Budget needed to implement a psychosocial intervention varies according to the setting, the availability of local resources, the amount of staff training required. Budget impact analysis needs to be performed in the context of scaling up. This represents significant short-term expenses which might be difficult in low resources settings, but efforts in ECD are known to turn great societal benefits in the long term.21 Time and commitment are also required form the key actors of the program (including health personnel, community health workers, district health authorities, traditional leaders, families and others).\n\nEnvironmental factors such as seasonal aspects and long distance to reach the households might prevent patients to receive a proper intervention. Moreover, family decision making process and social representations might hinder the capacities of the caregivers to access and benefit from this intervention. Therefore, the cultural adaptation, level of appropriateness, gender sensitivity considerations known to be crucial to intervention acceptability and sustainability need to be considered in the elaboration of the design.17\n\nWe believe that integration is necessary for scaling up psychosocial intervention. It refers to the level of system change needed in a system to integrate a new intervention into an existing infrastructure.22 Both the nutrition and MHPSS sectors develop scaling up strategies like WHO Mental Health Gap Action Programme23 aims at scaling up services for mental disorders for countries especially with low- and middle-income economies. Any additional intervention that could benefit to the child’s health and maximize cost-effectiveness of SAM treatment is of consideration, and more research is needed to draw lessons learned in terms of implementation and efficiency. Joint discussions and actions, among representatives of international agencies, research institutes and donors in the both nutrition and MHPSS sectors would facilitate integration and investment. Indeed, several findings show that engagement of all stakeholders (community representatives, policy makers, nutrition and MHPSS local experts and other implicated staff) is a determinant step at an earlier stage of the intervention testing and help with research uptake.24,25\n\nOur study has several limitations. Firstly, our questionnaire was in English language only, sent via e-mail to the author’s contacts. Consequently, our results might not be representative of all experts on this subject, which limits their generalizability. Secondly, the proposed combined intervention model was not tested. Furthermore, its acceptability by field workers and beneficiaries was not addressed by the present study. However, information gathered are relevant for future research and humanitarian programmes.\n\nCost-effectiveness and efficiency of an optimal combined intervention should then be assessed to provide decision-making support. Indeed, economic evaluations before issuing new or modified policies are required by donors, implementers and health decision makers.26,27 A psychosocial intervention effectively combined to nutrition treatment is unlikely to increase substantially structural costs (i.e. building and administrative costs). Main expenses would more likely consist in training, time allocation and equipment of the staff. Location of the intervention at the OTP or at the children’s home will presumably have an impact on global intervention cost and scalability. Beneficiary transport and opportunity costs would be lesser if the intervention occurred at home, but logistics and staff time cost would increase. A balance should be found to ensure an optimized effectiveness with the lowest possible intervention and beneficiaries’ costs. Once efficacy and cost-effectiveness evidence will be established, implementation studies will collect information on how to adapt those combined interventions to context and to make them sustainable in real-life conditions.27 Then, a robust and real-time monitoring and evaluation system should be in place to track the changes due to the implementation and adaptations required for a successful integrated intervention. Indeed, this would enable to 1) effectively plan for staff training, and adequate psychosocial activities duration, and resources, and 2) document advocacy activities to ensure sustained funding. Only in this manner could a combined psychosocial and nutrition intervention be sustainably scalable.17\n\n\nConclusion\n\nWhile most of the experts ranked this specific intervention as important, responses on how to implement it were not always consensual. This underlines again the need to have further debates with representatives of academia, NGOs and donors form both nutrition and MHPSS sectors. These results could guide further research on the impact of psychosocial interventions on SAM children’s health and development, and help designing innovative approaches to treat undernutrition.\n\n\nData availability\n\nOSF: Dieynaba N'Diaye, Cécile Salpéteur, Cécile Bizouerne, and Karine Le Roch. RawData_ExpertSurveyParticipantResponse_Deindentified.xlsx (Version: 1). http://doi.org//10.17605/OSF.IO/HA4T7.11\n\nThis project contains the following underlying data:\n\n- Raw data of the responses of the twenty participants approached for our fourteen-question online survey on the optimal design for a psychosocial intervention on severely acute malnourished children in humanitarian settings.\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 all the participants of this survey. They include but are not limited to: Nathalie Avril, Colleen Emary, Marko Kerac, Patricia Kariger, Sarah Rizk, Alexandra Rutishauser-Perera, Helene Schwartz, Fahmida Tofail, Inka Weissbecker, and Berhanu Nigussie Worku. We also thank Carine Magen Fabregat for her for critical feedback on the manuscript. The authors would like to state that this manuscript was presented as a poster in December 20218 at the 11th EPH European Public Health Conference and is available at: https://www.researchgate.net/publication/329371872_An_optimal_design_for_a_psychosocial_intervention_on_severely_acute_malnourished_children_in_humanitarian_settings_results_of_an_expert_survey.\n\n\nReferences\n\nSadler K, Kerac M, Collins S, et al.: Improving the Management of Severe Acute Malnutrition in an Area of High HIV Prevalence. J. Trop. Pediatr. 2008; 54: 364–369. Publisher Full Text PubMed Abstract \|\n\nUNICEF: UNICEF humanitarian action report 2009. Geneva:UNICEF, Office of Emergency Programmes;2009.\n\nValid International – Bringing about change in humanitarian practice\|Community-based Therapeutic Care (CTC): a field manual First edition. Oxford, UK:2006.\n\nDas JK, Salam RA, Saeed M, et al.: Effectiveness of Interventions for Managing Acute Malnutrition in Children under Five Years of Age in Low-Income and Middle-Income Countries: A Systematic Review and Meta-Analysis. Nutrients. 2020; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAshworth A: Efficacy and effectiveness of community-based treatment of severe malnutrition. Food Nutr. Bull. 2006; 27: S24–S48. Publisher Full Text\n\nPicot J, Hartwell D, Harris P, et al.: The effectiveness of interventions to treat severe acute malnutrition in young children: a systematic review. Health Technol Assess Winch Engl. 2012; 16: 1–316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLenters LM, Wazny K, Webb P, et al.: Treatment of severe and moderate acute malnutrition in low- and middle-income settings: a systematic review, meta-analysis and Delphi process. BMC Public Health. 2013; 13: S23. Publisher Full Text\n\nWHO: Guidelines for the inpatient treatment of severely malnourished children. WHO Regional Office for South-East Asia;2003.\n\nRuel MT, Alderman H; Maternal, Group CNS: Nutrition-sensitive interventions and programmes: how can they help to accelerate progress in improving maternal and child nutrition?. Lancet. 2013; 382: 536–551. Publisher Full Text\n\nDaniel AI, Bandsma RH, Lytvyn L, et al.: Psychosocial stimulation interventions for children with severe acute malnutrition: a systematic review. J. Glob. Health. 2017; 7: 010405–010405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nN’Diaye DS, Salpéteur C, Bizouerne C, et al.: Raw Data Expert Survey’s Participant Responses Deindentified.2021. Publisher Full Text Reference Source\n\nO’Brien BC, Harris IB, Beckman TJ, et al.: Standards for Reporting Qualitative Research: A Synthesis of Recommendations. Acad. Med. 2014; 89: 1245–1251. PubMed Abstract \| Publisher Full Text\n\nAshworth A, Ashworth A, Khanum S, et al.: Guidelines for the inpatient treatment of severely malnourished children. World Health Organization;2003.\n\nNahar B, Hamadani JD, Ahmed T, et al.: Effects of psychosocial stimulation on growth and development of severely malnourished children in a nutrition unit in Bangladesh. Eur. J. Clin. Nutr. 2008; 63: 725. PubMed Abstract \| Publisher Full Text\n\nSharma R, Gaffey MF, Alderman H, et al.: Prioritizing research for integrated implementation of early childhood development and maternal, newborn, child and adolescent health and nutrition platforms. J. Glob. Health. 2017; 7: 011002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKouam CE, Delisle H, Ebbing HJ, et al.: Perspectives for integration into the local health system of community-based management of acute malnutrition in children under 5 years: a qualitative study in Bangladesh. Nutr. J. 2014; 13: 22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamadani JD, Nahar B, Huda SN, et al.: Integrating early child development programs into health and nutrition services in Bangladesh: benefits and challenges. Ann. N. Y. Acad. Sci. 2014; 1308: 192–203. PubMed Abstract \| Publisher Full Text\n\nKirsh SR, Lawrence RH, Aron DC: Tailoring an intervention to the context and system redesign related to the intervention: a case study of implementing shared medical appointments for diabetes. Implement Sci IS. 2008; 3: 34–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeaux A, Osofisan W: A Review of Context Analysis Tools for Urban Humanitarian. Policy. 2016.\n\nLeon AC, Davis LL, Kraemer HC: The role and interpretation of pilot studies in clinical research. J. Psychiatr. Res. 2011; 45: 626–629. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack MM, Walker SP, Fernald LCH, et al.: Advancing Early Childhood Development: from Science to Scale 1. Lancet Lond Engl. 2017; 389: 77–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFixsen DL, Naoom SF, Blase KA, et al.: Implementation research: a synthesis of the literature.2005.\n\nWorld Health Organization: mhGAP operations manual: mental health Gap Action Programme (mhGAP).2018.\n\nOxman AD, Lewin S, Lavis JN, et al.: SUPPORT Tools for evidence-informed health Policymaking (STP) 15: Engaging the public in evidence-informed policymaking. Health Res Policy Syst. 2009; 7: S15. Publisher Full Text\n\nBrownson RC, Fielding JE, Maylahn CM: Evidence-based public health: a fundamental concept for public health practice. Annu. Rev. Public Health. 2009; 30: 175–201. Publisher Full Text\n\nBaltussen RM, Adam T, Tan-Torres Edejer T, et al.: Making choices in health: WHO guide to cost-effectiveness analysis.2003.\n\nMusgrove P, Fox-Rushby J: Cost-effectiveness analysis for priority setting. Dis Control Priorities Dev Ctries. 2006; 2.\n\nPinnock H, Barwick M, Carpenter CR, et al.: Standards for Reporting Implementation Studies (StaRI) Statement. BMJ. 2017; 356: i6795. Publisher Full Text" }	[ { "id": "247478", "date": "14 May 2024", "name": "Kieran S O'Brien", "expertise": [ "Reviewer Expertise Epidemiologist with a focus on child health in LMIC settings", "including SAM" ], "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 results of a survey conducted to determine the design of a psychosocial intervention integrated into existing severe acute malnutrition programs, which is a clear and needed first step in this process and thus a contribution to the literature in this area. In particular, more detail on how “humanitarian settings” was defined for the purposes of the survey and interpretation of the results is warranted given the framing of the study aims. The methods would also benefit from more detail on the process involved to select the experts, since this group drives the results. Similarly, the discussion needs consideration of the limitations of the sampling approach and the resulting low response. Additional specific comments are below.\nAbstract\nThird sentence. Unclear what the second half of the sentence is trying to say “modalities evidence on the impact…” Results sentence on duration could be rephrased for clarity – does this mean there was no consensus in ideal duration? Conclusions could be more specific to this study - can the authors propose any components based on their results, and what do they think specific next research steps should be?\n\nIntroduction\nFirst paragraph focuses on the lack of evidence of SAM treatment effectiveness, which feels slightly off topic since this paper focuses on integrating psychosocial interventions into SAM management. I suggest starting with a paragraph that discusses the evidence on the effectiveness of psychosocial stimulation to better make the case for why this should be considered.\nThen in the second paragraph the authors could briefly add more background on SAM burden/management.\n\nSecond paragraph – I suggest referring to the studies as non-randomized studies instead of controlled trials, the language is misleading otherwise.\nSame sentence – rephrase to say “effectiveness of including psychosocial stimulation in SAM programs” for clarity.\n\nThird paragraph – include citations.\n\nMethods\nFirst paragraph indicates that author contact lists were used to identify experts – authors of what? Was a literature search conducted for relevant paper to identify the authors to include? If so, this should be described in this section. The choice of sample drives the results of this study and so is important to describe in detail. Second paragraph – delete the parenthetical statement about women researchers working in an NGO. Expand on why the authors think that respondent characteristics wouldn’t impact results? It seems that the type of work someone focuses on, or their typical setting would impact responses.\nRelated to this, the sentence on engaging in reflexivity by reporting raw qualitative responses without editing could be expanded upon – I’m not sure this qualifies as reflexivity without more information, though it is a helpful gesture towards open reporting.\n\nDefinition of “humanitarian settings” used in the survey should be included, along with how this context was specifically considered in the development and interpretation of the survey. Explanation of how the specific components included in the survey were identified, since this is another major driver of the results.\n\nResults\nIt’s unclear how the authors got to the 10% recovery rate based on the data available online – please include a description in the methods of how this result was obtained.\n\nDiscussion\n\nClarifying how humanitarian settings differ from other SAM program settings and how this impacts the results, and their interpretation is needed. The sentence on environmental factors and seasonality seems to come out of nowhere – I didn’t see this mentioned in the results. Is this paragraph intended to discuss factors not considered in this study? If so, I would state that clearly. Similarly, the last paragraph on efficacy and cost-effectiveness feels a bit out of place in the context of the survey content – I can see why the authors want to discuss next steps, but I think this paragraph needs to first be grounded in what the authors found, and then they can expand from there into next steps. This also feels at odds with the conclusion paragraph, which states that there is not yet a consensus on the components of the intervention itself – consensus on what the actual intervention is would need to be the first step before exploring efficacy and cost-effectiveness. Perhaps a Delphi process would be a next step, to take the findings from the survey and extend it into a consensus-building activity that can be documented to define the optimal intervention. Limitations must include discussion of the low response rate and how this might have impacted results. Similarly, I think the authors can expand on why their group of experts might not be fully representative of this field.\n\nOther\nThe data files provided through OSF do not allow for full replicability as they appear incomplete (ex: only 3 respondents provided their background or current organization in this file, but the authors report in full where the respondents worked) and they are not in a true dataset that allows the viewer to see how the responses relate to each other from a single participant.\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?\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/10-1073
https://f1000research.com/articles/10-536/v1	05 Jul 21	{ "type": "Review", "title": "Nitric oxide for the prevention and treatment of viral, bacterial, protozoal and fungal infections", "authors": [ "Philip M. Bath", "Christopher M. Coleman", "Adam L. Gordon", "Wei Shen Lim", "Andrew J. Webb", "Christopher M. Coleman", "Adam L. Gordon", "Wei Shen Lim", "Andrew J. Webb" ], "abstract": "Although the antimicrobial potential of nitric oxide (NO) is widely published, it is little used clinically. NO is a key signalling molecule modulating vascular, neuronal, inflammatory and immune responses. Endogenous antimicrobial activity is largely mediated by high local NO concentrations produced by cellular inducible nitric oxide synthase, and by derivative reactive nitrogen oxide species including peroxynitrite and S-nitrosothiols. NO may be taken as dietary substrate (inorganic nitrate, L-arginine), and therapeutically as gaseous NO, and transdermal, sublingual, oral, intranasal and intravenous nitrite or nitrate. Numerous preclinical studies have demonstrated that NO has generic static and cidal activities against viruses (including β-coronaviruses such as SARS-CoV-2), bacteria, protozoa and fungi/yeasts in vitro. Therapeutic effects have been seen in animal models in vivo, and phase II trials have demonstrated that NO donors can reduce microbial infection. Nevertheless, excess NO, as occurs in septic shock, is associated with increased morbidity and mortality. In view of the dose-dependent positive and negative effects of NO, safety and efficacy trials of NO and its donors are needed for assessing their role in the prevention and treatment of infections. Trials should test dietary inorganic nitrate for pre- or post-exposure prophylaxis and gaseous NO or oral, topical or intravenous nitrite and nitrate for treatment of mild-to-severe infections, including due to SARS-CoV-2 (COVID-19). This review summarises the evidence base from in vitro, in vivo and early phase clinical studies of NO activity in viral, bacterial, protozoal and fungal infections.", "keywords": [ "Bacteria", "COVID-19", "fungus", "nitric oxide", "nitrate", "nitrite", "protozoa", "virus" ], "content": "Introduction\n\nNitric oxide (NO), an inorganic molecule, is generated endogenously by prokaryotes and eukaryotes from L-arginine by a family of NO synthase enzymes (NOS; Table 1.1).1 In higher animals, it is also generated by reduction of dietary and endogenous nitrate (NO3−) to nitrite (NO2−) and thence NO (Table 1.2). NO is a pleiotropic signalling molecule involved in vascular, neuronal and metabolic regulation and has multiple physiological effects including lowering blood pressure, increasing exercise performance, and reversing metabolic syndrome. Underlying these processes, NO modulates multiple cell types including leucocytes,2 platelets,3 endothelial cells and smooth muscle cells, and neuronal, cardiac and renal function. Three isoforms of NOS exist in eukaryotes: neuronal (nNOS, NOS1), inducible (iNOS, NOS2) and endothelial (eNOS, NOS3). In multicellular organisms, NOS1-3 produces NO that broadly mediates neurotransmission, cyto-toxicity and vascular regulation respectively. Within cells, NO interacts with mitochondrial respiration, activates metabolic regulatory pathways and reduces oxidative stress.\n\nAs people age, endothelial-derived vascular NO levels fall and so vascular function declines causing relative endothelial dysfunction, pro-platelet and pro-inflammatory effects, and increased smooth muscle proliferation. Vascular NO levels are even lower in people with established vascular disease, e.g. those with stroke.4,5 Numerous viruses (including adeno, Coxsackie, coronavirus, cytomegalovirus, echovirus, herpes simplex, human T-cell leukaemia virus type-1, human immunodeficiency virus, influenza, measles, mumps, polio) and bacteria (Leptospira spp.) can infect6–8 and damage endothelium and so further reduce vascular NO levels. This also appears to occur in SARS-CoV-2 infection.9\n\nMost physiological effects of NO are modulated by cyclic guanosine monophosphate (cGMP, second messenger),10,11 and terminated when cGMP is metabolised by phosphodiesterase-5 (PDE5). This combined L-arginine/nitrate-NO-cGMP-PDE5 system (or nitric oxide system) comprises one of two key vasculo-protective pathways, the other being the prostaglandin-cyclic adenosine monophosphate-phosphodiesterase-3 pathway (PG-cAMP-PDE3, or prostacyclin system, see below).\n\nThe NO system may be enhanced or stimulated exogenously with substrate (L-arginine, organic nitrate, inorganic nitrite or nitrate), NO gas, and PDE5 inhibitors. These can be inhaled or administered via transdermal, sublingual, oral, intranasal or intravenous routes (see below). Since endogenous NO generated by inducible NOS plays a key role in defence against multiple microbial pathogens (including viruses, bacteria, protozoa and fungi/yeast), this raises the possibility that exogenous NO might have therapeutic potential as a broad-spectrum antimicrobial, and this is the topic of this review.\n\n\nMethods for the review\n\nThere are numerous publications in this research area and our intention was not to perform a systematic review of these; rather we present exemplars from the research field. We identified publications relating to the effect of NO on viruses, bacteria, protozoa and fungi/yeasts from searches of our own reference libraries, PubMed and Google, and reference lists given in earlier reviews and commentaries.12–20\n\nThe primary searches for relevant studies for inclusion were done through PubMed from inception to 4 May 2021, in English with the following disease terms: “microbe” or “virus” or “bacteria” or “protozoa” or “fungi” or “yeast”. The results of these searches were crossed with the drug terms “nitric oxide” or “nitrite” or “nitrate”, and the identified abstracts were screened by one or more researchers. Earlier studies, including published reviews, were also identified from the files of the senior author. Studies included in vitro, in vivo and clinical trials. Although other drug classes, such as statins and angiotensin-converting enzyme inhibitors, enhance endothelial production of nitric oxide, their main effects are mediated through other pathways, and we have not assessed them here even though they may attenuate microbial disease severity.\n\n\nChemistry and biology of nitric oxide\n\nNO is a small diatomic hydrophobic colourless gas that diffuses easily and has a short half-life. With an unpaired electron (NO•), it is a free radical and so is chemically reactive and unstable in the presence of oxygen and superoxide producing reactive nitrogen oxide species (RNOS). In reality, the chemistry of NO is more complex and it exists in several redox forms: nitrosonium cation (NO+), NO (NO•) and nitroxyl anion NO−).21\n\nAs an inorganic molecule, its central role in biology as a signalling molecule was only discovered in the 1980s,22 the identification of which led to the Nobel Prize for Physiology and Medicine in 1998.23 Ironically, the medicinal use of NO in the form of glyceryl trinitrate (GTN) for angina prophylaxis antedates the modern understanding of the biological synthesis and role of NO by more than a century.24\n\n\nExperimental studies demonstrating nitric oxide inhibition of …\n\nNumerous preclinical in vitro studies have demonstrated that NO sources (stimulated endogenous NO, inorganic and organic nitrates, L-arginine) and PDE5-inhibitors can reduce replication in all seven types of virus as defined in the Baltimore classification (Table 2);25 this includes Class IV viruses (positive-sense RNA viruses) incorporating several coronaviruses26–31 including SARS-CoV-2.32–34 Most studies showed efficacy although neutral studies were reported for porcine reproductive and respiratory virus (an arterivirus, which is closely related to coronaviruses) and rhinovirus.35,36\n\nMultiple studies have assessed the effect of NO on bacteria and inhibitory effects have been seen across a wide range of gram negative, gram positive and acid-fast bacteria (Table 3). NO sources included L-arginine, NO, nitrite, organic nitrates, and endogenously-generated NO. Multiple mechanisms for efficacy have been reported, as discussed below.\n\nNO sources have been tested on both intracellular and extracellular protozoa (Table 4) with sources involving activated macrophages, sodium nitrite, glyceryl trinitrate, sodium nitroprusside (SNP) and S-nitroso-L-acetylpenicillamine (SNAP).\n\nThe effects of NO on several fungi and yeasts have been studied (Table 5). NO was donated exogenously through stimulating macrophages or as acidified nitrite. In vitro experiments demonstrated reduced replication whilst in vivo experiments in mice showed reduced infection.\n\nWhilst endogenous NO derived from eNOS and nNOS is physiologically active via its second messenger (cGMP), the antimicrobial effects of NO relate to its toxic effects when present at higher concentrations. Although it is technically challenging to measure free NO concentrations, studies suggest that NO concentrations derived from iNOS are 10–100× higher than those resulting from eNOS/nNOS (Table 6). NO concentrations resulting from exogenous administration lie between those from eNOS/nNOS and iNOS but approximate more closely to those from iNOS than eNOS. Importantly, much antimicrobial NO activity is likely to reflect the effects of derivative molecules rather than NO itself:\n\n• Nitric oxide (NO•). In general, bacteria deficient in low molecular weight thiols such as glutathione (e.g. Staphylococci spp.) are sensitive to attack by NO whereas those with high thiol concentrations are resistant to NO.\n\n• Peroxynitrite (OONO-, Table 1.3). The reaction between NO and superoxide means that NO synergises with the respiratory burst, another antimicrobial system present in phagocytic cells. Experimentally, this synergism can be inhibited with the addition of superoxide dismutase which converts superoxide into molecular oxygen and hydrogen peroxide (Table 1.4).\n\n• Peroxynitrous acid (ONOOH, Table 1.5), e.g. toxic to Escherichia coli.37\n\n• Nitrogen dioxide (NO2, Table 1.6), e.g. toxic to E. coli.37\n\n• Dinitrogen trioxide (N2O3, Table 1.7).\n\n• Dinitrogen tetroxide (N2O4, Table 1.8).\n\n• S-nitrosothiols (RSNO, e.g. S-nitrosoglutathione), e.g. toxic to E. coli and Salmonella enterica serovar typhimurium.38 RSNO reacts with protein sulphydryl groups changing their function. Thiol concentrations do not appear to determine sensitivity to peroxynitrite and S-nitrosothiols.\n\n• Dinitrosyl-iron ((2 RS)-Fe-(2 NO)). The reaction of NO with iron or iron–sulphur molecules can: inactivate enzymes such as aconitase (which converts citrate to isocitrate in the citric acid cycle), ribonucleotide reductase and ubiquinone reductase; increase free ferrous (Fe2+) which causes oxidative damage; and deplete iron stores.\n\n• RNOS (especially auto-oxidised products of NO).\n\nSince these molecules differ in their stability, reactivity, location and cellular diffusivity, the overall effect of NO will depend on the molecular species involved and its location.\n\nThe targets for NO and associated reactive nitrogen species are multitudinous:\n\n• DNA, through deamination of adenine, cytosine and guanine;39 cross-linking; breakage of strands; inhibition of DNA repair enzymes such as DNA alkyl transferases (and so preventing transfer of the guanine alkyl group to protein); and disruption of DNA replication by inhibition of ribonucleotide reductase;40 as in S. enterica and vaccinia virus.\n\n• RNA, through disruption of RNA replication by inhibition of viral ribonucleotide reductase.\n\n• Inhibition of mitochondrial function, specifically through inactivation of iron-sulphur complexes within respiratory chain enzymes.41\n\n• Protein modification at cysteine, methionine, phenylalanine, tryptophan and tyrosine residues, e.g. by RNOS. Such protein effects will reduce enzyme activity, as seen for DNA, proteases42 and mitochondrial function, as in Coxsackievirus.42\n\n• Limit late protein synthesis, e.g. through posttranslational modification of viral proteases. (Early protein translation/synthesis is not typically affected.)\n\n• Induction of lipid peroxidation.\n\n• Limit glutaminolysis by shuttling glutamine to glutathione synthesis, as in cytomegalovirus.43\n\n• Interaction with sulfhydryl-containing constituents of the bacterial cell.44\n\n• Disrupt zinc homeostasis, as in S. enterica.45\n\n• Limit virion assembly/particle formation.\n\n• Reduce bacterial adhesion to NO-releasing surfaces.46\n\nNitric oxide may also play an augmenting role as an antimicrobial agent. Examples include the adjuvant roles of NO when given with type I interferons in the treatment of DNA viruses47 and L-arginine when given with conventional chemotherapy in smear-positive TB.48\n\nIn addition, NO’s vasculo-active effects are likely to be beneficial in preventing infection and its severity, with NO:\n\n• Reversing endothelial dysfunction and so potentially reducing endotheliitis,9 as occurs in COVID-19.7,8\n\n• Reducing leucocyte function (e.g. adhesion, chemotaxis, phagocytosis);2 COVID-19 is associated with increased phagocyte counts.8,49\n\n• Reducing platelet activation and platelet–leucocyte conjugation and so reducing micro- and macro-thrombosis, as seen in COVID-19.8,50\n\n• Improving organ blood flow and perfusion through smooth muscle relaxation and vasodilation and so likely reversing infection-related vasoconstriction as seen in COVID-19,8 including in the pulmonary circulation.51\n\nThese actions of NO are all mediated via the second messenger cGMP.\n\nNO is produced by some bacteria, archaea and yeasts via several pathways including denitrification of nitrate to nitrite and then to NO52 and oxidation of L-arginine to NO and L-citrulline as catalysed by a bacterial nitric oxide synthase (bNOS), a process that can be inhibited by NOS inhibitors.53 Whilst eukaryotic NOS contains both catalytic and reductase domains, prokaryotic bNOS lacks the latter relying instead on other cellular reductases to generate NO; the one exception to this is the bNOS present in Sorangium cellulosum which does include a reductase module.\n\nIn contrast to the signalling role of NO in mammals, NO synthesis in bacteria has multiple functions which vary between antimicrobial species:54–57\n\n• Protection against oxidative stress with NO limiting thiol reduction and so the formation of hydroxyl radicals (Bacillus anthracis/subtilis, Staphylococcus aureus54) (Table 1.9).\n\n• Protection against oxidative stress with NO activating catalase (B. subtilis). Such defence will limit damage from phagocytic respiratory bursts.54\n\n• Protection against oxidative stress by reducing endogenous NO synthesis and increasing the expression of NO dioxygenase to detoxify NO (Candida albicans).56\n\n• Biosynthesis of toxins, e.g. thaxtomins (a phytotoxin) interfere with potato plant wall synthesis (Streptomyces turgidiscabies).54\n\n• Activation of aerobic and nitrate respiration to optimise growth (S. aureus).57,58\n\n• Protection against antimicrobial agents including amoxycillin, cefuroxime, gentamicin and novobiocin (B. anthracis/cereus/thuringiensis/weihenstephanensis, S. aureus),55 and azoles (Candida albicans).56\n\nThe production by some microbes of endogenous NO to protect against oxidative stress is ironic since hosts are using NO to try to destroy the microbe!\n\nMicrobial resistance to antibiotics is an increasingly common problem and has left some bacteria with few treatment options, e.g. drug-resistant Neisseria gonorrhoeae. Hence, it is vital to consider whether resistance to NO is innate in some microbes or can be acquired. As already highlighted, some microbes have an intrinsic ability to produce their own NO and so attenuate the effects of oxidative stress (e.g. B. anthracis/subtilis, C. albicans, S. aureus 54,56), activate aerobic respiration (S. aureus57) or protect against antimicrobial agents (B. anthracis/cereus/thuringiensis/weihenstephanensis, C. albicans, S. aureus 55,56).\n\nMicrobes may also have mechanisms for deactivating NO. One mechanism is via a NO reductase which reduces NO to nitrous oxide and then nitrogen, e.g. as occurs in fungi59 (Table 1.10). Bacteria have different NO reductases but similarly produce nitrous oxide,59 as seen in Pseudomonas aeruginosa.52 Loss-of-function mutations in NO reductase may be lethal, possibly because intracellular NO concentrations rise to toxic levels.\n\nA second mechanism for detoxifying NO is via NO dioxygenase oxidation to nitrate (Table 1.11). The pre-eminent NO dioxygenase is flavohaemoglobin,60 as present in bacteria (e.g. Salmonella enterica, S. aureus, Vibrio cholerae, Yersinia pestis,20,61,62) and yeasts. A related haemoglobin, truncated haemoglobin, detoxifies NO in mycobacteria. Of note, Mycobacterium leprae has undergone reductive genome evolution losing more than 2,000 genes, including some that protect against RNOS; as a result, M. leprae has fewer defences against NO than Mycobacterium tuberculosis.63 Bacterial lactate dehydrogenase also detoxifies NO, as seen in S. aureus.20 Importantly, these detoxifying enzymes only cope with low levels of NO and are not protective against high NO levels.\n\nAs a result, microbes show differing sensitivities to NO, as seen for common airways pathogens where sensitivity was ranked (sensitivity most to least): P. aeruginosa ~ C. albicans > S. aureus > Klebsiella pneumoniae ~ Staphylococcus epidermis.64\n\nHowever, there is little evidence that bacteria can acquire de novo resistance to NO, as confirmed in experiments on strains of E. coli, P. aeruginosa, S. aureus and Staphylococcus epidermidis.65 This property is unsurprising since NO has multiple mechanisms for antimicrobial activity and these are likely to be invoked orders of magnitude faster than any microbe can process metabolically, especially if protein synthesis is required. Equally, the main mechanisms for antibiotic resistance (drug inactivation, altered binding sites or metabolism and reduced drug permeability) are unlikely to be relevant to many NO sources. Whether viruses, protozoa and fungi can develop resistance to NO remains unclear.\n\n\nAdministering nitric oxide, donors and related compounds\n\nIn the presence of NOS, administration of L-arginine may enhance NO synthesis (Table 7) although intracellular L-arginine levels are not normally rate limiting and so administration may not have physiological effects.66 Although oral preparations of L-arginine are commercially available, consumption of high doses is associated with profuse diarrhoea (P Bath, personal observation).\n\nGaseous NO may be inhaled with the aim of improving pulmonary haemodynamics and killing microbes. Multiple trials are underway for COVID-19 prevention and treatment (Table 9). NO may also be created in real time by combining sodium nitrite and citric acid and administering this either as a nasal spray (for local therapy) or via nebuliser (for combined nasal and bronchial therapy).\n\nOrganic nitrates such as GTN, isosorbide dinitrate (ISDN) and isosorbide mononitrate (ISMN) are widely used in vascular medicine for the prevention and treatment of angina, treatment of chest pain in unstable angina and myocardial infarction, treatment of severe heart failure, and blood pressure lowering after cardiac surgery and in acute stroke (Table 7). There is increasing concern that chronic use of organic nitrates may cause major adverse cardiac events and death,67 reduce daily activity,68 and not improve quality of life or exercise capacity.68 Potential explanations include the development of tolerance, and induction of endothelial dysfunction and cell damage through oxidative stress, e.g. production of free radicals/peroxynitrite.69,70\n\nImportantly, organic nitrates only release NO in cells and tissues expressing mitochondrial aldehyde dehydrogenase-2.71 For example, SNP and SIN-1 inhibit monocyte chemotaxis whilst organic nitrates (ISDN, GTN and molsidomine) do not;72 this contrasts with smooth muscle cells which vasodilate with all five agents. Since aldehyde dehydrogenase-2 suffers from use-inactivation, nitrate tolerance (tachyphylaxis) and endothelial dysfunction develops70 and bioconversion only restarts following a nitrate-free period. Several in vitro studies have demonstrated the potential antimicrobial effects of organic nitrates (Table 7). Other non-organic nitrates include pentaerythritol tetranitrate and erythrityl tetranitrate.\n\nTherapeutic use of inorganic nitrite is limited with intravenous administration used in cyanide poisoning (British National Formulary). Topical acidified sodium nitrite has been shown to reduce cutaneous infections secondary to a variety of viruses and bacteria although its general use is probably limited by skin irritation and erythema (Table 7).\n\nNO may also be produced from dietary inorganic nitrate, as is present in high concentrations in green leafy and some root vegetables, e.g. spinach, lettuce, rocket, beetroot, celery, fennel, radish and Chinese cabbage.73 Nitrate is absorbed from the proximal gastrointestinal tract, excreted by salivary glands, reduced to nitrite by oral bacteria and then absorbed in the gastrointestinal tract. A number of bacterial species situated on the dorsal surface of the tongue perform this conversion via nitrate reductases.74 (In the absence of oxygen, nitrate and nitrite are commonly used by bacteria as terminal electron acceptors for respiration.75) Through this symbiotic relationship, the mammalian host provides the nutrients and the environment in return for nitrite production by bacteria.76\n\nAbsorbed and circulating nitrite is then further reduced to NO, a process that is enhanced in hypoxic or acidic conditions and by multiple mechanisms including deoxyhaemoglobin, deoxymyoglobin, xanthine oxidoreductase and endothelial nitric oxide synthase.77,78 As such, most effects of dietary nitrate will be vascular and perivascular. The beneficial vascular protective effects of vegetable consumption are very clear epidemiologically, as present in the classical Japanese diet,79 the Dietary Approaches to Stop Hypertension (DASH) diet,80 and the Mediterranean diet.81,82 Further, vegetable-derived nitrate may reduce the risk of gastrointestinal cancer.73,83,84 The benefit on cancer is at variance with oral consumption of nitrite. Although nitrite is not carcinogenic per se, the processing and cooking of nitrite-cured meat can form carcinogens such as N-nitroso compounds and heterocyclic aromatic amines. In contrast, carcinogens are not formed when eating raw vegetable-derived nitrate. A recent meta-analysis showed an increased risk gastric cancer with oral nitrite but reduced risk with oral nitrate.85 Dietary nitrate is known to modify the oral and gastric biome (Table 7).\n\nHigh dietary intake of nitrate is associated with many mechanisms that may have beneficial vascular, and potentially, antimicrobial effects. Experimentally, beetroot juice is often used as a potent source of dietary nitrate since dosing can be controlled and a nitrate-free placebo version is available for use in randomised controlled trials. Studies have shown that beetroot juice increases plasma nitrate and nitrite concentrations,86,87 that most vascular effects are mediated via the second messenger cGMP,87 tolerance does not develop (unlike with organic nitrates)73 and inorganic nitrate does not lead to free radical formation. In clinical studies, beetroot juice has been given over weeks and months86–91 and has been shown to have multiple effects with improved exercise performance (hence use by elite athletes)92; improved cognitive performance in older people92; vasodilation with reduced blood pressure89,90,92–95; antiplatelet and anti-leucocyte effects and reduced platelet-leucocyte conjugation86,89; improved endothelial function; reduced left ventricular volume;91 improved metabolic profile; and improved oral health.89 Beyond anti-inflammatory effects on blood cells, nitrite or nitrate reduce soluble pro-inflammatory factors including C-reactive protein, chemokine (C-X-C motif) ligand-1/2, endothelin-1, interleukins-1β/6/10/12p70, interferon-γ, monocyte chemoattractant protein and tissue necrosis factor-α.96 Dietary nitrate has profound metabolic effects and appears to have the potential for reversing the metabolic syndrome and have anti-diabetic effects.97 Overall, the pharmacological effects of beetroot juice have been demonstrated in younger and older people, and in people with cardiovascular disease, e.g. diabetes, obesity, hypertension, hypercholesterolaemia, heart failure and stroke.98 Importantly, inorganic nitrate (given as beetroot juice) may be taken by pregnant women.99 Experimentally, watermelon juice and chard gel may be used as an alternative source of dietary nitrate.100,101\n\nPDE5-inhibitors, such as dipyridamole and sildenafil, enhance the physiological effects of NO as mediated by cGMP. Whether these agents should have antimicrobial effects is unclear since they do not enhance NO levels per se; nevertheless, both drugs have exhibited antimicrobial activity (Table 7) and are being tested in COVID-19 trials (Table 9).102,103\n\nEndogenous NO production may also be stimulated externally. First, nasal breathing promotes the production of NO from the paranasal sinuses and this has bronchodilatory, vasodilatory and potential antimicrobial activities.104 This natural defence mechanism may be attenuated with mouth breathing, as occurs with increasing age and obesity. Second, ultraviolet radiation (UVA and UVB) stimulates the release of NO from both keratinocytes and melanocytes; NO has multiple effects including attenuation of free radical damage, melanogenesis, blood pressure lowering105 and potentially protection against skin infections.\n\nRecent research has focussed on the development of new antimicrobial NO delivery systems and some examples are listed:\n\n• NO microspheres, e.g. biodegradable poly (lactic-co-glycolic acid) spheres loaded with S-nitroso-N-acetyl-D-penicillamine.106\n\n• NO-releasing nanoparticles, with activity against Acinetobacter baumanii, C. albicans, Enterococcus faecalis, E. coli, K. pneumoniae, P. aeruginosa, S. aureus (MRSA), S. epidermidis, Trichophyton menatgrophytes.105,107\n\n• Modified chitosan, e.g. against Trypanosoma cruzi,108 E. coli, S. aureus, Streptococcus mutans.109\n\n• NO–metal complexes (zeolites), with activity against B. subtilis, Clostridium difficile, E. coli, P. aeruginosa, S. aureus (including MRSA).105\n\n• NONOoates (diazeniumdiolates), e.g. with activity against C. albicans, E. coli.105,110\n\n• NO coating of medical device surfaces and tubing,111,112 e.g. using S-nitroso-N-acetyl-D-penicillamine to kill Staphylococcus aureus and P. aeruginosa.\n\nNO sources can also be categorised by whether administration is local (e.g. cutaneous nitrite or intranasal preparations), systemic (e.g. dietary nitrate or L-arginine, oral isosorbide or sildenafil, sublingual GTN, intravenous GTN or SNP) or mixed local and systemic (transdermal GTN). Local administration allows high and potentially cidal concentrations of NO to be achieved without unwanted systemic effects. Intravenous formulations might allow for systemic infections to be treated.\n\nMost microbial studies presented above and in Tables 2-5 were performed in vitro and involved either inducing the L-arginine/NO pathway with cytokines (e.g. interferon gamma [IFN-γ] and/or lipopolysaccharide [LPS]) or with NO sources (such as NO gas, nitrite, 3-morpholinosydnonimine, S-nitroso-L-acetylpenicillamine or sodium nitroprusside). However, the inhibitory effect of NO on microbes in vitro does not represent the complex biochemical environment that they face in vivo including the presence of NO derivatives such as peroxynitrite, microbial production of NO, microbial resistance to NO and excess NO synthesis. Nevertheless, there are many ex vivo and clinical examples where NO has been effective. These issues are now discussed.\n\n\nNitric oxide for clinical infections\n\nAs already highlighted, oral bacteria (e.g. Corynebacterium pseudodiphtheriticum, Fusobacterium nucleatum, Nocardia spp., Prevotella melaninogenica, S. aureus, S. epidermidis, Veillonella spp.) are vital for the reduction of salivary nitrate to nitrite as part of the entero-salivary circulation; nitrite is further reduced to NO.113 This represents a symbiotic relationship between bacteria and the mammalian host; the host provides the nutrients and the environment in return for nitrite production,76 as in the absence of oxygen, nitrate and nitrite are commonly used by such bacteria as terminal electron acceptors for respiration.75\n\nOral consumption of nitrate and the resulting increase in nitrite in the oro-pharynx leads to salivary alkalinisation (pH ~7.0 to 7.5)114 and so reduction in detrimental bacteria and caries.74 Similarly, nitrate supplementation was associated with increased oral Rothia spp. and Neisseria spp, and diminished oral Prevotella spp. and Veillonella spp.; in parallel, plasma nitrite levels rose and systemic blood pressure fell.115 Salivary nitrite production is related to the abundance of oral-nitrate-reducing bacteria.116 In contrast, bacteria and yeast, in particular Lactobacillus spp., Streptococcus spp. and C. albicans, are key to the development of dental caries through the production of acid. Equally, antibiotics that kill nitrate-reductase-containing bacteria inhibit oral nitrite production and so increase the risk of oral thrush.117 Acidified nitrite has antibacterial activity against Helicobacter pylori in vitro,118 an experiment that likely mimics the scenario seen by these bacteria in the stomach after a nitrate/nitrite-rich meal.\n\nThe skin is a potent source of nitric oxide and production is increased with exposure to sunlight (specifically ultraviolet radiation) sufficient to lower blood pressure.119,120 Hence, skin-derived NO may form a natural dermatological antimicrobial defence. Numerous studies have demonstrated that topical acidified sodium nitrite reduces cutaneous infections due to a variety of viruses and bacteria (Table 7) although prophylaxis had to be continued in some cases since NO suppressed replication without necessarily being viro-toxic.121–126 Inorganic nitrate has been used for the treatment of human papilloma virus.127 Phase II clinical trials have found that acidified nitrite in cream reduced Leishmania major/tropica amastigotes and promastigotes with a reduction in cutaneous leishmaniasis123 and increased cure rates in tinea pedis.121 Novel NO agents are in development to treat skin conditions (Table 8).\n\nIn animal and human experiments, NO substrate (L-arginine) and a NO donor (SNP) has been shown to improve the mucociliary activity of the upper respiratory tract128 suggesting a modulatory role for NO in nasal barrier function and clearance. Novel NO agents building on this observation are in development (Table 8):\n\nEndogenous NO has potent pulmonary haemodynamic and bronchodilator effects physiologically. The importance of endogenous NO in preventing infection is apparent experimentally where inhibition of NO results in increased susceptibility to microbes including Leishmania spp., Mycobacterium spp. and Plasmodium spp.129 Similarly, NO sources are used therapeutically, for example sildenafil in the management of pulmonary hypertension (Table 7). In respect of airway epithelial cells, nitrite reduced P. aeruginosa biofilm growth.130 In infection, NO reduces pulmonary vascular resistance and intrapulmonary shunt, and improves oxygen partial pressure in patients with acute severe pneumonia.131 More specifically, inhaled NO improves arterial oxygenation enabling a reduction in inspired oxygen therapy and airway pressure support, and reduces lung infiltrates, in patients with severe acute respiratory syndrome (SARS).31 These findings continue after termination of NO therapy suggesting that NO has both pulmonary vasodilator and anti-SARS effects. Small uncontrolled clinical studies have suggested that iNO may be beneficial in COVID-19.132–136 iNO and novel NO agents are in development, primarily for COVID-19 at present (Table 8, 9).\n\nDipyridamole, a phosphodiesterase 5 inhibitor, may also have similar beneficial effects in severe COVID-19.33 A phase II clinical trial found that L-arginine might have beneficial effects when given on top of conventional therapy for tuberculosis (Table 7).48\n\nThere may also be a role for dietary nitrate/inorganic nitrite in the prevention and treatment of urinary tract infections. Many of the lower urinary tract opportunistic organisms (e.g. E. coli) possess nitrate reductases, this forming the basis of urine dipstick detection of nitrite. In acidic urine conditions, nitrite is reduced to NO with toxicity to bacteria; for example, transferring nitrite-rich urine containing E. coli to a more acidic environment (e.g. pH 5.5) dose-dependently inhibited bacterial growth,137 an effect potentiated by vitamin C. The antibacterial potency is comparable to conventional antibiotics such as trimethoprim and nitrofurantoin.\n\nThis approach has been tested by filling urinary catheter retention balloons with nitrite and ascorbic acid, resulting in measurable amounts of NO outside the membrane and effectively killing two strains of E. coli in the surrounding urine.138 A similar approach found decreased bacterial counts and prevented biofilm formation by P. aeruginosa, K. pneumoniae, and Enterobacter cloace (but not E. coli or S. aureus).139\n\nLast, instillation of bacillus Calmette-Guerin (BCG, an attenuated strain of Mycobacterium bovis) into the bladder is used for the treatment of superficial/non-muscle invasive bladder cancer and carcinoma in situ. BCG induces long-term increases in NOS activity in urothelial cells140,141 and the formed NO is toxic to the malignant cells. The use of BCG to provide non-specific protection against SARS-CoV-2 is to be tested142,143 although vaccination in infancy does not appear to protect against COVID-19 in adults.144\n\nNitrate (usually KNO3) and nitrite (NaNO2) have been used for millennia to preserve food, especially meat and fish.145 Food preparation leads to reduction of nitrate to nitrite, and nitrite inhibits bacterial growth, especially Clostridium botulinum, a key and severe cause of neurotoxin poisoning. Additionally, nitrite adds colour to food,58,146 flavour (in part by overcoming rancid tastes) and is an antioxidant.\n\nNO donors have also been investigated for eradicating or dispersing biofilm organisms. For example, GTN synergises with citrate and ethanol in eradicating biofilms (related to S. aureus, MRSE, P. aeruginosa and C. albicans) in an experimental catheter lock model.147 Similarly, isosorbide mononitrate synergised with antibiotics to disperse then kill S. aureus.148 An NO-releasing contact lens has been developed to treat microbial keratitis due to P. aeruginosa and S. aureus).149\n\nGTN may have improved outcome after infection in participants enrolled into the RIGHT-2 trial, a study where paramedics recruited patients with suspected stroke and randomised them to GTN versus sham. Overall, the trial was neutral.150 However, in a planned subgroup analysis of those participants with a final diagnosis of a non-stroke mimic, functional outcome was better with GTN.150 In a post hoc analysis of participants in this subgroup, GTN was associated with a better outcome in those with a final diagnosis of infections of the respiratory and urinary tracts which raises the possibility that NO was treating the infectious cause underlying the stroke mimic diagnosis.\n\nAs with the NO system, the prostaglandin-cyclic adenosine monophosphate-phosphodiesterase-3 (PG-cAMP-PDE3) system has similar vasculo-protective roles with anti-leucocyte, antiplatelet and anti-smooth muscle, and pro-endothelial effects. It is therefore interesting to note that prostaglandins (PGA1, PGJ2), including prostacyclin (PGI2 and analogues), have been reported to have antiviral effects.151–154 Whether drugs based on these155 or the PDE3 inhibitor, cilostazol, have efficacy against SARS-CoV-2 remains to be investigated. Further, endogenous NO and PGI2 work together in the vascular tree, and it is conceivable that their potential antimicrobial effects will similarly synergise. Their combination, in the forms of ISMN and cilostazol, have been tested after stroke156 but not yet reported for the prevention or treatment of infection.\n\nThe interaction between diet, nutrition state and vaccine effectiveness has been assessed in multiple studies, principally in low–middle income countries where vaccination is paramount, especially in children, and yet where malnutrition may be widespread. In a systematic review and meta-analysis of observational studies and randomised controlled trials, there was little suggestion that malnutrition had any effect on vaccine responses157; similarly, supplementation with vitamins and D, and iron and zinc, did not appear to modify responses. In preclinical studies, protein-energy malnutrition had limited influence on vaccine efficacy in mice.158 The effect of dietary nitrate levels on vaccine efficacy is unstudied.\n\nIf nitric oxide derivatives attenuate microbial infections, then the efficacy of vaccines based on live attenuated viruses and bacteria (such as measles, poliovirus, BCG) might be attenuated by treatment with NO. Although there are many factors known to alter vaccine effectiveness (e.g. age), the effect of NO has not been studied.\n\nMany infections cause long-term morbidity with chronic fatigue syndrome (CFS) and symptoms including fatigue, tiredness, myalgia, cognitive impairment and depression. Example associated microbes include Borrelia burgdorferi (Lyme disease), Chlamydia pneumoniae (community acquired pneumonia), Epstein–Barr virus (infectious mononucleosis), human herpes virus 6 (exanthema subitem), human immunodeficiency virus (AIDS), polio virus, SARS-CoV-1 virus (SARS), SARS-CoV-2 (long-COVID) and West Nile virus (fever).159–161 Although CFS may represent chronic or latent infection, it is more likely to reflect the presence of post-infectious chronic inflammation. Hypothetically, these patients might benefit from inorganic nitrates in view of their positive effects on exercise performance (elite athletes take beetroot juice for this purpose) and cognition,73,92 and potentially antimicrobial effects, a question that needs addressing (Table 7). A phase II trial of L-citrulline is studying this approach in patients with post-polio syndrome.162\n\nDuring severe infection, sepsis (defined as “life-threatening organ dysfunction caused by a dysregulated host response to infection”) often develops. Septic shock is a subset of sepsis and is a leading cause of death worldwide.163 It manifests as hyper- or hypo-pyrexia, altered mental state, hypotension, tachycardia, tachypnoea, hypoxia, anuria and/or lactataemia. This can occur with many infections due to:\n\n• Gram negative bacteria: Bacteroides fragilis, C. pneumoniae, Enterobacter spp., E. coli, Haemophilus influenzae, Klebsiella spp., Legionella spp., Neisseria meningitidis, Proteus spp., P. aeruginosa, Yersinia pestis.61\n\n• Gram positive bacteria: Clostridium spp., Enterococcus spp., Listeria monocytogenes, Staphylococcus spp., Streptococcus agalactiae/pneumoniae/pyogenes\n\n• Viral: Adenovirus, Coronaviruses, Dengue viruses, Ebola virus, Enteroviruses, human immunodeficiency virus, Influenza virus (A and B), haemorrhagic fever viruses, Parechoviruses.164,165\n\n• Fungi: Candida spp.166\n\n• Protozoa: Plasmodium falciparum, Schistosoma mansoni.167\n\nTypically, autoamplification of circulating cytokines (so-called cytokine storm) leads to excess NO synthesis, mostly derived from inducible NOS, leading to high circulating NO levels and the development of septic shock. In these circumstances, treatment with exogenous NO might be inappropriate. Trials of inhibiting endogenous NO synthesis with NOS-inhibitors in critically ill patients with sepsis have been reported although, disappointingly, did not improve outcome; indeed, the non-specific NOS-inhibitor, NG-methyl-L-arginine hydrochloride (L-NMMA, 546C88), was associated with increased death.168 It is not clear why inhibiting NO synthesis was ineffective but non-selective NOS inhibitors were used meaning that both toxic (iNOS) and beneficial (eNOS) sources of NO were inhibited; pharmacologically, such inhibitors will have reduced cardiac output, organ perfusion and tissue oxygenation. In the absence of licensed selective iNOS inhibitors, perhaps the analogous approach used in the management of hyperthyroidism using block (with carbimazole) and replace (thyroxine) might be effective, i.e. block NOS activity and replace with a low dose of a NO donor. That excess NO is dangerous does not mean that pharmacological doses of NO cannot be effective (Figure 1, Tables 6, 11) since all effective interventions in medicine have an inverted “U” dose response.\n\n\n\n1. Reduced eNOS-derived NO related to dietary insufficiency, older age, vascular disease\n\n2. Normal eNOS-derived vascular NO\n\n3. iNOS-derived NO or low/moderate dose exogenous NO source\n\n4. iNOS-derived NO in septic shock or high dose exogenous NO source\n\nSome infections have opposing in vitro and in vivo responses to NO. For example activated macrophage-derived NO or NO donors such as SNAP reduced Trypanosoma brucei proliferation in vitro169,170 whereas endogenous iNOS-derived NO suppressed protozoa-antigen specific T-cell proliferative responses and so worsened infection, at least in infected mice.170 Intracellular protozoal infections are unlikely to be affected in this manner since macrophage-derived NO would be able to act directly on pathogens such as Leishmania major.171\n\nOther infections do not appear, at least in vitro, to induce iNOS. For example, Cryptococcus neoformans failed to induce NOS in primed macrophages,172 apparently due to a lack of TNF-α secretion, probably because the polysaccharide capsule masked the signal for TNF-α secretion. Interestingly, non-encapsulated mutants of C. neoformans did induce endogenous NOS.\n\nHigh levels of iNOS activation were antimicrobial in studies of malaria. Based on monocyte-derived mRNA levels in circulating blood, uncomplicated malaria was associated with increased levels of iNOS activation in contrast to patients with severe malaria who had lower levels.173\n\nOver recorded history, most epidemics and pandemics have resulted from viral infections including Ebola (viral haemorrhagic fever), influenza (H1N1, H2N2, H3N2, H3N8), HIV-1 (AIDS), polio (poliomyelitis), smallpox, yellow fever, zika or corona (OC43, MERS-CoV, SARS-CoV-1/2) viruses. Bacterial pandemics have resulted from Vibrio cholerae (cholera), S. enterica (typhoid fever) and Yersinia pestis (plague). Studies in vitro have reported findings suggesting that NO can reduce infection for some of these pathogens (Tables 2, 3) but information appears to be lacking for smallpox, yellow fever, zika and cholera (Table 10).\n\nWith multiple pandemics over the last 100 years, it is only inevitable that further ones will occur and some, like COVID-19, will comprise a “global catastrophic biological risk”.174 Global pandemics will most likely be caused by a respiratory-spread virus that crosses over from animals such that humans have no inherent immunity to it. Likely candidates include orthomyxoviruses (especially influenza A viruses such as H7N9), paramyxoviruses (e.g. measles, mumps, croup), pneumovirus (e.g. human metapneumovirus), coronaviruses and picornaviruses (especially rhinoviruses and enteroviruses). All of these have had strains that have crossed from animals to humans. This emphasis on RNA viruses is because DNA viruses tend to have lower mutation rates and, therefore, evolve more slowly and are less likely to escape the human immune system within the first rounds of infection. Nevertheless, DNA viruses, such as pox or herpes viruses from great apes or monkeys, do have the potential to jump species. Non-viral causes of pandemics are less likely since most bacteria will be treatable with broad-spectrum antibacterial agents, most fungi are thermally restricted, and prions would require massive food contamination (and only spread slowly).174 Protozoa are usually thermally restricted although global warming may allow malaria to spread more widely in temperate zones.\n\nUnfortunately, pandemics/epidemics may co-exist as seen with SARS-CoV-2 and dengue in Brazil,175 and both with S. enterica in Pakistan;176,177 in part, this reflects increasing travel with aircraft providing a portal for numerous microbes.178 Of theoretical concern was the potential for COVID-19 and epidemic influenza to co-exist during winter in the Northern hemisphere, this possibly leading to a dramatic increase in deaths.179 Nevertheless, ‘flu rates were very low in both southern and northern hemisphere 2020 winters, presumably due to hands, face, space, mask and fresh air measures. All-in-all, the absence of a true broad-spectrum of antiviral agents is a major concern180 and a potential agent such as NO with antimicrobial effects that extend beyond viruses would be most welcome.\n\nOne possible explanation for the observation that COVID-19 outcomes are worse in older people, males, black or Asian ethnicity, and those with co-morbidities such as diabetes, hypertension, stroke and chronic lung disease,181 is that these groups have lower vascular NO activity4,5,9 and so mount a sub-optimal host response against infection. Increasing NO availability is therefore a potential therapeutic strategy. Several NO sources have potential relevance to preventing and treating COVID-19. L-arginine, sodium nitrite, GTN, SNP, NO and dipyridamole each have clinical antimicrobial activity and can be administered, variously, orally, intravenously or as NO gas in the intensive care unit. Transdermal GTN, and oral ISMN, dipyridamole and sildenafil may be administered in the community or hospital. Of these, NO gas, dipyridamole and sildenafil are already being tested for preventing or treating COVID-19 (Table 9). It remains to be determined if increasing dietary nitrate may be a cost effective and safe intervention of widespread health relevance for the prevention of COVID-19 and, indeed, other emerging, pandemic, epidemic or endemic infections. Recent trial evidence provides indirect supporting evidence for the potential anti-SARS-CoV-2 effect of NO. First, dexamethasone and tocilizumab reduced death in patients in intensive care units,182,183 and these agents and NO share anti-inflammatory effects. And second, interferon-ß reduced the need for intensive care in COVID-19 patients;184 type I interferons increase iNOS activity and so have antimicrobial effects, as seen with L. major and Burkholderia pseudomallei.185,186\n\n\nDiscussion and conclusions\n\nNitric oxide is a fundamental molecule with wide-ranging and potent vascular, anti-platelet, anti-inflammatory and tumoricidal effects. Further, there is a large volume of literature spanning the last 30+ years demonstrating that NO also has potent in vitro antimicrobial effects on a wide variety of viruses, bacteria, protozoa, fungi and yeasts; these are supported by a modest number of in vivo studies. Further, several positive clinical phase II trials of NO have been reported in viral, bacterial, protozoa and fungal infections, these relating particularly to skin and respiratory infections administered by cream and gas respectively. Although not from randomised trials, there is also evidence that dietary nitrate modifies the oral biome and so reduces dental caries.\n\nHowever, these results cannot be considered persuasive on their own. First, few neutral or negative studies have been reported suggesting that there may be a risk of publication bias. Second, conflicting data in some dual-protocol studies with positive in vitro and neutral/negative in vivo data suggest that although NO is antimicrobial per se, the local tissue environment may overcome or reverse this effect. Third, organic nitrates can suffer from tolerance and may lead to the generation of reactive NO species such as peroxynitrite and S-nitrosothiols which might exacerbate rather than attenuate infection. Fourth, resistance may develop although this seems unlikely to be a generic issue, not least because NO levels can change, and be changed, much faster than any microbe can raise defensive mechanisms. Fifth, some microbes can produce their own NO and use this to resist the oxidative stress induced by external NO and its derivatives. Sixth, excess NO production is associated with the development of septic shock which might suggest that any NO is ineffective. Potentially, unsuccessful trials of non-selective NOS-inhibitors in severe sepsis may have confused the issue, perhaps by suggesting that treatment with NO is not important in infection. Seventh, positive clinical studies have been performed in environments where very high local concentrations of NO can be achieved and without the risk of reactive responses, in particular on the surface of tissues such as cream on the skin, dietary nitrate in the mouth, nitrite in the stomach, NO gas in the lungs and nitrite in the bladder; whether NO is effective as an antimicrobial within tissues and the vascular tree remains to be determined.\n\nThere are many sources of NO suitable for studying the prevention and treatment of milder infections in the community and hospital (e.g. topical sodium nitrite, oral NO donors such as ISMN, or oral PDE5 inhibitors such as dipyridamole or sildenafil), and treatment of serious infections in hospital (e.g. intravenous L-arginine, sodium nitrite or NO donors such as GTN or SNP, of NO gas). NO may also be delivered via a high nitrate diet, thus offering a widely available and inexpensive public health approach to potentially reducing and attenuating the severity of infections worldwide. This approach has the added advantage that such diets are already known to reduce vascular disease and some cancers, and possibly other inflammatory diseases and dementia.\n\nIn summary, the wealth of in vitro data suggest that NO has generic antimicrobial effects. However, some data suggest that NO may be ineffective or even hazardous and these reinforce our view for the need for large scale clinical trials of NO donors in the community and hospitals to prevent and treat infections. Although such studies need to focus urgently on the COVID-19 pandemic (especially with the lack of broad spectrum antiviral agents180), other pathogens also need to be targeted. However, patients with established septic shock should not be administered NO donors to avoid exacerbating vascular collapse. 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PubMed Abstract \| Publisher Full Text\n\nAdams LB, Hibbs JB, Taintor RR, et al.: Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine. J Immunol. (Baltimore, Md: 1950) 1990; 144(7): 2725–9. PubMed Abstract\n\nHayashi S, Chan CC, Gazzinelli RT, et al.: Protective role of nitric oxide in ocular toxoplasmosis. Br J Ophthalmol. 1996; 80(7): 644–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGross NT, Nessa K, Camner P, et al.: Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus. Med Mycol. 1999; 37(3): 151–7. PubMed Abstract\n\nLane TE, Wu-Hsieh BA, Howard DH: Antihistoplasma effect of activated mouse splenic macrophages involves production of reactive nitrogen intermediates. Infect Immun. 1994; 62(5): 1940–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowning JF, Kachel DL, Pasula R, et al.: Gamma interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine- and tumor necrosis factor-dependent mechanisms. Infect Immun. 1999; 67(3): 1347–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenci E, Romani L, Mencacci A, et al.: Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans. Eur J Immunol. 1993; 23(5): 1034–8. PubMed Abstract \| Publisher Full Text\n\nAlspaugh JA, Granger DL: Inhibition of Cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis. Infect Immun. 1991; 59(7): 2291–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStasko N, McHale K, Hollenbach SJ, et al.: Nitric Oxide-Releasing Macromolecule Exhibits Broad-Spectrum Antifungal Activity and Utility as a Topical Treatment for Superficial Fungal Infections. Antimicrob Agents Chemother 2018; 62(7). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall CN, Garthwaite J: What is the real physiological NO concentration in vivo? Nitric Oxide. 2009; 21(2): 92–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOhta K, Rosner G, Graf R: Nitric oxide generation from sodium nitroprusside and hydroxylamine in brain. Neuroreport. 1997; 8(9-10): 2229–35. PubMed Abstract \| Publisher Full Text\n\nBátai I, Kerényi M, Tekeres M: The growth of bacteria in intravenous glyceryl trinitrate and in sodium nitroprusside. Anesth Analg. 1999; 89(6): 1570–2. PubMed Abstract \| Publisher Full Text\n\nBarraud N, Storey MV, Moore ZP, et al.: Nitric oxide-mediated dispersal in single- and multi-species biofilms of clinically and industrially relevant microorganisms. Microb Biotechnol. 2009; 2(3): 370–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller C, McMullin B, Ghaffari A, et al.: Gaseous nitric oxide bactericidal activity retained during intermittent high-dose short duration exposure. Nitric Oxide. 2009; 20(1): 16–23. PubMed Abstract \| Publisher Full Text\n\nJones ML, Ganopolsky JG, Labbé A, et al.: A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis. Appl Microbiol Biotechnol. 2010; 87(2): 509–16. PubMed Abstract \| Publisher Full Text\n\nTonew M, Tonew E, Mentel R: The antiviral activity of dipyridamole. Acta Virol. 1977; 21(2): 146–50. PubMed Abstract\n\nBooth L, Roberts JL, Cash DR, et al.: GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease. J Cell Physiol. 2015; 230(7): 1661–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberts JL, Tavallai M, Nourbakhsh A, et al.: GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases. J Cell Physiol. 2015; 230(10): 2552–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardwick JB, Tucker AT, Wilks M, et al.: A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane. Clin Sci (Lond). 2001; 100(4): 395–400. PubMed Abstract\n\nRiccio DA, Schoenfisch MH: Nitric oxide release: part I. Macromolecular scaffolds. Chem Soc Rev. 2012; 41(10): 3731–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin H, Yang L, Ahonen MJR, et al.: Nitric Oxide-Releasing Cyclodextrins. J Am Chem Soc. 2018; 140(43): 14178–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadrearhami Z, Nguyen TK, Namivandi-Zangeneh R, et al.: Recent advances in nitric oxide delivery for antimicrobial applications using polymer-based systems. J Mater Chem B. 2018; 6(19): 2945–59. PubMed Abstract \| Publisher Full Text\n\nSharma K, Sengupta K, Chakrapani H: Nitroreductase-activated nitric oxide (NO) prodrugs. Bioorg Med Chem Lett. 2013; 23(21): 5964–67. PubMed Abstract \| Publisher Full Text\n\nHibbard HAJ, Reynolds MM: Synthesis of novel nitroreductase enzyme-activated nitric oxide prodrugs to site-specifically kill bacteria. Bioorg Chem. 2019; 93: 103318. PubMed Abstract \| Publisher Full Text\n\nAlvarez RA, Berra L, Gladwin MT: Home Nitric Oxide Therapy for COVID-19. Am J Respir Crit Care Med. 2020; 202(1): 16–20. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "93464", "date": "27 Sep 2021", "name": "Khosrow Kashfi", "expertise": [ "Reviewer Expertise Pharmacologist with a strong background in biochemistry and intermediary metabolism." ], "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\nNitric oxide (NO) is one of the ten smallest molecules found in nature. It is released intracellularly when L-arginine is oxidized by the enzyme nitric oxide synthase (NOS), of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme regulated by negative feedback and release low fluxes of NO over a short period regulating neural and vascular function, respectively. The third isoform (iNOS, NOS2) is calcium-independent, inducible, produces supra-physiological concentrations of NO, and is involved in immune surveillance. NO can also be produced through the reduction of nitrite/nitrate under low oxygen conditions.\nNO has important roles in normal physiology, exemplified by regulation of vascular relaxation, control of inflammation by inhibiting NF-κB activation, and suppression of pro-inflammatory mediators in mast cells, macrophages, and vascular smooth muscles. In addition, NO regulates blood flow, modulates platelet and leukocyte activation, adhesion, and aggregation.\nIn this review, the authors have focused on NO's role in defense against multiple microbial pathogens [including viruses, bacteria, SARS-CoV (COVID-19), protozoa, and fungi/yeast]. In addition, they have highlighted the possibility that exogenous NO might have therapeutic potential as a broad-spectrum antimicrobial. Finally, they have also summarized various NO donating/releasing platforms.\nIn general, I do not think that there is a \"right\" or \"wrong\" way of presenting a review, as long as it is accurate, balanced, and gives adequate historical background. I found this review to be quite compelling and of interest to those starting in this field and an update for the seasoned investigators. In short, it is an authoritative review. For the novice, the review gives the directions for further in-depth reading. Finally, I would like to make some suggestions to the text for the authors to consider as I believe it would add to the overall body of this work.\nThroughout the text, when you talk about “NO may be taken as dietary substrate (inorganic nitrate, L-arginine)….\", please also discuss L-citrulline as a source of NO. We recently reviewed this topic and you may want to use this review as a source to expand on this1.\n\nIn Table 1, please add L-citrulline as a substrate and give the appropriate biochemical equation.\n\nUnder Therapeutic inorganic nitrite and nitrate, please consider adding the use of nitrite/nitrate in animal models of wound healing. Refer to Afzali et al. 20202 for an introduction to this.\n\nUnder novel nitric oxide agents, please add RRx-001, which is a novel NO modulator3.\n\nAs summarized in the review, dietary intake of foods such as beetroot that are rich as a source of nitrate have many health benefits. And as mentioned, \"Dietary nitrate has profound metabolic effects and appears to have the potential for reversing the metabolic syndrome and have anti-diabetic effects.\". However, although the beneficial metabolic effects of inorganic nitrate and nitrite in type 2 diabetes mellitus have been documented in animal experiments, this is not the case for humans. Perhaps this should be mentioned on page 18 of the review4.\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": [ { "c_id": "7307", "date": "22 Oct 2021", "name": "Philip Bath", "role": "Author Response", "response": "Reviewer 1 responses We thank Dr. Kashfi for his most helpful and considered comments and respond to each as below. In this review, the authors have focused on NO's role in defense against multiple microbial pathogens [including viruses, bacteria, SARS-CoV (COVID-19), protozoa, and fungi/yeast]. In addition, they have highlighted the possibility that exogenous NO might have therapeutic potential as a broad-spectrum antimicrobial. Finally, they have also summarized various NO donating/releasing platforms. In general, I do not think that there is a \"right\" or \"wrong\" way of presenting a review, as long as it is accurate, balanced, and gives adequate historical background. I found this review to be quite compelling and of interest to those starting in this field and an update for the seasoned investigators. In short, it is an authoritative review. For the novice, the review gives the directions for further in-depth reading. Finally, I would like to make some suggestions to the text for the authors to consider as I believe it would add to the overall body of this work. Throughout the text, when you talk about “NO may be taken as dietary substrate (inorganic nitrate, L-arginine)….\", please also discuss L-citrulline as a source of NO. We recently reviewed this topic, and you may want to use this review as a source to expand on this1. We have added L-citrulline wherever dietary L-arginine is mentioned (abstract, text and tables), and added two references: Flam et al. 2007, Bahadoran et al. 2021. In Table 1, please add L-citrulline as a substrate and give the appropriate biochemical equation. We have added L-citrulline as a substrate and the relevant equations. Under Therapeutic inorganic nitrite and nitrate, please consider adding the use of nitrite/nitrate in animal models of wound healing. Refer to Afzali et al. 20202 for an introduction to this. We have added a comment relating to the reference of Afzali et al. 2020, and the reference itself. Under novel nitric oxide agents, please add RRx-001, which is a novel NO modulator3. We have added a comment relating to RRx-001 using a reference from Oronsky et al. 2020, and the reference itself. As summarized in the review, dietary intake of foods such as beetroot that are rich as a source of nitrate have many health benefits. And as mentioned, \"Dietary nitrate has profound metabolic effects and appears to have the potential for reversing the metabolic syndrome and have anti-diabetic effects.\". However, although the beneficial metabolic effects of inorganic nitrate and nitrite in type 2 diabetes mellitus have been documented in animal experiments, this is not the case for humans. Perhaps this should be mentioned on page 18 of the review4. We have added two references relating to beetroot juice improving insulin sensitivity and reducing blood glucose (Wootton et al. 2014, Beals et al. 2017)." } ] } ]	1	https://f1000research.com/articles/10-536
https://f1000research.com/articles/9-1156/v1	21 Sep 20	{ "type": "Research Article", "title": "Pneumococcal carriage and antibiotic susceptibility patterns in mother-baby pairs in a rural community in Eastern Uganda: a cross-sectional study", "authors": [ "Gabriel Madut Akech", "Mercy Naloli", "Paul Sebwami", "Patrick Kazibwe", "Maureen Atwikiriize", "Julius Onyait", "Paul Oboth", "Julius Nteziyaremye", "Rebecca Nekaka", "Jacob Stanley Iramiot", "Gabriel Madut Akech", "Mercy Naloli", "Paul Sebwami", "Patrick Kazibwe", "Maureen Atwikiriize", "Julius Onyait", "Paul Oboth", "Julius Nteziyaremye", "Rebecca Nekaka" ], "abstract": "Background: Pneumonia poses a significant threat to the lives of children below five years old worldwide, contributing to a high number of hospitalizations and death. Morbidity and morbidity are especially common in children under five and the elderly, although any age group can be affected. This study aimed to estimate pneumococcal carriage and determine antibiotic susceptibility patterns of the pneumococci isolated from mother-baby pairs in Ngora district after the rollout of the pneumococcal vaccine. We hypothesized that high carriage of Streptococcus pneumoniae in mothers leads to carriage in their babies and hence a greater chance of contracting pneumonia. Methods: Consecutive sampling was used to select 152 mother-baby pairs from community visits and those seeking care at the health facility. We collected nasal swabs from both baby and mother for culture and sensitivity testing using the Kirby-Bauer’s agar disc diffusion method. Results: This study found that there was a low prevalence of pneumococcal carriage in the mother-baby pair in Ngora district. We also observed high rates of microbial resistance to penicillin, which is the first-line drug for the management of pneumonia in Uganda. Conclusions: The relationship between pneumococcal carriage and immunization status suggests that the pneumococcal vaccine is protective against pneumococcal carriage. Resistance of S. pneumoniae to commonly used antibiotics was high.", "keywords": [ "Pneumococcal carriage", "mother-baby pair", "antibiotic susceptibility pattern", "immunization with PCV 10", "Eastern Uganda." ], "content": "Introduction\n\nPneumonia poses a significant threat to the lives of children below five years worldwide, contributing to a high number of hospitalizations and death1–3. Morbidity and morbidity is especially common in children under five and the elderly, although any age group can be affected4,5. One third of the overall global childhood mortality due to pneumonia has been attributed to Streptococcus pneumonia4. Developing countries are now faced with the double burden of community-acquired and nosocomial pneumonia, with both the under-funded health care systems and individual caretakers of the affected patients bearing negative economic consequences6. Streptococcus pneumoniae has been reported to be the most commonly isolated bacteria in both community-acquired and nosocomial pneumonia5. A high proportion of mortality due to community-acquired pneumonia has been documented, with up to 81% of these deaths occurring outside of hospital due to challenges related to access to health care in low- and middle-income countries7. The evolution of antimicrobial resistance among organisms causing pneumonia has made the consequences of childhood pneumonia even worse.\n\nOver two million children die annually from pneumonia each year, accounting for almost 1 in 5 under five deaths in low- and middle-income countries on average, as compared to 1 in 66 children in high-income countries8.\n\nPneumonia, together with diarrhea and malaria, was responsible for the deaths of 2.2 million children under five in 2012 in Sub-Saharan Africa, accounting for a third of all deaths in under-fives in this region9. In Uganda, pneumonia kills around 24,000 children below the age of five every year2. The overall pneumococcal carriage among children under five was estimated to be 56% when the PCV-10 and PCV-13 coverage in Uganda was 42% and 54%, respectively4. With improved PCV-10 and PCV-13 vaccine coverage, most of the invasive serotypes should be covered4. Pneumococcal carriage among mothers may lead to pneumococcal infections in children by droplet infection. This study aimed to estimate pneumococcal carriage and determine antibiotic susceptibility patterns of the pneumococci isolated from mother-baby pairs in Ngora district, Eastern Uganda.\n\n\nMethods\n\nWe obtained ethical approval for this study from Mbale Regional Referral Hospital Research and Ethics Committee (MRRHREC060418). A letter of introduction to the study was obtained following approval from the Busitema University Faculty of Health Sciences community education program office and used by the researchers to seek permission from the Medical Superintendent of Ngora District Health Centre IV. Written informed consent for participation was obtained from participants preceding to the actual data collection exercise. Access to collected data was restricted to persons directly involved in the study only. Participants were free to withdraw their consent to participate in the study at any stage of the study. Such a decision did not affect the medical care they received or possible participation in future research studies in any way. The procedures were verbally explained to the research participants by the researchers and those who agreed to continue with the study could consent and participate.\n\nA cross-sectional study was carried out in Ngora district Health Centre IV and Ngora Health Center IV catchment area from 7th April to 5th May 2018. It which serves a population of approximately 142,487. Ngora district is one of the districts in Teso sub-region and was part of Kumi district until 2010, when it was established as a separate district by a Uganda parliamentary act. Ngora district covers an area of approximately 715.9 square kilometers and is predominantly inhabited by the Iteso and Kumam ethnicities. According to the Ugandan healthcare hierarchy of organizations, a Health Center IV is expected to serve a population of up to 100, 000 people, meaning at its current capacity, this health facility operates above the level of a Health Center IV10.\n\nChildren under five years old and their mothers were selected in a ‘mother-baby pair’ to determine the relationship between carriage of pneumococci in the baby, prevalence of pneumococci in mothers and the child’s immunization status. The inclusion criteria were mother –baby pairs from selected villages in the community, and those who visited Ngora Health Center IV for routine immunization with babies under the age of five years. For this study, we defined a baby as any person under the age of five years and a mother was considered as either biological or any other female in direct care of the baby for at least four weeks. This was aimed at comparing samples from the baby, and the person in closest contact with the baby for the longest duration of time in the most recent past to allow presumed cross transmission/cross-infection. Exclusion criteria were babies above five years, and those presented by male care takers as those were presumed to spend less time with the babies. Mothers who did not consent or opted out at any stage of the research were excluded as well. The purpose of the research was explained to the mothers and they were asked to voluntarily participate in the research.\n\nConsecutive sampling was used. Every participant meeting the criteria of inclusion was selected at the young child clinic and in randomly selected villages in the community until the required sample size was achieved. This method is relatively easy to employ and reduces the chances for intentional and unintentional manipulation by staff, or errors due to confusion. A total of 152 mother-baby pairs fulfilling the inclusion criteria were sampled. Sample size was estimated using the Kish and Leslie formula developed in 1965.\n\n\n\nDemographic data was captured using a pre-tested questionnaire. The questionnaire was pre-tested with 10 mother-baby pairs at Mbale regional referral hospital Young Child Clinic (YCC) because the mother-baby pair in Mbale hospital had homogenous characteristics with our target population in Ngora district. After pre-testing of the questionnaires, some questions were rephrased for clarity. The Uganda national immunization guidelines require all children to receive their first PCV dose at six weeks (1.5 months), second dose at 10 weeks (2.5 months) and third dose at 14 weeks (3.5 months). Therefore, in the questionnaire, full immunization was defined as any child 14 weeks (3.5 months) of age and above, who had received all the three PCV doses as stipulated in the national immunized schedule. Partial immunization included children above six weeks (1.5 months) of age who had received less than three doses of PCV, whether off schedule, or still on schedule as per the national immunization guidelines. All children below five years of age who had not received any single dose of PCV including those at below six weeks (still on schedule) were categorized as not immunized.\n\nNasal pharyngeal specimens were collected from the posterior nasopharynx of the mother and the baby using sterile cotton swab sticks moistened with 0.9% physiological saline. Sample collection was carried out at the young child clinic in Ngora Health Centre IV and at the selected villages in the community by qualified hospital laboratory technicians. Separate swabs were used to collect samples from a mother and a baby in a mother-baby pair. To prevent sample contamination, the swab was placed in a casing containing Amies transport medium and immediately placed into a cool box containing ice packs for transportation to Busitema University Microbiology laboratory for culture and susceptibility testing within 12 hours.\n\nSamples were cultured on sheep blood agar and chocolate agar, followed by 24 hours of incubation at 37°C anaerobically. The isolates were identified morphologically by colony appearance and Gram staining. Optochin sensitivity and bile solubility testing were conducted on colonies that were potentially identifiable as S. pneumoniae by alpha-haemolytic appearance on the culture media and lancet shaped Gram-positive cocci appearing in pairs.\n\nA 0.5 McFarland standard of S. pneumoniae was made from a 24-hour subculture by suspending colonies in sterile normal saline and inoculated by swabbing onto a plate of Mueller-Hinton agar supplemented with 7% sheep blood for susceptibility testing. Antibiotic susceptibility to penicillin G (1U), chloramphenicol (30µg), tetracycline (10µg), clindamycin (2µg), erythromycin (30µg) and ceftriaxone (30µg) was determined using modified Kirby-Bauer agar disc diffusion methods and the disc zone diameters were interpreted using the Clinical and Laboratory Standards Institute Guidelines12.\n\nCollected data were entered in Microsoft Excel 2010, cleaned, coded and imported to SPSS Version 16.0 statistical package for analysis. Data were analyzed using descriptive statistics, frequencies and bivariate analyses (cross-tabulations). The primary outcome was pneumococcal carriage and the secondary outcome was antibiotic resistance. Statistical frequency distribution tables and graphs were used for data presentation in terms of proportions, absolute values, percentages and confidence intervals for point approximations at 95% level of confidence, with P<0.05 considered as statistically important.\n\nLaboratory procedures were performed by laboratory scientists under close supervision of a clinical microbiologist to ensure quality results were obtained. Data was double entered into Microsoft Excel for accuracy and reliability. ATCC 49619 S. pneumoniae was used as a control strain for quality assurance during isolation and drug susceptibility testing.\n\n\nResults\n\nAt the young child clinic, a total of ninety-three pairs were sampled, two of whom were excluded because the babies were presented by male care takers, and one mother opted out due to fear of discomfort associated with the procedure for sample collection. Ninety pairs were recruited. In the community, a total of sixty-nine pairs were sampled, five were excluded because they presented children above five years of age, while two were presented by male caretakers. Sixty-two were recruited, making a total of 152 mother-baby pairs.\n\nThe study participants comprised of 152 mothers and 152 babies. Of the 152 babies, 74 were male and 78 were female with the age range of 0–59 months. The youngest mother was 16 years, whereas the oldest was 44 years. None of the mothers who participated in the study reported having formal employment13.\n\nDuring the study, 304 samples were collected; 152 from the mothers and 152 from the children, making 152 mother-baby pairs. All samples were cultured, and antibiotic susceptibility was carried out on the isolated pneumococci. Out of 152 samples from the mothers, only five (5/152) isolates of pneumococci were obtained whereas seven (7/152) were isolated from the babies. Only one mother-baby pair (1/152) was found to be colonized with pneumococci in both mother and baby and the rest of S. pneumoniae colonized either the mother or baby.\n\nDuring data collection, immunization status of the baby was categorized as fully immunized, not immunized and partially immunized among different age groups (Figure 1). There was high immunization coverage among the children above 12 months old but lower in the 3.5-<12 age group.\n\nThe antibiotic susceptibility testing was done on positive isolates for both mother and baby.\n\nGenerally, a high trend of anti-microbial resistance was observed among the S. pneumoniae isolated (Table 1). The highest resistance patterns were recorded with chloramphenicol (50%) and tetracycline (50%), whereas the lowest resistance was recorded in clindamycin (17%).\n\nBabies that were fully immunized had a lower likelihood of being colonized by S. pneumoniae than their non-immunized counterparts P<0.05. Other factors examined by this study were not significantly associated with colonization with S. pneumoniae among the babies.\n\n\nDiscussion\n\nWe determined the prevalence of pneumococcal carriage and factors associated with colonization of pneumococci in a mother-baby pair in our study. Out of the 304 nasal swabs cultured, only 12 (3.95%) were positive for pneumococci, seven (4.61%) in children under five and five (3.29%) in mothers. We report a low carriage of pneumococci among mothers and babies that were included in this study. In contrast, a previous study in Iganga/Mayuge reported high carriage rates of over 50% in children aged under five years4. In the Iganga/Mayuge study, participants were selected on the basis of presentation with pneumonia symptoms as defined by WHO guidelines, as opposed to our study, which included all children that fulfilled our selection criteria and did not take into account signs and symptoms of pneumonia. In a similar study carried out in Kenya, 90.0% of children were colonized with pneumococci. Both the Iganga/Mayuge and Kenyan studies were carried out prior to the introduction of PCV-10, accounting for the difference in carriage observed in our study. Different studies have shown varied carriage rates of pneumococci among children under five in Uganda and elsewhere4,14,15, with most of them reporting a higher carriage rate than reported in our study. A systematic review reported the carriage rate in Africa to range between 21–94%16, with more studies done among children than in adult population. The high immunization coverage for PCV-10 in Ngora district could further explain the low carriage rate of pneumococci in our study as opposed to the Iganga/Mayuge study, which indicated a high carriage rate of 56% at a lower immunization coverage of 42% for PCV-10 and 54% for PCV-13. There was a statistically significant relationship between the pneumococcal carriage and immunization status of the babies in our study (Table 2). Pneumococcal carriage is a prerequisite for disease; therefore, our findings suggest that full immunization with PCV-10 is protective against pneumococcal carriage and hence pneumonia caused by S. pneumoniae. Several studies have reported a decrease in the burden of invasive pneumococcal disease and serotype distribution since the introduction of the PCV vaccines17–20. The immunization coverage for the first dose of PCV-10 (PCV1) in Ngora district in the current study was 97.78% (133/136), and 2.22% (3/136) of children above six weeks had not received PCV1. Of the 152 participants, 10.53% [16] were children below six weeks and were therefore not eligible for immunization with PCV10. The immunization coverage for PCV3 was 90.99% (101/111). Children below fourteen weeks who had not received PCV3 were excluded from the denominator because they were not eligible.\n\nWe also report a low carriage rate of pneumococci among the mothers. A similar study in coastal Kenya indicates that pneumococcal carriage was associated more with children under five than adults15. The low carriage rate of pneumococci among adults has been attributed by other studies to the development of natural immunity21. The upper respiratory tract apparently appears a disadvantageous niche for S. pneumococci due to the development of mucosal host defenses such as sIgA22,23. Also vaccination with PCV has resulted in the development of herd immunity in the adult population against S. pneumoniae15. Cases of pneumococcal colonization are, however, reported to rise in the elderly population due to immune senescence24, with many countries not considering the importance of immunization to this group of people.\n\nIn our analysis, there was no statistically significant association between carriage of pneumococci and sex/gender of the child. This finding is similar to the results of a systematic review in Africa, which noted that there was no association between pneumococcal carriage and gender16, although one study associated pneumococcal carriage with males and the other that reported association of carriage with females.\n\nA trend of antimicrobial resistance was observed in chloramphenicol, tetracycline and erythromycin. Other studies have similarly reported resistance of pneumococci to commonly used antibiotics25,26. In a study of erythromycin-resistant S. pneumoniae, 81% of the isolates were resistant to tetracycline and 76% were multi-drug resistant, whereas 12% were resistant to clindamycin, tetracycline, chloramphenicol and kanamycin combined25. As opposed to our study, low resistance rates to tetracycline, erythromycin, chloramphenicol and ceftriaxone were reported in Tanzania26. Again, contrary to our findings, an earlier study in Uganda reported no resistance to erythromycin and ceftriaxone27, indicating the emergence of antimicrobial resistance against those drugs, which may be attributed to the irrational use of antibiotics in Uganda and also due to the fact that such drugs are given empirically since there is no laboratory capacity to carry out culture and sensitivity studies.\n\nWe were not able to serotype the pneumococci isolated to determine the circulating serotypes and to link pneumococcal carriage to development of the disease.\n\n\nConclusions and recommendations\n\nWe report low pneumococcal carriage in the mother-baby pair in Ngora district. There was no significant relationship between pneumococcal carriage in the mother and prevalence in the baby. The relationship between pneumococcal carriage and immunization status suggest that PCV-10 is protective against pneumococcal carriage. Resistance of S. pneumoniae to commonly used antibiotics was high.\n\n\nData availability\n\nOSF: Pneumococcal carriage and antibiotic susceptibility patterns in mother-baby pairs in a rural community in Eastern Uganda: a cross-sectional study. https://doi.org/10.17605/OSF.IO/H9X7R13\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 gratefully acknowledge our research participants who provided useful information for this work. We are also grateful to the Ngora district local government leadership for the support and good will towards this project.\n\n\nReferences\n\nMartins AL, Nascimento Dda S, Schneider IJ, et al.: [Incidence of community-acquired infections of lower airways among infants]. Rev Paul Pediatr. 2016; 34(2): 204–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZar HJ, Madhi SA, Aston SJ, et al.: Pneumonia in low and middle income countries: progress and challenges. Thorax. 2013; 68(11): 1052–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSigaúge B, Verani JR, Massora S, et al.: Burden of invasive pneumococcal disease among children in rural Mozambique: 2001-2012. PLoS One. 2018; 13(1): e0190687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLindstrand A, Kalyango J, Alfvén T, et al.: Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate? PLos One. 2016; 11(11): e0166018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdler N, Weber H, Gunadasa I, et al.: Adherence to therapeutic guidelines for patients with community-acquired pneumonia in Australian hospitals. Clin Med Insights Circ Respir Pulm Med. 2014; 8: 17–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNair GB, Niederman MS: Nosocomial pneumonia: lessons learned. Crit Care Clin. 2013; 29(3): 521–46. PubMed Abstract \| Publisher Full Text\n\nle Roux DM, Zar HJ: Community-acquired pneumonia in children - a changing spectrum of disease. Pediatr Radiol. 2017; 47(11): 1392–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nlevine O, Dinleyci EC: Pneumonia: The forgotten killer of children. Annotated. 2010; 4(10): 27–8. Reference Source\n\nGreen RJ, Kolberg JM: Neonatal pneumonia in sub-Saharan Africa. Pneumonia (Nathan). 2016; 8(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmelia A, Walter A, Emmanuel A, et al.: Awareness of antimicrobial resistance among Primary Health Care workers in Buyende district, rural eastern Uganda. Microbiol Res J Int. 2017; 22(5): 1–11. Publisher Full Text\n\nNantanda R, Tumwine JK, Ndeezi G, et al.: Asthma and pneumonia among children less than five years with acute respiratory symptoms in Mulago Hospital, Uganda: evidence of under-diagnosis of asthma. PLoS One. 2013; 8(11): e81562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInstitute CaLS: Performance Standards for Antimicrobial Susceptibility Testing. 2014; 2014: M100–S25. Reference Source\n\nIramiot JS, Akech GM, Naloli M, et al.: Pneumococcal carriage and antibiotic susceptibility patterns in mother-baby pairs in a rural community in Eastern Uganda: a cross-sectional study. 2020. http://www.doi.org/10.17605/OSF.IO/H9X7R\n\nRutebemberwa E, Mpeka B, Pariyo G, et al.: High prevalence of antibiotic resistance in nasopharyngeal bacterial isolates from healthy children in rural Uganda: A cross-sectional study. Ups J Med Sci. 2015; 120(4): 249–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdullahi O, Nyiro J, Lewa P, et al.: The descriptive epidemiology of Streptococcus pneumoniae and Haemophilus influenzae nasopharyngeal carriage in children and adults in Kilifi district, Kenya. Pediatr Infect Dis J. 2008; 27: 59–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUsuf E, Bottomley C, Adegbola RA, et al.: Pneumococcal carriage in sub-Saharan Africa--a systematic review. PLoS One. 2014; 9(1): e85001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuevara M, Barricarte A, Gil-Setas A, et al.: Changing epidemiology of invasive pneumococcal disease following increased coverage with the heptavalent conjugate vaccine in Navarre, Spain. Clin Microbiol Infect. 2009; 15(11): 1013–9. PubMed Abstract \| Publisher Full Text\n\nKellner JD: Update on the success of the pneumococcal conjugate vaccine. Paediatr Child Health. 2011; 16(14): 233–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams SR, Mernagh PJ, Lee MH, et al.: Changing epidemiology of invasive pneumococcal disease in Australian children after introduction of a 7-valent pneumococcal conjugate vaccine. Med J Aust. 2011; 194(3): 116–20. PubMed Abstract \| Publisher Full Text\n\nHo PL, Chiu SS, Ang I, et al.: Serotypes and antimicrobial susceptibilities of invasive Streptococcus pneumoniae before and after introduction of 7-valent pneumococcal conjugate vaccine, Hong Kong, 1995–2009. Vaccine. 2011; 29(17): 3270–5. PubMed Abstract \| Publisher Full Text\n\nRamos-Sevillano E, Ercoli G, Brown JS: Mechanisms of Naturally Acquired Immunity to Streptococcus pneumoniae. Front Immunol. 2019; 10: 358. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinsker U, Lees JA, Hammond AJ, et al.: Immune exclusion by naturally acquired secretory IgA against pneumococcal pilus-1. J Clin Invest. 2020; 130(2): 927–941. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCroucher NJ, Campo JJ, Le TQ, et al.: Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening. Proc Natl Acad Sci U S A. 2017; 114(3): E357–E366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Buynder P, Booy R: Pneumococcal vaccination in older persons: where are we today? Pneumonia (Nathan). 2018; 10(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkdoğan Kittana FN, Mustak IB, Hascelik G, et al.: Erythromycin-resistant Streptococcus pneumoniae: phenotypes, genotypes, transposons and pneumococcal vaccine coverage rates. J Med Microbiol. 2019; 68(6): 874–81. PubMed Abstract \| Publisher Full Text\n\nMoyo SJ, Steinbakk M, Aboud S, et al.: Penicillin resistance and serotype distribution of Streptococcus pneumoniae in nasopharyngeal carrier children under 5 years of age in Dar es Salaam, Tanzania. J Med Microbiol. 2012; 61(Pt 7): 952–9. PubMed Abstract \| Publisher Full Text\n\nJoloba ML, Bajaksouzian S, Palavecino E, et al.: High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda. Int J Antimicrob Agents. 2001; 17(5): 395–400. PubMed Abstract \| Publisher Full Text" }	[ { "id": "73502", "date": "01 Dec 2020", "name": "Wilber Sabiti", "expertise": [ "Reviewer Expertise I have expertise in Microbiology", "Molecular biology and Immunology. I understand all aspects of the paper. It will be great for authors to explain their sampling method and why they chose it." ], "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 sought to decipher the relationship between pneumococcal carriage and PCV-10 vaccine. They asked whether the vaccine had an impact on the carriage of pneumococcal bacteria among the under-five children. In a cross-sectional study involving participants from Ngora district health centre IV and its catchment area, they showed a significant reduction of carriage following vaccination. Although the sample size was small in relation to the population covered by Ngora health centre IV, having a baseline carriage prior to vaccination and prevalence in the area helped give credence to the findings and conclusions. Nevertheless, it is not clear whether the authors used purposive or random sampling. Random sampling would have been better for such a sample in a large population. The article is well written, easy to read and follow and the conclusions are reflective of the findings. The discussion is well balanced putting the findings into context of other studies done before. The study adds evidence to the value of pneumococcal vaccine and I strongly recommend a follow on study covering the whole country. The addition of an immunological analysis arm to the study would serve as evidence that the observed associations are indeed due to vaccination.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7223", "date": "18 Oct 2021", "name": "Jacob Stanley Iramiot", "role": "Author Response", "response": "Thank you very much for that very useful feedback" } ] }, { "id": "76579", "date": "11 Jan 2021", "name": "Adnan Al-Lahham", "expertise": [ "Reviewer Expertise Streptococcus pneumoniae resistance", "epidemiology and typing" ], "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 article \"Pneumococcal carriage and antibiotic susceptibility patterns in mother-baby pairs in a rural community in Eastern Uganda: a cross-sectional study\" should be a good one since it contains results of the pneumococcal carriage in the rural area of eastern Uganda. comments to the article:\n\nRegarding the abstract: It should contain at least the summary of the results obtained but has no results at all, and it has some mistakes like morbidity and morbidity. One more comment is that the background starting to talk about pneumonia which has nothing to do with the research results. A third comment on the abstract: it contains only text and the results are only two lines.\n\nThe Introduction contains repeated data as in the abstract background, and it talks mainly about the pneumonia not carriage.\n\nIn the methodology: There are mistakes like Nasal Pharyngeal, not Nosopharyngeal. Secondly, in the laboratory procedures, it is stated that samples were cultured anaerobically which is wrong and the CLSI reference from 2014 is not the recent one.\n\nIn the results: a. 152 samples were taken and cultured from mothers and children as pairs. As a result, only 12 isolates were obtained. This result is not accepted. b. Why immunization status, since there is no data showing these cases are vaccinated or not. c. The table with the sensitivity shows only the 12 strains which is not ok since the method is not valid for the isolation. d. There are no data available about the serotypes or the macrolide resistant phenotypes or genotypes\n\nAs a result: This paper is not eligible for indexing and cannot be accepted from my side.\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? 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? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "7224", "date": "18 Oct 2021", "name": "Jacob Stanley Iramiot", "role": "Author Response", "response": "The abstract has been improved for a better read. A thorough review of grammar and other language issues has been done. There have been a couple of mentions of pneumococcal carriage as a precursor to pneumonia in children which should better inform the audience. The authors mentioned the CLSI 2014 because that was the version available and used by the team then. The authors acknowledged that they were not able to do serotyping in the limitation section of the manuscript. About the validity of the methods, it is not clear to the authors what the reviewer really means by this because standard methods and procedures were followed and as available in our setting." } ] } ]	1	https://f1000research.com/articles/9-1156
https://f1000research.com/articles/9-1310/v1	10 Nov 20	{ "type": "Systematic Review", "title": "Breastfeeding assessment tools for at-risk and malnourished infants aged under 6 months old: a systematic review", "authors": [ "Concetta Brugaletta", "Karine Le Roch", "Jennifer Saxton", "Cécile Bizouerne", "Marie McGrath", "Marko Kerac", "Jennifer Saxton", "Cécile Bizouerne", "Marie McGrath", "Marko Kerac" ], "abstract": "Background: Many small and malnourished infants under 6 months of age have problems with breastfeeding and restoring effective exclusive breastfeeding is a common treatment goal. Assessment is a critical first step of case management, but most malnutrition guidelines do not specify how best to do this. We aimed to identify breastfeeding assessment tools for use in assessing at-risk and malnourished infants in resource-poor settings. Methods: We systematically searched: Medline and Embase; Web of Knowledge; Cochrane Reviews; Eldis and Google Scholar databases. Also the World Health Organization (WHO), United Nations International Children’s Emergency Fund (UNICEF), CAse REport guidelines, Emergency Nutrition Network, and Field Exchange websites. Assessment tool content was analysed using a framework describing breastfeeding ‘domains’ (baby’s behaviour; mother’s behaviour; position; latching; effective feeding; breast health; baby’s health; mother’s view of feed; number, timing and length of feeds). Results: We identified 29 breastfeeding assessment tools and 45 validation studies. Eight tools had not been validated. Evidence underpinning most tools was low quality and mainly from high-income countries and hospital settings. The most comprehensive tools were the Breastfeeding, Evaluation and Education Tool, UNICEF Baby-Friendly Hospital Initiative tools and CARE training package. The tool with the strongest evidence was the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form. Conclusions: Despite many possible tools, there is currently no one gold standard. For assessing malnourished infants in resource-poor settings, UNICEF Baby-Friendly Hospital Initiative tools, Module IFE and the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form are the best available tools but could be improved by adding questions from other tools. Allowing for context, one tool for rapid community-based assessment plus a more detailed one for clinic/hospital assessment might help optimally identify breastfeeding problems and the support required. Further research is important to refine existing tools and develop new ones. Rigorous testing, especially against outcomes such as breastfeeding status and growth, is key.", "keywords": [ "Breastfeeding", "Assessment Tools", "Infants" ], "content": "Introduction\n\nProtecting breastfeeding has been described as the single most effective child survival intervention (UNICEF, 2009; WHO, 2007). It also plays a key role in reducing the global burden of undernutrition (The Lancet Series, 2008) and is one of 13 priority interventions highlighted by the international ‘Scaling Up Nutrition’ movement (SUN, 2010). Despite this, suboptimal breastfeeding practices are common, accounting for significant morbidity and 804,000 deaths per year - 11.6% of all deaths in children aged under 5 years worldwide (Black et al., 2013). The greatest burden of mortality and morbidity is in low income countries. High background mortality and high rates of undernutrition and communicable disease all make the protective effects of breastfeeding critical. With collapses in infrastructure and normal societal networks, emergency affected populations are particularly vulnerable if breastfeeding is not supported and problems are not quickly identified and addressed.\n\nWhilst the importance of breastfeeding is widely recognised, supporting it can be challenging. Under the overall heading of ‘Promoting proper feeding for infants and young children’, the World Health Organization (WHO) lists several areas of work including: the Baby-Friendly Hospital Initiative (BFHI) (WHO/UNICEF, 2009a); promotion of exclusive breastfeeding; and the International Code of Marketing of Breast-milk substitutes. These initiatives are aimed at population level breastfeeding support; there is good evidence of their effectiveness (Beake et al., 2012). More challenging is how to help those who fall through these population ‘safety nets’; when an individual mother-infant pair presents with an established problem. Managing very small infants, those with growth failure and other high-risk characteristics is particularly complex. Breastfeeding problems are common in this group but there are many other potential underlying causes and contributory factors (Goh et al., 2016). Breastfeeding problems may be a primary cause or secondary to other causes. There is also a wide and complex spectrum of breastfeeding problems ranging from a simple positioning difficulty leading to insufficient milk intake, milk insufficiency perception, early complementary feeding introduction, to secondary milk insufficiency due to maternal depression, due in turn to lack of social support at home (Amir & Ingram, 2008; Moore et al., 2012; Pannu et al., 2011; WHO/UNICEF, 1994).\n\nThis review arose from a project exploring the Management of (Nutritionally) At-risk Mothers and Infants aged under 6 months (MAMI) Project (ENN/UCL/ACF, 2010b). The goal of the original MAMI Project was to investigate the management of malnourished infants under six months of age in resource-poor and humanitarian settings, and to contribute to evidence-based, better practice guidelines to improve practice. The project identified that the burden of infant less than 6 months’ undernutrition is significant: worldwide, 3.8 million infants are severely wasted; 4.7 million are moderately wasted (Kerac et al., 2011). Since breastfeeding difficulties are associated with undernutrition (Gagliardi et al., 2012; Gribble et al., 2011) (Gribble et al., 2011) and exclusive breastfeeding in infants under 6 months, a common treatment goal (ENN/UCL/ACF, 2010a), the report also examined breastfeeding assessment as part of overall infant assessment. It found no ‘gold-standard’ breastfeeding assessment tool that catered for inpatient and community settings. This is a critical gap; correct ‘diagnosis’ of a breastfeeding problem is vital to inform appropriate support and treatment. Building upon and updating the work of the MAMI Project, this current review thus aims to: a) identify and profile currently available breastfeeding assessment tools; b) discuss their potential application for assessing at risk and malnourished infants under 6 months (i.e. to determine the link between breastfeeding problems and malnutrition in a particular individual; to describe the nature of that breastfeeding problem). Informed assessment is critical to targeted intervention of support.\n\n\nMethods\n\nBreastfeeding assessment tools were defined as: documented guidance for clinicians, nurses, midwives, community health workers and carers on how to observe and/or assess the breastfeeding performance. These could take the form of checklists, questionnaires, algorithms, indices, history taking forms or listing of the specific aspects of breastfeeding that should be assessed.\n\nInclusion criteria: We included articles that: tested or used breastfeeding assessment tools; integrated at least one clinically relevant maternal or child outcome (e.g. duration of breastfeeding, infant weight gain); reported on tool performance. Articles describing complex interventions that included breastfeeding support could only be included if it was clear which tool had been used, and if breastfeeding assessment had been explicitly mentioned in the intervention description. There were no study design restrictions.\n\nExclusion criteria: Tools that focused just on artificial feeding (i.e. use of a breastmilk substitute) or that were designed for women after breast augmentation/reduction surgery were not considered in this review. Also excluded were tools that involved complex and expensive technology that are not designed for routine clinical use in resource poor settings (e.g. those using electromyographic methods; direct measurements of breastmilk composition; web-based tools; software to measure sucking strength/effectiveness; ultrasound measures of milk removal/swallowing). Tools that focused on wider breastfeeding support (e.g. employer support) rather than the actual process of breastfeeding were also excluded as were those focused solely on change in health worker knowledge, attitude or practice as an outcome. The literature search was restricted to English language articles with human subjects.\n\nDatabases and search terms: Articles were identified by searching electronic database Medline and Embase via Ovid interface (full search strategy in Extended data (Kerac et al., 2020)). Key words and MeSH terms were selected by the review on The Lancet Breastfeeding Series (The Lancet Series, 2016) and a recent similar review on feeding assessment tools (Howe et al., 2008). Searches were finalised in March 2018. This updated an earlier search done as part of the original MAMI project performed on PubMed, Web of Knowledge, Cochrane Review, Eldis and Google scholar databases which concluded in November 2013. In that original search, highly relevant journals were also searched directly: Maternal and Child Nutrition, International Breastfeeding Journal, Journal of Human Lactation, and BMC Family Practice. Reference lists and the ‘related articles’ were used to identify further articles. A standard two-stage search strategy was used: initial screening of titles and abstracts; detailed review of full articles secondly. Since tools were few but varied, risk of bias was not formally scored for each individual study but is discussed under ‘limitations’ for studies as a whole.\n\nTo understand and characterise the tools we also examined:\n\nThere are several aspects or ‘domains’ of breastfeeding. Knowing which are affected helps guide appropriate subsequent treatment. We used an established framework (Moran et al., 2000) to characterise which aspects of breastfeeding the assessment tools assessed. These included: baby’s behaviour (e.g. alertness to feed), mother’s behaviour (e.g. watches and listens for baby’s cues), positioning (e.g. baby facing mother), attachment (e.g. lower lip turned outward on breast), effective feeding (e.g. sucking, swallowing, jaw movement and signs of milk release), health of the breast (e.g. nipple trauma), health of the baby (e.g. alert), and mother’s experience (e.g. feels strong suction). We added another domain on number, timing and length of feeds. We also noted any other domains identified by individual studies.\n\nStudies were grouped according to type of evidence presented. One group looked at prediction of later breastfeeding status. Another assessed test-retest, inter-rater reliability and sensitivity and specificity of tools. A final group of studies focused on assessment tools used to directly improve breastfeeding technique or experience.\n\n\nResults\n\nFrom a total of 15,649 titles and abstracts screened, a final count of 52 papers describing 29 distinct breastfeeding assessment tools were identified (Figure 1).\n\nDetails of the 29 tools identified are summarised in Table 1.\n\nExclusions and reason for those are presented in web-appendix (Extended data (Kerac et al., 2020)). We were unable to get sufficient information about two tools: The LATTM (Cadwell et al., 2004) and the Prague Newborn Behaviour Description Technique (Sulcova & Tisanska, 1994) so we could not include them in the final review.\n\nOf the 29 tools identified: 22 (76%) were developed in high-income countries and used in 31 studies carried out in high-income countries and four (14%) tools were developed in low and middle-income countries. Sixteen tools (55%) were developed for hospital settings. Of these, 24 (83%) tools were designed and/or tested for use in infants less than 6 months with breastfeeding problems; none of these were specifically designed for or tested on at risk and malnourished infants less than 6 months.\n\nTable 2 shows that most tools covered a number of different domains but only one, the Breastfeeding Evaluation and Education Tool (Tobin, 1996), covered them all.\n\nOther tools covering a wide range of domains were the Baby-Friendly Hospital Initiative (BFHI) guidelines (UNICEF, 2010; WHO/UNICEF, 2009a) and the CAse REport guidelines (CARE guidelines) (CARE, 2004). The BFHI and CARE guidelines also highlighted other items that could be useful for future testing: positions for low birth weight babies, differentiating between ‘perceived’ and ‘real’ milk insufficiency, mother’s health, and the use of BMS and dummies/pacifiers. The World Health Organization/United Nations International Children’s Emergency Fund (WHO/UNICEF) B-R-E-A-S-T-Feed Observation Form covered seven domains, missing out ‘health of the baby’ and ‘mother’s view of the feed’ (WHO/UNICEF, 1994). Additional domains identified by other tools included: mother’s comfort level, previous breastfeeding experience, other foods/liquids being given to the baby, loss of >10% of birth weight, hypertension and delivery type (Darmstadt et al., 2009; Dongre et al., 2010; Hall et al., 2002; Mannan et al., 2008; Milligan et al., 1996; Palmer et al., 1993).\n\nIn total, 12 (41%) tools had been tested for their ability to predict breastfeeding outcomes (Table 3).\n\nThe present studies either tested the tools or tested the intervention or tested both. The tools with the most studies testing their ability to predict breastfeeding outcomes during an intervention study were the LATCH (n=5), the WHO/UNICEF B-R-E-A-S-T-Feed observation form (n=6) and the BAS tool (n=4). The BAS was consistently predictive in all studies, although as shown in Table 2, it covers the least number of breastfeeding domains. There were mixed findings for the LATCH tool: three studies observed positive findings, and two reported limited ability of the tool to predict breastfeeding outcomes. The WHO/UNICEF B-R-E-A-S-T-Feed Observation Form was predictive of breastfeeding outcomes in three studies, but was not predictive of exclusive breastfeeding in a fourth study. Two further studies described the determinants of poor scores on the WHO/UNICEF B-R-E-A-S-T tool including repeated crying, colic history, shorter sleeping episodes and regurgitation (Yalcin & Kuskonmaz, 2011), and primiparity, cracked nipples, mastitis, preterm and low birth weight babies and poor suckling (Goyal et al., 2011).\n\nThe extent of tool testing varied substantially; 8 tools had no validation studies: Infant Feeding in Emergencies (IFE) Module 2 (ENN et al., 2007), Breastfeeding Evaluation and Education Tool (Tobin, 1996), Systematic Assessment of the Infant at the Breast (Shrago & Bocar, 1990), CARE guidelines (CARE, 2004), Via Christi, and tools identified by Walker (Walker, 1989), (Cadwell, 2007) and Righard & Alade, 1992 (Righard & Alade, 1992). Of the remaining 21 tools, we identified 45 validation studies. Of these, 32 were observational studies; 6 were randomised or cluster randomised controlled trials, two reported time trends; and 1 reported intervention baseline and endline data without a control group.\n\nThe BAS tool had four validation studies, all of which show positive results for the tool, in terms of ability to identify those at risk of breastfeeding cessation, and moderate sensitivity and specificity (Gianni et al., 2006; Hall et al., 2002; Mercer et al., 2010; Zobbi et al., 2011). The evidence to support the use of the Essential Nutrition Actions Framework tool is weak in terms of validation (i.e. no control group; not clear if the tool was routinely used) (Guyon et al., 2009). IBFAT also had a low inter-rater reliability. Furthermore, most studies were low quality (e.g. small sample size and observational designs) and were also conducted exclusively in high income settings (Furman & Minich, 2006; Matthews, 1988; Matthews, 1991b; Riordan & Koehn, 1997; Schlomer et al., 1999).\n\nNine tools were tested for test-retest and inter-rater reliability in eight studies - one study compared three tools. Two tools performed well: the Integrated Management of Childhood Illness (IMCI) showed good sensitivity and high specificity in highlighting breastfeeding problems judged against clinician assessments (Darmstadt et al., 2009); the Mother Infant Breastfeeding Progress Tool (MIBPT) showed high inter-rater agreement (Johnson et al., 2007). There were mixed findings for the remaining tools. Details of these studies are in Table 4.\n\nFew studies tested the use of tools to correct breastfeeding technique or to improve breastfeeding experience. These are shown in Table 5.\n\n\nDiscussion\n\nOur review identified a number of breastfeeding assessment tools which could be used in the management of our target group of at-risk and malnourished infants aged under 6 months. Though none of the tools were developed for or tested on this group directly, characterising them and understanding the underlying evidence-base allows for better informed decisions about which might be the most helpful for future programme use.\n\nRegarding the coverage of breastfeeding domains, only one tool (BEET) achieves full coverage of all the key assessment domains. The tools that achieve the widest coverage (IFE Module 2, BEET, and WHO/UNICEF B-R-E-A-S-T-Feed Observation Form and UNICEF/WHO Breastfeed Observation Aid) are generally those which have been developed with resource-poor low and middle income countries in mind. Although these tools are based on extensive clinical and field experience, they suffer from lack of validation research and miss some important domains (e.g. WHO/UNICEF B-R-E-A-S-T-Feed Observation Form misses health of the baby, IFE Module 2 misses positioning). These shortfalls could be addressed with minor modifications in the short term and with appropriately designed studies soon after to help determine which domains are the most important and relevant to patient care. Only 11 tools assess mothers’ own behaviour towards the baby: this is telling about her psychosocial status and can inform management. It is important to consider and account for such gaps since an infant may be effectively breastfed but at risk and malnourished for another reason, e.g. related to child health status or maternal factors. A challenge validating breastfeeding assessment tools is the lack of a ‘gold standard’ treatment option for at-risk and malnourished infants less than 6 months. This makes validation studies a challenge methodologically since it is difficult to separate out the performance of an assessment tool from the effectiveness of the subsequent management strategy in averting adverse nutrition/morbidity outcomes. It is likely that different tools and different levels of management will be appropriate to different settings, e.g.\n\nIn primary healthcare / community settings: simple and rapid breastfeeding assessment tools, associated with easy-to-deliver interventions and to prompt referral for more specialised support. For use by community healthcare workers who may have limited training and experience.\n\nIn secondary healthcare / outpatient clinic settings: more detailed tools could be appropriate but would need more training and staff with more background skills, expertise and time to deliver.\n\nIn tertiary-level inpatient settings: more complex assessments would be appropriate to identify more complex problems. These could be delivered by more highly trained healthcare staff such as nurses and doctors.\n\nNo single tool meets all these needs. Which tool is more appropriate to a given setting and individual mother-infant situation is itself an important question that warrants further testing and exploration.\n\nFor immediate use, whilst refining current tools and developing new future ones, the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form, the aids in Module 2 on IFE and UNICEF/WHO Breastfeed Observation Aid, offer the most promise for programmes targeting at-risk and malnourished infants aged under 6 months.\n\nIn future research testing current and new tools, there is a need to agree on the most appropriate outcomes for validation studies targeting at-risk and malnourished infants under 6 months. The fact that so many tools exist, and that they cover such a wide range of feeding outcomes and domains arguably reflects uncertainly and lack of consensus about how best to assess the effectiveness of breastfeeding. For example, must there always be sufficient infant weight gain associated with other measures of effective feeding? Most current evidence comes from high-income countries and hospital settings. For use in tackling the significant global burden of malnutrition in infants aged less than 6 months, this is a problem. More tools for low income countries and for community settings are urgently needed (Moran et al., 2000; Mulder, 2006; Riordan, 1998; Riordan & Koehn, 1997).\n\nAnother key finding of our review was the variable - and overall low - quality of evidence underpinning existing breastfeeding assessment tools. Often the evidence-base for a particular tool is unclear, particularly their effectiveness in identifying specific breastfeeding problems and facilitating a resolution. Prospective and ideally randomised studies testing tools’ ability to do this are important in the future (Da Costa et al., 2008). Simple checklists have been shown to be powerful if used consistently in clinical settings (Haynes et al., 2009; Pronovost et al., 2006). There is therefore an argument to develop checklist-based tools that can be incorporated into routine breastfeeding assessment, to maximize the chances of resolving breastfeeding problems. These should also be able to discriminate between different types of breastfeeding problems and lead clearly to specific interventions.\n\nWe found that tools varied in their level of complexity, and their scoring systems. This may make individual tools relevant only for specific contexts. For example, three tools involve two stages: IFE Module 2 includes a simple rapid assessment, followed by a full assessment (ENN et al., 2007); the BFHI guidelines may include initial use of the breastfeeding assessment form, leading on to the UNICEF/WHO breastfeed observation aid if necessary (UNICEF, 2010; WHO/UNICEF, 2009a); the IMCI algorithm includes both a brief history taking and observations of the breastfeed (Mannan et al., 2008). This is potentially a good thing. Rather than one tool trying to do everything, different tools for different levels of assessment could be helpful: e.g. a quick, basic tool for use in the community to identify and correct ‘simple problems and identify referral need, complemented by a more detailed tool if problems are suspected or identified; another more detailed one for clinic/hospital use assessing more serious and complex problems flagged by the first tools. Tool developers need to consider what the key contact points with infants are, and the associated opportunities and capacities with these contact points. Coupled with this must be the capacity to respond to any problems identified. To address breastfeeding in high mortality/morbidity settings, tools need to consider not just physiological issues and techniques around breastfeeding, but also the wider social and psychological factors, which may be contributing to or perpetuating a problem (Galipeau et al., 2017).\n\nFrom this review, Baby Friendly Hospital tools, the Module 2 IFE and WHO/UNICEF B-R-E-A-S-T-Feed Observation Form, have emerged as potentially useful for use in humanitarian settings with at-risk and malnourished infants under 6 months. They require a short training and they are easy-to-use. Baby Friendly Hospital tools and the Module 2 IFE could benefit from adaptation by adding the missing components that we would be considered useful for humanitarian contexts. While BFHI has become a ‘gold standard’ for maternity care in hospital setting, the effectiveness of the training course has been assessed but the evaluation of the breastfeeding assessment form requires more studies. Equally, these tools could be combined (e.g. by adding questions from one tool to another) in a way that might improve the quality of breastfeeding assessment, and that would take into account the specific needs and limitations of contexts with a high burden of undernutrition. It will be important to ascertain the feasibility of community health workers using these tools.\n\nBased on coverage of domains, appropriateness to target population and setting, and underlying evidence, WHO/UNICEF B-R-E-A-S-T-Feed Observation Form appears to be the most suitable for assessing at risk and malnourished infants aged under 6 months. In two Danish RCTs, health visitors were trained to conduct home visits incorporating breastfeeding assessment and classification of technique problems (Kronborg & Vaeth, 2009; Kronborg et al., 2007). One study found a 14% lower breastfeeding cessation rate amongst intervention participants, and greater confidence of mothers that their breast milk was sufficient. However, the other found no difference in exclusive breastfeeding rate or a reduction in breastfeeding problems - this may be due to a single corrective intervention being insufficient to resolve breastfeeding problems. The authors argued for on-going breastfeeding support to ensure breastfeeding problems are truly resolved. This idea is corroborated by a third Brazilian hospital-based RCT with a low socioeconomic population, which found no impact of a single breastfeeding assessment and correction on exclusive breastfeeding rates, breastfeeding technique or breastfeeding problems 30 days post-partum (De Oliveira et al., 2006). A further RCT in Brazil also used the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form but included a greater number of home visits (n=6). This observed a 39% increase in any breastfeeding, and a significant increase in exclusive breastfeeding. One limitation of this study is that it is difficult to unpick the effect of the breastfeeding observation and corrective advice from the other interventions during the home visit (Leite et al., 2005). This underlines the importance of not just having a good tool, but using it to maximum effect i.e. not just conducting a single assessment and correction, but providing on-going support through community outreach (Imdad et al., 2011). What is most encouraging about the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form is its apparent usability in routine clinical settings, with relatively short training if conducted for the use of the test only. As the tool is part of a broader training on breastfeeding counselling, it is recommended to explore the whole manual, but it is also possible to adapt to the situation’s needs. It would still be valuable to do further validation of this tool and possibly extend the tool components to include aspects of the baby’s health, as identified in the section on coverage of breastfeeding domains.\n\n\nWays forward\n\nAs well as standard validation studies, new tools or those initially developed in/adapted from resource-rich settings should be assessed for cultural relevance and sensitivity before they are considered for use in resource-poor developing country/humanitarian settings. This formative work should ideally precede detailed validation or intervention studies. Validity is likely to vary according to target patient group and studies should therefore be sufficiently powered to explore subgroups. Tools that are designed to assess breastfeeding in healthy, well-nourished infants are not necessarily as good or adequate for assessing sick or undernourished ones. As none of those tools presented above were developed and tested in malnourished children and since these infants are at particularly high risk of morbidity and mortality, specific tools should consider the needs of infants aged less than 6 months with malnutrition – the group who inspired this review in the first place. Since there are many factors potentially underlying or contributing to malnutrition, we believe that tools for this group should be part of a wider assessment of the mother-infant dyad and take an appropriately broad perspective by considering other factors known to impact on infant nutrition e.g. maternal mental health, maternal illness, and maternal malnutrition.\n\n\nLimitations\n\nWe acknowledge the limitation of our review. Firstly, it was restricted to articles written in English; there may be useful breastfeeding assessment tools published in other languages that were not captured.\n\nSecondly, it is possible that we missed some studies, e.g. those using a broader approach to improving infant feeding may not have explicitly mentioned breastfeeding assessment tools as part of their intervention protocol; those which were using a tool in a programme but were not in the title or abstract clearly evaluating/testing the tool itself; those that may have had relevant content (e.g. maternal psychosocial status) but did not meet the inclusion criteria of one clinically relevant maternal or child outcome.\n\nThird, we did not explicitly grade the quality of individual studies – this was felt not to add significant extra value to our review since observational studies, which comprised great majority of papers identified, are by definition low quality compared to intervention/RCT type designs. Quality grading would not have helped differentiate between more/less valuable tools, since the quality of evidence underpinning them all was generally low.\n\nFinally, we found few tools explicitly targeted to our setting and main patient group of interest. This is not ideal since it means applicability had to be extrapolated based on our judgement rather than on hard data.\n\nDespite these limitations, we do not believe that the overall direction or message arising from our findings are affected.\n\n\nConclusion\n\nIn this review of breastfeeding assessment tools for resource poor settings and targeting the assessment of malnourished infants less than 6 months, we have identified many possible but few stand-out ‘gold standard’ options. This represents an important evidence gap and highlights an urgent need for future research. The many different tools that we did find arguably show that one tool alone is unlikely to be suitable or even desirable. Tools must strike the right balance between simplicity, feasibility of use and minimal training requirements without losing the depth of information required to help healthcare workers and the women they are working with address breastfeeding difficulties. Thus, different tools for different levels of the health care system are needed: simple, quick-to-use tools for initial triage and problem identification in the community; more sophisticated tools for use in secondary and tertiary care settings where initial attempts at support have failed. Supplementary items such as pictures of good latch, and materials to help mothers and health workers understand the nature of breastfeeding problems (e.g. ‘take action cards’ (Dongre et al., 2010)), may be helpful. For any tool at any level, it is important that it leads to clear corrective actions. A “diagnosis” or “problem label” by itself is not always useful. Hence, future tools might give appropriate weight to problems, which can most readily be solved, or those which have the biggest short and long term impact. Research on breastfeeding assessment tools needs to consider such impacts – again, good test inter- and intra-observer validity is necessary but not alone sufficient to make a ‘good’ tool. It must help improve key outcomes like breastfeeding status and infant growth. Robustly designed studies in the contexts in which they will be used are essential.\n\nFinally, we note that time will be needed to develop and test better future breastfeeding assessment tools. Yet support for women and their infants is urgently needed now. Not having an ideal tool is not a reason to defer breastfeeding assessment of at risk and malnourished infants under 6 months. There are great opportunities at present to collect and report good quality operational data using tools that are currently available. Expanding the current literature on breastfeeding assessment will be of great benefit to future tool developers. More importantly, focus on this area will also raise the profile of and directly benefit breastfeeding as a key child nutrition, health and survival intervention.\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\nLSHTM Data Compass: Breastfeeding assessment tools for at-risk and malnourished infants aged under 6 months old: a systematic review, https://doi.org/10.17037/DATA.00001881 (Kerac et al., 2020).\n\nThis project contains the following extended data:\n\n- Tools excluded from the second stage of the literature search\n\n- Full search strategy\n\nLSHTM Data Compass: PRISMA checklist for ‘Breastfeeding assessment tools for at-risk and malnourished infants aged under 6 months old: a systematic review’, https://doi.org/10.17037/DATA.00001881 (Kerac et al., 2020).\n\nData are available under the terms of the Creative Commons Attribution-NonCommercial 2.0 UK license (CC BY-NC 2.0 UK).", "appendix": "Acknowledgements\n\nWe are thankful to Professor Andrew Seal, UCL Institute for Global Health for his support and we are also thankful to Anne-Dominique Israel, Senior Nutrition and Health advisor at Action contre la Faim for supporting the initiative.\n\n\nReferences\n\nAdams D, Hewell S: Maternal and professional assessment of breastfeeding. 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PubMed Abstract \| Publisher Full Text\n\nZobbi VF, Calistri D, Consonni D, et al.: Breastfeeding: validation of a reduced Breastfeeding Assessment Score in a group of Italian women. J Clin Nurs. 2011; 20(17–18): 2509–2518. PubMed Abstract \| Publisher Full Text" }	[ { "id": "74628", "date": "01 Dec 2020", "name": "Kerstin E. Hanson", "expertise": [ "Reviewer Expertise Pediatric and nutrition programming and case management in low-resource and humanitarian settings." ], "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 review by Brugaletta et al. addresses the important gap in tools and evidence for effective case management of at-risk and malnourished infants aged under 6 months old in low-resource and humanitarian settings. The authors focus specifically on the availability and quality of breastfeeding assessment tools for use in this population. They start with an excellent introduction highlighting the importance of this theme – describing the essential role of breastfeeding in protecting the health and lives of children and the subsequent place it has in global priority interventions, and the challenges that remain in properly addressing persistently suboptimal breastfeeding practices.\nResults of the review are presented in clear tables, summarizing important features of the various tools identified. The authors provide a clear breakdown of these features into the following categories: context, coverage of breastfeeding domains, ability to predict breastfeeding outcomes, evidence underpinning the tools, and ability to correct breastfeeding technique or improve breastfeeding experience. The analysis also addresses not only technical or academic features of the tools, but also “real world” implementation issues, and ability to bring about the desired outcomes - improved breastfeeding.\nThe discussion highlights strengths and gaps of individual tools, as well as the overall “collection” of tools identified. The authors are clear to state that none of the tools were directly developed for or tested on at-risk and malnourished infants aged under 6 months, nor do any of the tools fully meet the various needs in terms of categories outlined above. The authors do nevertheless identify three tools that could be used “for immediate use, whilst refining current tools and developing new future ones”.\nWe appreciate the overall approach to this review – identifying and analyzing current tools, recognizing that we do not currently have an ideal tool, explaining the key gaps and ways forward, and importantly – providing temporary best options. Minor suggestions to consider in subsequent versions:\nIn the abstract the authors list the following as part of their search: the World Health Organization (WHO), United Nations International Children’s Emergency Fund (UNICEF), CAse REport guidelines, Emergency Nutrition Network, and Field Exchange websites as parts of the search. These are not mentioned in the database and search terms of the methods section. They are perhaps listed in one of the references, but it might be helpful to include them in this later section of the manuscript as well.\n\nIn figure 1 it is unclear where the “Handsearch Papers” fit. This part of the search could be expanded upon in the methods section.\n\nUnder context the authors state: “Of the 29 tools identified: 22 (76%) were developed in high-income countries and used in 31 studies carried out in high-income countries and four (14%) tools were developed in low and middle-income countries.” What about the 3 tools, not included in the 22 developed in high-income countries and the 4 developed in low- and middle-income countries?\n\nThe introduction does a nice job addressing the particular challenges associated with “managing very smalll infants, those with growth failure and other high-risk characteristics”. It also highlights the complex spectrum of breastfeeding problems including potential underlying causes and contributory factors, including but not limited to maternal wellbeing and social support. Although these essential topics are touched upon very briefly in the results and discussion sections, and in a bit more detail in the ways forward section, the review could benefit with expansion of these critical topics.\n\nIn the discussion section the authors note that only one tool, BEET, achieves full coverage of all the key assessment domains. Yet, this tool is not included amongst those listed as potentially useful for immediate use; it could be useful to note why this is the case.\n\nIn the initial paragraphs of the discussion, the authors suggest: “For immediate use, whilst refining current tools and developing new future ones, the WHO/UNICEF B-R-E-A-S-T-Feed Observation Form, the aids in Module 2 on IFE and UNICEF/WHO Breastfeed Observation Aid, offer the most promise for programmes targeting at-risk and malnourished infants aged under 6 months”. Later, under the heading Which tools for resource-poor, high-undernutrition settings, they suggest: “From this review, Baby Friendly Hospital tools, the Module 2 IFE and WHO/UNICEF B-R-E-A-S-T-Feed Observation Form, have emerged as potentially useful for use in humanitarian settings with at-risk and malnourished infants under 6 months.“ Referring to the tools listed in the tables, we imagine that the Baby Friendly Hospital tools and UNICEF/WHO Breast Observation Aid noted above are referring to the same tool. If so, the same naming convention should be used in both instances for clarity.\n\nThere are a number of minor typos and grammatical errors throughout the paper and tables that should be corrected.\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": "6714", "date": "25 May 2021", "name": "Concetta Brugaletta", "role": "Author Response", "response": "Thank you very much for taking the time to read the manuscript and share your comments and constructive advice. 1. Regarding your first comment where you point out that we mentioned the World Health Organization (WHO), United Nations International Children’s Emergency Fund (UNICEF), CAse REport guidelines, Emergency Nutrition Network, and Field Exchange websites as parts of our search but we didn't mentioned these in the database and search terms; we have now clarified this in the methods search under the databases and search terms paragraph as follow: “We also included hand search papers form grey literature, WHO and ENN websites”. 2. Regarding your second comment for figure n1 where was unclear where the “Hand search Papers” fit; we amended figure n1 in the new version of the manuscript. We also add a reference Preferred Reporting items for Systematic Reviews and Meta-Analyses (PRISMA) diagram of literature search results. Diagram retrieved from: http://prisma-statement.org/PRISMAStatement/FlowDiagram.aspx 3. Regarding your request of clarification for the paragraph “Of the 29 tools identified: 22 (76%) were developed in high-income countries and used in 31 studies carried out in high-income countries and four (14%) tools were developed in low and middle-income countries.\" Three tools are missing such designations\"; The tools that do not give any information about country of origin (described in the table 1 as not specified) are: - Baby-friendly Hospital Initiative (BFIH) Worldwide (When the Baby-friendly Hospital Initiative was conceived in the early 1990s in response to the 1990 Innocenti Declaration on the Protection, Promotion and Support of Breastfeeding call for action, there were very few countries that had dedicated Authorities or Committees to oversee and regulate infant feeding standards.) - Essential Nutrition Action Message (Guyon & Quinn 2011) Low and middle-income countries - Infant Feeding in Emergency (IFE) (ENN 2007) Low and middle-income countries - Neonatal Eating Assessment tool (NeoEAT) This is a USA based organisation: AWHONN, the Association of Women's Health, Obstetric and Neonatal Nurses. https://www.awhonn.org/ We amended the information above in table n1 of the new version of the manuscript. In the text of the new version of the manuscript we clarify the follow information: “Of the 29 tools identified: 22 (76%) were developed in high-income countries and used in 31 studies carried out in high-income countries, six (21%) tools were developed in low and middle-income countries and one (3%) was developed worldwide”. 4. Regarding your comment on considering complex spectrum of breastfeeding problems including potential underlying causes and contributory factors, including but not limited to maternal wellbeing and social support; It is a great suggestion; the number of words required does not allow us to expand on this very interesting and crucial topic. We have however added a reference of the MAMI website, which is regularly being updated and a note saying “The mother-infant dyad is at the heart of approaches to treat malnutrition, but wider family and community relationship are also important but cannot be treated extensively in this review”. (ENN/LSHTM, 2021)https://www.ennonline.net/ourwork/research/mami https://www.ennonline.net/mami/practice 5. Regarding your comment on the discussion section where only one tool, BEET, achieves full coverage of all the key assessment domains. Yet, this tool is not included amongst those listed as potentially useful for immediate use; Thank you for your comments. The justification is in the paragraph on evidence underpinning tools: ‘The extent of tool testing varied substantially; 8 tools had no validation studies: Infant Feeding in Emergencies (IFE) Module 2 (ENN et al., 2007), Breastfeeding Evaluation and Education Tool (Tobin, 1996)’. This is why we do not include for immediate use. We have added a note in the text “only one tool (BEET) achieves full coverage of all the key assessment domains, but there was no validation study at our knowledge”. 6. Regarding your comment on request of clarification if the Baby Friendly Hospital tools and UNICEF/WHO Breast Observation Aid are referring to the same tool. We would like to explain their difference and the rational for our subtle considerations: - The Baby Friendly Hospital tools: is a checklist (12 to 14 items) designed with the aim to identify area of problem and give advice. These tools take in consideration health professional background and day of life of the baby and one can also be self-administered. This means there are 4 slightly different tools available: for mother and midwife, for mother and health visitor, for neonatal and for mother alone. The domain covered are: baby’s and mother behaviour, positioning, lactating, effective feeding, breast health, baby health, mothers view (in addition these tools look at urine and stools, formula). (https://www.unicef.org.uk/babyfriendly/baby-friendly-resources/implementing-standards-resources/breastfeeding-assessment-tools/). - The UNICEF/WHO Breastfeeding Observation Aid is a checklist of identify dichotomous items ( 42 items/5 scales) list signs that represent that BF is going well versus possible difficulties. It cover similar breastfeeding domains of the BFH tools but is a more simple checklist and doesn’t offer possible solutions. (https://www.scribd.com/document/353627133/Breastfeed-Observation-Job-Aid) This is why we maintained 2 different names and we advise to use the BFHT for resource-poor, high undernutrition settings where it is useful to have alongside the assessment also some initial advice. 7. Regarding your advice to proofread the paper to improve the overall writing; we have proofread and now we think this looks fine in this last of the manuscript." } ] }, { "id": "78113", "date": "18 Feb 2021", "name": "Sandra Fucile", "expertise": [ "Reviewer Expertise Oral feeding in critically ill infants." ], "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\nBrugaletta et al.'s, review of breastfeeding assessment tools for assessing at-risk and malnourished infants in resource-poor settings provides a comprehensive literature search of available tools for this highly vulnerable population. The authors reveal there is no ‘gold standard’ tool available for at-risk and malnourished infants in resource-poor settings. However, they highlight three ready available tools, the Breastfeeding, Evaluation and Education Tool, UNICEF Baby-Friendly Hospital Initiative tools and CARE training package, that can be used with this population and emphasize the need for refining or developing new breastfeeding tools to meet the needs of infants in resource-poor settings.\nOverall, the authors provide a very thorough introduction with a clear rationale for undertaking this study. The authors perform a systematic in-depth literature search, which included seven online database resources. The authors identified 29 breastfeeding assessment tools and 45 studies related to the tools' psychometric properties. They found that the evidence and psychometric properties of the tools was low quality and mainly from high-income countries. The strengths and weakness of these 29 breastfeeding tools were described in terms of the tool content of breastfeeding domains, predictive validity, reliability, and evidence underlying the content each tool. The tables provided clearly synthesize and integrate the strengths and weakness of each tool. In the discussion, the authors address the limitations of the study and bring to light the drawbacks of current available tools in achieve the defined outcome in this study.\nThe findings from this review are clinically significant and I have minor suggestions:\nI encourage the authors to thoroughly reread the manuscript to ensure there are no grammatical and editorial errors.\n\nFigure 1 appears to be adopted from the PRISMA framework, ensure that this is referenced.\n\nUse of terminology, the authors refer to Table 3 as validation studies, I recommend rewording to use predictive validity studies.\n\nI recommend including specific definitions for high vs low income countries, define at-risk infants, define malnourished infants either in the introduction or methods section.\n\nThe authors note 29 tools were identified, 22 were developed in high income countries, 4 low income countries. Three tools are missing such designations.\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": [ { "c_id": "6713", "date": "25 May 2021", "name": "Concetta Brugaletta", "role": "Author Response", "response": "Thank you for taking the time to read the review and write your suggestion. Regarding your advice to proofread the manuscript to ensure there are no grammatical and editorial errors; We have proofread and we think this looks fine in the new version of the manuscript. Regarding your advice to ensure the reference on Figure 1; we added the reference at the base of the figure in the new version of the manuscript. Regarding your suggestion on the use of a different terminology for Table 3; We hope it is okay to stick with validation studies as it is commonly used in the literature. For reference, see: https://www.equator-network.org/reporting-guidelines-keyword/validation-studies/ Regarding your recommendation to include a specific definitions for high vs low income countries, define at-risk infants, define malnourished infants either in the introduction or methods section; We added the following references that will help clarifying the definition and the concept as follow: - High and low income countries: as classified by The World Bank Fantom, Neil; Serajuddin, Umar. 2016. The World Bank's Classification of Countries by Income. Policy Research Working Paper;No. 7528. World Bank, Washington, DC. © World Bank. https://openknowledge.worldbank.org/handle/10986/23628 License: CC BY 3.0 IGO. - At-risk infants: https://www.ennonline.net/ourwork/research/mami Regarding your comment \"29 tools were identified, 22 were developed in high-income countries, 4 low income countries. Three tools are missing such designations\"; The tools that do not give any information about country of origin (described in the table 1 as not specified) are: - Baby-friendly Hospital Initiative (BFIH) Worldwide (When the Baby-friendly Hospital Initiative was conceived in the early 1990s in response to the 1990 Innocenti Declaration on the Protection, Promotion and Support of Breastfeeding call for action, there were very few countries that had dedicated Authorities or Committees to oversee and regulate infant feeding standards.) - Essential Nutrition Action Message (Guyon & Quinn 2011) Low and middle-income countries - Infant Feeding in Emergency (IFE) (ENN 2007) Low and middle-income countries - Neonatal Eating Assessment tool (NeoEAT) This is a USA based organisation: AWHONN, the Association of Women's Health, Obstetric and Neonatal Nurses. https://www.awhonn.org/ We amended the information above in table n1 of the new version of the manuscript. In the text of the new version of the manuscript we clarify the follow information: “Of the 29 tools identified: 22 (76%) were developed in high-income countries and used in 31 studies carried out in high-income countries, six (21%) tools were developed in low and middle-income countries and one (3%) was developed worldwide”." } ] }, { "id": "77936", "date": "19 Feb 2021", "name": "Nurul Husna Mohd Shukri", "expertise": [ "Reviewer Expertise Mother-infant signalling", "relaxation therapy during breastfeeding", "breast milk hormones" ], "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 compares different breastfeeding assessment tools to identify suitable tools to be used among at-risk and malnourished infants. The review also evaluates the breastfeeding assessment tools by comprehensively discussing both advantages and limitation of each tool and its reliability, as well as its validity in assessing breastfeeding outcomes. This review emphasizes the need for future tools to suit different breastfeeding management levels and settings, from primary to tertiary settings.\nThe authors clearly outline the study limitations and provide important suggestions in developing comprehensive and target-setting specifics of future breastfeeding assessment tools. All tables are clear and well-organised.\nOverall, this review critically analyses various important criteria of breastfeeding assessment tools in different settings, addressed important key gaps, and provides suggestions in establishing a better version of the tools in the future. All of these would provide a significant value to the literature.\nNevertheless, there are a few minor suggestions to consider:\nIt is suggested to describe the key term used or provide a table showing all Medical Subject Heading (MeSH) and keyword used for literature search.\n\nClarify or describe the term at-risk and malnourished infants.\n\nDifferentiate or explain the difference/similarity between breastfeeding outcomes and performance.\n\nIt is suggested for authors to proofread the paper to improve the overall writing.\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": [ { "c_id": "6712", "date": "25 May 2021", "name": "Concetta Brugaletta", "role": "Author Response", "response": "Thank you very much for taking the time to read the review and for all your comments, which we find it constructive and useful to clarify some concepts as below. 1. Regarding your first comment on describe the key term used or provide a table showing all Medical Subject Heading (MeSH) and keyword used for literature search; The search-strategy is deposited at LSHTM Data Compass as part of the Extended Data. Extended data contains tools excluded from the second stage of the search, full search strategy and PRISMA checklist. This was pointed out in the reference “ Kerac M, Brugaletta C , Le Roch K: Breastfeeding assessment tools for at-risk and malnourished infants aged under 6 months old: a systematic review. [Data Collection]. London School of Hygiene & Tropical Medicine, London, United Kingdom.2020. http://www.doi.org/10.17037/DATA.00001881” We have added the link in the methods to make it easier to access. 2. Regarding your second comment to clarify or describe the term at-risk and malnourished infants; “A group with higher risk of mortality and morbidity are the small and nutritionally at risk infants under six months of age compared to the infant that achieve optimal growth. At a population level, small and nutritionally at-risk children are those identified as wasted, stunted and underweight and a combination of these (ENN/LSHTM, 2021)”. We added a reference in the text of the larger project that clarify this concept: https://www.ennonline.net/ourwork/research/mami 3. Regarding your third comment on differentiate or explain the difference/similarity between breastfeeding outcomes and performance; We decided to modify the phrasing and replaced by: ‘how to observe and/or assess the breastfeeding performance with outcomes’. 4. Regarding your suggestion for authors to proofread the paper to improve the overall writing; We have now proofread and think this looks fine in this last version. the major changes are: Table 2: corrected ‘behaviour’ Table 3: corrected ‘postnatal’; ‘paediatric’ Table 4: corrected ‘analysis’ Text and table 1: corrected ‘lactating’ (https://www.macmillandictionary.com/dictionary/british/lactate Text corrected ‘paediatric’" } ] } ]	1	https://f1000research.com/articles/9-1310
https://f1000research.com/articles/10-1068/v1	21 Oct 21	{ "type": "Research Article", "title": "COVID-19 forecast for 13 Caribbean countries using ARIMA modeling for confirmed, death, and recovered cases", "authors": [ "Debjyoti Talukdar", "Vrijesh Tripathi", "Debjyoti Talukdar" ], "abstract": "Background: The rapid spread of the Covid-19 virus in the Caribbean region has led to increased surveillance with an increasing trend of confirmed cases of COVID-19 in 13 Caribbean countries. Our study aims to analyze the impact of Covid-19 (SARS nCoV-2) in 13 Caribbean countries in terms of the number of confirmed cases, deaths, and recovered cases. Methods: The study uses the ARIMA model based on the time series pattern according to data retrieved from John Hopkins University. The data were analyzed using Stata 14 SE software for the period January 22, 2020, and August 16, 2021, and forecasted till December 31, 2021. All chosen models were compared with other models in terms of various factors like AIC/BIC, log-likelihood, p-value significance, coefficient < 1, and 5% significance. The ACF and PACF graphs were plotted to reduce bias and select the best-fitting model. Results: The results show the predicted trend in terms of confirmed, death, and recovered cases of COVID-19 for 13 Caribbean countries. The projected ARIMA model forecast for the period December 16, 2021, to December 31, 2021, shows 2470272 (95% CI 2438965 - 2501579) confirmed cases, 27220 (95% CI 26886 - 27555) deaths, and 818105 (95% CI 818085 - 818125) recovered cases related to Covid-19. The final ARIMA model chosen for confirmed COVID-19 cases, several deaths, and recovered cases are ARIMA (9,2,4), ARIMA (1,2,1), and ARIMA (1,2,1), respectively. Conclusions As per the results of the forecasted COVID-19 models, there is a steady rise in confirmed, death, and recovered cases during the period June 1, 2020, until November 30, 2020, and April 1, 2021, until June 15, 2021. It shows an increasing trend for confirmed and recovered COVID-19 cases and a slowing of the number of deaths.", "keywords": [ "SARS nCoV-2", "COVID-19", "ARIMA Forecast", "COVID-19 in the Caribbean", "Confirmed Cases", "SARS nCoV-2 Death", "Recovered COVID-19 cases" ], "content": "Introduction\n\nAccording to the World Health Organization (WHO) and the Caribbean Public Health Agency (CARPHA), the ongoing pandemic caused by the SARS nCoV-2 virus is a grave concern worldwide, including in the 13 Caribbean countries, namely Antigua and Barbuda, Bahamas, Barbados, Cuba, Dominica, Dominican Republic, Grenada, Haiti, Jamaica, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, and Trinidad and Tobago. The risk of transmission of the COVID-19 virus in the Caribbean is high due to the high inflow of tourists and the return of nationals from all over the world. The islands also have a large number of inter-island migrants who returned to their mother states. Interestingly, the islands also have in-built advantages in controlling the pandemic because individual islands can be isolated and inter-island spread can be controlled. However, the region has particular disadvantages because its small population size causes its data to be overseen in global discussions. The Caribbean is home to only 0.56% of the world population. The number of confirmed COVID-19 cases is comparatively less compared to the U.S which has more than nine million confirmed cases till Oct 15, 2020. There is an inevitable risk of a second wave, as new cases can emerge in the region due to the opening of borders. In addition, there is the problem of illegal migrants in many of these countries who are unwilling to approach the health system once they realize that they need help. Porous borders are an unacknowledged source of worry in this crisis.\n\nWHO, the Pan American Health Organization (PAHO), CARPHA, local health administrations, and other international organizations have developed risk assessment levels that establish COVID-19 information centers in different regions1,2. All CARPHA member states have been advised to increase surveillance mechanisms, including random tests, lockdown measures, increase public awareness, and implement national preparedness plans. PAHO, based in Trinidad and Tobago, Barbados, and Jamaica, is proactively collaborating with global organizations including the WHO and the various Ministries of Health in each of the Caribbean countries to convey information concerning the latest developments, public health policies, lockdown measures, therapeutics, and diagnostic modalities including an overview of rapid testing among vulnerable populations3. PAHO is also collaborating with various Caribbean countries regarding the state of public awareness, the use of masks, maintenance of social distancing, and relaxation of lockdown norms in a phased manner4–6. Regional and international health authorities are assisting with infection control strategies, essential drugs, and the supply and training of essential health care workers. The Caribbean Community (CARICOM) along with its member states has been working with the chief medical officers and local health administrators of various Caribbean countries to establish COVID-19 testing facilities, quarantine zones, and the National Emergency Operations Center for COVID-19 briefings7. In addition to the health sector, the growing COVID-19 pandemic has also affected tourism and businesses in the Caribbean.\n\nTo assess the spread of the SARS nCoV-2 virus in the region, we intend to project a short-term forecast in terms of the number of confirmed cases, deaths, and recovered cases in the Caribbean region. Data analysis involves the auto-regressive integrated moving average (ARIMA) model which forecasts future values in a time series based upon its past values, lags, and forecast errors. The predicted forecast will help CARPHA, PAHO, and other regional health organizations in the Caribbean organize resources and better prepare to assess risk in terms of community transmission from imported cases, coordinate regional preparedness with Incident Management Teams (IMT) and prepare travel guidelines for local populations.\n\n\nMethods\n\nThe data for the Stata analysis is freely available for academic and research purposes through John Hopkins University GitHub Repository. It is one of the largest online communities for health care professionals, health institutions, and medical researchers. The data is maintained by John Hopkins University in collaboration with the Center for Systems Science and Engineering (CSSE) and technical support from ESRI and John Hopkins University Physics Laboratory8,9. The data include information concerning confirmed, death, and recovered cases of COVID-19 from 13 Caribbean countries, namely Antigua and Barbuda, Bahamas, Barbados, Cuba, Dominica, Dominican Republic, Grenada, Haiti, Jamaica, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, and Trinidad and Tobago (in alphabetical order). The time frame of the data starts from January 22, 2020, since situation reports from WHO about SARS nCoV-2 were published starting from January 21, 2020. Our study has been forecast until December 31, 2021.\n\nThe data consist of time series patterns with information concerning province/state, country, longitude, latitude, and confirmed COVID-19 cases for each respective country. Similar time series data were provided for COVID-19 deaths and recovered cases, respectively, from January 22, 2020, to August 16, 2021. The data was compiled in a Microsoft Excel spreadsheet, merged using a ‘sum’ argument entered as a formula for each specific date from January 22, 2020, and forecast until December 31, 2021. It was converted to .dta format for analysis using Stata 14 SE software ARIMA time series analysis. The analysis can be performed using PSPP software, written in C. It has a conventional command line with a graphical interface. The software uses the GNU Scientific Library and it is freely available for use. To declare the data as time series, the date variable was formatted using the Stata command ‘format’ and ‘%td’ in DDMMYYYY and quarterly format. It also involves the Stata command 'tsset', which declares the data as time series for a while using lead and lag operators. Also, the Stata command ‘tsfill’ was used to fill out the gaps in the time series model if there are any. The stationarity of the time series model was checked using the Stata ‘d’ differencing operator10. The differencing operator generates lags between current and previous values. Second-order differencing was performed using the ‘d2’ operator based on the lag values of the first-order differencing. Graphical representation of first-order and second-order differencing was made using two-way line plots using the Stata command ‘two-way’ and ‘tsline’ as shown in Figure 1. It is also very useful to check the stationarity of the time series using graphical plotting as second-order differencing plots diminish around zero. These plots are essential to check the time series stationarity as they fit numerically on the x and y-axis respectively (Figure 1, Figure 2).\n\nBased on the graphical representation, the ARIMA model can be formed using ACF and PACF graphs using the Stata command ‘ac’ and ‘pac’ using first-order differencing and second-order differencing variables, respectively, as shown in Figure 2. ACF plots involve autocorrelation of prior values in a time series with various lags11,12. MA can be derived from ACF plots graphically which are involved in building ARIMA models for further analysis. In an ACF graph, the lines situated in the shaded region are within acceptable regions (95% CI) while the lines outside the shaded region are lags that are autocorrelated in a series and the values are taken up as MA for ARIMA modeling. ACF plots are based upon Barlett’s formula for MA(q) processes involving pointwise confidence intervals. Similarly, PACF graphs are based upon the selection of the partial autocorrelation in the selected time series. It involves a confidence interval calculated using standard error 1/ (the root of n). The graph may also include residual variance for each lag. The values of AR are derived from PACF graphs which can be used for ARIMA model forecasting. The value of I depends on the differencing of the first order (I=1) or second-order (1 = 2). The models are chosen based upon smaller AIC/BIC values, which indicates a better fitting model. Other features like log-likelihood ratio, p-value significance, and coefficient < 1 and 5% significance can also be checked to compare various models13–16. The survey was analyzed using regular regression and post-stratification methods. It involved predictive analysis and health informatics with the help of Farr Institute wherein complex and adaptive modeling techniques were used for large-scale data mining, statistics, and analysis.\n\n\nResults\n\nBased on our data, out of the 13 Caribbean countries, the highest number of confirmed cases of COVID-19 is seen in the Dominican Republic, followed by Cuba, Jamaica, Haiti, Trinidad, and Tobago, Bahamas, Barbados, Antigua, and Barbuda, Grenada, St. Lucia, St. Vincent, and the Grenadines, Dominica, St. Kitts and Nevis, in this order17. Similarly, Caribbean countries with the highest number of death cases reported are the Dominican Republic, followed by Cuba, Haiti, Bahamas, Jamaica, Trinidad and Tobago, Barbados, Antigua and Barbuda, in this order. The highest number of recovered patients were from the Dominican Republic, followed by Cuba, Trinidad and Tobago, Jamaica, Barbados, Bahamas, Haiti, St. Lucia, Antigua and Barbuda, Dominica, Grenada, St. Vincent and the Grenadines, in this order. Our study demonstrates the future trend of the COVID-19 crisis in the Caribbean and its impact on health services and local communities.\n\nOur data analysis with autoregressive integrated moving averages (ARIMA) modeling for 13 Caribbean countries predicts during the period Nov 16, 2021 - Nov 30, 2021, 2102437 (95% CI 2073255 - 2131619) confirmed cases, 23284 (95% CI 22971 - 23597) deaths and 817887 (95% CI 817871 - 817903) recovered COVID-19 cases; 2281189 (95% CI 2251323 - 2311055) confirmed cases, 25199 (95% CI 24879 - 25518) deaths and 817989 (95% CI 817971 - 818006) recovered COVID-19 cases during the time frame - December 1, 2021, to December 15, 2021. Similarly, during the period December 16, 2021 - December 31, 2021, 2470272 (95% CI 2438965 - 2501579) confirmed cases, 27220 (95% CI 26886 - 27555) deaths, and 818105 (95% CI 818085 - 818125) recovered COVID-19 cases are projected, respectively. The final ARIMA model chosen for confirmed COVID-19 cases, several deaths, and recovered cases are ARIMA (9,2,4), ARIMA (1,2,1), and ARIMA (1,2,1) respectively. These models were compared with other potential models such as ARIMA (4,2,1), ARIMA (7,2,2), ARIMA (2,1,1), ARIMA (2,1,5), ARIMA (3,1,1), in terms of AIC/BIC (Akaike Information Criterion/Bayesian Information Criterion), the significance of the p-value, coefficient < 1 and 5% significance and logarithmic likelihood ratio. The AIC/BIC values for confirmed cases are reported as 8316.06 and 8385.62, respectively. The best-fitting models in our study have the lowest A1C/BIC values in comparison to other potential models. The p-value is considered significant if it's less than 0.05. The AIC/BIC values for the number of deaths and recovered COVID-19 cases have AIC/BIC values - 3917.15/3934.54 and 13558.08/13571.12, respectively. The current and projected trends are shown in Figure 3 for COVID-19 number of confirmed cases, deaths, and recovered cases, respectively.\n\nThe projected ARIMA forecast shows a linear increase for COVID-19 confirmed cases along with an increase in recovered patients in the 13 Caribbean countries. The time series was checked for stationarity through first-order and second-order differencing along with the Dickey-Fuller test. The results were graphically plotted using PACF and ACF to derive AR and MA for forecasting model building (Table 1). Data analysis for the time series model was checked for stationarity as shown in Figure 1 using Stata differencing operators, and graphs were plotted along the axis for the time frame from January 22, 2020, to December 31, 2021. Figure 2 shows the ACF and PACF models for the number of confirmed cases, deaths, and recovered cases of COVID-19 plotted to find the best fitting model based on Bartlett's formula for the MA (q) 95% CI bands. Figure 3 shows current and projected forecast trends for final COVID-19 selected models - confirmed cases ARIMA (9,2,4), number of deaths ARIMA (1,2,1), and recovered cases ARIMA (1,2,1), respectively. Table 1 offers a comparison of various models along with the significance of the p-value and the AIC / BIC values. Table 2 shows the current and projected forecasts for the Caribbean region in terms of the number of confirmed cases, deaths, and recovered cases of COVID-19 (Table 2).\n\np < 0.05 p < 0.01 **p < 0.001\n\n\nLimitations\n\nThe study was limited to data regarding COVID-19 confirmed, death & recovered cases from 13 Caribbean Countries. We were unable to access information on vulnerable populations or demographic information, and therefore the study is limited to projecting only a general trend about Covid-19 for the Caribbean.\n\n\nDiscussion\n\nOur study forecasts the likelihood of the pandemic over the next two months. It shows an increasing trend for confirmed and recovered COVID-19 cases and a slowing of the number of deaths. This would reflect the gradual easing of the lockdown restrictions. This is consistent with a study conducted by Sumner et al., 2020, who argue that the economic impact of COVID-19 will be drastic, specifically in the Latin America and the Caribbean (LAC) region, with potential short-term health and financial consequences18. Another study by Simbana-Rivera et al., 2020, performs a comparative analysis of the burden of COVID-19 among Latin American and Caribbean countries following government norms such as social distancing and wearing masks to slow the spread of the virus among local communities19. Our study focuses exclusively on the Caribbean and should help Caribbean countries not only prepare themselves for the upcoming impact of the Covid-19 spread but also reduce its economic impact on the very fragile economies of the smaller islands.\n\nOur approach has been validated by several other studies that have used ARIMA modeling for short-term predictions. A study by Benvenuto et al., 2020, focuses on the application of ARIMA modeling on the COVID-19 epidemic. It adds that various institutions will benefit from this forecast as ARIMA modeling involves incidence descriptive analysis of data along with incidence forecast and overall trend analysis20. Another article by Dehesh et al., 2020, shows the importance of prediction models and analyzing the trend of Covid-19 as a global pandemic for various countries16. Chintalapudi et al., 2020 recommend a data-driven model approach and highlight the significance of government policies based upon forecasting and modeling, which can help to reduce COVID-19 cases in Italy. The study commends preventive measures like regular hand washing, disinfection, wearing masks, travel restrictions, and suspension of public gatherings based upon modeling and forecasts21. Although most Caribbean countries are dependent on tourism as a major economic activity, we recommend a guarded approach to open borders the already weak economies may not be able to sustain a fresh wave of the pandemic.\n\nA study by Jenkins et al. discusses Caribbean trade relations with China in terms of foreign direct investment and its impact on the economy22. The consequences of the COVID-19 outbreak have significantly impacted Caribbean countries, even as China continues to be a prominent supporting partner. Another study by Rodrguez-Morales, MacGregor and, Kanagarajah, 2020 on COVID-19 discusses the importance of carefully assessing the situation based on scientific knowledge sharing and coordinated efforts to revitalize and uplift Caribbean communities amid the COVID-19 crisis23. We agree with them and recommend that as countries gear up to open the borders, they should introduce enhanced screening/ monitoring at entry and exit ports.\n\n\nConclusion\n\nThe outbreak of COVID-19 is a grave concern in the Caribbean region specifically due to the high risk of transmission among the local community. CARPHA has issued certain guidelines for its member states, including the 13 Caribbean countries based on WHO recommendations as mentioned in our study. Our cumulative projected ARIMA model for COVID-19 positive patients, deaths, and recovered cases in the 13 Caribbean countries offer insight for the government and health organizations operating in the region to assess pandemic preparedness plans and revise risk levels based on current and forecasted trends.\n\n\nData availability\n\nGithub: COVID-19 Data Repository by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, https://github.com/CSSEGISandData/COVID-19. The data was accessed from Jan 22, 2020 until Aug 16, 20212.\n\nThe data is maintained by John Hopkins University in collaboration with the Center for Systems Science and Engineering (CSSE) and technical support from ESRI and John Hopkins University Physics Laboratory.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe are thankful to STATA Corp. LLC for their support.\n\n\nAuthors’ contributions\n\nDebjyoti Talukdar: Investigation, Writing- Original draft preparation, Conceptualization, Methodology, Software, Visualization, Data curation\n\nDr. Vrijesh Tripathi: Supervision, Software, Validation, Writing- Reviewing and Editing, Visualization\n\n\nReferences\n\nCarpha.org: CARPHA - Novel Coronavirus. 2020; (accessed 10 Oct 2020). Reference Source\n\nDong E, Du H, Gardner L: An interactive web-based dashboard to track COVID-19 in real-time The Lancet infectious diseases. 2020; 20(5): 533–534. http://www.doi.org/10.1016/S1473-3099(20)30120-1\n\nWHO: Coronavirus disease 2019 (COVID-19) situation reports. (accessed Oct 8, 2020). Reference Source\n\nCARICOM: CARICOM-Caribbean Community. CARICOM, 2020; (accessed 8 Oct 2020). Reference Source\n\nThe Caribbean Council: COVID 19 Will Pass – The Economic Impact Could Be Longer Lasting. The Caribbean Council, 2020; (accessed 12 Oct 2020). Reference Source\n\nGhinai I, McPherson TD, Hunter JC, et al.: First known person-to-person transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the USA. Lancet. 2020; 395(10230): 1137–1144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaho.org: Coronavirus Disease (COVID-19). PAHO/WHO Pan American Health Organization, 2020; (accessed 15 Oct 2020). Reference Source\n\nCoronavirus (COVID-19). Centers for disease control and prevention, 2020; (accessed Oct 12, 2020). Reference Source\n\nStata.com: Title Syntax. Stata Date Time Display Format, (accessed Oct 8, 2020). Reference Source\n\nStata.com: Tsfill. Stata, (accessed Oct 8, 2020). Reference Source\n\nStata.com: Partial correlogram with confidence intervals. (accessed Oct 8, 2020). Reference Source\n\nStata.com: Correlogram with confidence intervals. (accessed Oct 10, 2020). Reference Source\n\nCheung YW, Lai KS: Lag order and critical values of the augmented Dickey–Fuller test. J Bus Econ Stat. 1995; 13(3): 277–80. Publisher Full Text\n\nBaum CF: Stata: The language of choice for time-series analysis? The Stata Journal. 2005; 5(1): 46–63. Publisher Full Text\n\nBenvenuto D, Giovanetti M, Vassallo L, et al.: Application of the ARIMA model on the COVID-2019 epidemic dataset. Data Brief. 2020; 29: 105340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDehesh T, Mardani-Fard HA, Dehesh P: Forecasting of COVID-19 Confirmed Cases in Different Countries with ARIMA Models. medRxiv. 2020. Publisher Full Text\n\nJohns Hopkins Coronavirus Resource Center: Coronavirus COVID-19 Global Cases by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). 2020; (accessed Oct 8, 2020). Reference Source\n\nSumner A, Hoy C, Ortiz-Juarez E: Estimates of the Impact of COVID-19 on Global Poverty. UNU-WIDER, 2020; 800–9. Reference Source\n\nSimbaña-Rivera K, Gómez-Barreno L, Guerrero J, et al.: Interim Analysis of Pandemic Coronavirus Disease 2019 (COVID-19) and the SARS-CoV-2 virus in Latin America and the Caribbean: Morbidity, Mortality and Molecular Testing Trends in the Region. medRxiv. 2020. Publisher Full Text\n\nChaurasia V, Pal S: COVID-19 Pandemic: ARIMA and Regression Model-Based Worldwide Death Cases Predictions. SN Comput Sci. 2020; 1(5): 288. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChintalapudi N, Battineni G, Amenta F: COVID-19 virus outbreak forecasting of registered and recovered cases after sixty day lockdown in Italy: A data driven model approach. J Microbiol Immunol Infect. 2020; 53(3): 396–403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJenkins R, Peters ED, Moreira MM: The impact of China on Latin America and the Caribbean. World Development. 2008; 36(2): 235–53. Publisher Full Text\n\nRodríguez-Morales AJ, MacGregor K, Kanagarajah S, et al.: Going global - Travel and the 2019 novel coronavirus. Travel Med Infect Dis. 2020; 33: 101578. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "155343", "date": "24 Nov 2022", "name": "Eric Ocran", "expertise": [ "Reviewer Expertise Applied Statistics and Statistics of Extremes" ], "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 sought to employ the ARIMA model to forecast the number of confirmed COVID-19 cases, the number of COVID-19 deaths and the number of recovered COVID-19 cases in 13 Caribbean countries. The results showed an increasing trend in the confirmed and recovered COVID-19 cases.\nThe results obtained in the paper are exciting, but the following concerns must be addressed:\nThe authors appear to be justifying the method they employed in the discussion session with the following references:\nBenvenuto D, Giovanetti M, Vassallo L, et al.: Application of the ARIMA model on the COVID-2019 epidemic dataset. Data Brief. 2020; 29: 105340\nDehesh T, Mardani-Fard HA, Dehesh P: Forecasting of COVID-19 Confirmed Cases in Different Countries with ARIMA Models. medRxiv. 2020.\nChintalapudi N, Battineni G, Amenta F: COVID-19 virus outbreak forecasting of registered and recovered cases after sixty day lockdown in Italy: A data driven model approach. J Microbiol Immunol Infect. 2020; 53(3): 396–403\nHowever, I suggest the authors move these references to the introduction session. That is, the justification should be done in the introduction session.\n\nThe manuscript needs language editing. For example, the sentence \"we recommend a guarded approach to open borders the already weak economies may not be able to sustain a fresh wave of the pandemic\" is unclear.\n\nI do not see the relevance of the reference \"Jenkins R, Peters ED, Moreira MM: The impact of China on Latin America and the Caribbean. World Development. 2008; 36(2): 235–53\" in the discussion.\n\nFrom Figure 2, the ARIMA model for recovery is ARIMA(6,2,4), but on page 5, you have stated ARIMA(1,2,1). Please check the inconsistency and correct it.\n\nThe authors presented the results for the stationarity test after presenting the results on the ARIMA models. I suggest you present the stationarity results before presenting the results on the ARIMA models.\n\nOn page 6, the authors claim,' the projected trend is linear, I disagree with that statement.\n\nThe authors should check their in-text citation style. The in-text citation style is not consistent.\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? Partly", "responses": [] }, { "id": "223017", "date": "15 May 2024", "name": "Namita Rath", "expertise": [ "Reviewer Expertise Organizational Behaviour", "Sustainability", "Human Resource Management", "Psychology", "Work Behaviour." ], "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 latest literature cited is from 2020. It may be due to the fact that COVID-19 studies have ceased to be of any additional research value. It would have been of interest to see a comparison of forecasted results with the actual incidents to see the accuracy of the models.\nFigure 3 attempts to show actual and forecasted trends which don't look in a satisfactory agreement. It would have been helpful to see quantitative estimates of the fit using parameters such as MSE, MAPE etc. The X axis labels are not clear and should be modified.\nSmall typographic errors need be corrected such as Caption of Table 2 and 'AIC' typed as 'A1C'.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1068
https://f1000research.com/articles/10-1067/v1	20 Oct 21	{ "type": "Data Note", "title": "Prevalence and associates of depression among rural and urban Rwandan mothers and their daughters 26 years after the 1994 genocide against the Tutsi", "authors": [ "Celestin Mutuyimana", "Cindi Cassady", "Vincent Sezibera", "Epaphrodite Nsabimana", "Cindi Cassady", "Vincent Sezibera", "Epaphrodite Nsabimana" ], "abstract": "Background: Mental disorders continue to be a challenge for Rwandan society, especially for women after the genocide against the Tutsi. The risk of inheritance of mental disorders is eminent. We therefore conducted a study on the prevalence of depression among grandmothers and their daughters using quantitative data. This paper explains in detail why and how the dataset was created, and it describes the dataset itself. This will allow readers to easily access and use the data. Methods: A sample of 309 dyads of mothers and daughters was recruited. Data were collected using the Mini International Neuropsychiatric Interview, Life Events Questionnaire and the Social Demographics Questionnaire. Data was analyzed using descriptive statistics, chi-square test, logistic regression, and one-way ANOVA.", "keywords": [ "Depression", "dataset", "grandmothers", "daughters", "Rwanda", "genocide", "rural", "urban" ], "content": "Introduction\n\nData are one of the most important and crucial aspects of any research study. Scientific research is based on collecting and analysing data. Datasets are essential to promote the development of data analysis and sharing is an emerging practice of scientific communication and facilitates the advancement of science by making data accessible, verifiable and reproducible (Rowhani-Farid et al., 2017). In this regard, we created a dataset after data collection to facilitate hypothesis evaluation through data analysis and data sharing. The aim of the study was to examine the prevalence of depression in Rwanda after the genocide in three groups of grandmothers and their adult daughters who had a child of their own. We examined women who were targeted during the genocide and referred to as \"genocide survivors\", those who were in Rwanda during the genocide but were part of the \"non-target group\", and those women who were outside Rwanda during the genocide and referred to as \"1959 returnees\". Given that more than half of Rwanda's population live in rural areas (National Institute of Statistics of Rwanda, 2018), this study was also interested in examining the impact of urban vs. rural residence on the prevalence of depression among participants.\n\n\nMaterials and methods\n\nParticipants were selected according to their genocide survival category. 309 Dyads of mothers and their daughters from genocide survivors, non-targeted and 1959 returnees were selected in rural provinces and in the city of Kigali. Data were collected by a team of 6 local clinical psychologists with clinical backgrounds and experience in data collection. Prior to data collection, they received 2 days of training that enabled them to make a clinical diagnosis of depression using MINI and ethical considerations in data collection.\n\nTo reach participants, data collectors were assisted by local authorities who referred them to households with participants who met the study criteria. Participants were approached and the interview was conducted with those who agreed to participate. The interview was conducted in Kinyarwanda, the local language, in a secure room prepared for a face-to-face clinical interview. The interview was conducted in the participant's home or in a nearby office of the local leader. Interview duration ranged from 15-20 min. The sample consisted of 309 dyads of mothers and their adult daughters, including 103 dyads of genocide survivors, 111 of non-targeted, and 95 dyads of 1959 returnees.\n\nMothers participants had to be Rwandan citizens and permanent residents of the country, a mother with an adult daughter who was able to complete a clinical interview. The daughter met the study criteria if she lived with her mother on a full-time basis or lived until she married.\n\nParticipants with communication difficulties, or who had experienced a mental health crisis at the time of the study, or who had recently experienced a traumatic event, or who refused voluntary participation were excluded. Ethical clearance for this study was obtained from the University of Rwanda' Institutional Review Board, College of Medicine and Health Sciences No 72/CHMS/IRB/2020. Written informed consent was obtained from the respondents before the interview.\n\nThe Mini International Neuropsychiatric Interview (MINI) questionnaire (APA, 2013) (https://harmresearch.org/index.php/product/mini-international-neuropsychiatric-interview-mini-7-0-2-13/) (An open-access alternative tool is the Patient Health Questionnaire (PHQ-9) (Kroenke et al., 2001). The tool is available at https://patient.info/doctor/patient-health-questionnaire-phq-9.) and socio-demographic questionnaire were completed with mothers from rural and urban areas of Rwanda. Participants also completed Life Events Questionnaires DSM-5 (Weathers et al., 2013) and a short form of the Perception of Parents Questionnaire (Pasquali et al., 2012). Data were entered into an online system tool (Aspa-net: vision studio 2008. Dotnet framework 3.5) (open-access software providing an equivalent function: Microsoft Excel) during fieldwork from which data were retrieved for statistical analysis in SPSS version 27 (https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-27) (As an open-access software alternative to SPSS, data is also stored in JASP version 0.14.1 format).\n\nThe dataset was created with 86 variables. Since the sample was composed of the grandmothers and their adult daughters who completed the questionnaires separately, we created one dataset. The first 309 cases are the data for the grandmothers while from 310 to 618 are the data for their daughters. The last four variables, which include depression, place of residence (urban or rural), generation (grandmother or daughter), and survivor status (genocide survivor, non-survivor, and 1959 returnee), were coded separately to facilitate the analysis (Mutuyimana et al., 2021b).\n\nBefore the analysis was performed, relevant assumptions of the statistical analysis were checked. First, it was verified that there were no missing values or outliers. The exploratory data showed that there were no missing values or outliers. Examination of the independence of the independent variables revealed that there was no multicollinearity and all independent variables were not highly correlated. To obtain the socio-demographic characteristics of the participants, a descriptive statistical analysis was performed. To test the difference in trauma exposure between the three survival categories of mothers and daughters, the one-way test ANOVA was used. Since the results indicated significant differences, a Tukey HSD post-hoc test was conducted to find these specific differences. A chi-square test was performed to examine prevalence differences of depression in the sample of mothers and daughters by survival category and place of residence. To test associates of major depression, a four-level hierarchical logistic regression was performed for the sample of mothers and a six-level logistic regression for the sample of daughters. Depression score of the mothers and daughters, were the dependent variables. The common independent variables in both samples were survival status, residence, and trauma exposure, while mothers' depression and trauma exposure in the daughters' sample were additional independent variables. Each independent variable was entered into the model individually. Age, education level, marital status and occupation were entered together as covariates at the last level.\n\nData validation was performed using SPSS version 27 to identify suspect and invalid cases, variables, and data values. At the first level, basic checks were performed, including maximum percentage of missing values (100), maximum case in a single category (100), maximum percentage of categories with a count of 1 (90), maximum standard deviation (0), and maximum coefficient of variation (.001). At this level, all cases passed the test. At the second level, rules were defined for individual variables (Name of variable, type of variable, and specify the minimum and maximum value of each) to see if there is no variable that falls outside the defined rules and all cases passed the required test. At the third level, cross-variable validation rules were created, displayed and modified and no violation was found, only gender violated the rules, but this was because the participants were only female. At the last level we saved the output and it was found that all cases did not violate any rules and the data was validated for analysis (Mutuyimana et al., 2021c). The names of the variables represent the limitation of the dataset.\n\n\nData availability\n\nZenodo: Questionnaires used in the study rural-urban depression prevalence in Rwanda https://doi.org/10.5281/zenodo.4914716 (Mutuyimana et al., 2021a)\n\nThis project contains the following underlying data:\n\nQuestionnaires: rural urban depression prevalence (Kinyarwanda/English)\n\nZenodo: ‘Rural-urban differences in the prevalence of depression among Rwandan grandmothers and their daughters 26 years after the 1994 genocide against the Tutsis’,\n\nhttps://doi.org/10.5281/zenodo.4723384 (Mutuyimana et al., 2021b).\n\nThis project contains the following underlying data:\n\nData file 1. Depression prevalence.sav\n\nData file 2. Depression prevalence.jasp\n\nZenodo: ‘Output of analysis for Urban-rural depression prevalence in Rwanda’, http://doi.org/10.5281/zenodo.4741243 (Mutuyimana et al., 2021c).\n\nThis project contains the following underlying data:\n\nData file 1. Depression prevalence analysis.spv\n\nData file 2. Output analysis excel format.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0)\n\n\nConsent\n\nWritten informed consent was obtained from the participants before the interview. Ethical clearance for this study was obtained from the University of Rwanda' Institutional Review Board, College of Medicine and Health Sciences No 72/CHMS/IRB/2020.", "appendix": "Acknowledgements\n\nThe authors would like to thank the participants of this study as well as the data collectors including Jonas Bikorimana, Christelle Ishimwe, Munganyinka Claudine, Etienne Kayumba, Albert Nahishakiye and Emile Bambara. Thanks to Remy Twagirimana for his contribution in managing the online software system.\n\n\nReferences\n\nAmerican Psychiatric Association (APA): Diagnostic and statistical manual of mental disorders (5th ed.). Arlington, VA: Author. 2013. Reference Source\n\nKroenke K, Spitzer RL, Williams JB: The PHQ-9: validity of a brief depression severity measure. J Gen Intern Med. 2001; 16(9): 606–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutuyimana C, et al.: Questionnaires used in the study rural-urban depression prevalence in Rwanda. Zenodo. 2021a. http://www.doi.org/10.5281/zenodo.4914716\n\nMutuyimana C, et al.: Rural-urban differences in the prevalence of depression among Rwandan grandmothers and their daughters 26 years after the 1994 genocide againstthe Tutsis. Zenodo. 2021b. http://www.doi.org/10.5281/zenodo.4723384\n\nMutuyimana C, et al.: Output of analysis for Urban-rural depression prevalence in Rwanda. Zenodo. 2021c. http://www.doi.org/10.5281/zenodo.4741243\n\nNational Institute of Statistics of Rwanda (NISR): The Rwanda Multidimensional Poverty Index Report. 2018. Reference Source\n\nPasquali L, Gouveia VV, Santos WS, et al.: Perceptions of Parents Questionnaire: Evidence for a Measure of Parenting Styles. Paidéia (Ribeirão Preto). 2012; 22(52): 155–164. Publisher Full Text\n\nRowhani-Farid A, Allen M, Barnett AG: What incentives increase data sharing in health and medical research? A systematic review. Res Integr Peer Rev. 2017; 2: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeathers FW, Litz BT, Keane TM, et al.: The PTSD Checklist for DSM-5 (PCL-5) Scale. The National Center for PTSD. 2013. Reference Source" }	[ { "id": "249920", "date": "05 Mar 2024", "name": "Peter Tammes", "expertise": [ "Reviewer Expertise Holocaust", "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 study addresses an important health topic (depression and generational inheritance of depression after genocide) and provide a relevant dataset to study this health problem. The Introduction should start with addressing the health problem and gaps in knowledge. In the next paragraph in the introduction, the authors should make clear the (un)availability of data and the need for the creation of an accessible dataset. Thereafter the authors should state their aim – creation of a dataset and the opportunity to do so. Since this paper is a Data Note the authors should shift their focus on the creation and description of the created dataset – something like a data profile to inform and persuade researcher to use the dataset. The examination of prevalence and testing hypotheses should be addressed in separate research paper(s).\n\nThe sentences about the 3 groups – genocide survivors, non-target groups, and 1959 returnees are important but should be moved to the Materials and Method section. In the Material/Methods section, more information is needed on: -selection process of mothers and daughters: who were eligible and why; in the article the authors sometimes speak of grandmothers but at other times of mothers – consistency and clarity are needed here. Besides, what if a mother had multiple daughters, which daughter was selected and why (always eldest, for example). -data collection, by whom, when, and how. -non-response: number of refusals and reason if possible; this provides information about potential bias in the dataset. -sample size of subgroups (genocide survivors, non-target groups, 1959 returnees) In the section on Measures, much more information needs to be provided on the outcome variable or issue under study, namely depression. How is that measured and are other studies such as those on the Holocaust and post-WWII depression or related illnesses among Jews consulted. Is it a constructed variable and what are the underlying factors? The authors should provide some preliminary descriptive statistics on these variables. One of the important explanatory factors is ‘who was targeted’ (survivor status). Provide the sample sizes of these 3 groups. In this section the authors should also provide some descriptive statistics on sociodemographic of participants, preferable separately for mothers and daughters and/or by survivor status group such as on age, education, employment, marital status, religion, (other) heath issues. If available, also on living area such as ethnically composition (mixed or homogeneous). Is there some information collected on fathers/husbands during the interview such as their age, mental status, employment – if so, mention that. These factors could be (other) explanatory factors or be included in the analyses as confounders. The section on data validation should come earlier and may be described after the data cleaning/inspection section (on missing info, etc). Also, the Consent section should be moved forward – perhaps as a subsection in the Material section. More info should be given about anonymizing the data, where the data is stored, who is responsible for the dataset, who might have access and how researchers can get access. In the final section – a conclusion or discussion section, the authors should discuss the opportunities for research, for example addressing a few testable hypotheses, and some other perspectives such as improving healthcare for victims with depression. Perhaps, follow-up data collection if that is relevant. After restructuring/re-writing the paper, the authors should rethink their paper title, reflecting the building of a dataset. There are many data or cohort profiles in epidemiological journals for inspiration to restructure this paper.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? No\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] }, { "id": "275628", "date": "17 Jun 2024", "name": "Simone Honikman", "expertise": [ "Reviewer Expertise Public health", "mental health", "perinatal mental health", "implementation science", "social determinants of mental 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\nThank you for asking me to review this submission. The authors describe how mental health issues persist as a major challenge in Rwandan society, especially among women who survived the Tutsi genocide. There is a significant risk of these mental disorders being passed down to subsequent generations. The manuscript provides an explanation of on open access dataset's creation and its features, facilitating access and use by other researchers.\nI have some suggestions to improve the manuscript.\n\nIt is not clear why the researchers have not reported on the prevalences and associations themselves, either in this or in prior papers. The authors should suggest how the data may be used by others, e.g. for cross-comparisons in other settings, to contribute to systematic review data, to conduct sub-analyses exploring particular variables etc. The terms ‘grandmothers and daughters’ and ‘mothers and daughters’ are used interchangeably – I suggest consistent use. Abstract: The sentence: ‘The risk of inheritance of mental disorders is eminent.’ The authors seem to be making the important point about the intergenerational impact of trauma on mental health status. However, I suggest the wording should be amended as ‘inheritance’ implies genetic mechanisms and the term ‘eminent’ is inappropriate here. ‘Data’ should consistently be used in the plural, e.g. Data were analyzed… Do not start a sentence with a number, rather e.g. We recruited 309 dyads of…. Recruitment of participants\nthere is unnecessary repetition of the numbers per category in the second sentence and at the end of the second paragraph. Third paragraph – who is grandmother vs mother vs daughter is confusing What is the rationale for why the ‘daughter’ needed to live with her mother?\n\nMeasures\nThe first three lines are not constructed as a sentence. From this, it is not clear whether you used the MINI or the PHQ-9 or both. If both, why” Much more description, rationale and validation information are required for the tools described. I am very puzzled by the ‘Perception of Parents Questionnaire’ – it seems to have been developed in Brazil for adolescents – looking at the wording, I am not clear how this tool would be cross-culturally valid in the Rwandan setting. For the MINI usage – was only the depression module used? Information on translation processes should be included too.\n\nThe section that starts ‘before the analysis was performed’ – should be labelled ‘Analysis’ and this section should follow ‘Data validation’. Again is it not clear why the results are not shared. I am not able to comment on the data validation and analysis sections and suggest a statistician be asked to review this.\nI feel this work has potential to contribute importantly to a critically important field, but substantial improvements would be required first.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "289445", "date": "03 Jul 2024", "name": "Emmanuel Habumugisha", "expertise": [ "Reviewer Expertise Trauma 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\nWhile reading the title of this work you think really on the trauma transmission from parent to the child especially from mother to daughter and the role or associations between depression observed with other variables like place of residence (urban/rural area). Authors are changing terms mother and grandmother in different parts like in title mother-daughter but in abstract grandmother-daughter and this may confuse, the author has clarified the sample used because this may imply the involvement of grandchildren and think transmission mode (trauma related symptoms from grand-mother to daughter, daughter (mother) to daughter (child) or grand-mother to grand-daughter). If the grandmother, mother/daughter and grand-daughter/daughter has meaning, the methodology especially sample should be changed from dyad to triad and study the co-factors. Authors didn't present in their work the prevalence of depression among target population or state it in previous studies to show the gap so that further studies have to tackle or work on, I will advise them to clarify the gap and the full clarification of the importance of creating dataset.\nI suggest the authors to clarify the population where they drawn samples and the used techniques and I want them to show the results as they mentioned that data were analysed so that in the introduction readers may be pushed to go further for checking or not their replicability.\nI want authors to show target group (that calls authors to conduct the study or creating dataset) among three groups if there was a control group(s) among three groups (survivors, non-targeted during genocide and returnees). In the test administration, the setting was not common for participants (samples) as some were met at their home and others at state houses or offices this wouldn't affect the results or quality of data? I would like authors to clarify the data collection procedures, if possible, clarifying the measurements (define the test used, their goals and the reliability) Authors has to present some findings and discuss a little bit on it and call for other researches to work on the study limits\nAuthors need to rethink on key words (what about grand-mothers)?\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1067
https://f1000research.com/articles/10-1065/v1	20 Oct 21	{ "type": "Method Article", "title": "Health status classification model for medical adherence system in retirement township", "authors": [ "Abubaker Faisal Abubaker Sherif", "Wooi Haw Tan", "Chee Pun Ooi", "Yi Fei Tan", "Abubaker Faisal Abubaker Sherif", "Chee Pun Ooi", "Yi Fei Tan" ], "abstract": "Medical adherence and remote patient monitoring have gained huge attention from researchers recently, especially with the need to observe the patients’ health outside hospitals due to the ongoing pandemic. The main goal of this research work is to propose a health status classification model that provides a numerical indicator of the overall health condition of a patient via four major vital signs, which are body temperature, blood pressure, blood oxygen saturation level, and heart rate. A dataset has been prepared based on the data obtained from hospital records, with these four vital signs extracted for each patient. This dataset provides a label associating each patient to the number of medical diagnoses. Generally, the number of diagnoses correlates with the patient's medical condition, with no diagnoses indicating normal condition, one to two diagnoses suggest low risk, and more than that implies high risk. Thus, we propose a method to classify a patient’s health status into three classes, which are normal, low risk and high risk. This would provide guidance for healthcare workers on the patient's medical condition. By training the classification model using the prepared dataset, the seriousness of a patient's health condition can be predicted. This prediction is performed by classifying the patients based on their four vital signs. Our tests have yielded encouraging results using precision and recall as the evaluation metrics. The key outcome of this work is a trained classification model that quantifies a patient's health condition based on four vital signs. Nevertheless, the model can be further improved by considering more input features such as medical history. The results obtained from this research can assist medical personnel by providing a secondary advice regarding the health status for the patients who are located remotely from the medical facilities.", "keywords": [ "Medical adherence", "home hospitalization", "machine learning" ], "content": "Introduction\n\nMedical adherence is described as a patient's willingness to follow their doctor's treatment plan by taking the medications recommended to them.1 Failure to follow a treatment plan might result in negative clinical outcomes and a significant rise in hospitalisation cost.2 According to a recent study, most patients do not follow their doctors' advice, resulting in a considerable rise in hospitalizations and medical visits.3\n\nMany researchers and healthcare firms have been focusing on home hospitalization technology in recent years.4 Many approaches are used to identify medical adherence: some employ blood testing, some employ patient self-assessment feedback, and others employ pill dispensers to determine whether the medicine was taken. These solutions can give important information on a patient's behaviour, but they are ineffective, as some solutions are costly to operate, while others do not provide information on the type of illness.\n\nBy proposing a method for continuous monitoring of the patient's vital signs, this study intends to develop a solution that can aid doctors in monitoring the patient's health state and medication intake. We used machine learning to determine the patient's health status and match it to the pharmaceutical consumption schedule defined by doctors. Major vital indicators are taken by off-the-shelf medically approved devices. The data is logged and analysed, and the result is presented to doctors in the form of a suggestion, which they may use to alter medicine dose or frequency, or to schedule a doctor visit for the patient.\n\nA lot of research has been focused on medical adherence and home hospitalisation.5–12 Patients' in-hospital days were reduced when they were given a home hospitalisation option.5 The abilities to remotely monitor the patient's health state and check their adherence to prescriptions are critical factors for enabling home hospitalisation. Tripathi et al.6 proposed to gather information about the patient via tracking sensors and wearable devices, then transmitting the data to an Internet server, where the decision is taken whether to contact family members, an ambulance, or clinical aid. To enable a smart health environment, Zulkifli et al.7 created a health monitoring and information system. Patients may use their cell phones to submit feedback to physicians, and doctors will reply to their reports to determine if the patient needs an appointment or can continue therapy from home.\n\nDue to the benefits of reducing hospital stays, cutting treatment costs, and freeing up doctors' time, home hospitalisation has garnered a lot of attention. Early release care, which permits patients to stay at home for a portion of their inpatient therapy, was studied by Hernández et al.8 They urged nurses to visit these patients on a regular basis and record their vital signs in an online system that doctors could access. Federman et al.9 assessed hospitalised patients and sent some of them home to undergo treatment. Nurses and health experts evaluated patients' vital signs before asking all patients to rate their treatment experience, with the results indicating that those who received therapy at home obtained a higher rating. Sherif et al.10 suggested a system to facilitate home hospitalisation, by tracking patients' medicine consumption using integrated electronics. Patients were instructed to record their medication adherence using an alarm button that was linked to a monitoring dashboard. While this strategy is useful for the monitoring of patients, it cannot assure that the medicines have been taken.\n\nSimilarly, Daramola et al.11 used a smartphone application to report medicine consumption and relied on patient self-reporting. Kumar et al.12 presented a similar method for measuring medication adherence using medicine dispensers. However, these approaches do not guarantee that the medicines have been consumed. To overcome these limitations, this paper intends to verify medicine intake by monitoring patients' vital signs and health status regularly.\n\nThe study's main goal is to improve patients' medication adherence without the need of special technology, nurse or caregiver monitoring. Thousands of people have been hospitalised because of the current COVID-19 pandemic. Many healthcare institutions have run out of resources to handle the rising number of patients.13 Thus, most people with minor symptoms have been recommended to stay at home and monitor their health.13 This pandemic has highlighted the critical necessity for home hospitalisation. Consequently, we propose a complementary tool that assists doctors in monitoring patients’ health from home.\n\nThis study received ethical approval from the ethical Review Board of the Multimedia University, Technology Transfer Office, Malaysia (EA0962021).\n\nAs healthy patients’ data was collected retrospectively and anonymised, written informed consent was waived.\n\n\nMethods\n\nThe method proposed in this paper consists of two sections. The first section is data preparation and pre-processing, while the second section is creating a classification model to identify the health status category based on the vital signs.\n\nBlood pressure, body temperature, heart rate and blood oxygen saturation level can be considered as good indicators to predict the health status of the patient. The initial stage in data preparation was to assemble a labelled dataset collected by physicians to anticipate the patient's health status. We adopted a publicly available medical dataset called Medical Information Mart for Intensive Care III (MIMIC-III) Clinical Database, which contains health data from over 40,000 patients hospitalised in intensive care units.14 The dataset consists of numerous tables with patients’ information, such as vital signs and doctor diagnoses. The caregivers documented the data collected on an hourly basis. However, the data from a single patient is saved in different tables across the dataset. As a result, the data must be restructured before they can be used for model training. Patients who had more than four daily measurements, including blood oxygen saturation, blood pressure, body temperature, and heart rate, as well as a doctor's diagnosis, were selected. These readings were divided into two classes: the first class included those with two or fewer diagnoses while the second class comprised those with more than two diagnoses. To predict the health status of the patients, we needed to train the machine learning model with data from healthy people, who were not diagnosed with any diseases. Since vital sign data from healthy people were not widely available, we decided to collect these data with the help of a general physician. The resulting dataset consists of three classes: the first class corresponding to healthy people; the second class includes patients with up to two diagnoses; the third class includes patients with more than two diagnoses.\n\nThree multiclass classification models were proposed to help doctors by providing a secondary opinion based on vital signs, to determine how serious a patient’s health condition is. The prediction was based on the labelled dataset, which consisted of three classes. The classifiers were chosen, based on the size of the dataset, were k-nearest neighbour classifier, linear support vector machine (SVM) classifier, and logistic regression classifier. Scikit-learn v. 0.23 was used to train these models, and is well-known for its classification methods.15 The purpose of the classifiers was to forecast the patient's health status class based on their vital signs. Diagnoses used in our prepared dataset were related to heart disease, respiratory sickness and hypertension, which the doctors would subsequently verify or reject in order to offer feedback data that could be used to train a more accurate model.\n\n\nResults and discussion\n\nA well-labelled and filtered dataset with five variables and one label was prepared. The mean values for blood oxygen saturation level, diastolic blood pressure, systolic blood pressure, heart rate, and body temperature were used as variables in the prepared dataset, as well as a label that indicated whether the measurements were normal, abnormal or abnormal with a serious condition. The variables included in the prepared dataset are listed below:\n\n• Diastolic blood pressure\n\n• Systolic blood pressure\n\n• Heart rate\n\n• Blood oxygen saturation level\n\n• Temperature\n\n• Label\n\nA total of 1,382 rows were collected after filtering data for those patients who had been diagnosed with diseases related to the vital signs, including only those who had more than three readings per day. A similar approach was utilised to get 102 “normal” readings manually with the aid of a medical expert. Both tables were then concatenated; the patient ID was removed, and labels reading \"0\" were assigned to normal readings, while \"1\" was assigned to abnormal readings up to two diagnoses, and “2” was assigned to abnormal readings with more than two diagnoses.\n\nAlthough we had collected as many data for healthy people as possible, the dataset was still imbalanced, with the majority of the data samples coming from the MIMIC-III dataset, resulting in biasing.\n\nAs shown in Table 1, the dataset consisted of 102 rows of healthy data samples (Class 0), 813 rows of low-risk data samples (Class 1) and 569 rows of high-risk data samples (Class 2). A 10% fraction of the data samples was used for testing. The rest of the dataset was split using repeated stratified K-Fold cross-validation, with four splits, and ten repetitions. One significant aspect of this validation technique is its balanced training weights and its testing of data while handling unbalanced data, since the percentage of observations with a particular categorical value is the same.14 Multiple splits and repetitions allowed the training and evaluation of different iterations of the same model, with training and test outcomes differing depending on the sampling data. After the data were divided into training and test sets, we trained three distinct models to assess their performance. These processing stages are shown in the system architecture of the proposed solution as depicted in Figure 1.\n\nThe first trained model was linear SVC. The settings of the hyperparameters used were ‘C’ set to 1, ‘max_iter’ set to 1000 and ‘intercept_scaling’ set to 1. This model exhibited a relatively low performance in predicting patients with more than three diagnoses, as shown in the confusion matrix shown in Table 2. The evaluation results in terms of precision and recall for the first model are presented in Table 3.\n\nThe second model trained was based on logistic regression, constructed with ‘max_iter’ set to 1000 and ‘C’ set to 1. As shown in Table 4 and Table 5, the logistic regression model demonstrated a modest performance in relation to the previous classifier with, better precision and recall for Class 2. Nevertheless, the recall for Class 1 was considerably lower.\n\nThe third model was the k-nearest neighbour classifier, trained with 'n_neighbors' set to 5, 'weights' set to ‘uniform’ and using Euclidean distance. The confusion matrix generated by applying the k-NN classifier on the test data is shown in Table 6. The results for the trained model are presented in Table 7. The performance of the model is on par with the first model, but worse than the second model for Class 2 in terms of recall.\n\nAfter training three different classification models, we trained a majority-vote classifier, which aggregated the predictions of the three trained models and made the predictions based on the class that obtained the greatest number of votes. The results are presented in Table 8 and Table 9.\n\nIn this study, the majority-vote classifier was chosen according to its overall performance in terms of precision and recall, in comparison to the results of the other three trained models. Further improvements will be made in the future regarding imbalanced data training, or by adding more minority data to the dataset. The positive results suggest that the proposed classification model is feasible in predicting severity of health condition, especially between normal people and those with medical risks.\n\n\nConclusions\n\nThe goal of this study was to improve medical adherence among patients who get therapy at home, by tracking their health status using supervised machine learning models. We are confident that the proposed solution will increase medicine adherence and provide a way to enable home hospitalisation, which is now in great demand. The trained model can predict how serious a patient's health status is, as determined by the four measured vital signs. Clinicians should be able to accept or reject the model's predictions, providing feedback which can then be utilised to train and enhance another version of the model. To further improve the performance of the model, additional input variables such as medical history can be considered.\n\n\nData availability\n\nThe experiments in this work were carried out using data from the MIMIC-III Clinical Database: https://doi.org/10.13026/C2XW26.14\n\nZenodo: Five vital signs of normal people, https://doi.org/10.5281/zenodo.5549632.16\n\nThis project contains the anonymised vital signs data from healthy patients collected retrospectively.\n\nAnalysis code available from: https://github.com/abubakerSherif/A-Health-Status-Classification-Model/tree/v1.0\n\nArchived analysis code as at time of publication: https://doi.org/10.5281/zenodo.5551854\n\nLicense: MIT", "appendix": "References\n\nHo PM, Bryson CL, Rumsfeld JS: Medication adherence: its importance in cardiovascular outcomes. Circulation. 2009; 119(23): 3028–3035. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization, Healthtopics: chronicdiseases.Reference Source\n\nSpan P: A dose of confusion.2011.Reference Source\n\nPolese F, Carrubbo L, Caputo F, et al.: Managing healthcare service ecosystems: abstracting a sustainability-based view from hospitalization at home (HaH) practices. Sustainability. 2018; 10(11): 3951. Publisher Full Text\n\nHeidenreich PA, Ruggerio CM, Massie BM: Effect of a home monitoring system on hospitalization and resource use for patients with heart failure. Am. Heart J. 1999; 138(4): 633–640. Publisher Full Text\n\nTripathi V, Shakeel F: Monitoring health care system using internet of things – an immaculate pairing. 2017 International conference on next generation computing and information systems (ICNGCIS). IEEE. 2017; pp 153–158.\n\nZulkifli FY, Mustika IW: Development of monitoring and health service information system to support smart health on android platform. 2018 4th international conference on nano electronics research and education (ICNERE). IEEE. 2018; pp 1–6.\n\nHernaÃÅndez C, Aibar J, Seijas N, et al.: Implementation of home hospitalization and early discharge as an integrated care service: a ten years pragmatic assessment. Int. J. Integr. Care. 2018; 18(2), 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFederman AD, Soones T, DeCherrie LV, et al.: Association of a bundled hospital-at-home and 30-day postacute transitional care program with clinical outcomes and patient experiences. JAMA Intern. Med. 2018; 178(8): 1033–1040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSherif S, Tan WH, Ooi CP, et al.: LoRa driven medical adherence system. Bull Electr Eng Inf. 2020; 9(6): 2294–2301. Publisher Full Text\n\nDaramola O, Nysaulu P: A digital collaborative framework for improved tuberculosis treatment adherence of patients in rural settings. Cape Town, South Africa:Open innovations (OI);2019; 297–303. Publisher Full Text\n\nKumar MP, Nelakuditi UR: IoT and I2C protocol based M-health medication assistive system for elderly people. IEEE 16th India Council international conference (INDICON), Rajkot, India. 2019; pp 1–4. Publisher Full Text\n\nNundy S, Patel KK: Hospital-at-home to support COVID-19 surge – Time to bring down the walls.2020.Reference Source\n\nJohnson AEW, Pollard TJ, Shen L, et al.: MIMIC-III, a freely accessible critical care database. Scientific Data. 2016; 3: 160035. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: machine learning in python. J. Mach. Learn. Res. 2011; 12(Oct): 2825–2830.\n\nSherif A, Tan WH, Ooi CP, et al.: Five vital signs of normal people [Data set]. Zenodo. 2021. Publisher Full Text" }	[ { "id": "234456", "date": "19 Feb 2024", "name": "Jesper Mølgaard", "expertise": [ "Reviewer Expertise Continuous vital sign data", "postoperative complications" ], "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 of this article report on the results of using three different statistical methods for discriminating between 3 patient categories. They propose this model for helping healthcare workers understand which patients need medical attention and which do not. And doing it to reduce the duration of hospital admission is a noble endeavour. It is also great to see the vital signs dataset specifically collected for this study being available for other researchers, and I commend the authors for doing so. Here are some comments that I hope will improve the quality of the article:\nMajor points: Focus of the study is a bit unclear. Three focus points are introduced in the introduction and motivation part of the article:\n“…this paper intends to verify medicine intake by monitoring patients' vital signs and health status regularly.” “we propose a complementary tool that assists doctors in monitoring patients’ health from home.” “The study's main goal is to improve patients' medication adherence without the need of special technology, nurse or caregiver monitoring.”\nIs the focus either to verify medicine intake, to assist in monitoring patients’ health, or to improve medication adherence – or all three at once? Also if the authors solely wanted to “verify medicine intake” – why not propose a medicine monitoring device instead of a patient-monitoring device. The authors define 3 sets of vital values, which are not very well defined, and deserves more fleshing-out, especially the collection of vital values from the healthy cohort:\nHealthy patients:\nSingle point values from (which?) patients collected (where and when?) by a general physician. (some of the information is available in the supplementary data for the article)\nModerately diseased: Continuous values from the MIMIC-III dataset for patients with 0-2 diagnoses. Seriously diseased: Continuous values from the MIMIC-III dataset for patients with 3+ diagnoses.\nThe authors find a precision-recall value of 1 for predicting healthy patients, which is not very surprising, given the difference in patient populations (healthy at-home values vs. actively diseased ICU values), and is perhaps more a reflection of the fact that these values are taken from two different datasets. For the ICU patients, did the authors adjust for ICU patients with ventilator support, vasopressor therapy, dialysis treatment, moribund patients?\nSmall issues: The title of the article is not very informative: “Health status classification model for medical adherence system in retirement township” – whereas the actual article reports the validation of 3 different statical methods for discriminating between 3 levels of disease.\nThe authors have chosen to not adhere to the traditional Introduction-Method-Results-Analysis-Discussion (IMRAD) format and have made a result + (discussion) section. But I would have liked to have their results put into context of the current body of related studies.\nA lot of descriptions that from my understanding belong the ‘methods’ section of the article are written in the ‘results’ – section “The classifiers were chosen, based on the size of the dataset” – How did the size of the dataset help to choose which classifiers were used?\nI do not feel that figure 1 adds much to the understanding of the system, and should be revised, or removed. Also, maybe I can’t see the figure 1 legend. But I think some of the icons require attribution to the relevant artist at flaticons.com (or other relevant site.).\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? No\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? 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/10-1065
https://f1000research.com/articles/9-34/v1	23 Jan 20	{ "type": "Opinion Article", "title": "Intrinsic negative feedback as a limiting factor for the evolution of higher forms of intelligence", "authors": [ "Stefan T. Arold" ], "abstract": "Longstanding scientific efforts have been dedicated to answer why and how our particular intelligence is generated by our brain but not by the brain of other species. However, surprisingly little effort has been made to ask why no other species ever developed an intelligence similar to ours. Here, I explore this question based on genetic and paleontologic evidence. Contrary to the established view, this review suggests that the developmental hurdles alone are not high enough to explain the uniqueness of human intelligence (HI). As an additional explanation I propose that HI is normally not retained by natural selection, because it is, under most conditions, an intrinsically unfavourable trait. This unfavourableness, however, cannot be explained by physical constraints alone; rather, it may also be rooted in the same emotional and social complexity that is necessary for the development of HI. Thus, a major obstacle towards HI may not be solely the development of the required physical assets, but also to cope with harmful individual, social and environmental feedback intrinsically associated with this trait.", "keywords": [ "iterative evolution", "convergent evolution", "cognition", "complexity", "gene expression", "sustainability" ], "content": "Introduction\n\nOur particular type of intelligence is commonly believed to be the central feature that has allowed humans to become one of the most abundant mammals on Earth. Yet, although our intelligence is such a powerful and generally useful trait, it has never been paralleled in any other organism. As an explanation, we generally assume that our cognitive uniqueness results from the major difficulty in achieving the required level of developmental sophistication. And after billion years of competitive evolution, we humans were the first species advanced enough to surmount these difficulties and to accumulate all features necessary for achieving this higher intelligence. Here, I investigate this conjecture and its underlying assumptions. For this purpose, I deliberately and necessarily adopt a very narrow anthropocentric definition for human (or human-like) intelligence (HI) as “the intelligence that enables development of advanced technology-based societies like ours”. Within this definition, ‘intelligence’ does not only refer to ‘computing power’, but also to the full ensemble of cognitive and character traits required.\n\nFirst, underlying this conjecture is a worldwide trend toward ever higher levels of intelligence, a trend that reached a critical threshold in the Pliocene when HI was finally developed in humans1,2. Evidence for such a trend is weak. While a global trend toward bigger brains has been observed in several instances, it does not hold true for all taxonomic levels1–4. It is unclear if this trend, which is weak and concomitant with an increase in diversity and afflicted by exceptions, is the result of directed evolution or random drift3. It also appears that this trend, if it indeed exists, is a much weaker driving force than are the forces that are linked to adaptation to a specific niche5.\n\nThe argument that Homo sapiens was the first to reach an HI-enabling level of developmental sophistication evokes a linear view of evolution that posits that basal forms develop into ever more advanced and intelligent organisms. However, paleontologic and genetic evidence shows that evolution is not an orderly step-by-step advancement; rather, it is characterised by consecutive waves of radiation of species (many of which become dead-end groups) into the same ecological niches6. During this iterative process of consecutive adaptations, many sophisticated characteristics (such as vision, flight, echolocation, burrowing or re-adaptation to water) evolved several times, successively and independently, in different species6–11 (Figure 1). Even mammalian hallmark features, such as the middle ear and tribosphenic (crushing and biting) molars, evolved not once but several times in different mammals6,12. This convergence level of evolution indicates that the geological time scale is sufficiently large compared to the biological reproduction and diversification rates that even complex anatomical and molecular features can be reproduced if they enhance the chances of a species’ survival. Since our type of intelligence appears to be such a powerful and versatile development for the survival of a species, why has no other organism acquired HI?\n\nLeft: Skeletal changes associated with evolution of the human brain. (A) Rhinopithecus (stub-nosed monkey), (B) Australopithecus, (C) Homo erectus, (D) Homo neanderthalensis, (E) Homo sapiens. Note that also other anatomical features are likely to be required for human intelligence (HI) (for example those enabling tool-use). Middle: Skeletal changes associated with the evolution of whales. Adaptations to the aquatic habitat include: Removing of external appendices (ears, genitals, hind legs); changes of skin; loss of hair; stream-lined body shape; development of a sealable blowhole on top of the head, flippers, tail flukes and a dorsal fin; evolution of a flexible rib cage; increased organ-selective oxygen storage in the body; increased anaerobic capacity of muscles; capacity to slow down pulse (bradycdia); capacity of resting (‘sleeping’) one half of the brain at a time; tail-first birth of babies; development of echolocation (odontoceti). (A) Diacodexis, (B) Pakicetcus, (C) Ambulocetus, (D) Dorudon, (E) Balaena. Taken from 27. Right: Convergent evolution for wing development. (A) pterosaurus, (B) bat, (C) bird, (D) insect, (E) flying fish. Note that although vertebrate forelimbs adaptions are functionally convergent, they are not anatomically convergent. Insect wings are formed from a totally different organ. Although flying and re-adaptation to water are not equivalent to the development of HI on all levels, they might serve as comparison in terms of ‘evolutionary difficulty’ with respect to the required profound reshaping or combination of existing structures (including alterations in bone structure, muscle, metabolism, respiration, vision or connective tissue).\n\nThe previous section highlights the importance of ecological niches in directing the evolution of specific traits. Hominid evolution is indeed likely to have been driven by particular changes or variability in climate, although the exact nature and importance of these changes are a matter of debate13. More specifically concerning our cognition, coping with environmental changes has been suggested as a factor that drives brain evolution, within the cognitive buffer hypothesis14. Nonetheless, the climate changes evoked are not sufficiently rare or specific to be a serious limiting factor for the development of HI in other species. Moreover, similar environments, or environmental changes, have failed to promote the emergence of HI in other species. Additionally, if the competitive advantage and driving force of HI development lies in overcoming dependence of the environment14, then this advantage would profit many species, and hence is not a limiting factor. Finally, the rapid and almost worldwide spread of Homo species shows that its type of intelligence can be used under many conditions and is not an adaptation limited to a unique environmental ecological niche or geological condition that existed only at the precise time and place of the origin of humans.\n\nCompared to the brain structure and brain-body ratios of other animals, the human brain is, of course, exceptionally big and complex in terms of layered structure, interconnectedness and neuronal diversity. But is its structure unique and sophisticated enough to justify that our brain capacity was never paralleled through adaptation and convergent evolution? Accumulated evidence suggests that the answer is no. Neuroscientists have established that the brain structures of birds and mammals are never simple. The anatomical differences between species reflect the animals’ adaptations to a particular niche, not the lack of sufficient time for their brains to become more complex5. In fact, the human brain is less evolved (in the sense of being altered with respect to the brains of stem mammalians) than the aye-aye (Daubentonia madagascariensis) brain15. Rather, the human brain is more or less a scaled-up version of a non-human primate brain16. Its size and structure are principally the result of simply extending the high growth rate of foetal brains into early infancy17–20. This prolonged growth was achieved by the modification of gene expression patterns of a few regulators21,22, a common mechanism for species differentiation (for example,23–25). Of course, the scaling-up of the primate brain did not linearly affect all components, and the enhanced increase of the neocortex was certainly key to generating HI. Yet, altering the gene expression pattern of a single transcription factor (Pax6) in mice to a human-like pattern suffices to obtain a primate-like increase in neural progenitors, notably basal radial glia, and ultimately in the number of neurons produced, which is thought to underlie the evolutionary expansion of the neocortex22. Human brain performance was additionally boosted by slightly increasing the ratio of astrocytes to neurons, a tendency already observed in other higher mammals26. This ratio present in humans is, however, similar to that seen in other primates16. In terms of genetic modifications, the development of the human brain therefore appears to be based on extending already existing features through gradual and common mechanisms, rather than being the result of a developmental quantum leap.\n\nSuch genetic evolutionary mechanisms are not restricted to primates. For example, it was the expansion of a handful of gene families and genome rearrangements in an otherwise standard invertebrate gene background that allowed octopuses to produce profound morphological and neurological changes28. These changes sufficed to produce a large and sophisticated nervous system in addition to profound morphological innovations for their vision, arms and embryogenesis, resulting in a dramatically increased cognition and behavioural richness in octopuses as compared to worms, molluscs and other lophotrochozoans. Thus, the emergence of our highly specific human cognition has been achieved through continuity on a genetic level, accessible in principle also to other species.\n\nSince the human brain originated through extensions of primate brain features, can HI originate only from the brain structure of apes (Hominoidea)? If this is the case, then apes would have had to evolve first before HI occurred, and this condition may help to explain the late and single occurrence of this type of intelligence. However, it was not only other primate species (such as capuchins), but also elephants, cetaceans and certain birds that independently evolved cognitive and social characteristics that are very similar to those of apes (including self-consciousness, grief, altruism, play, envy, compassion and abstract numerical competence), despite having fundamentally different brain architectures29–35. Although the overall brain architecture differs, convergent evolution can produce obligatory neuronal adaptations in unrelated genera. For example, von Economo neurons, which have been related to complex social behaviour, have independently evolved in primates, whales, elephants and racoons36. Because of neuroplasticity, such convergent evolution is even easier for the brain than for other organs. Accordingly, our particular intelligence is not based on unique characteristics; rather, it arises from a combination and enhancement of abilities found in other vertebrates37. In fact, our large brains and particular intelligence appear to be independent of our ape phylogeny; rather, they result from convergent processes similar to those that produced avian and New World monkey brains38. Taken together with the finding that in some cognitive tasks monkeys can be outperformed by other animals, including birds39,40 and even fish41, scientific evidence does not support the unilinear view that only the brain structure of apes is suited to produce HI.\n\nBig brains are energetically costly organs. Moreover, because of its neuronal density and neuron/glial cell ratio, a human brain requires more energy than for example a rodent brain of the same size42. Could the unique development of HI in humans be explained by our species being the only one capable of coping with the high metabolic demand of a brain required for generating HI?\n\nThere are many ways of overcoming metabolic limitations. These include shifting to higher-energy food sources (such as nuts, honey or meat, especially from invertebrates like molluscs or insects); external transformation of food to a more edible state (through grinding, fermentation, cooking or other types of food processing); more efficient use of existing food sources through simple tools and/or strategies (for transport, butchery, or storage); adaptive genetic changes to improve the digestive system (and associated microflora), or to diminish body energy expenditure [such as changes in skin coating (e.g. fur or feathers) or locomotion (e.g. bipedalism, architecture of limbs and feet)]; increasing foraging efficiency through intra-species organisation (e.g. for hunting, gathering, carcass defence or food processing); or shifting to habitats richer in high-protein food sources. A subset of these strategies was sufficient to provide the increased energy demand throughout hominid evolution (see ref. 43 and discussion therein). These strategies (including the cognitive capacities to cook44) do not require features that are only found in humans, making sufficient subsets of them also accessible to other species. Moreover, as the brain of an organism becomes bigger and more powerful, the enhanced cognitive capacities will increasingly enable this organism to implement strategies to enhance energy intake. The cognitive pay-offs of big brains should therefore allow alleviation of the associated energetic cost. In conclusion, while the high energy demand of an HI-capable brain is an important roadblock for the development of HI, the physical and intellectual strategies required to overcome this hurdle are not only accessible to the human body plan. And although the adaptations required to sustain a big brain are profound, they are paralleled, for example, by the metabolic and physical adaptations required for powered flight, which has evolved multiple times in different taxa (Figure 1).\n\nIt appears safe to assume that HI would not be possible without a powerful language. Compared to other animals, the human language appears to distinguish itself in many ways, including the large amount of symbols used (‘vocabulary’), the complex compositional, hierarchical and recursive syntax, and the need to learn the complete language from scratch during an individual’s lifetime45. It has been suggested that language evolution and efficient tool use have stimulated each other mutually46. More generally, it might be that the way of thinking that our language enables is what makes us human47,48. It is therefore important to discuss whether the uniqueness of HI can be explained by the uniqueness of our language.\n\nAdopting the particular viewpoint of this assay, we then have to ask whether the production of HI-supporting language would be possible for other species. Let us first consider the requirements for producing a sufficient vocabulary. On a purely anatomical or mechanistic level, a HI-supporting language might be any sort of expression not limited to speech (e.g. twitter, tapping, signs, gestures, visual cues) that can produce a sufficient vocabulary space. This requirement does not seem to be restrictive. Moreover, some evidence suggests that the increase in human brain size preceded the development of finely articulated speech49. It is therefore not the capacity for an articulate vocalisation, but only a predisposition for it, that is essential to the evolution of HI.\n\nLet us now consider language syntax and acquisition with respect to human uniqueness. It has indeed been proposed that such properties of the human language are neither specific to language nor to humans47,48. Already Darwin had recognized the similarity between birdsong and human speech50. The process and the underlying neuronal circuits by which a young bird learns the songs from an adult ‘tutor’ show indeed strong parallels with speech acquisition in human infants on behavioural, neural and genetic levels51,52. Moreover, common genetic key players have been identified, including the FOXP2 gene53,54. The hierarchical combination of ‘words’ bears resemblance to semantic combinations in primate calls or compass headings in honeybees55,56. It has therefore been suggested that the human language results from the combination of separate, pre-existing simple systems that may have evolved for other functional tasks57.\n\nIn conclusion, while the exact behavioural, neural and molecular links between language, intelligence and behaviour are still emerging and under debate, significant characteristics of human language appear accessible to other species, including non-primates, and are not tied to a specific brain anatomy or size. Thus, current evidence suggests that the evolution of a HI-supporting language is not a feature strictly limited to humans or the human brain anatomy or speech production.\n\nHI poses requirements to a candidate species beyond developing and coping with the appropriate brain anatomy, associated energy demands and language. For example, HI would be most useful in an organism that can use tools and has a complex social system that allows transmitting and accumulating acquired knowledge and culture. Importantly, these individual features synergise with each other (e.g. 46,58) and may need to evolve as an ensemble. While producing one individual feature may not be particularly difficult, and although combination of pre-existing traits is a common evolutionary mechanism, it could still be that the combined likelihood of assembling all required features in one species is sufficiently small to explain HI’s uniqueness. Unfortunately, it is currently impossible (i) to test which sub-ensemble of characteristics is a strict prerequisite for HI development (as opposed to characteristics which can be developed en route towards HI), and (ii) to quantitate the likelihood for the occurrence of such an ensemble in one animal. Thus, we can only approximate this question by asking if the presence of HI-enabling features and capacities was uniquely found in early hominids. In other words, is HI development such a rare event because of a shortage of suitable candidate species?\n\nTool use is well documented in many animals and is neither restricted to primates (it has been observed in sea otters, elephants and dolphins) nor to mammals (more than 120 cases of tool use in 104 bird species have been reported)59–62. Many species of dinosaurs, Triassic archosaurs or marsupials were/are bipeds with free, articulate limbs, affording them great potential for tool use9, many relatively small, birdlike dinosaurs, such as Troodon, possessed a grasping hand with an opposable digit63, whereas dolphins use tools even though they have no limbs to grasp the tools. Child caring, intra-species communication and even cultural transmissions are documented not only in primates, but also other vertebrates (such as meerkats and fish) and maybe even in insects (e.g., teaching behaviours have been suggested in ants)64. Social complexity as a potential driver for enhanced brain size and social intelligence (the ‘social complexity hypothesis’58) has been reported in diverse non-human vertebrates (including lemurs, dolphins, parrots and spotted hyenas65–69, suggesting that social complexity can drive brain evolution in diverse species. With respect to the importance of language, as discussed above, many species possess means of communication which might have the potential to evolve, within a few million years, into a sufficiently complex vocabulary and syntax to synergise with an emerging cognition. Thus, great apes, dolphins and some bird species have reached a high sophistication in terms of vocabulary, semantics, syntax and symbolic references, together with their cognitive capacities32,70,71\n\nFinally, development of HI does not appear restricted to organisms with sizes similar to humans. Although the brain-to-body size (encephalization) appears to correlate roughly with cognitive capacity, brains do not have to be above a particular absolute size to produce high levels of intelligence72. For example, despite having a smaller brain than chimpanzees and australopithecines, Homo floresiensis, the Indonesian small-bodied ‘Hobbit’ species, was capable of manufacturing tools as advanced as those produced by Homo species with a three times larger brain73. Moreover, the complex cognitive abilities of some bird species (including tool use, episodic-like memory, predicting the behavior of conspecifics based on own experiences and self-recognition) are produced by brains that are less than 10 g in weight74. Thus, HI candidate species are not limited to those animals that have comparable sizes to humans.\n\nIn summary, while it is currently impossible to evaluate the evolutionary likelihood for combining the required features into a HI-capable animal, it is clear that many organisms besides humans readily combined several characteristics that could support HI (such as tool use, cultural transmissions, size of groups or body). During development towards HI, these characteristics can in principle be developed further, and completed, as it has been the case in our lineage. And although the combination of features found in early hominids before brain expansion (i.e. free limbs, capacity for grasping, social structure and tool use) are not common, they are not unique either, and it is therefore unlikely that HI was developed only once because no other species would have been a suitable candidate.\n\nAccording to the findings discussed above, the development of HI is not complex enough to fully explain that it has never been reproduced in any other species than Homo sapiens. If HI were within reach of convergent evolution, as my survey suggests, then an intriguing possible explanation for the fact that HI nonetheless evolved only once would be that HI is normally an overall unfavourable feature that is sooner or later sanctioned by selection. Yet, the physical constraints associated with HI appear to be manageable, and the human body is not special enough to suggest that no other organism could have evolved to cope with those constraints. If there is an unfavourable aspect of HI, then it must arise from somewhere other than these physical constraints.\n\nCould it be that some of the same mental and behavioural characteristics that are necessary for the development of HI might become increasingly unfavourable as a species evolves towards HI, creating a negative feedback loop? There is certainly a positive correlation between the proximity of species to HI and the emotional and structural complexity of their individuals and societies (where emotional complexity is defined as having emotional experiences that are broad in range and well differentiated). This correlation is apparent in species with very different brain structures and sizes (cetaceans, elephants, apes, Cebus monkeys and some birds29,30,32,71,75), suggesting that it is an intrinsic hallmark of HI.\n\nBehavioural studies on capuchin monkeys may provide an anecdotal illustration of this concept. These New World monkeys independently evolved an intelligence with certain characteristics similar to that of great apes and humans32. Owing to their particular amalgam of a strong cognitive capacity and an emotional and almost pugnacious character, capuchins have developed tools and strategies that allow them to forage for food that is impossible for most other animals to attain. Their complex social system stimulates learning, the emergence of culture and the establishment of an efficient communal defence against predators32. This type of intelligence, however, produces a society in which individuals spend an excessive amount of time engaged in complex nonproductive or even counterproductive social activities (such as allomothering, non-reproductive sex, apparently non-profit harassment of other animals or harmful “games” such as eyeball poking) and in violent or lethal aggression (the major cause of death for a capuchin monkey is an altercation with another capuchin monkey)32. If a further increase in the monkey’s para-HI intelligence requires increasing the same behavioural and character features that cause counterproductive comportments, then the resulting negative feedback may block development of HI. Conversely, if a candidate species deviates too much from these characteristics, then HI may not result, despite a suitable cognitive power.\n\nIn humans, an illustration for the negative effects of HI-associated cognitive complexity is provided by the high incident of cognitive diseases. For example, schizophrenia has been suggested to be ‘the price that Homo sapiens pays for language’76. It has also been shown in animals and humans that the more polymorphic tri-nucleotide repeats are present in the gene Hdh (which codes for the protein huntingtin), the higher the capacity of this gene to promote the neural tube formation required for complex brains, but also the higher the probability to develop fatal neurodegenerative Huntington’s disease77. More generally, the genes regulating synaptogenesis and neuronal circuit formation have been associated with an increased risk of mental illnesses78,79. Based on autism spectrum disorders, it was suggested that brain networks involved in HI-required cognitive skills, such as language and complex social behaviour, have less compensatory mechanisms, and are hence less robust, than more ancient biological functions79. As a final example, the rise of complex diseases (i.e. diseases caused by a combination of genetic, environmental and lifestyle factors) has been linked to the rapid genetic, geographic, dietary and cultural changes associated with HI80. The impact of neurological pathologies increases in high-cognition species, and cumulates in humans, where currently about one in four individuals is affected (source: World Health Organization, 2001). The incidence of mental disorders continues to grow, and with it its individual, social and economic impacts (World Health Organization, 2019). Accordingly, mental health costs are now the highest single source of global economic burden in the world81. Thus, the increased instability and lability inherently associated with a trajectory towards HI might produce a negative feedback loop counteracting the evolution of HI.\n\nAdditionally, there might be a different type of negative feedback loop intrinsic to HI—HI might simply allow a species to become so successful in exploiting food resources that these resources become exhausted. A non-human example is provided by long-tailed macaques living on islands in Thailand. These monkeys developed stone tools that allow foraging on shellfish. Over time, however, this technology is so efficient that the macaques severely deplete the shellfish populations on the islands. This triggers a feedback loop where diminishing prey size results in reduced stone size82. The authors suggest that continuation of this pattern leads to a point where this technology is no longer beneficial or useful to the island macaques, ultimately leading to extinguishing of this technology.\n\n\nConclusion and outlook\n\nA specie’s intelligence is a multidimensional characteristic adapted to maximise the specie’s survival. My analysis suggests that the genetic adaptations required for development of HI might have been within reach of more species than only Homo sapiens. However, all other species that have radiated into high-intelligence niches have stagnated at sub-HI levels, often for many million years; only the Homo lineage has crossed this barrier in a very short time. The fact that no other species has reached HI appears unsatisfactorily explained by physical constraints of HI. A possible additional explanation may be that the main barrier towards HI is not only the development of the required physical assets, but also that negative feedback from social, behavioural and molecular complexity, as well as negative environmental feedback make the development of HI increasingly unfavourable.\n\nHow could Homo overcome this barrier? I speculate that key features may have been that our brain originated very rapidly as an exaptation, not adaptation83, together with an ensemble of favourable anatomical changes (e.g., skeletal proportions, dental function and the respiratory system), in a species with extremely low population densities (typically about 10 individuals per 100 km284). This origin may have initially allowed Homo to develop the brain structure required for HI, while circumventing or sufficiently attenuating immediate negative feedback associated with the use of HI in a socially complex populous environment where intra-species competition is the biggest threat.\n\nHowever, as we are becoming overly abundant, and use our brain to its full potential to succeed within our complex societies, the negative side effects become increasingly challenging. Despite the development of psychological, moral, behavioural (‘self-domestication’), pharmacological and technological solutions, we are knowingly and consciously pursuing our unsustainable development that is rapidly destroying the resources upon which we critically depend85,86. However, globally engaging in the actions required to change our current trajectory and achieve long-term sustainability appears contrary to human nature.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nI thank all colleagues and anonymous reviewers who have contributed to this manuscript through stimulating discussions, and by providing comments and counterarguments. I thank KAUST Research Support Services for editorial assistance (Virginia. A. Unkefer) and for figure design and preparation (Ivan Gromicho).\n\n\nReferences\n\nJerison HJ: Brain evolution: new light on old principles. Science. 1970; 170(3963): 1224–1225. PubMed Abstract \| Publisher Full Text\n\nKruska DC: On the evolutionary significance of encephalization in some eutherian mammals: effects of adaptive radiation, domestication, and feralization. Brain Behav Evol. 2005; 65(2): 73–108. 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Reference Source" }	[ { "id": "65187", "date": "03 Jul 2020", "name": "Rafael Bretas Vieira", "expertise": [ "Reviewer Expertise Spatial navigation", "neuro-electrophysiology", "behavioral psychology", "consciousness", "bodily self-awareness." ], "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 discusses the uniqueness of human intelligence (HI) despite its apparent evolutionary advantages. Although the theme has been tirelessly revisited by many authors, a new theory is offered, that higher intelligence is disadvantageous and kept from developing through negative feedback. The author briefly goes through the main molecular and ecological arguments for the appearance and advantages of high intelligence, showing that those are not especially uncommon or slow in evolutionary history. Therefore, none could fully justify the evolution of HI, suggesting it to be generally an unfavorable trait.\nThe theory is well constructed and the references support the hypothesis. Despite that, there are a few points in the manuscript that warrant attention:\nPage 3 - \"Brain size, structure, and complexity\" The author affirms that \"In terms of genetic modifications, the development of the human brain therefore appears to be based on extending already existing features through gradual and common mechanisms, rather than being the result of a developmental quantum leap\" (page 3, para 5, line 33). The example given a few lines before, of Pax6, as well as other well known genes, such as the human-specific ARHGAP11B, despite being relatively small changes, result in significant leaps in brain development. The later example of octopuses also seems to indicate a sudden change, from the moment molluscs lost their shells, which still supports the paragraph conclusion that the genetic mechanisms that allow intelligence are available to other species.\nPage 5 - \"Language and intelligence\" In general, this session is not clear about language being a requirement for the development of HI, a mere consequence of it, or something in between, with some contradictions. For example \"It is therefore not the capacity for an articulate vocalization, but only a predisposition for it, that is essential to the evolution of HI\" (page 5, para 5, line 11) and \"current evidence suggests that the evolution of a HI-supporting language is not a feature strictly limited to humans or the human brain anatomy or speech production\" (page 5, para 7, line 6). The conclusion is especially confusing when declaring \"HI-supporting language\" while the manuscript appears to state that language is not a prerequisite for HI. About the passage: \"Let us now consider language syntax and acquisition with respect to human uniqueness. It has indeed been proposed that such properties of the human language are neither specific to language nor to humans (47,48)\" (page 5, para 6, line 1). The references cited (47, 48) indicate that there may be a human-only syntax, either through universal grammar or simply an advanced form of syntax allowed by the human FLB.\nPage 7 - \"Conclusion and outlook\" Despite mentioning the possible negative feedback from molecular complexity, I couldn't find any examination of it in the main text. For reference: \"the main barrier towards HI is not only the development of the required physical assets, but also that negative feedback from social, behavioural and molecular complexity, as well as negative environmental feedback make the development of HI increasingly unfavourable\" (page 7, para 4, line 11). I wonder if there is a link between HI and sexual selection. Although intelligence as a simple fitness indicator would be equally accessible to other animals as well, cultural and behavioral products could also have influenced mating preferences.\nFinally, the text mentions the possible negative feedback of niche construction: page 7, \"there might be a different type of negative feedback loop intrinsic to HI—HI might simply allow a species to become so successful in exploiting food resources that these resources become exhausted\" (page 7, para 3, line 1) and again on the conclusion \"we are knowingly and consciously pursuing our unsustainable development that is rapidly destroying the resources upon which we critically depend\" (page 7, para 6, line 6). Despite that, HI also generates powerful positive feedback with niche construction, in which the environmental changes produced by HI favor HI itself.\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": [ { "c_id": "5815", "date": "19 Aug 2020", "name": "Stefan Arold", "role": "Author Response", "response": "I thank Dr. Atsushi and Dr. Vieira for their time and efforts to critically evaluate my manuscript, and for their very constructive feedback. In the following I provide a point-by-point reply to their comments, outlining also the changes I have made to the manuscript. I hope that my explanations (given below, which are accessible to readers) and manuscript changes respond to the reviewer’s comments in a satisfactory manner. Should issues remain, I’d be happy to further discuss and integrate these. Reply to comment on Page 3 –“ Brain size, structure and complexity”The reviewers refer to my statement that \"In terms of genetic modifications, the development of the human brain, therefore, appears to be based on extending already existing features through gradual and common mechanisms, rather than being the result of a developmental quantum leap\" (page 3, para 5, line 33). They note that the “relatively small changes [in genes] result in significant leaps in brain development.”I thank the reviewers for this comment that identifies a poor formulation in my essay. Indeed, I argue (in agreement with the reviewers) that the apparently big anatomical and cognitive changes were caused by only relatively small genetic changes. Consequently, I have corrected the wording into: “rather than being the result of a genetic quantum leap.” Reply to comment on Page 5 - \"Language and intelligence\"The reviewers note that “this session is not clear about language being a requirement for the development of HI, a mere consequence of it, or something in between, with some contradictions”, and they give specific examples for this contradiction. My position is that ‘full-blown’ HI needs a powerful communication system; however, Australopithecines and very early homo forms did certainly not have the language necessary to produce modern legislation, Shakespearean theatre or comprehensive gene ontologies. Hence, although a powerful communication system is a requirement for HI, it can (and probably will) be developed while a species develops HI. I agree that some of my formulations lacked precision, and I have now amended the text to enhance the clarity of this message:“we then have to ask whether the development of HI-supporting language would be possible for other species”.“It was therefore not the pre-existence of an articulate vocalisation, but only a physiological potential for it, that was sufficient for allowing the evolution of HI in humans”.“Thus, current evidence suggests that the capacity for developing a HI-supporting system of communication is not a feature strictly limited to humans or the human brain anatomy or speech production.” I thank Dr. Atsushi and Dr. Vieira for pointing out the mismatch between references and statement in the passage “let us now consider language syntax […]47,48”. Indeed, these references should not have been mentioned at this position, because the references for this statement follow in the ensuing discussion (references 50-57). Therefore, in the revised version, I have deleted the references 47,48 at this position (they are still used in the preceding passage) and put a colon instead of the full-stop for additional clarity. Reply to comment on Page 7 - \"Conclusion and outlook\"The reviewers rightly ask: “Despite mentioning the possible negative feedback from molecular complexity, I couldn't find any examination of it in the main text.”. Indeed, in the initial version, ‘molecular complexity’ refers to the extension of protein-level amino-acid repeats that are linked to both, increased cognition, but also increased psychopathology. I gave the protein huntingtin as an example. However, I agree that such repeat extensions (which are frequently linked to cognition and neurological pathologies) are not adequately described as ‘molecular complexity’. Moreover, the genetics of mental illness are only emerging very slowly, with different studies often reaching different conclusions (for a summary, please see M. Marshall, Nature 581, 19-21 (2020) doi: 10.1038/d41586-020-00922-8). Hence, it is also premature for general correlations between complexity and psychopathology on a molecular level.However, over the last decade, a good consensus has been reached that mental disorders can involve altered connectivity in our highly complex brains (e.g. Fornito and Harrison, Front. Psychiatry, 27 July 2012 https://doi.org/10.3389/fpsyt.2012.00072). My manuscript already refers to it in “HI as an intrinsically unfavourable trait”, p7, in the two sentences following the mentioning of huntingtin (“[…]the genes regulating synaptogenesis and neuronal circuit formation have been associated with an increased risk of mental illnesses. […] brain networks involved in HI-required cognitive skills, such as language and complex social behaviour, have less compensatory mechanisms, and are hence less robust, than more ancient biological functions”. Hence, to respond to the reviewers’ comment, I have changed ‘molecular complexity’ into ‘neuroanatomical complexity’ in this sentence. The reviewers also ‘wonder if there is a link between HI and sexual selection’. This is an interesting suggestion, and my thoughts on it are as follows: Interspecies variations in HI would not change the outside appearance and appeal of one individual with respect to a rival. However, above-average HI might indirectly influence mating preferences, for example through resulting in a higher in-group social status, better food sources (and hence physical fitness), and, at a more advanced stage, more attractive cultural/ornamental items. However, given the wide-spread occurrence of sexual selection across animal species, I don’t see how sexual selection would uniquely favour HI in homo, but not in any other species. Hence, while sexual selection might have an influence, this influence would not be specific enough to humans to justify that HI has only emerged in our lineage. In their final comment, the reviewers refer to my discussion on the possible negative feedback of niche construction. In particular, they refer to my discussion of data showing the unsustainable nature of HI in its current ‘implementation’ in modern humans, ultimately making HI a maladaptive feature [“HI might […] allow a species to become so successful in exploiting food resources that these resources become exhausted\"; and “we are knowingly and consciously pursuing our unsustainable development that is rapidly destroying the resources upon which we critically depend\"]. The reviewers suggest that “despite that, HI also generates powerful positive feedback with niche construction, in which the environmental changes produced by HI favour HI itself.”In response, I argue that both statements do not contradict each other. Rather, they consider different effects resulting in this feedback mechanism.Niche construction theory states that acquired features that allow a species to favourably transform its environment can be selected for during evolution. If a species can use its intelligence to ‘consciously’ and favourably alter its environment, then this would certainly result in positive feedback for the development of HI. The dam-building beaver is an obvious example for ‘intelligent’ niche construction (as opposed to, say, cyanobacteria or earthworms). Hence, I agree with the reviewers and existing literature (e.g. doi:10.1098/rstb.2010.0306) that ‘conscient’ niche construction can boost the development of HI. However, two aspects are important in the context of my essay: Firstly, nothing indicates that the positive feedback between HI and the development of ‘conscient’ niche construction is limited to humans. Hence, niche construction would not explain the main focus of my manuscript, namely the uniqueness of HI in humans. To take into account the reviewer’s comments, and to directly clarify this point, I have added the general aspect of niche construction in my revised manuscript (see: Lack of suitable environment?: “Additionally, if the competitive advantage and driving force of HI development lies in overcoming dependence of the environment 14 , or enables favourable niche construction, then this advantage would profit many species, and hence is not a limiting factor. “). Secondly, while niche construction may support the development of increased cognition, this synergy will become overall maladaptive if the changes to the environment deplete the food resources. As a non-human example, I have mentioned the study on island populations of long-tailed macaques. Obviously, through HI, humans were able to consciously alter their environment more than any other animal (cyanobacteria might have altered our planet even more than humans, but not ‘consciously’). However, as I note in my manuscript, available studies overwhelmingly conclude that these HI-enabled environmental changes are unsustainable because they are “rapidly destroying the resources upon which we critically depend”. Hence, the environment-transforming capacity of HI is now critically endangering the survival of our species—and this is the ‘increasingly negative side effects’ of our HI that I refer to in the ‘Conclusion and Outlook’ section." } ] } ]	1	https://f1000research.com/articles/9-34
https://f1000research.com/articles/10-441/v1	03 Jun 21	{ "type": "Software Tool Article", "title": "FDRestimation: Flexible False Discovery Rate Computation in R", "authors": [ "Megan H. Murray", "Jeffrey D. Blume", "Jeffrey D. Blume" ], "abstract": "False discovery rates (FDR) are an essential component of statistical inference, representing the propensity for an observed result to be mistaken. FDR estimates should accompany observed results to help the user contextualize the relevance and potential impact of findings. This paper introduces a new user-friendly R pack-age for estimating FDRs and computing adjusted p-values for FDR control. The roles of these two quantities are often confused in practice and some software packages even report the adjusted p-values as the estimated FDRs. A key contribution of this package is that it distinguishes between these two quantities while also offering a broad array of refined algorithms for estimating them. For example, included are newly augmented methods for estimating the null proportion of findings - an important part of the FDR estimation procedure. The package is broad, encompassing a variety of adjustment methods for FDR estimation and FDR control, and includes plotting functions for easy display of results. Through extensive illustrations, we strongly encourage wider reporting of false discovery rates for observed findings.", "keywords": [ "false discovery rate", "multiple comparisons", "adjusted p-value", "null proportion estimation", "R Package" ], "content": "Introduction\n\nThe reporting of observed results is not without controversy when multiple comparisons or multiple testing is involved. Classically, p-values were adjusted to maintain control of the family-wise error rate (FWER). However, this control can come at the cost of substantial Type II Error rate inflation, especially in large-scale inference settings where the number of comparisons is several orders of magnitude greater than the sample size. Large scale inference settings occur frequently in the analysis of genomic, imaging, and proteomic data, for example. Recently, it has become popular to control the false discovery rate (FDR) instead of the FWER in these settings because its Type II Error rate inflation is much less severe. The FDR is essentially the propensity for a finding to be mistaken i.e., the propensity for a non-null claim to be, in fact, wrong.\n\nControlling the FDR at or below a specific level, say γ, does not imply that the Type I Error rate, per-comparison or family-wise, is also controlled at the same level. The increase in Type I Errors that is allowed by FDR control is accompanied by fewer Type II errors. Moreover, different approaches to controlling the FDR allow for different degrees of error trade-off. And software for implementing these approaches vary widely in their scope, options, and accessibility. In addition, methods for controlling the FDR, which use the classical rejection-testing framework, are often confused with the methods used to provide an estimate of the FDR for a particular result.\n\nThe FDRestimation package distinguishes between methods for FDR control and methods for FDR estimation, and it allows the user to easily access complex statistical routines for computing the desired quantity. The plotting functions allow users to visually assess results and differences between methods. We should note that the base package function stats::p.adjust is now frequently used to compute the estimated FDR, however stats::p.adjust actually reports the adjusted p-values for FDR control, and these are not always the same thing. More on this important distinction later. Our package also provides a wide range of methods for estimating the FDR, estimating the proportion of null results, and computing the adjusted p-values. We hope by clearly illustrating the usage of our package in routine settings that these FDR methods will become more accessible and gain even more popularity in routine practice.\n\nWe begin with a simple example to fix ideas. Table 1 shows five unadjusted (raw) p-values for experimental features along with their corresponding Z-values. The third column lists the Benjamini-Hochberg adjusted p-values to be used for FDR control (Benjamini and Hochberg, 1995). Controlling the FDR at level γ amounts to selecting all of the adjusted p-values in column 3 that are below γ. Note here that the adjusted p-values are monotonically increasing, just like the raw p-values, but inflated.\n\nExample with 5 features using the Benjamini-Hochberg adjustment and assuming a two-sided normal distribution.\n\nIf the goal is to control the FDR at 5%, then only the first feature would be declared interesting and selected. Throughout the paper, we use the term “interesting” to describe features that are selected by a procedure with FDR control. We do not use the term “significant” in order to avoid confusion with those features that would have been selected from by a procedure with strict Type I Error control.\n\nThe fourth column presents FDR estimates for each feature. As we show later, there are several ways to invert the FDR control procedures to yield an estimate of the FDR. Our package performs this inversion for most popular methods. The FDRs here were obtained by inverting the Benjamini-Hochberg FDR control procedure, and so we will refer to them as the BH FDRs (Benjamini and Hochberg, 1995). In practice we find these estimates to be the most context useful when making scientific decisions about which findings to pursue.\n\nImportantly, these are clearly not identical to the BH adjusted p-values nor are they even monotone. The non-monotonicity results from the group-wise p-value adjustment procedure (”step-up”) and the non-smooth estimate of the p-value mixture distribution, which is needed for FDR estimation. The important insight is that the set of features that are selected by the FDR control procedure is not equivalent to the set of feature whose individual FDR is less than the control threshold. For example, if the FDR threshold was γ = 0.07, then the first four features would be selected by BH to control the group-wise FDR at 7%. However, only the first and fourth features have estimated false discovery rates below 0.07, and thus only these two features would be reported as having a false discovery propensity less than 7%. Note that both approaches come from the same Benjamini-Hochberg machinery, and thus have the same distributional assumptions. The distinction between adjusted p-values and estimated FDRs are critical here.\n\nBecause FDRs are only estimates, and because there are a variety of estimation approaches, it helps to have a feature-specific benchmark for each FDR. The fifth column provides such a benchmark; it displays a well-known lower bound on the FDR assuming a Gaussian posterior and a null proportion of 50%. These assumptions are relatively benign for reasons we discuss later and represent a “best-case” scenario. This benchmark shows two things: (1) the adjusted p-values are a poor substitute for the FDRs, and (2) the smoothness of the FDR estimation approach is important.\n\n\nMethods\n\np-value based approaches\n\nLet p1,…,pm be the individual unadjusted p-values derived for each of m different features or tests. For clarity, the ith p-value is for the ith feature and has not been adjusted for any multiple testing. It is sometimes referred to as the “uni-variate” p-value. The sorted or ranked p-values are represented by p(1),…,p(m) where p(1) is the smallest, p(m) is the largest and with p(k) is the kth ranked p-value.\n\nLet γ be the false discovery rate threshold for interesting findings. This threshold is context specific, and is either set by the researcher or according to a community standard. This threshold is specified a priori when performing FDR control procedures, but it need not be specified for FDR estimation procedures. The Benjamini-Hochberg algorithm for FDR control is to find the largest index, say k, such that\n\nThis can be written compactly k=max[i:p(i)≤γi/m]. Then all features with p(1),…,p(k) are deemed interesting at the FDR γ threshold and considered “findings”. This is called a “step-up” procedure because not all of the rejected features will have unadjusted p-values that meet the above criterion. Only the largest of them must meet that criterion. Because this is a “step-up” procedure, the adjusted p-values will depend on the raw p-values from other features. The Benjamini-Hochberg adjusted p-value for the ith feature is notated in this paper by p~i and defined in Equation (2), where := means “is defined as”.\n\nThese adjusted p-values are monotone increasing in raw p-value ranking, so one can directly compare p~i to γ to see if a particular feature would be rejected as null for the FDR threshold γ. Importantly, the feature specific FDR estimates need not be monotone. To see this, re-arrange Equation (1) as follows in Equation (3).\n\nThe derivation of FDR is described in the following section. This shows that the BH procedure is, in effect, estimating the feature specific FDR as FDRi. See also Efron LSI for motivation for this definition (Efron, 2013). Because estimation of the feature specific FDR does not include group-wise control of the FDR, the “step-up” monotonicity condition does not apply. Thus, feature specific FDR estimates such as FDRi are not always monotone in raw p-value ranking.\n\nA consequence of this dichotomy is that an individual feature may be rejected at FDR γ level by the BH algorithm even though its feature specific FDR estimate is actually greater than γ. This is largely a consequence of the smoothness of the FDR estimates and the fact that they can have substantial variability. Note that there are several methods for estimating the FDR, and some methods may be better suited to certain contexts. Our package offers several methods for FDR estimation, as described in later sections of this paper.\n\nTo illustrate we simulated real data from 100 hypothesis tests and captured the 100 raw p-values. For context, 80 of these p-values were generated from a uniform distribution (and hence under the null) while the other 20 were generated from a skewed distribution representing the alternative. Results are computed using our p.fdr function, which we detail later. The raw p-values are displayed in Figure 1 as black points; Figure 2 shows only the 20 features with the smallest ranked raw p-values. The black sloped line is the BH rejection line from Equation (1). Also included in the plot are the BH adjusted p-values (blue stars), the BH FDR threshold for interesting findings (blue horizontal line), and the BH FDR estimates (red points).\n\nIn Figure 2 we see that exactly 8 of the adjusted p-values fall below our threshold of interest (blue line, set here to 0.06). Therefore, the BH FDR procedure that controls the group-wise FDR identifies the 8 smallest p-values as interesting findings. However, notice the non-monotonicity of the individual FDRs. Only the first and last of the 8 lowest FDRs are less than 0.06.\n\nFrom these results it should be clear that the feature-specific FDRs and the BH adjusted p-values have different purposes and interpretations. To emphasize, when a feature is identified as ‘interesting’ by an FDR control procedure, it does not always follow that the feature’s individual propensity to be a false discovery is less than the desired threshold. Both quantities must be computed, as the tasks are not always exchangeable.\n\nZ-value based approaches\n\nFor FDR estimation, it is often helpful to transform the p-values p1,…,pm to into Z-values z1,…,zm using the standard normal quantiles. For example, zi = Φ−1(1 − pi) for one-sided p-values or zi = Φ−1(1 − pi/2) for two-sided p-values. Efron explains the rationale as an attempt to leverage the distributional properties of a set of Gaussian random variables (Efron, 2013). Note that these Z-values are not intended to be the original test statistics. We will adopt Dr. Bradley Efron’s formulation as described here (Efron, 2013).\n\nWe begin with the classic two-group model, which assumes each of the m features is either null (distribution known) or alternative (distribution unspecified), but that this status is unknown. As a group the combined data can be used to provide an estimate of the mixture distribution, where the mixing proportion (π0) is also unknown. Let f0(z) be the probability density function of the z-values when they come from the true null distribution and f1(z) be the probability density function of the z-values when they come from the alternative distribution. Then F0(⋅) and F1(⋅) denote the probability of rejection for any subset Z of the real line such that,\n\nWith mixing or null proportion π0, the proportion of non-null features is simply π1 = 1 − π0. The mixing distribution function is\n\nWhen working with Z-values, it is reasonable to use a gaussian distribution for the theoretical null probability density function, so that f0(z) ∼ N(0,1) (Efron, 2013). When estimating the FDR, is it also common to assume that π0 = 1 because doing so results in a conservative estimate of the FDR. Then, an application of Bayes famous theorem yields:\n\nSubstituting the natural empirical estimate of the mixture distribution F(Z) results in empirical Bayes estimates the global FDR Equation (6) (Benjamini and Hochberg, 1995) (Efron, 2013). For example, the obvious empirical estimate of the mixing distribution function is the step function F^(Zi)=rank(pi)/m. Notice that the right hand side of Equation (1) then looks like γ⋅F^(Zi) or γ times the step function. In some settings smoothing F^(Zi) can be beneficial. Very often it is assumed π0 = 1 and F0(Z)=1−Φ(Z) for one-sided tests. An advantage of estimating the FDR from the right hand side of Equation (6) is that one only needs to accurately estimate the mixture distribution function to get good estimates of the FDR and this does not require the independence of the z-values.\n\nFigure 3 and Figure 4 show the application of this framework using the same simulated data as in the last example (100 tests, 80 truly null). In the z-space, the null distribution is now the standard normal and the alternative distribution was set to N(2,1) (of course this is unknown, in practice). Figure 3 shows these densities overlaid on a histogram of the raw data. The blue curve indicates the null density, the red curve indicates the alternative density, and the black curve is the mixture density with π0 = 0.8. Clearly, the blue curve does not fit the histogram well, with a much shorter right tail than the histogram shows. So, assuming all 100 tests come from the null distribution does come with a penalty.\n\nFDR Z-values plot.\n\nFigure 4 displays the relationship between the Z-values and various FDR quantities. The black dots show the raw p-values (y-axis) versus their Z-value (x-axis); the red dots show the estimated FDRs (y-axis) versus their Z-value (x-axis); and the blue stars show the BH adjusted p-values (y-axis) versus their Z-value (x-axis). This is the comparable plot to Figure 1, where the x-axis has been changed from p-value ranking to z-scale. The usefulness of this plot is that is shows what the desired FDR quantity is for a given Z-value. This provides context for our FDRs and adjusted p-values.\n\nHere we see that Z-values greater than 2.85 and less than -2.5 have adjusted p-values less than 0.06 (blue threshold line, horizontal). This means in order to control the group-wise FDR, one would identify features with these Z-values as “interesting”. Notice that the Z-value above 4 has a FDR less than 0.06. Also the Z-value of 2.9 has a FDR less than 0.06. In practice, we find that the display in Figure 1 is more intuitive for non-statisticians, but that Figure 4 provides some essential insight into the stability and smoothness of the estimation procedure.\n\nLower bound on the FDR\n\nThe previous section introduced an empirical Bayes estimator for the FDR, which has become one of the most popular estimates. However, there are many different approaches for estimating the FDR. We have found it helpful in practice to be able to benchmark the magnitude of the FDR under known conditions in order to provide a contrast for estimators that rely heavily on distributional assumptions. This lower bound can help to contextualize findings and illuminate differences masked by empirical assumptions.\n\nOur preferred benchmark is a well-known lower bound on the posterior probability of the null (hypothesis) under a gaussian model. This lower bound depends only on the data for the feature or test of interest and it does not borrow strength across features (for better or worse). Hence, it can also be used when only a single test is performed, i.e. when only a single p-value is available. In our experience, the gaussian assumption tends to have minimal influence because sampling distributions tend to be symmetric.\n\nThe lower bound arises as follows. Let the joint density of data from a single feature be g(X1,…,Xn\|θ) where θ is a parameter of interest. The likelihood function is Ln(θ) ∝ g(x1,…,xn\|θ) and denote the maximum likelihood estimator as θ^n. Recall that the null hypothesis is H0 : θ = θ0. Let π0 = P(null) be the prior probability of the null and let z be the observed test statistic of the null hypothesis. Then, a lower bound on the posterior probability, P(null\|x1,…,xn), which is effectively the FDR, is given by\n\nThe first inequality holds because ∫g(x1,...xn\|θ1)h(θ1)dθ1leqg(x1,...xn\|θ^n) for all θ1 ∼ h(θ1). Note that ∫h(θ1)dθ1=π1 by definition. The second approximation comes from the general asymptotic behavior of a classical likelihood ratio test, where −2logL(θ0)L(θ^n)∼χ12=[N(0,1)]2 for one-dimensional parameters. This lower bound is similar to that derived and explored by Berger (1985). Our function uses default odds, π1/π0 = 1, reasonable in many circumstances, which easily can be changed. As the z-statistic approaches zero, the lower bound approaches 1/2, as would be expected.\n\nFor illustration, consider feature 4 in Table 1. Feature 4 has an observed p-value of 0.051, but has a univariate gaussian lower bound on the FDR of 0.13 =(1+exp(1.9512/2))-1. In this case the BH estimated FDR is 0.064, substantially below the lower bound. This discrepancy in estimates is due to differing underlying assumptions. In contrast, feature 2 has a p-value of 0.049 and FDR of 0.122, very close to its lower bound. Although feature 4 has nearly the same p-value as feature 2, its BH FDR is nearly half that of feature 2. The univariate gaussian lower bound is helpful for identifying when FDR estimates may be optimistic, as in the case above. Similarly, we see that the adjusted p-values can be much less than the lower bound, which is another reason why they should not be mistaken for FDR estimates.\n\nThe computation of adjusted p-values and FDRs for each method follows a similar intuitive approach. First, estimates of the FDR for each feature are obtained using the preferred method, e.g. Benjamini-Hochberg or Benjamini-Yekutieli. Step-up or step-down adjustments are not applied at this stage. Next, adjusted p-values are obtained from the estimated FDRs by applying the step-up or step-down adjustment that is associated with the method. The step adjustment is necessary for error control but not for FDR estimation. For methods that do not have a step-up or step-down component, e.g. Bonferroni, the adjusted p-values and FDRs will be the same. The distinction between the estimated FDRs and the adjusted p-values is an important one that is routinely confused in practice.\n\nNote that all estimates of adjusted p-values and FDRs are forced to be 1 or less. Also, when ranks are used in our package the ties.method = \"random\". This means for example that if the 4 smallest p-values in a vector tie in value then they will be assigned ranks 1,2,3,4 randomly. The user can change the ties method in the input to the function.\n\nBelow we illustrate this with the remaining five methods (BH is discussed above).\n\nBenjamini-Yekutieli\n\nBenjamini-Yekutieli (BY) is a step-up method for controlling the false discovery rate under arbitrary dependence (Benjamini and Yekutieli, 2001). For a pre-specified dependence structure, there exits an adjustment function called c(m) that is used to modify the Benjamini-Hochberg estimate of the FDR. For example, in the case of flexible positive dependence, the function c(m)=∑j=1m1j is used. Then, the threshold criteria is to find the largest index i such that Equation (8) holds, which is a scaled version of the BH criterion given in Equation (1).\n\nThis can be written compactly k=max[i:p(i)≤γ⋅i/(m⋅c(m))] or for non-ordered vectors of p-values k=maxrank(pi):pi≤γ⋅rank(pi)/(m⋅c(m)). Then all features with p(1),…,p(k) are deemed interesting at the FDR γ threshold and considered “findings”. Recall that Benjamini-Hochberg procedure uses the step function (F(p(i)) = i/m) as its implicit empirical estimate of the mixing distribution function (CDF) Check this notation. The Benjamini-Yekutieli procedure amounts to simply using a modified estimate for the CDF, namely (F(p(i)) = i/(m ⋅ c(m))).\n\nMathematically, the adjusted p-values and estimated FDRs are\n\nComparing this form to the general formula for the FDR in Equation (6), we see that the BY correction amounts to changing the estimate of the mixture distribution F(Z) from [rank(pi)/m] to [rank(pi)/(m ⋅ c(m))] to account for dependence. Note that we have avoided using the ordered notation for False discovery rate estimates, say FDR(i), because although those estimates are dependent on ordered p-values the FDR estimates themselves do not have to be monotonic.\n\nHere we see the BY FDRs, or red points, jump above and below the 0.06 threshold in ranks 1 to 6. Then in ranks 7 and greater the red dots remain above the threshold and quickly are adjusted to the value of 1. The positive dependence correction causes these BY FDRs to be closer to 1, or more conservative.\n\nBonferroni\n\nThe Bonferroni correction controls the family wise error rate (FWER) (Bonferroni, 1936). We include it in our function because of its popularity in multiple adjustments even though it is not directly related to FDR. For this method we would reject the null hypothesis for each pi≤γm in order to control the FWER at ≤ γ level. In our functions the adjusted p-values and adjusted FDRs will always be identical for this method.\n\nFrom this form we see that the Bonferroni correction amounts to changing the estimate of the mixture distribution F(Z) to [1/m].\n\nSidak\n\nThe Sidak or Dunn-Sidak correction controls the family wise error rate (FWER) (Šidák, 1967). This correction method is exact for tests that are independent, it is conservative for tests that are positively dependent, and it is liberal for tests that are negatively dependent. For this method is slightly less strict then the traditional Bonferroni method. For each pi≤γSid=1−(1−γ)1m reject the null hypothesis in order to control the FWER at ≤ γ level. In our functions the adjusted p-values and adjusted FDRs will always be identical for this method.\n\nFrom this form we see that the Sidak correction amounts to changing the estimate of the mixture distribution F(Z) to [pi/(1 − (1−pi)m)] assuming F0(Z) = pi.\n\nHolm\n\nThe Holm method, also known as the Holm-Bonferroni method, controls the FWER and is less conservative and therefore uniformly more powerful than the Bonferroni correction (Holm, 1979). For this method we use the step-down procedure which would reject the null for those rankings 1,…,(k − 1) such that k is the smallest ranking where:\n\nFrom the above equation we see that it relies on the ranking or j that means our function’s outputted adjusted p-value and FDR can be different.\n\nFrom this form we see that the Holm correction amounts to changing the estimate of the mixture distribution F(Z) to [1/(m + 1 −rank(pi))].\n\nHochberg\n\nThe Hochberg method uses the same equation as the Holm method, Equation (15) (Hochberg, 1988). However for this method we use the step-up procedure. This means we would reject the null for those rankings 1,…,j such that j is the largest ranking where:\n\nThis change from the step-down to the step-up procedure results in the Hochberg correction being more powerful than the Holm method.\n\nFrom this form we see that the Hochberg correction is the same as the Holm and amounts to changing the estimate of the mixture distribution F(Z) to [1/(m + 1 −rank(pi))].\n\nNull proportion (π0) estimation\n\nThe proportion of truly null features (π0), also known as the mixing proportion, is an important component of the FDR estimate that can be a strong driver of the estimate. While generally not identifiable, reasonable estimates of π0 can be obtained under certain conditions. Many of the popular FDR estimation routines take a conservative approach by setting π0 = 1, which results in a larger, i.e. conservative, FDR estimates.\n\nThe default in p.fdr is to assume that π0 = 1. However, users are able to set the null proportion to a particular value or specify an estimation routine to estimate π0 from the data. Many methods have been proposed for estimating the mixing proportion π0 in a two-component mixture. p.fdr includes several of these methods such as Storey, Meinshausen, Jiang, Nettleton, and Pounds (Storey and Tibshirani, 2003; Meinshausen, Rice, et al., 2006; Jiang and Doerge, 2008; Nettleton, Hwang, Caldo, and Wise, 2006; Pounds and Morris, 2003). In next section, we propose a new approach that we call “Last Histogram Height”. This new approach is simple, appears to have excellent performance over a wide range of scenarios, and less computationally intensive that Storey’s approach, which is quite popular. An evaluation and comparison to existing approaches is described in the subsequent subsection.\n\nLast Histogram Height\n\nUnder the null, a test statistic for a feature, say a Z-value, is standard normal. As such, the corresponding p-value has a uniform distribution over the unit interval. Therefore, if all the features were null, we would expect an empirical histogram of the observed p-values to be approximately flat. Moreover, we see that the distribution of non-null p-values tends to be shifted toward zero.\n\nThe “Last Histogram Height” method uses the bin height of p-values near 1 to estimate the true proportion of null features. We rely on the assumption that larger p-values are more likely to be come from null features. Let bin heights be H1,H2,…,HB, where B is the total number of bins. When B = m (m is the number of features) and all features are null, we would expect Hi ≈ 1 for all i = 1,…,B. The caveat is estimating bin height is sensitive to the choice of bin width. However, we have found that Scott’s normal reference rule tends to work very well for this method (Scott, 1979).\n\nWhen π0 < 1, the empirical distribution of the p-values (as shown by the histogram) will not be uniform over the unit interval. Departure from the uniform becomes easier to detect as π0 moves further from 1 because the histogram shape quickly deviates from a uniform appearance. An example is presented in Figure 5, which shows a histogram of raw p-values from our simulated example of m = 1000 features. The red horizontal line is drawn at the height of the last bin, HB. In this approach HB is our “null height” and HB ⋅ B is an estimate of the total number of null features. We then divide that by the number of total features (m) to estimate the null proportion (see Equation (21)):\n\nSimulated histogram of p-values.\n\nThe approach works because we would expect π0 * m/B null p-values to be in each bin. This simple method performed well over many different simulation settings, as we described in the next section. It is also relatively free of constraining assumptions on the alternative distribution. We note that this approach can also be viewed as a form of central matching, as discussed by Efron (Efron, 2013), with center mass 1B and a very small bin width. The “Last Histogram Height” algorithm is as follows:\n\nStorey\n\nStorey et al. (2003) propose an iterative procedure for estimating π0. This procedure is popular and tends to have good performance characteristics over a wide range of scenarios. Storey’s method relies on the fact that null p-values are uniformly distributed. As such, the bin height of p-values greater than 1/2 should give a conservative estimate of the null proportion. But there is nothing magical about 1/2, so Storey uses a tuning parameter. Let λ identify “large” p-values, e.g., #pi > λ where i = 1,…,m, such that the estimate of the null proportion π^0(λ), can be tuned by λ to yield a desirable bias-variance trade-off. Storey smoothes π^0(λ) before tuning, which provides some numerical stability. Note that for the “Last Histogram Height” approach, the bin height closest to one is used to estimate the null proportion, which is conceptually similar to using limλ→1π^0(λ) as Storey does. Storey’s algorithm for estimating π0 is as follows:\n\nComparison\n\nBelow in Figure 6 are three plots showing the range of behavior of the six methods for estimating the null proportion that are included in our R package. These plots show the arrogate behavior of each method for estimating π0 over 1000 simulations where the methods are used on a set of 100 features. A standard normal distribution was used for null features and three different alternative distributions were examined for alternative features (three different plots). The x-axis represents the true π0 used to generate data and ranges from 0 to 1. The y-axis represents the average estimate π0 (over the 1,000) simulations for each of the six methods.\n\n“Last Histogram Height” and Storey’s method preformed the best across these scenarios (and others not shown here). They routinely produce the closest estimates of the true null proportion. Although we only display three different mixture distributions for a set of 100 features here, we tested 12 different mixture distributions over three different features set sizes to confirm our results. We also tested the mean squared error and the results are well represented by the three examples given here. Our recommendation is to use the default of setting π0 = 1 when the majority of features are expected to be null or nearly null. But in cases where the null proportion is likely to be different from one (say less than 0.95 or 0.9), then the “Last Histogram Height” algorithm tends to perform the best.\n\nFDRestimation is a user-friendly R package that directly computes and displays false discovery rates from p-values or z-scores under a variety of assumptions. The following sections will explain the primary functions in this package and illustrate how to implement them.\n\nThis p.fdr function is used to compute FDRs and multiple-comparison adjusted p-values from a vector of raw p-values. The stats package function stats::p.adjust is similar in that it will produce multiple-comparison adjusted p-values. However, stats::p.adjust returns the BH adjusted p-value labeled as the FDR estimate. Strictly speaking this is inaccurate, because the BH FDR estimate should not have the forced monotonicity that its adjusted p-values must have. In addition, when estimating the FDR, our FDRestimation::p.fdr function allows adjustments of key assumptions that are not adjustable in the stats::p.adjust implementation (they are set to the simplest, most popular options).\n\nThis FDRestimation::p.fdr function allows for the following adjustment methods: Benjamini-Hochberg, Benjamini-Yekutieli (with both positive and negative correlation), Bonferroni, Holm, Hochberg, and Sidak (Benjamini and Hochberg, 1995; Benjamini and Yekutieli, 2001; Bonferroni, 1936; Holm, 1979; Hochberg, 1988; Šidák, 1967). It also allows the user to specify the threshold for important findings, the assumed pi0 value, the desired pi0 estimation method, whether to sort the results, and whether to remove NAs in the imputed raw p-value vector count (stats::p.adjust actually counts NAs as viable features in its Bonferroni adjustment). Table 2 shows all of the inputs for this function and their descriptions.\n\nThe underlying methods for estimating the null proportion can be set by using the “estim.method” and “set.pi0” arguments. The default value of “set.pi0” is 1, meaning it assumes that all features are null features. Accordingly, this approach will yield conservative estimates of the FDR. Alternatively, and less conservatively, one can attempt to estimate the null proportion from the data. To do this, we recommend using “Last Histogram Height”, as it was the simplest routine and one of the most accurate in our simulations (Murray, 2020).\n\nHere we see an example of how to use this FDRestimation::p.fdr function in R. We simulate 100 features with a true null proportion of 80%.\n\n\n\nThe function will return a list object of the p.fdr class. In Figure 7 we see this list object from the first five p-values for with the following components (Murray, 2020).\n\n• fdrs A numeric vector of method adjusted FDRs.\n\n• Results Matrix A numeric matrix of method adjusted FDRs, method adjusted p-values, and raw p-values.\n\n• Reject Vector A vector containing Reject.H0 and/or FTR.H0 based off of the threshold value and hypothesis test on the adjusted p-values.\n\n• pi0 A numeric value for the pi0 value used in the computations.\n\n• threshold A numeric value for the threshold value used in the hypothesis tests.\n\n• Adjustment Method The string with the method name used in computation(needed for the plot.fdr function).\n\nThe get.pi0 function is used to estimate the null proportion from the raw p-values. The user can choose one of six different methods included in our function: Last Histogram Height, Storey, Meinshausen, Jiang, Nettleton, and Pounds (Storey and Tibshirani, 2003; Meinshausen et al., 2006; Jiang and Doerge, 2008; Nettleton et al., 2006; Pounds and Morris, 2003). The user may also change the methods of determining the number of histogram breaks, which is an essential component for many of the methods implemented here. Table 3 shows function arguments and their descriptions.\n\nHere we see an example of how to use this get.pi0 function in R. We used the simulated data from above sim.data.p where the true null proportion was set to 80%. In the first example, for the purposes of the estimation routine, π0 was set to a single value with the set.pi0=0.8 argument (1 is the default). Alternatively, we can use one of the six estimation methods in get.pi0 instead of specifying π0 a priori. Below is an example where we set the estimation method to \"last.hist\" (i.e., “Last Histogram Height”). In that case, the get.pi routine returned an estimate of null proportion of 0.95.\n\n\n\nThis plot.p.fdr function is used to plot the results of p.fdr. By default, the adjusted FDRs, adjusted p-values and raw p-values are plotted along with two threshold lines to help contextualize the points. Any combination of p-values and thresholds can be removed from the plot. The user can set the axis limits, the location of the legend, the title of the plot and the plotting symbols and colors. Table 4 shows all the function arguments and their descriptions.\n\nHere we see an example of the plot.p.fdr function in R. We used our simulated data sim.data.p, where the a true null proportion was 80%, for illustration. Figure 8 show the default plot, and Figure 9 zooms in on an interesting subset of findings.\n\n\n\nThis article was written using R version 4.0.3 (2020-10-10 on https://cran.r-project.org/bin/windows/base/old/4.0.3/) and FDRestimation version 1.0.0. The FDRestimation R package is available from CRAN and works on R versions 3.4 and above.\n\nThe package can be installed from CRAN using the following code:\n\n\n\n\nConclusions\n\nWe encourage the use of FDR methods and desire to illuminate the importance of contextualizing important findings. Our package provides useful and easy tool for those want to compute the false discovery rate, analogous to the role that stats::p.adjust plays for multiple comparison adjustments in everyday practice. Importantly, we hope it is now clear that p-value adjustments are not interchangeable with FDRs. In addition, FDRestimation package clearly delineates between methods for FDR control and methods for FDR estimation, while still allowing the user to choose from many different inputs and assumptions for their data. The more flexibility the user has at their disposal with these methods, better interpretations and applications will result.\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\nR package FDRestimation is available from CRAN: https://cran.r-project.org/package=FDRestimation.\n\nSource code available from: https://github.com/murraymegan/FDRestimation.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.4684221.\n\nLicense: MIT + file LICENSE", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank the Vanderbilt University Biostatistics SEDS Lab for help discussing these ideas.\n\n\nReferences\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: A practical and powerful approach to multiple testing. J Royal Statistical Society. 1995; 57(1): 289–300. Reference Source\n\nEfron B: Large-Scale Inference: Empirical Bayes Methods for Estimation, Testing, and Prediction. Cambridge University Press; 2013. ISBN 9780511761362.\n\nBenjamini Y, Yekutieli D: The control of the false discovery rate in multiple testing under dependency. Annals Stat. 2001; pages 1165–1188.\n\nBonferroni C: Teoria statistica delle classi e calcolo delle probabilita. Pubblicazioni del R Istituto Superiore di Scienze Economiche e Commericiali di Firenze. 1936; 8: 3–62.\n\nŠidák Zbyněk: Rectangular confidence regions for the means of multivariate normal distributions. J Am Stat Assoc. 1967; 62(318): 626–633.\n\nHolm S: A simple sequentially rejective multiple test procedure. Scandinavian J Stat. 1979; pages 65–70.\n\nHochberg Y: A sharper bonferroni procedure for multiple tests of significance. Biometrika. 1988; 75(4): 800–802. Publisher Full Text\n\nStorey JD, Tibshirani R: Statistical significance for genomewide studies. Proc Natl Acad Sci U S A. 2003; 100(16): 9440–9445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeinshausen N, Rice J, et al.: Estimating the proportion of false null hypotheses among a large number of independently tested hypotheses. Annals Stat. 2006; 34(1): 373–393. Publisher Full Text\n\nJiang H, Doerge RW: Estimating the proportion of true null hypotheses for multiple comparisons. Cancer informatics. 2008; 6: 117693510800600001. PubMed Abstract \| Free Full Text\n\nNettleton D, Hwang JTG, Caldo RA, et al.: Estimating the number of true null hypotheses from a histogram of p values. J Agri Biol Environmental Stat. 2006; 11(3): 337. Publisher Full Text\n\nPounds S, Morris SW: Estimating the occurrence of false positives and false negatives in microarray studies by approximating and partitioning the empirical distribution of p-values. Bioinformatics. 2003; 19(10): 1236–1242. Publisher Full Text\n\nScott DW: On optimal and data-based histograms. Biometrika. 1979; 66(3): 605–610. Publisher Full Text\n\nMurray M: Fdrestimation.Nov 2020. Reference Source" }	[ { "id": "86958", "date": "07 Jul 2021", "name": "Ted Westling", "expertise": [ "Reviewer Expertise Non-parametric efficient estimation" ], "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 emphasizes the necessity of distinguishing between the computation of adjusted p-values for FDR control and FDR estimation. A variety of commonly used methods for FDR estimation are introduced in the paper and the implemented package makes the methods easily accessible. The authors also provide a new method for estimation of the proportion for the null hypothesis. A simulation study verifies the new method works well when compared with other existing methods. The authors also have detailed interpretations of the functions in the package, together with some example code in the following section. Overall, the paper develops in a logical way with replicable results.\nWe have several comments and questions about the article and package:\n1. All three functions have one common argument, ”z values”, which can either be a vector or a string. When we tried to pass only one vector to the argument (without providing p-values) to compute the FDR, the function didn’t work. we had to pass the p-values corresponding to the z-values to make it work. Given that there’s a z-value based approach for FDR estimation, is it possible to make the function work whenever it takes a numerical vector for the argument ”z values”?\nFor example, if we run the following code: ``` z <- runif(10) p.fdr(zvalues = z) ``` It will give me an error message \"Error in TRUE %in% (pvalues > 1 \| pvalues < 0) : argument \"pvalues\" is missing, with no default\", unless we run it this way: ``` p.fdr(pvalues = pnorm(z)) ```\n2. The lower bound on FDR is introduced in the paper as one way of identifying optimistic FDR estimations. Is it reasonable to incorporate this lower bound into the output of the function ”p.fdr”?\n3. In terms of algorithm 1, is it necessary to store the heights for every bin? Is the height of the last bin alone enough?\n4. In figure 6, when the true null proportion is 1, in which case the null distribution has a uniform distribution, seems to be biased downward. Shouldn’t the histogram reflect the uniform distribution better than other skewed distributions (when the proportion is less than 1), thus a better estimation of ?\n5. Though it is claimed that the last histogram height method has the minimum MSE, Figure 6 only shows the bias of the estimators. It would be better if there’s another figure showing the variance and/or MSE for all estimators.\n6. Please add axis labels and interpretable legends to every figure.\n7. The default method when there are ties in the ranks is important. The authors set the default to “random”, which has the downside that the results may differ on the repeated running of the same code. Can the authors discuss this choice? In addition, we think if there are ties, the functions should provide a warning or message about the presence of ties and how they are being handled.\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? Yes", "responses": [ { "c_id": "7287", "date": "19 Oct 2021", "name": "Megan Murray", "role": "Author Response", "response": "Thank you very much for your time and suggestions. We have incorporated those suggestions and revised accordingly, which resulted in an improved paper. Some key issues were noted, and we clarify those below. This is a subtle but important issue. The false discovery rates and adjusted p-values are all computed using the provided ‘pvalues’ vector. The z-values are used only for graphing in the z-value space and for computing the lower bound on the FDR. Remember that the z-values alone do not uniquely determine the p-value; it matters if the p-values are two-sided, one-sided in either direction, or adjusted for continuity or multiple looks. Accordingly, we found it simpler to require the p-values instead of hard-coding certain assumptions. We added a warning message for when the ‘pvalues’ vector is missing. It reads: “Error: The ‘pvalues’ vector is required for all FDR computations.” This is already included in the function. The lower bound is applied in all cases where the user has only supplied a single p-value. We have changed the class name of the output for this situation to improve user clarity. Perhaps. However, we call the graphics package function “hist()”, which automatically computes and stores every bin height. So, changing this would not improve the run time. However, the all the bin heights do not have to be stored for this method. When the true null proportion is one, all of methods produce estimates that are biased downward (largely because the estimates are capped at 1). Accordingly, there is no unbiased estimate of the null proportion. Moreover here “better” is open for debate, as there is a bias-variance trade-off among the methods. We have included the plots of the MSE in new Figure 7 so readers can assess for themselves the relative improvements. In our opinion, ‘Last Histogram Height’ and ‘Storey’ methods were the two methods that performed best over the widest range of cases. We have submitted an updated version with a few axis labels and interpretable legends added. For Figures 1,2,4,8, and 9 we do not label the y-axis label to avoid confusion between the adjusted p-values and raw p-values (the y-axis space is the unit interval). Thank for your input here. We added the suggested warning to alert the user when ties are present. We chose the default “random” because in most cases the order of the p-values vector is not contextually meaningful. To address the reproducibility issue, the user can set a seed before running the function and reproduce the exact results." } ] } ]	1	https://f1000research.com/articles/10-441
https://f1000research.com/articles/10-1063/v1	18 Oct 21	{ "type": "Research Article", "title": "Detection of CYP2C192_ allele among Helicobacter pylori -infected patients in two tertiary hospitals of Khartoum, Sudan, 2019", "authors": [ "Azza Abbas", "Sawazen Malik", "Bushra Sulieman", "Khalid Enan", "Bushra Sulieman", "Khalid Enan" ], "abstract": "Background: CYP2C192 has been identified as the most common allelic variant of CYP2C19 affecting the response to Proton pump inhibitors (PPI). This study aims to detect CYP2C192 allele in H. pylori-infected Sudanese population, owing its probable effect on H. pylori eradication. Methods: Antral biopsies was collected from 30 patients attending endoscopy units. Extraction of DNA was performed through QIAamp® DNA Mini Kit. Samples were screened for Urease C (UreC) gene of H. pylori using conventional PCR. Detection of CYP2C192 was performed in positive H. pylori samples using Real time-PCR. Results: The mean age of patients was 40.7 (±20.2 SE). Positive samples for UreC were 24 (80%) samples. Among them, four samples (16.6%) were found positive for CYP2C192 allele presence. Gender was found to be statistically associated with the presence of the allele (p < 0.05). Conclusion: This study illustrates that CYP2C192 is of modest prevalence among H. pylori-infected Sudanese population. The determination of genotypic and allelic frequencies of CYP2C19 gene among different populations will provide data to be used to personalize treatment according to individual genetic profile, and minimize the possible adverse side effects of CYP2C19 substrates.", "keywords": [ "CYP2C192", "Helicobacter Pylori", "Gastritis", "Sudan" ], "content": "Abbreviations\n\nGI: gastrointestinal\n\nH. pylori: Helicobacter pylori\n\nIM: Intermediate metabolizer\n\nNM: Normal metabolizer\n\nPM: Poor metabolizer\n\nPPI: Proton pump inhibitors\n\nRM: Rapid metabolizer\n\nUM: Ultra rapid metabolizer\n\nUreC: Urease C\n\n\nIntroduction\n\nHelicobacter pylori (H. pylori) is considered the most common bacteria diagnosed in chronically infected stomachs in patients with chronic gastritis.1 It is infecting more than half of the population worldwide.2 In Sudan, it was found in 80% of patients with Barrett's esophagus and gastritis3 and 56.3% among Sudanese children.4\n\nH. pylori eradication is inevitably important, as it has been classified as a class 1 human carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO).5 Stomach cancer in Sudan was among the 10 most common primary sites of cancer.6 Moreover, H. pylori is related to chronic gastritis, duodenal and gastric ulcers, and MALT lymphoma.7\n\nThe first line regimens used are triple and quadruple therapy.8 They include proton pump inhibitors (PPI) along with other antibiotics. However, high rate of unsuccessful eradication was reported worldwide,9 and the most important causes of treatment failure of H. pylori were found to be clarithromycin resistance and CYP2C19 polymorphism.10 CYP2C19 allelic variations are considered the keystone and the most addressed pharmaco-genetic factors affecting the response to PPI.11\n\nCYP2C19 gene is a member of the CYP450 gene family, which is located at the 10q24.1–10q24.3 locus of chromosome 10.12 It is required in metabolizing of several important therapeutic drugs, including PPI, proguanil, propranolol, imipramine, and numerous other drugs.13 CYPs’ allelic constitution varies according to ethnic and individual variations. This will affect the efficacy of drugs and disease susceptibility.14\n\nAround 25 genetic variants have been detected in CYP2C19, precisely in the exonic region.15 CYP2C19 phenotype is classified into ultra-rapid (UM), rapid (RM), normal (NM), intermediate (IM) and poor metabolizer (PM), depending on the allelic constitution of an individual, and affecting the metabolism of a range of xenobiotics.11\n\nCYP2C19 allelic variation affect H. pylori eradication regimen in many ways. Studies showed that lower eradication rates of H. pylori were observed in NM compared to IM and PM.11 Moreover, lower doses of CYP2C19 substrates will be required for patients with IM and PM phenotypes, due to the risk of overexposure and adverse effects of standard doses.16\n\nCYP2C192 has been identified as the most common allelic variant of CYP2C19, and it’s considered as the main allele contributing to the PM phenotype.12 It is located in exon 5, where a splice mutation generates a cryptic splice site, causing the formation of aberrant mRNA, which is translated as non-functional protein.17 CYP2C19 3, 4, 5, 6, 7, 8 are other non-functioning alleles, but they are less frequent.18\n\nPM phenotype which entails carrying two copies of non-functioning alleles is present in about 2-5% of European and African nations, and 15% of Asians.19 IM phenotype which requires only one copy of a non-functioning allele is present in 25-35% of Europeans and African nations, and 45%-50% of Asians.19\n\nIn Sudan, this allele has not been tackled among H. pylori-infected Sudanese population. Thus, this study aims to detect CYP2C192 allele among this population, owing to its probable effect on H. pylori eradication, and the high frequency of this mutant allele in various ethnicities.\n\n\nMethods\n\nConvenient sampling of patients visiting endoscopy units of two tertiary hospitals of Khartoum city in the period of May and June 2019 was executed. The study included 30 patients who were undergoing oesophago-gastro-duedenoscopy (OGD), indicated for upper gastrointestinal (GI) symptoms, and accepted to participate in the study. Samples were analyzed and stored at the Virology department of Central laboratory, Ministry of Higher Education and Research, Sudan.\n\nA single antral gastric biopsy was collected from each patient after taking informed verbal consent. A questionnaire composed of three sections; details of sample collection, socio-demographic data, and clinical symptoms and signs related to H. pylori history were used to collect patient’s information. Samples were collected in phosphate buffer saline medium for reservation and stored at −20°C in 1.5 ml cryo-tubes.\n\nGenomic extraction and primers and probes sequences\n\nSamples were processed and extracted using QIAamp® DNA Mini Kit (250) 51306 (QIAGEN, Germany). First, samples were cut into small pieces to decrease the lysis time, then they were transferred into 1.5 ml microcentrifuge tubes. 20 μl of proteinase K was added and samples were incubated at 56°C until the tissue was completely lysed. Brief centrifugation was then performed, followed by adding 200 μl of Buffer AL, mixed by pulse-vortexing for 15 seconds, and incubated at 70°C for 10 minutes. This was followed by adding 200 μl of ethanol (96–100%), which was mixed by pulse-vortexing for 15 seconds, and briefly centrifuged. The mixture then was transferred to QIAamp Mini spin column and centrifuged at 6000 × g (8000 rpm) for one minute. Then QIAamp Mini spin column was placed in a clean 2 ml collection tube, and the remaining filtrate was discarded. 500 μl of Buffer AW1 was then added and centrifuged at 6000 × g (8000 rpm) for one minute, and QIAamp Mini spin column was placed in a clean 2 ml collection tube, and the remaining filtrate was discarded. This was repeated using 500 μl of Buffer AW2, but centrifuged at full speed for three minutes. The QIAamp Mini spin column was then placed in a new collection tube and centrifuged at full speed for one minute. Then the spin column was placed in a 1.5 microcentrifuge tube, and 200 μl Buffer AE was added, and incubated at room temperature for one minute, then centrifuged at 6000 × g (8000 rpm) for one minute.\n\nThe quantity and quality of the extracted DNA were examined spectrophotometrically. Primers and probes sequences are mentioned in Table 1.\n\nHelicobacter pylori detection\n\nConventional PCR was performed for the detection of Urease C (UreC) gene in H. pylori.20 The reaction mixture was 20 μl comprising of 4 μl of Solis BioDyne Master Mix (5× FIREPol® Master Mix Ready to Load), 0.5 μl of each forward and reverse primers, 5 μl of template DNA, and 15 μl of H2O.\n\nDNA amplification was carried out as follows:\n\n1. Denaturation at 94°C for five minutes in the first cycle, followed by annealing for 30 seconds at 65°C.\n\n2. Extension for 30 seconds at 72°C. The extension for the last cycle was increased to five minutes to ensure complete extension of the amplified fragments.\n\n3. Denaturation for 30 seconds at 94°C for a total of 35 PCR cycles. Then the PCR products were resolved by 2% agarose gel electrophoresis. The band size was 297 base pairs.\n\nReal-time PCR for CYP2C192 detection\n\nDetection of CYP2C192 was performed among positive H. pylori samples through Real-time PCR (Genebank No. NG_008384.2). The reaction mixture was 10 μl comprising of 2 μl of Solis BioDyne Master Mix (5× HOT FIREPol® Probe Universal qPCR Mix), 0.4 μl of each forward and reverse primers, 0.2 μl of the probe, 3 μl of template DNA, and 4 μl of H2O. Initial activation was at 95oC for five minutes, followed by 15-second denaturation at 95oC, and one minute annealing at 600 for 45 cycles.\n\nInformed consent was taken verbally form each patient, and patient’s parents in case of minors (below 18 years), and detailed full information about the research purposes and procedures was explained. Verbal consent was performed and approved by the ethics committee due to the cultural stigma in the population regarding the signed procedures as contract-like documents. Also, time and setting of taking the consent was limited, as it was executed after the patient is prepared for the endoscopy procedure.\n\nEthical approval was obtained from the ethical committee of Central Laboratories, Ministry of Higher education and Research (Ref. No: CL/85/2019). Consent was also taken from hospitals managers and research committee. Patient information was held with a high level of confidentiality.\n\nThe collected data were entered into Microsoft Excel database and analyzed using Statistical Package for Social Sciences (SPSS) version 23 (SPSS Inc., Chicago, Illinois, USA). Descriptive statistics were reported as frequencies and percentages for categorical variables, and continuous variables were described as mean ± standard deviation (SD). Chi-square test was used to test the significant difference among categorical variables, and p-value at 0.05 was considered significant.\n\n\nResults\n\nA total of 30 patients were enrolled to participate in the study. Patients were selected from two tertiary hospitals of Khartoum city from endoscopy sessions. All patients were performing OGD for various indications, such as alarming upper GI symptoms and refractory GI symptoms. All patients had not taken any antibiotics or PPI in the last month.\n\nThe mean age of patients was 40.7 (±20.2 SE). Ranging from 9 to 89 years old. Gender was almost divided equally. More information is mentioned in Table 2.\n\nPatients were mostly suffering from vomiting as the main symptom, followed by epigastric pain, regurgitation, and heartburn, respectively (Figure 1).\n\nSOB: shortness of breath.\n\nEndoscopic findings were mainly gastritis.32 Duodenal and gastric ulcers were only found in 6.6% and 3.3%, respectively. No patients had a lesion suggestive of malignancy (Figure 2).\n\nOnly seven of the participants had known H. pylori profiles, which five of them were previously found to be positive for the infection. Stool antigen test and serology were the test of choice for their investigations. Only two patients were treated for the infection.\n\nOut of the 30 investigated biopsies, 24 (80%) were found positive for UreC gene of H. pylori. Only those 24 UreC positive samples were explored for CYP2C192 allele. Four samples (16.6%) were found positive for the allelic mutation.\n\nThe four samples were all of female gender, originally from Northern state, and residing in Khartoum. Their mean age was 42.5 (±22 SD), ranging between 21-74 years old. Three of them showed endoscopic appearance of gastritis, and two of them suffered from epigastric pain. Gender was found to be statistically associated with the presence of the allele (p < 0.05) using Chi-square test of significance. Age, origin, and education have shown no association with the presence of the mutant allele.\n\n\nDiscussion\n\nCYP2C19 gene variants have been investigated in different communities including Europeans, Asians, and Africans,21 but it has not been well-studied among the Sudanese population. CYP2C192 allele is considered the most common allele as reported by many studies, and it accounts for poor metabolism of PPI, which helps in the eradication of H. pylori, yet cause many adverse drugs effects22; hence this study aimed at the detection of the CYP2C192 allele among H. pylori-infected patients, in which 30 patients undergoing OGD for various indications were selected from two tertiary hospitals in Khartoum city. Among them, females were 53.3%, and males were 46.7%. Patients were mostly suffering from vomiting as the main symptom, followed by epigastric pain, regurgitation and heartburn, respectively.\n\nOut of the 30 investigated biopsies, 24 (80%) samples were positive for the Urease C gene of H. pylori. Those positive samples were explored for CYP2C192 allele and 4 (16.6%) of the samples were positive for the allelic mutation.\n\nEradication of H. pylori is challenging nowadays due to many reasons including antibiotics resistance, poor compliance, high gastric acidity, and CYP2C19 polymorphism.23 PPI is a main part of the eradication regimen, and the main enzyme involved in PPI metabolism (except Rabeprazole) is CYP2C19; hence PPI effect depends on the genetic variants of CYP2C19.23\n\nSeveral studies revealed significant inter-ethnic differences in CYP2C192 variants. Approximately similar inferences of our results were reported among Beninese population24 and Zimbabwean population,25 but less frequency occurred among Ghanaian population (6%),26 Tunisian population (9%),27 and Egyptians (4%).28 Significantly higher frequencies were reported among Japanese (19-23%), Chinese (15%), and Koreans (13%).12\n\nOur study reveals a significant association between gender and CYP2C192 allele detection (p < 0.05). This is consistent with a study done among the Korean population which showed that females had significantly lower metabolic rates than males (p < 0.0001), but such a gender difference was not reported among Swedes.29 Caucasians also showed no evidence of gender difference.30 In this study, neither age nor ethnicity was found to be associated with allelic variant presence (p > 0.05), but worth noting that all of the positive samples were originally from the Northern state of Sudan, which are considered originating from Arab ancestry. This is inconsistent with a study was done in Sudan in 2018,31 showing a significant association with ethnicity (p = 0.048), but Arabs were also found to be the highest ethnicity to possess the mutant allele.\n\nThe findings of our study have to be seen in the light of some limitations. The sample size was small due to many reasons such as the refusal of some patients to be part of the study and the cost and unavailability of the materials used in the detection of CYP2C192 allele.\n\n\nConclusion\n\nYet, this study illustrates that CYP2C192 is of modest prevalence among H. pylori-infected Sudanese population. Physicians in Sudan should consider this allele as a determinant of suitable drug dosage, which would improve the regimen’s effectiveness. The determination of genotypic and allelic frequencies of CYP2C19 gene among different populations will provide data to be used to personalize treatment according to individual genetic profile, and minimize the possible adverse side effects of CYP2C19 substrates.\n\n\nData availability\n\nNCBI GenBank: H. pylori samples through Real-time PCR, https://www.ncbi.nlm.nih.gov/nuccore/NG_008384.2.\n\nFigshare: Detection of CYP2C192 allele among Helicopacter pylori-infected patients in two tertiary hospitals in Khartoum-Sudan: https://doi.org/10.6084/m9.figshare.14333138.v1.32\n\nThis project contains the following underlying data:\n\n- DATA.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors’ contributions\n\nDrs. Abbas and Malik conceived, designed, acquired, and analyzed the data. Drs. Sulieman and Enan participated in its design and coordination. All authors have read the article critically and approved the final manuscript.", "appendix": "References\n\nOleastro M, Ménard A, Santos A, et al.: Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori. J Clin Microbiol. 2003 Jan 1; 41(1): 397–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuggiero P: Helicobacter Pylori and Inflammation. Curr Pharm Des. 2011; 16(38): 4225–4236. PubMed Abstract \| Publisher Full Text\n\nAhmed HH, Mudawi HMFS: Gastro-oesophageal reflux disease in Sudan: a clinical endoscopic and histopathological study. Trop Gastroenterol. 2004; 25(3): 135–138. PubMed Abstract\n\nSalih KMA, Elfaki OA, Hamid YHM, et al.: Prevalence of Helicobacter Pylori among Sudanese children admitted to a specialized children hospital. Sudan J Paediatr. 2017; PubMed Abstract \| Free Full Text\n\nSchistosomes, liver flukes and Helicobacter pylori. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson JA, Cavallari LH: Pharmacogenetics and Cardiovascular Disease — Implications for Personalized Medicine. Pharmacol Rev. 2013; 65(3): 987–1009. PubMed Abstract \| Publisher Full Text\n\nAbd SA, Rahem A, Elhag WI: Molecular Detection of Helicobacter pylori in Drinking Water in Khartoum State (Sudan). Afr J Med Sci. 2018; 3(5): 3–8.Reference Source\n\nVu N, Nguyen H, Tran N, et al.: CYP2C19 genetic polymorphism in the Vietnamese population. Ann Hum Biol. 2019; 46(6): 491–497. PubMed Abstract \| Publisher Full Text\n\nLähdevirta J: Nephropathia epidemica in Finland. A clinical, histological and epidemiological study. Ann Clin Res. 1971; 3(Suppl. 8): 154 pp. PubMed Abstract Reference Source\n\nLu C-Y, Shih H-Y, Kuo F-C, et al.: CYP2C19 polymorphism influences Helicobacter pylori eradication. World J Gastroenterol. 2016; 20(43): 16029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllabi AC, Gala J, Desager J, et al.: Genetic polymorphisms of CYP2C9 and CYP2C19 in the Beninese and Belgian populations. Br J Clin Pharmacol. 2003; 56: 653–657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDandara C, Masimirembwa CM, Magimba A, et al.: Genetic polymorphism of CYP2D6 and CYP2C19 in east- and southern African populations including psychiatric patients. Eur J Clin Pharmacol. 2001; 57: 11–17. PubMed Abstract \| Publisher Full Text\n\nKudzi W, Dodoo AMJ: Characterisation of CYP2C8, CYP2C9 and CYP2C19 polymorphisms in a Ghanaian population. BMC Med Genet. 2009; 10(1).Reference Source\n\nAbdelhedi R, Bouayed N, Alfadhli S, et al.: Characterization of drug-metabolizing enzymes CYP2C9, CYP2C19 polymorphisms in Tunisian, Kuwaiti and Bahraini populations. J Genet. 2015; 94(4): 765–770. PubMed Abstract \| Publisher Full Text\n\nEllison C, Abou El-Ella S, Tawfik M, et al.: Allele and Genotype Frequencies of CYP2B6 and CYP2C19 Polymorphisms in Egyptian Agricultural Workers. J Toxicol Environ Health. 2012; 75(4): 232–241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamsjö M, Aklillu E, Bohman L, et al.: CYP2C19 activity comparison between Swedes and Koreans: effect of genotype, sex, oral contraceptive use, and smoking. Eur J Clin Pharmacol. 2010; 66(9): 871–877. PubMed Abstract \| Publisher Full Text\n\nHagg S, Spigset ODR: Influence of gender and oral contraceptives on CYP2D6 and CYP2C19 activity in healthy volunteers. Br J Clin Pharmacol. 2001; 51(2): 169–173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhalil SI, Elkhawad AO: A study of CYP2C19 * 2, * 3 and * 7 in different Sudanese ethnic groups and their response to omeprazole based triple therapy in Khartoum. Sudan. Biomed Res. 2018; 29(13): 2743–2765.Reference Source\n\nAbbas A, Malik S, Suleiman B, et al.: Detection of CYP2C19*2 allele among Helicopacter pylori-infected patients in two tertiary hospitals in Khartoum-Sudan [Internet]. figshare . 2021 [cited 2021 Oct 11]. Reference Source" }	[ { "id": "124017", "date": "04 Mar 2022", "name": "Mitsushige Sugimoto", "expertise": [ "Reviewer Expertise Upper GI", "H. pylori", "GERD", "Gastric 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\nGeneral:\nIn this study, the authors evaluated the rate of CYP2C192 allele in H. pylori infected Sudanese population. In this study, authors showed that 16.6% of patients have CYP2C192 allele.\nMajor comments:\nSerious problem of this study is loss of sample power. Therefore, it is unclear whether 16.6% of Sudanese population have CYP2C192 allele.\n\nAuthors should detect CYP2C19 3 and 17 alleles among this population.\n\nPlease use H. pylori in italic.\n\nWhy did authors use UreC gene for detection of H. pylori infection?\n\nIn general, most of patients infected with H. pylori have chronic gastritis. Why 30% in this study?\n\nIn Sudan, it was found in 80% of patients with Barrett's esophagus and gastritis (ref 3) and 56.3% among Sudanese children (ref 4). However, there is no data about Barrett's esophagus in this study.\n\nCYP2C19 gene variants have been investigated in different communities including Europeans, Asians, and Africans, but it has not been well-studied among the Sudanese population. Please compare rate of CYP2C19 2 allele with previous studies in the Sudanese population and this study.\n\n16.6% of patients have CYP2C192 allele. How about rates of poor metabolizer (2/2)?\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": [] }, { "id": "324969", "date": "19 Sep 2024", "name": "Marek Mirowski", "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\nThank you for giving me opportunity to review article entitled “Detection of CYP2C192_ allele among Helicobacter pylori -infected patients in two tertiary hospitals of Khartoum, Sudan, 2019”. After reading, some questions arose:\nHelicobacter pylori should be written in italics. In the methodological part, paragraph Real-Time PCR for CYP2C192 last line on page 4, the annealing temperature should be 600C. I am not convinced about the socio-demographic data included in Table 2, which includes occupations and education - are such data necessary?\n\nThe Authors themselves are aware of the small size of the study group and note this fact in the paragraph before the conclusions. However, this research may document the tendency observed by the Authors. According to the order of citing articles in the text, the article published in References with number 32 should have number 21, and the numbering of articles from number 21 onwards should be changed by one number up.\nIn summary, I think that the article presents new information related to the occurrence of the CYP2C192 allelic variant in H. pylori infected Sudanase population and should be indexed.\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/10-1063
https://f1000research.com/articles/10-1062/v1	18 Oct 21	{ "type": "Study Protocol", "title": "Project PaThWay: protocol for a school-based health promotion intervention for prevention of non-communicable diseases (NCDs) behavioral risk factors", "authors": [ "Shalini Bassi", "Deepika Bahl", "Vinod Gajanan Shah", "Arun Kandasamy", "Melissa Blythe Harrell", "Shreela V Sharma", "Monika Arora", "Deepika Bahl", "Vinod Gajanan Shah", "Arun Kandasamy", "Melissa Blythe Harrell", "Shreela V Sharma", "Monika Arora" ], "abstract": "Background: Prevention of non-communicable diseases (NCDs) and their behavioral risk factors (tobacco use, unhealthy diet, physical inactivity, harmful use of alcohol) among children and adolescents have garnered paramount importance under the Sustainable Development Goals. Methods: Project PaThWay is a school-based, two years, multi-component intervention to prevent key behavioral NCD risk factors among school-going children (classes 6-8th; 10-14 years) from private and public schools across two Indian cities (Pune and Bengaluru). We assessed the baseline knowledge, attitude, and behavior related to NCD risk factors (unhealthy diet, physical inactivity, and tobacco use) of the participating students through a survey. The intervention was developed and designed for implementation by the trained teachers and similar-age peers, as facilitators. The teachers and peer leaders were trained through organising school-level orientation workshops for implementation of intervention activities, after baseline assessment. Year 1 of the intervention focuses on the knowledge and learnings and year 2 on enhancing the life-skills (leadership, communication, refusal, health advocacy, etc.). Regular monitoring visits by the project team to ensure intervention activities are being carried out as planned and providing continuous support. The end line evaluation will be done after the completion of two years’ intervention to evaluate the effectiveness of the Project PaThWay intervention. Outcome measures will include improved knowledge, positive attitude, improve behaviors related to diet, physical activity, and tobacco use, and enhanced skills in handling NCD risk factors. A process evaluation will explore several aspects of Project PaTHWay intervention (fidelity, dosage, reach, adaptations), social validity (acceptability, feasibility, utility). Conclusion: Project PaTHWay, having a multiple-component intervention, may offer the best chance for success, as it addresses multiple risk factors using multi-pronged strategies. The agents of intervention implementation are trained teachers and similar-age student peer leaders (as facilitators), one of the successful and effective approaches in school-based interventions globally and in India.", "keywords": [ "Non-communicable diseases", "behavioral risk factors", "schools", "children" ], "content": "Introduction\n\nNon-communicable diseases (NCDs) are the world’s leading public health challenge in the twenty-first century, contributing to poor health, economic and life loss, impaired quality of life, and poor social development, equally in high and low-resource countries1,2. The burden of NCDs is also on the rise in India accounting for 65 percent of deaths in 2019 across all age groups3. India is estimated to lose approximately US$ 3.55 trillion by 2030 if the four major NCDs (diabetes, cardiovascular disease, chronic respiratory disease, and cancer) are not addressed4.\n\nMost NCDs are attributable to four common modifiable behavioral risk factors: tobacco use, harmful use of alcohol, unhealthy diet, and physical inactivity5. NCDs have been viewed as a problem of adulthood, but children, adolescents, and young people are equally at risk, as these behavioral risk factors are etched during the early years of life i.e. childhood and adolescence. Children and adolescents are aggressively targeted through the marketing of unhealthy products (e.g. tobacco, alcohol, and foods high in fat, sugar, and salt), particularly in low and middle-income countries6. Furthermore, many of them grow up in an environment not conducive for the adoption of healthy lifestyles (e.g. participation in physical activities, availability, and accessibility of healthy foods)7,8. Thus, investing in the health and wellbeing of children and adolescents through primary prevention efforts is an imperative step for the success and sustainability of our future generation.\n\nMore than half of the NCDs burden may be averted by health promotion and prevention initiatives9,10. Health promotion at school has been considered an effective intervention strategy that can go a long way in preventing and controlling communicable and NCDs by formulating school health policies, healthy physical and social environment, skill-based health education, school health services, and parents and community links11. School is an ideal platform for health promotion interventions, creating a favorable environment for inculcating health promoting behaviors12,13 as the information reaches a captive audience, including children, their families, and school staff14. Schools also provide learning through peers and a social environment that sets norms and can have a substantial influence on health-promoting behaviors and educational outcomes15. Students can be effective advocates for creating a healthy school and can become “Agents of Change” for the community.\n\nThe multi-component, school-based health promotion interventions, developed using evidence-informed theories and models of health promotion, when implemented efficiently, resulted in positive outcomes, e.g., reduction in body weight, body mass index16, reduced screen time17, reduction in unhealthy food consumption18, increased refusal skills and self-efficacy19. Taking into account the existing evidence, school-based, health promotion intervention under Project PaTHWay has been developed, to comprehensively address key behavioral NCD risk factors (unhealthy diet, physical inactivity, and tobacco use) among school-going children from two Indian cities (Pune and Bengaluru). The Project PaTHWay’s intervention is well aligned with the newly launched National School Health programme under the Ayushman Bharat Scheme of the Ministry of Health and Ministry and Human Resource Development, Government of India. The National School Health programme addresses the need for a comprehensive preventive and promotive health education programme for children of government and government-aided schools20,21. Given the Indian government’s current priority on improving students' health through educational environment, Project PaTHWay intervention is a timely initiative considering both public and private school students are equally vulnerable for indulging in an unhealthy lifestyle.\n\nProject PaTHWay described in this paper, may offer the best chance for success, as it addresses multiple risk factors using multi-pronged strategies. Project PaTHWay encompasses the development, implementation, and evaluation of a school-based intervention to prevent behavioral risk factors for NCDs (i.e., unhealthy diet, physical inactivity, and tobacco use) among school-going children (classes 6–8th; 10–14 years). The intervention is designed for implementation by the trained teachers and similar-age peers as facilitators. This approach has been adopted as it has been successful in our previous school-based tobacco control programme22, and nutritional interventions13. The specific objectives of Project PaTHWay are to: conduct a baseline evaluation to assess students’ knowledge, attitude, and behaviors (KAB) relevant to key behavioral NCD risk factors; develop a school-based, multi-component intervention for improving knowledge and bringing a positive change in attitude and behaviors of students, teachers, parents, and community by promoting healthy behaviors such as healthy diet, physical activity and non-use of tobacco; implement and evaluate the utility, feasibility, and acceptability of the developed intervention for enhancing the KAB of the target population. This protocol paper describes the conceptual model for PaTHWay; its intervention components (e.g., curricula); and implementation and evaluation plan.\n\n\nProtocol\n\nProject PaTHWay is quasi-experimental with a pre and post-evaluation.\n\nProject PaTHWay targeted school-going students (classes 6–8th; 10–14 years) from twenty schools across Pune and Bengaluru, India. Pune is located in Maharashtra and Bengaluru in Karnataka. These cities are highly urbanised and densely populated23. These states have been selected in Project PathWay as both Maharashtra (71.7%) and Karnataka (72.4%) have the highest NCD burden compared to the national average (64.9%)3. These schools were purposively selected to represent different socio-economic strata (private schools: middle to higher socio-economic status; public schools: lower socio-economic status) within these two cities.\n\nFollowing the selection of the schools, participants (students) were recruited. All students studying in sixth grade (n=1238; Pune: n=806; Bengaluru: n=432) from these twenty schools were eligible and invited to participate in Project PaTHWay. To invite students to participate in Project PaTHWay, letters were sent out to parents or guardians informing them of the programme and their right to opt-out during the programme. The letter also explained that all students would be involved in the programme if they give their assent. In the first year of the programme (2018-19), students of sixth grades were enrolled from these twenty schools and followed up for two years (through 7th and 8th grades, in 2019-2020 and 2020-21, respectively).\n\nProject PaTHWay is a school-based, skill-oriented intervention, developed for teachers and student peer leaders, to deliver theme-based activities for preventing behavioral risk factors (unhealthy diet, physical inactivity, and tobacco use) of NCDs. The intervention package (curricula) was developed based on a review of evidence-based interventions13,24,25, the social-ecological model26, findings from the students’ baseline KAB assessment27, and formative research conducted with various stakeholders, including, teachers (n= 28), students (n=62), officials from state health and education department (n=15) and civil society representatives (n=4). The development of the intervention package was also guided by the members of the Technical Advisory Committee, formulated for this study, consisting of national and international members with domain expertise. The experts having background in epidemiology, public health, and designing and evaluating behavioral change interventions for youth.\n\nThe curricula comprise of twenty activities on four themes (health, diet, physical activity, and tobacco) with an average delivery time of 35–40 minutes for each session, over a two-year implementation period (Table 1). Year 1 of the intervention focuses on the knowledge and learnings and year 2 on enhancing life-skills (leadership, communication, refusal, health advocacy, etc.). A vital component of the intervention is to provide a supportive environment at school (increase availability of healthy food options in and around schools, healthy canteen menus, supportive physical activity environment, a tobacco-free environment within and around schools) and at home (increased availability of healthy food options at home, promoting healthy breakfast, promoting physically active environment at home, nurture smoke/smokeless tobacco-free norms at home) to sustain healthy behavior change (Figure 1: Project PaTHWay’s Intervention Model).\n\nThe intervention activities comprise of teacher-led discussions at the beginning and end of each session, and peer-led small group activities (e.g., ice-breakers, card games, comic book, role plays, worksheets, case studies, preparing healthy birthday menu, letter writing for a newspaper, etc.) as the main focus of each activity. The intervention was designed considering the active engagement of students and peer leaders. As an extension of the classroom activities, a series of school-level activities (mock parliament on health, health-related art competitions, activities advocating for tobacco-free school), and competitions (poster-making, slogan writing, health quiz, etc.) were incorporated as part of the intervention. Theme-based posters, audio-video films were also developed as part of the intervention material to complement the classroom activities and reinforce the message. Postcards and health calendars were also incorporated into the curricula, for sending home through the children to sensitise their parents, to disseminate information and messages on healthy lifestyle practices among families. All the programme resources (manuals, posters, audio-video films) were translated into the regional languages i.e. Marathi (Pune) and Kannada (Bengaluru), to facilitate understanding and uptake of the learning resources.\n\nThe trained teachers and similar-age student peer leaders, as facilitators, are the agents of Project PaThWay intervention implementation. The teachers and peer leaders were trained by the research team having expertise in designing, implementing, and evaluating behavioral change interventions. These teachers and peer leaders were trained through separately organised school-level orientation workshops at the start of the programme. These workshops primarily involved an introduction to the curriculum, programme learning resources, briefing of each session, and its delivery. The teachers and peer leaders were given comprehensive training manuals that provided the background to each session, steps for implementation of intervention activities, all necessary teaching material and resources (card games, comic books, and worksheets). In total, 65 teachers and 74 peer leaders were trained in 20 school-level workshops.\n\nThe intervention sessions are scheduled fortnightly to avoid disruption of their academic calendar. Each participating school shared the roster for the implementation of activities with the study team in advance. Year 1 intervention activities (2019–2020) were implemented physically by the trained teachers, facilitated by student peer leaders, in schools in the presence of the project PaTHWay team. Multiple monitoring visits were made by the project team to ensure intervention activities were being carried out as planned and to provide continuous support. After completion of each intervention activity, project team members recorded the attendance for each activity, the number of peer leaders who facilitated the activity with teachers, and the duration to complete the activity. During our second year of intervention implementation, COVID-19 hit India. Hence, the implementation of the year two intervention was modified to conduct virtually (Zoom/Google Meet/ Teams), due to the closure of schools, following the Government of India’s lockdown protocols to prevent the COVID-19 transmission. The implementation of year 2 intervention through virtual sessions is only happening in private schools, as reaching out to the public schools is challenging due to the digital divide (weak mobile networks, lack of access to mobile phones, and low digital comfort). No intervention sessions were held virtually in public schools considering this challenge.\n\nAll baseline measures were completed before the implementation of the intervention, and follow-up will occur after the completion of intervention implementation. All the measures at baseline were assessed through a questionnaire. This questionnaire was developed based on the socio-ecological model28, adapting measures from reliable instruments that have been validated with adolescents in India29–32 and questionnaires used by the authors of this paper in previously conducted studies in India22,33,34. The questionnaire was pre-tested to assess its validity (face and content) and reliability (internal reliability of attitude construct) before the commencement of data collection. An English version of the questionnaire was administered in private schools, and Kannada and Marathi versions were administered in public schools in Bengaluru and Pune, respectively.\n\nThe key baseline findings suggest that the knowledge about tobacco use being harmful was higher than the knowledge about a healthy diet and the importance of physical activity. Differences in these behaviors by gender, school type showed that both boys and girls of private and public schools are vulnerable for indulging in an unhealthy lifestyle practices27. The same survey will be administered at the end line (after completing the intervention) to evaluate the effectiveness of intervention. Due to the COVID-19 pandemic, the end line survey is planned virtually. The survey will be implemented only with the students who will receive the complete two-year intervention (physically and virtually). A positive increase in the KAB of students relevant to the behavioral risk factors targeted by this programme is expected at the end of two years. We anticipate that there will be changes in:\n\nKnowledge and skills among students\n\n○ Increase percentage with correct knowledge about healthy lifestyles (healthy eating, physical activity, no tobacco use)\n\n○ Improved critical thinking skills, refusal skills, decision-making skills\n\n○ Increase percentage of those who know about the consequences of tobacco use and the benefits of quitting\n\n○ Improved skills to: (a) resist social influences to use tobacco (i.e., develop resistance skills); and (b) promote tobacco-free environments in their homes, and communities (i.e., develop advocacy skills)\n\n○ Increased knowledge about government efforts such as Adolescent Friendly Health Clinics for seeking clinical and counseling services\n\nBehaviors of students\n\n○ Increase percentage of students with daily breakfast consumption\n\n○ Increase in the frequency of fruits and vegetables consumption per day\n\n○ Increase in the frequency of whole-grain consumption per day\n\n○ Increase in the frequency of milk and milk products consumed per day\n\n○ Decrease frequency of intake of sugar-sweetened beverages (SSBs)\n\n○ Decrease consumption of outside unhealthy food\n\n○ Increase in frequency of reading nutritional labels\n\n○ Increase percentage participating in at least 60 minutes of moderate-to-vigorous physical activity per day\n\n○ Decreased percentage engaged in sedentary activities (> 2 hours per day)\n\n○ Reduce experimentation and decrease susceptibility to tobacco use (cigarette, bidis smokeless tobacco, and e-cigarette)\n\nThe in-depth evaluation of project PaThWay’s reach, feasibility, acceptability, dose, and fidelity will be measured through process evaluation, as described in Table 236. The process evaluation data will be collected using: (1) forms to document attendance at the orientation workshops (teachers and peer leaders) and intervention sessions (students participation), (2) post-training satisfaction survey (teachers and peer leaders), (3) feedback forms for each intervention activity (teachers and peer leaders), (4) focus group discussions after completion of the intervention to capture the acceptability of programme components, implementation challenges and facilitators, programme improvement suggestions and any unintended consequences of the programme (students, their parents, and teachers), (5) observations of implementation of intervention activities (study team members), and (6) data collection through the end line survey to measure the participation of students in intervention activities.\n\nAll quantitative and qualitative data will be stored in the lock and key computers at the Public Health Foundation of India. The Data Manager, along with the Research Assistant, will be responsible for cleaning and coding the quantitative data and transcribing qualitative data.\n\nThe analysis will specify random effects at the school (cluster) and class levels. The primary analysis will only use the intervention (dummy coded on class level) and stratification variables (on school level) as independent variables. Sensitivity analyses will be conducted for adding pretests and imputation of missing data. If subgroup analyses (gender and city) are to be conducted these will be defined in the statistical analysis plan as well. The process data will be analysed using a framework36 to measure the extent to which Project PaTHWay intervention was delivered or received as planned, participant interactions and responses to intervention, effectiveness, its suitability and sustainability for translation into routine practice.\n\nPermissions for implementation of the study were obtained from the Maharashtra State Board of Secondary and Higher Education and the Karnataka Secondary Education Examination Board. Approval from authorities at schools, written active informed parental consent and student assent were also sought. Information sheets were sent home from the schools to parents of all eligible students, wherein the Project PaTHWay details were mentioned. The consent stated the permission for data collection, scientific publications, dissemination in conferences by maintaining the confidentiality of the study participants and anonymity of the collected data. The study protocol was reviewed and approved by the Institutional Ethics Committees at Public Health Foundation of India (TRC-IEC-373/18) and NIMHANS (NIMHANS/EC-[BEH.SC.DIV]15th MEETING/2018).\n\nAdverse events, defined as a negative, emotional and behavioral occurrence, or sustained deterioration in a research participant, will be captured as part of the study. Any adverse event reported in the study will be informed to the Institutional Ethics Committee of PHFI by the Principal Investigator (MA) and of NIMHANS by the regional Investigator (AK).\n\nSeveral activities will be undertaken to support the dissemination of study findings:\n\n1) by publishing research articles in peer-reviewed journals for public health researchers and academia to fill the existing research gap,\n\n2) disseminating the findings in national and international conferences, webinars, seminars, and all NCD and educational policy forums (at central and state level).\n\nThe participants will be anonymised in all dissemination activities.\n\nOngoing. The year 2 of intervention implementation is ongoing and endline data is yet to be collected.\n\n\nDiscussion\n\nThis paper described the protocol of a quasi-experimental, school-based, multi-component, health promotion intervention designed for school students in India. There is a need to amplify health promotion education activities and context-specific health intervention materials for students by engaging parents and communities. The COVID-19 pandemic is the most crucial global health calamity of this century as well as the most formidable challenge humanity has faced since World War II37. Children and adolescents are highly vulnerable to the impacts of the pandemic, but these ramifications are manifold and require immediate attention38. The children and adolescents are at more risk due to reduced physical inactivity and unhealthy eating, due to confinement at home. Thus, Project PathWay is a timely initiative for preventing NCD behavioral risk factors by engaging teachers, peer leaders and students. To the best of our knowledge, this is the first of its kind intervention developed for school children that comprehensively capture all the behavioural NCD risk factors. The Project PaThWay intervention developed is suitable to the context as it considers the perception of various stakeholders (findings from formative research), existing knowledge, attitude, and behaviors of the target group (findings from baseline assessment), and review of evidence-based interventions. We envisage that intervention may improve knowledge and bring a positive change in attitude and behaviors of students, teachers, parents, and other adults (at the community level), by promoting healthy behaviors (healthy diet, physical activity, and non-use of tobacco) and reducing the risk of NCDs. Earlier, strategies addressing NCD risk factors emphasised secondary prevention and treatment, but now the shift in approach is primary prevention of NCDs that would slow the exponential growth of NCD burden by impeding the development of risk factors39. The management of NCDs and their risk factors have also gained a prominent position under the Sustainable Development Goals and its targets40. Relying entirely on the treatment option in developing countries like India is not an effective strategy, rather focusing on strengthening protective factors at a young age provides an adequate opportunity for reducing the NCD burden41.\n\nThere are few limitations as the study design is quasi-experimental so the statistical association emerging at the end of the study may not imply causality. The schools were not randomly selected from the population but were representative of the mix of types of schools (Public and Private) in these two cities and hence limits the generalisability of the study findings.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nWorld Health Organisation: Noncommunicable Diseases Progress Monitor 2015. Geneva: 2015; (accessed 27 Jul 2021). 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "98227", "date": "14 Dec 2021", "name": "Neha Bakshi", "expertise": [ "Reviewer Expertise Nutrition", "Liver disease", "Non communicable disease", "clinical nutrition" ], "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 good study, indeed it's the need of the hour to focus intervention strategies on children for a healthy future generation. Schools are an apt place to imbibe discipline related to healthy living and eating among children.\nA little more explanation may be provided by the authors regarding tools/ techniques that will be used to impart knowledge on reading food labels and advertising among the study participants.\nAuthors can explain with already existing data on-screen time in support of this sentence\"There is an increased dependency on digital media which has increased the screen time among children\".\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-1062
https://f1000research.com/articles/10-1060/v1	18 Oct 21	{ "type": "Opinion Article", "title": "NMDA receptor modulation and severe acute respiratory syndrome treatment", "authors": [ "Blaise M. Costa" ], "abstract": "N-Methyl-D-aspartate (NMDA) subtype of glutamate receptors is expressed in the human lungs and central nervous system. NMDA receptor potentiation could increase calcium ion influx and promote downstream signaling mechanisms associated with cellular contractions that are disrupted in severe acute respiratory syndrome. Pharmacological effects generated by triggering glutamate receptor function in the brain, coupled with concurrent stimulation of the respiratory tract, may produce a synergetic effect, improving the airway smooth muscle function. A novel multipronged intervention to simultaneously potentiate NMDA receptors expressed both in the central nervous system and airway muscles would be helpful for the treatment of severe acute respiratory syndrome that deteriorates peripheral and central nervous system function before causing death in humans.", "keywords": [ "NMDA", "severe respiratory syndrome (SARS)", "GluN2D", "Potentiator" ], "content": "Expression of NMDA receptors in the human lungs\n\nThe Human Proteome Project identified abundant expression of NMDA receptor subunits in various organs outside the CNS, including lungs, esophagus, and T-helper cells.1 This finding corroborates a large number of previous reports on the extraneuronal expression of NMDA receptors in various animals.1,4–12 NMDA, when applied to perfused tracheal segments of guinea pigs, increased resting muscle tone and enhanced the contractile response to acetylcholine.13,14 In whole guinea pig lungs, when administered through the trachea, NMDA increased airway perfusion pressure and this increase was abolished by NMDA receptor channel blocker MK-801(4). Following systemic MK-801 administration, adult cats developed apneusis.15,16 In addition, recent studies reveal the critical role of endogenous glutamate in NMDA receptor function during acute lung injury and airway inflammation.4,14,17,18 An NMDA receptor blocker could impair fetal rat lung development.19,20 NMDA receptor activation mediates lung fibroblast proliferation and differentiation in hyperoxia-induced chronic lung disease in newborn rats.20 Acute lung injury, acute respiratory distress syndrome and severe acute respiratory syndrome (SARS) all imply the occurrence of lung injury resulting from direct or indirect respiratory insult.21\n\nThe expression of GluN1/2A and 1/2B subtypes were not confirmed in the lung cells, whereas the GluN1/2C subtype was found to be expressed in peripheral and middle-lobe lung samples.4 The GluN1/2D subunit was predominantly expressed in the peripheral, gas-exchange zone of the lungs and in alveolar macrophages; this expression was upregulated in lungs treated with NMDA.4 GluN1 and all four GluN2 subunits were also expressed in the human pulmonary artery smooth muscle cells.22 Overall, these findings indicate that NMDA receptors could control the respiratory tract function in vertebrate animals.23\n\n\nKinetics of NMDA receptor subtypes\n\nGlutamate, with concurrent binding of the co-agonist D-serine or glycine, activates NMDA receptors that non-selectively conduct ions across the cells at depolarizing membrane potential which unbinds the otherwise blocking Mg2+ ions. NMDA receptor mediated transport of calcium and sodium ions into the cytoplasm is essential for excitatory cellular events that result in human airway smooth muscle contraction.24 Each non-GluN1 subunit confers distinct spatiotemporal expression and biophysical properties that result in varying agonist affinity, magnesium sensitivity, ion conductance, activation kinetics, open probability, mean open time, cellular localization, and downstream signaling mechanisms.2 In general, diheteromeric NMDA receptors (GluN1/2) exhibit deactivation time constants that span about a 50-fold range, with the following order (from fastest to slowest): NR2A < 2C < 2B << 2D.25 The GluN1/2A subunit-containing NMDA receptor deactivation time constant is about ~50 ms, GluN1/2B ~400 ms, GluN1/2C ~290 ms and GluN1/2D is >1second.25 Since GluN1/2C&D subunits of NMDA receptors are predominantly expressed in the lung epithelial cells and macrophages, and are the slowest channel (among other glutamate receptors) to deactivate, these receptors can conduct a large amount of calcium and sodium ions into the cells and trigger cellular contractions.25–27\n\n\nAntiviral properties of drugs acting on NMDA receptors\n\nOne of the clinically used antiviral agents, amantadine, is a potent NMDA receptor antagonist.28 This drug is also an FDA-approved drug of choice (brand name, Gocovri®) for the treatment of dyskinesia in patients with Parkinson’s disease. An analog of amantadine, memantine (brand name, Namenda®), is one of two FDA-approved clinically used drugs for the treatment of moderate to severe symptoms of Alzheimer’s disease. Since both amantadine and memantine are chemically similar adamantane derivatives, memantine also exerts antiviral effects as previously reported.29 Presumably, these effects could be a collective outcome of activities on host cell glutamate receptors and viral proteins like M2-viroporin.30\n\n\nNovel NMDA receptor modulators\n\nIn recent years, a variety of NMDA receptor modulators have been identified, and they exhibit a broad spectrum of subunit selectivity and mechanisms of action.31–35 These compounds have been largely studied for their activities in neuronal NMDA receptor populations, with the aim of developing treatments for neurological and psychiatric disorders; however, these compounds and their analogs might have therapeutic potential for non-CNS disorders, but this has not yet been explored.\n\nThrough our ongoing NMDA receptor drug discovery project, we have identified a compound from PubChem (CID# 3794169), coded as CNS4, and studied its activity on NMDA receptors.36 CNS4 selectively potentiates GluN1/2D receptor currents up to 8-fold, when activated by 100 μM glycine and 0.3μM glutamate, and produces minimal effects on GluN1/2A or 1/2B receptors.36 CNS4 has a variety of other biological activities as reported by the National Center for Advancing Translational Sciences (NCATS); for example: an inconclusive anti-viral activity against influenza-A virus non-structural protein-1 (PubChem AID# 2326); anti-malarial, as an inhibitor of apical membrane antigen-1 of Plasmodium falciparum (AID# 720542); antiprotozoal, as an inhibitor of fructose 1,6- bisphosphate aldolase from Giardia Lamblia (AID# lamblia (2451); and inhibition of nuclear receptor ROR-gamma in the immune cells (AID# 2551 & 2546). The chemical structure of CNS4 and more details on its activities are available at PubChem.\n\n\nA multipronged approach to treat SARS\n\nAn NMDA receptor modulator with antiviral properties could serve as a novel treatment strategy for SARS. Potentiating NMDA receptor activity in the lung epithelial cells will increase calcium ion influx and promote downstream signaling mechanisms associated with cellular contractions that are possibly impaired during SARS. Pharmacological effects generated by triggering neuronal NMDA receptor function, coupled with concurrent potentiation of NMDA receptors expressed in the respiratory tract, could synergistically improve airway smooth muscle contractions. Further, a variety of neurological symptoms were clinically diagnosed in hospitalized COVID-19 patients.37,38 Neuropathogenesis could occur due to the neurologic injury resulting from systemic dysfunction,39 dysregulated renin-angiotensin aldosterone system,40 proinflammatory reactions,41,42 para-infectious and post-infectious triggers,43 and direct viral invasion of the nervous system.44–46 As a well characterized neuropsychiatric drug target,47 with the potential to improve lung function, NMDA receptors could be an ideal focal point for future pharmacological interventions of COVID-19. Clinical conditions involving hypoxia increase blood glutamate concentration by promoting transaminase activity that generates α-keto acids.48–50 Further, neuronal glutamate excitotoxicity induces paralysis in mice after infection by a human coronavirus.51 The connection between disruption in glutamate homeostasis and pathogenesis of various neurological and psychiatric disorders has been extensively studied in the past three decades. Therefore, optimizing glutamatergic signal transmission through neuronal and non-neuronal cell types, that express the major glutamate receptor subtypes like NMDA receptor, could be an appropriate strategy to reduce the extent of lung damage caused by SARS. In this perspective, compounds that modulate NMDA receptors based on glutamate concentration would be an ideal starting point for the development of a treatment approach involving modulation of glutamate signaling at both nerve cells and non-neuronal cells. CNS4 and other recently identified novel glutamate concentration biased NMDA receptor modulators34,35 could serve as lead candidates in the development of clinically useful compounds to treat COVID-19 or other SARS caused by various pathological conditions. Future studies should be carried out in this direction to test this hypothesis.\n\n\nConclusion\n\nAn increasing body of evidence suggests the expression of functional NMDA receptors in the lungs and their critical role in glutamate induced acute lung injury and acute respiratory distress syndrome.4,13,14,17–20 Despite its direct role in lung injury, little effort has been taken to develop NMDA receptor based therapeutic strategies for the treatment of lung diseases. With the revolution in glutamate receptor pharmacology in the past decade that yielded a variety of chemical tools to modulate NMDA receptors,31–36 and a COVID-19 pandemic that kills humans by primarily affecting lung function, this could be a suitable time to start working on a novel drug target for SARS treatment.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Acknowledgements\n\nDr. Katherine Jamison is acknowledged for reading this article.\n\n\nReferences\n\nKim MS, Pinto SM, Getnet D, et al.: A draft map of the human proteome. Nature. 2014; 509(7502): 575–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTraynelis SF, Wollmuth LP, McBain CJ, et al.: Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 2010; 62(3): 405–96. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerszyk RE, Swanger SA, Shelley C, et al.: Biased modulators of NMDA receptors control channel opening and ion selectivity. Nat. Chem. Biol. 2020; 16(2): 188–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCosta BM, Kwapisz LC, Mehrkens B, et al.: A glutamate concentration-biased allosteric modulator potentiates NMDA-induced ion influx in neurons. Pharmacol. Res. Perspect. 2021; 9(5): e00859. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPezzini A, Padovani A: Lifting the mask on neurological manifestations of COVID-19. Nat. Rev. Neurol. 2020; 16(11): 636–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMao L, Jin H, Wang M, et al.: Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China. JAMA Neurol. 2020; 77(6): 683–90. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "142691", "date": "25 Jul 2022", "name": "James Pearle", "expertise": [ "Reviewer Expertise Pulmonologist" ], "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 severe acute respiratory distress syndrome, cellular contractions may be disrupted.\n\nBy triggering glutamate receptor function in the brain and with stimulation of the respiratory tract, a synergetic effect may improve airways smooth muscle function and neurologic function.\nStimulation of NMDA receptors in the brain and in the airway muscles could be beneficial in the treatment of severe respiratory distress syndrome and associated neurologic disease.\nThe authors excellently outline how an NMDA receptor modulator with antiviral properties could be a novel treatment strategy for SARS.\nThis avenue of treatment has the potential to prevent or treat the neurological dysfunction often associated with SARS.\nAt the same time, NMDA receptor modulation may improve pulmonary status by improving smooth muscle airways function, often disrupted in this syndrome.\nThe multifaceted potential benefits of stimulating glutamate receptors could involve both the lungs and the nervous system.\nThe authors have clearly and concisely outlined the rationale and hypothesis for the use of NMDA modulators in the treatments of SARS and associated neurologic dysfunction.\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": "157840", "date": "12 Dec 2022", "name": "Jaewon Ko", "expertise": [ "Reviewer Expertise Synaptic neuroscience." ], "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 short article states that modulation of an NMDA receptor (NMDAR) function with anti-viral properties could be beneficial for treating disorders that involve NMDAR dysfunctions. The author first summarized known facts for distinct NMDAR subunits expressed in lung cells (i.e., expression levels and kinetics). The author then introduced amantadine (a potent NMDAR antagonist) and its analog (memantine) that have been clinically used for treatment of dyskinesia patients. The author also detailed a novel NMDAR modulator (i.e., CNS4) that has a possibly therapeutic potential for non-CNS disorders and SARS.\nI believe that the author has done a wonderful job in concisely providing a logical opinion: a newly developed NMDAR modulator could be clinically promising for treating various non-neurological disorders that are linked to NMDAR dysfunctions.\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/10-1060
https://f1000research.com/articles/10-1059/v1	18 Oct 21	{ "type": "Research Article", "title": "Distribution of imposter syndrome among medical students of Bangladesh: a cross-sectional study", "authors": [ "Md. Shahjalal", "Md Nafiul Alam Khan", "Faroque Md Mohsin", "Shahariar Rokon", "Riaz Rahman", "Mohammad Morshad Alam", "Rashidul Alam Mahumud", "Md Nafiul Alam Khan", "Faroque Md Mohsin", "Shahariar Rokon", "Riaz Rahman", "Mohammad Morshad Alam", "Rashidul Alam Mahumud" ], "abstract": "Background: Imposter syndrome (IS), suffering from self-doubt and fear, despite clear accomplishment and competencies, is often detected in medical students and adversely affects the well-being of the student. This study aimed at assessing the prevalence of IS among public and private medical students in Bangladesh.\nMethods: This study was a cross-sectional design among medical students in Bangladesh. Data were collected between February to July 2020 through snowball sampling technique across medical colleges in Bangladesh. Relative risk ratios (RRRs) with 95% CI were calculated to investigate the magnitude of association between imposter syndrome exposure and explanatory variables.\nResults: A total of 500 students participated in this study with approximately 47% and 53% of students studying at public and private medical colleges, respectively. Around 32% of medical students were exposed to IS (47% of public and 53% of private medical college students). Medical students were the most significantly associated with IS for third (RR: 1.487, CI: 1.068-2.071) and fourth-year students (RR: 1.493, CI: 1.043-2.136). Overall, we found that respondents aged 22 to 25 were 3.6% (RR:1.036, CI:0.801-1.339) more likely to be suffering from IS than their younger counterparts.\nConclusion: Third and fourth-year medical students, in particular, require more care than others; teachers and authorities should provide them with proper guidance and care, encourage them, and thus grow their self-reliance and confidence.", "keywords": [ "Imposter Syndrome", "Bangladesh", "Medical College", "Young Imposter Scale", "Self-doubt." ], "content": "Introduction\n\nImposter syndrome (IS) is characterized by a feeling of not belonging, out-of-placeness, and the belief that one’s perceived competence and success by others is undeserved. Usually, this is considered an individual problem that should be addressed by keeping a record of accomplishments as a reminder of progress (Breeze, 2018; Mak et al., 2019; Hendriksen, 2015). In late 1978, clinical psychologists Pauline Clance and Suzanne Imes first cited the IS which arbitrated the relationship between perfectionism and anxiety and partially influences perfectionism and depression (Clance & Imes, 1978)\n\nA more recent systemic review conducted in the U.S. in 2020 found that the prevalence of IS ranged from 9% to 82% in the general population (Bravata et al., 2020), while another review conducted in the U.S. in 2020 found that it ranged from 22% to 60% among physicians and from 33% to 40% among trainee physicians (Gottlieb et al., 2020). In the United States, recent studies on IS have found that 57% of pharmacy students (Sullivan & Ryba, 2020) and 15% of medical students have IS (Holliday et al., 2020). Indeed, IS is becoming a growing public health issue both globally and regionally. For instance, the prevalence of IS among medical students has been found to be 30% in the US (Villwock et al., 2016), 45.7% in Malaysia (Ikbaal & Salim Musa, 2018), and 47% in Pakistan (Qureshi et al., 2017).\n\nSeveral studies suggest that IS has a strong correlation with general psychological distress (Bravata et al., 2020; Sullivan & Ryba, 2020; Wang et al., 2019) and demographic and personal characteristics, including age (Bravata et al., 2020; Holliday et al., 2020; Sullivan & Ryba, 2020), gender (Gottlieb et al., 2020; Holliday et al., 2020; Rosenstein et al., 2020), and academic year (Gottlieb et al., 2020; Ikbaal & Salim Musa, 2018; Qureshi et al., 2017).\n\nThe evidence indicates that medical students experienced moderated-to-strong IS (Levant et al., 2020), which has psychological and academic consequences (Ikbaal & Salim Musa, 2018; Qureshi et al., 2017; Villwock et al., 2016). The move to the clinical phases of study can be particularly challenging and cause students to suffer from low confidence during this time (Burstein et al., 1980; Niemi & Vainiomäki, 2006). In addition, many studies confirmed that IS often negatively affects medical students’ physical and academic well-being (Ikbaal & Salim Musa, 2018; Levant et al., 2020; Villwock et al., 2016). Thus, they may miss out on opportunities because they do not recognize their potential.\n\nThere have been many studies on IS among medical students conducted across the world. However, no study has yet been published on IS among medical students in Bangladesh. Therefore, this study aims to draw attention to the growing need for more mental health research within Bangladesh. The current study focused on evaluating the prevalence of IS, exploring the frequency distribution of IS in different institutions, and assessing the factors contributing to IS among public and private medical college students. The survey provides insight into areas of commonality and differences between IS in students.\n\n\nMethods\n\nEthical approval was obtained from the Institutional Review Board (IRB) at North South University, Dhaka, Bangladesh. The study objectives were explained to each participant and confidentiality, and anonymity was assured, and written informed consent was obtained from respondents before proceeding.\n\nThis study was conducted using a cross-sectional survey assessing the presence of IS among medical college students in Dhaka city. Data were collected from medical colleges (public and private) in Dhaka between February 2020 and July 2020. Our study did not include any students studying in medical colleges located outside of Dhaka city, Bangladesh. We collected more samples than the recommended minimum sample size for better accuracy and results. So, throughout our survey, we collected a sample size of 500 students selected from public and private medical colleges in Dhaka. Our survey was conducted using a snowball sampling method (a non-probability sampling method) to achieve the desired sample size. The medical colleges chosen for the study are attended by students from across the country, allowing this study to provide an understanding of the prevalence of IS among medical students across Bangladesh. Participants of the study were the students of both public and private medical colleges ranging from 1st to 5th year of study. Initially, we identified some potential IS cases among our respondents and asked them to recommend other people for the study. After this, we contacted them via email or mobile phone and collected data through face-to-face interviews. The eight items from the Young Imposter Syndrome (YIS) scale (Villwock et al., 2016) were used to assess whether the participant had IS or not. The scale was in the form of eight questions, and a student was considered to have IS if they answered 5 or more questions as “Yes”.\n\nPublic and private medical colleges represent the whole community of medical students, and our study aimed to find out the underlying risk of having IS among medical college students. This condition is less discussed in Bangladesh, so we aimed to reveal such mental conditions and associated risks within the study group.\n\n\nMeasures\n\nIn our study, we used IS as the dependent variable. The dependent variable contains two categories, “Yes” (has IS) and “No” (does not have IS). We defined them as follows:\n\nIn our survey, we used Young Imposter Syndrome (YIS) scale to assess the presence of IS. The scale was in the form of eight questions. A student was considered to have IS if they answered 5 or more questions as “Yes” in the YIS scale, otherwise considered “No”.\n\nParticipants’ sociodemographic factors and some academic data were considered as independent variables. The sociodemographic variables included gender (male, female), age group (18-21, 22-25 years), smoking status (current, past, never), and living with family (yes, no). Body Mass Index (BMI) was categorized using the WHO BMI standard scale (Nuttall, 2015). The economic condition was determined by the monthly family income (MFI) of the respondents. The MFI was categorized as ≤20,000 BDT (approximately US$237), 21,000-30,000 BDT, 31,000-40,000 BDT, ≥41,000 BDT. This MFI interval was a self-imposed category. The academic year was categorized as first, second, third, fourth, or fifth, and the reasons for studying in medical college were classified as own preference, family preference, failing to qualify for another department or better job opportunities. Each response was dichotomized (yes, no). All relevant information was directly collected from each participant.\n\nIn our study, we collected the desired data through snowball sampling. This sampling method was used because of the lack of a sampling frame and the unavailability of targeted samples. Using the (Israel, 1992) formula (i), we obtained the minimum sample size required for the study.\n\nWhere, n0 is the initial sample size, z is the standard normal variate, and p is the (estimated) proportion of the population with the attribute in the question, i.e., p + q = 1, d is the allowed maximum error in estimating a population proportion. Considering the degree of accuracy, d = 0.05 and p as the approximate proportion of YIS (p = 0.47), taken from a related study conducted in Pakistan (Qureshi et al., 2017). So, the calculated sample size was 382; however, we targeted more subjects to be included than the required sample size. Finally, we collected complete information from 500 unique samples for our study, and incomplete questionnaires were not considered for analysis.\n\nData were collected through a face-to-face interview. Participants were reassured that all the information collected would be kept strictly confidential and would not be used for anything other than research purposes. Written consent was taken for the study from the participants. After this, we briefly introduced the student to our research and presented them with the questionnaire. The response was scribed immediately. The collected data was compiled for further processing and analysis. After cleaning the raw data, we had 500 complete observations for our study.\n\nStatistical analysis was performed with Statistical Package for Social Science (SPSS) version 26 (IBM Corporation, Armonk, NY, USA). And Stata 16.0 for windows (Stata Corp LP, 4905 Lakeway Drive College Station, USA). Throughout our study, we collected only the completed questionnaire and discarded any incomplete questionnaire. To observe the background of the study, we used frequency distribution which is presented in Table 3. For the bivariate study, we used a relative risk ratio (RRR) with a 95% confidence interval to determine the risk of having IS, as presented in Table 4.\n\n\nResults\n\nWe selected 500 students among the medical colleges, and the questionnaires were collected from each individual during face-to-face meetings. Therefore, the response rate was 100%. Out of the 500 participants, 233 (46.6%) were public, and 267 (53.4%) were private medical college students. Detailed information on how respondents addressed all eight questions (Villwock et al., 2016) is displayed in Table 1.\n\nThe students who answered “Yes” for five or more out of eight questions were considered positive for IS. In total, 61 (32.2%) out of 500 students answered “Yes” for five or more questions, and 339 (67.8%) answered “No” according to the definition of the YIS scale. A breakdown of YIS scores in relation to the institution is given in Table 2.\n\nFor univariate analysis, we used frequency distributions and percentages to compare the variables within the study, as presented in Table 3.\n\nIn our study, 161 (32.2%) out of 500 observations were suffering from YIS, while the rest of the respondents (339; 67.8%) were students without YIS. This indicates that around one-third of our total respondents were experiencing symptoms of YIS. Approximately 53% of our respondents were from private, and 47% were from public medical colleges. Most of the respondents were young; 58% were between 18 and 21 years old, and 42% were between 22 and 25 years old. Overall, 269 (53.8%) out of 500 respondents were male, and 231 (46.2%) were female. The majority of participants (191; 38.2%) of our study had a healthy BMI (between 18.5 and 24.9). Most of our respondents (208; 41.6%) replied that they study medical science because of their own preference, and 197 (39.4%) study because their family wanted them to. Most of our respondents (361; 72.2%), were non-smokers, and the rest were smokers or had smoked in the past. Most of our respondents (306; 61.2%) lived without family, and the rest lived with the family.\n\nIn order to find out the risk of having YIS, we calculated relative risk along with 95% confidence interval, and this is presented in Table 4.\n\nNote: RRR=relative risk ratio; CI= confidence interval; 1: reference; BDT: Bangladesh Taka.\n\nIn our study, respondents who study in public medical colleges were 1.21 times or 21% (RR: 1.219, CI: 0.946-1.572) more at risk of having IS than private medical colleges. Respondents aged 22 to 25 years are 3.6% (RR: 1.036, CI: 0.801-1.339) more at risk of having IS than their younger counterparts. Our study found that female respondents were 4.1% (RR: 1.041, CI: 0.807-1.343) more at risk of having IS than male respondents. Surprisingly, we found that students who had a lower than healthy BMI score (between 18.5 and 24.9) were 9.6% (RR: 1.096, CI: 0.649-1.849) more at risk of having IS. We also found that students whose BMI was higher than the healthy range were at lower risk (RR: 0.986, CI: 0.739-1.315 and RR: 0.666, CI:0.459-0.967) of having IS. Students in their second year had a lower risk of having IS (RR: 0.711, CI: 0.352-1.435) than those in their first year. However, third, fourth, and fifth-year students have 48% (RR: 1.487, CI: 1.068-2.071), 49% (RR: 1.493, CI: 1.043-2.136), and 43% (RR: 1.434, CI: 0.999-2.058) greater risk of having IS than first-year students. Respondents who first choice was a subject other than medicine and had failed to qualify, had a 13% greater risk (RR: 1.137, CI: 0.755-1.714), of having IS than those who took medical science as their desired field of study. Students whose MFI was between 31000-40000 BDT had a 10% (RR: 0.902, CI: 0.486-1.675) reduction in risk of IS compared to those who had a MFI between 21000-30000 BDT. Furthermore, students whose MFI was ≥41000 BDT had a 23% (RR: 0.777, CI: 0.425-1.420) reduction in risk compared to those who had a MFI ranging from 21000-30000 BDT. In our study, we also found that students who smoked were 13% (RR: 1.133, CI: 0.816-1.575) more susceptible to IS than those who had never smoked, but past smokers had relatively low risk (RR: 0.982, CI: 0.650-1.484) of having IS, compared to those who have never smoked. Finally, respondents who lived with family were 4.1% (RR: 1.041, CI: 0.803-1.349) more at risk of having IS than those who didn’t live with the family.\n\n\nDiscussion\n\nIS affects medical students from all different medical colleges and circumstances. In this study, we found nearly one-third (32.2%) of the respondents have IS, 85 out of 184 were male, and 76 out of 155 were female students. Whereas, in a study conducted by Qureshi et al., in 2017 in Pakistan, almost half (47.5%) of the participants had IS, among 143 students where 23 were males, and 45 were females (Qureshi et al., 2017). Generally, female students were more likely to had IS. In some research, women were twice as likely to suffer from it than men (Farzana et al., 2017; Maqsood et al., 2018; Qureshi et al., 2017). Our observations were also similar, and the female respondents had more IS (32.9%) than males (31.6%). The leading cause was that women tended to address IS by confronting their concerns and working harder to address the concerns, whereas men tended to avoid areas where they felt uneasy (Villwock et al., 2016).\n\nAccording to our study, fourth-year students were more likely to suffer from IS, with 39.7% meeting the criteria, the largest among all academic years. Most likely, fourth-year students faced more pressure than others because of the increased academic curriculum, worries about study and performance, and concerns about the future (Farzana et al., 2017; Villwock et al., 2016). A prior study of IS conducted in 2017 showed that among Pakistani medical college students, 23.1% of respondents had an IS score of 1 (IS score <5 means does not possess IS, ≥5 means they might possess IS), with males making up 27.1%, and females 20.2%. In the same study, 4.9% of all the students had an IS score of 4, among which 5.1% were male, and 4.8% were female (Qureshi et al., 2017). Similarly, in our study, the most frequented IS score was 4, with 30.8% of respondents scoring 4. Of those participants, 27.9% were public, and 33.3% were private medical college students. Only 0.8% of students scored 8 (meaning they might possess IS), with 0.9% from public and 0.7% from private medical schools.\n\nAccording to Eva et al., private medical college students were more relaxed and confident than public medical college students (Eva et al., 2015). However, our study suggested that both public and private medical college students lacked confidence and suffered from IS. Indeed, in our study, 60.7% of private and 52.4% of public medical college students answered “yes” to the question “Do you live in fear of being found out, discovered, or un-masked?”.\n\nAn earlier study conducted in the U.S. in 2016 showed that almost 25% of male and nearly 50% of female students analysed had IS and that IS was significantly associated with burnout or dropout (Villwock et al., 2016). Every year, many students drop out or burn out and suffer from anxiety and other psychological problems (Eva et al., 2015).\n\nAccording to the findings of Maqsood et al. (2018) the fourth-year medical students suffered more severely with IS than others, reflecting our own findings. They also found that 27.41% of the third year and 46.31% of fourth-year medical students had moderate IS and that 64.51% of the third year and 47.36% of fourth-year students had severe IS (Maqsood et al., 2018). In 2003, a survey of the mental health of medical students in Iran examined the age and health status of the respondents and using the 12-item General Health Questionnaire (GHQ-12) (Montazeri et al., 2003), 30.6% of first-year and 30.6% of fourth-year medical students were suffering psychological distress (Hendriksen, 2015).\n\nA study conducted in 2012 among medical college students in Malaysia observed that 14.8% of total students were underweight (12.2% males and 17.0% females), 14.8% were overweight (males 13.7% and females 15.7%), 15.9% were pre-obese (BMI>30kg/m^2) (18.3% of males and 13.8% of females), and 5.2% were found to be obese (males 9.2% and 1.9% females) (Hameed et al., 2019). Our study has identified similar findings; 5.2% of total respondents were underweight (BMI<18.5kg/m2), 38.2% were normal weight (BMI=18.5−24.9kg/m2), overweight and pre-obese made up 31.8% (BMI=25−29.9kg/m2) and 24.8% were found to be obese (BMI=30&>30kg/m2). We also noticed that 38.5% of total underweight students had a positive IS score (IS score ≥ 5), the largest among the different BMI classes. In contrast, only 23.4% of total obese students had IS.\n\nThe generalisability of our findings on IS among medical college students studying in Dhaka, to other city students is unknown. Studying only medical college students in Dhaka, may have resulted in an underestimation of the coverage of IS. Similarly, investigating the underlying causes was beyond the scope of our study. Our study presents the risk of IS at the time of our assessment, however, the situation in Bangladesh has likely further deteriorated because of the COVID-19 pandemic. Given that the COVID-19 situation has forced Bangladesh’s education system to close for nearly two years, there is a greater need that ever to conduct surveys to monitor mental health issues such as IS amongst students. Our presented data and our risk analysis showed higher BMI, and higher MFI coincided with lower risk of IS. So, ensuring appropriate diet and proper socio-economic improvement, may help reduce the risk of IS and other mental health problems among medical students. The hope is that this study will encourage more research on this topic and a greater understanding of the impact of IS on various aspects of the social, academic, and daily life of those who suffer from it.\n\n\nStrength and limitations\n\nThis study advances knowledge in several ways. This research is among the first to examine the risk of IS among medical college students in Bangladesh, which is not a commonly discussed syndrome within this country. This research also addresses further need for inquiries into the psychological predictors of IS, helping to understand the reasons and the causes behind IS. IS is an increasingly prevalent psychological syndrome. However, it’s possible, when looking for participants for a mental health study, potential volunteers might be hesitant to participate due to the personal nature of the topic. As a result, snowball sampling was utilized in this study to reach the population, because it is difficult to collect samples using other sampling methods.\n\nWhile this study makes several contributions to IS research, there are some limitations. This study used the snowball sampling method, which is a non-probability type sampling method. The researchers had little control over the sampling approach. We only interviewed those who were recommended by the previous respondents. Therefore, the sampling approach was limited and subjective. There might be some sampling bias present in this study because of the nature of the sampling method. Because of the sampling bias, our samples might not represent the true distribution of the population. Similarly, our study only sampled Dhaka city; it would’ve been better if we’d studied the entire country. Consequently, we would have been able to get a better picture of the distribution of IS and the underlying factors that influence it.\n\n\nConclusion\n\nOur analysis revealed a serious public health issue for medical college students in Bangladesh. As a result, students may suffer from depression and, in some cases, even commit suicide (Qureshi et al., 2017). Public and private medical institutions should take that matter seriously and establish various programs, organizations, counseling teams, and other activities to help identify students suffering from IS and support them. It is evident that students, especially those in their third and fourth year of medical college, need more care than others to ensure they stay focused on their goals. Faculty and fellow students should guide, mentor, and encourage IS suffers to produce self-confident and self-sufficient students.\n\n\nData availability\n\nMendeley: Shahjalal, Md et al., 2021 Imposter Syndrome Data Set. http://doi.org/10.17632/znb42mw6fy.3 (Shahjalal, Md et al., 2021a)\n\nThis project contains the following underlying data:\n\n- Date file 1. (Complete survey responses, XLSX format)\n\nMendeley: Shahjalal, Md et al., 2021 Distribution of imposter syndrome (IS) among medical students of Bangladesh: A cross-sectional study: Copy of questionnaire.\n\nhttp://doi.org/10.17632/znb42mw6fy.3 (Shahjalal, Md et al., 2021b)\n\nThis project contains the following extended data:\n\n- Copy of questionnaire used in study\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBravata DM, Watts SA, Keefer AL, et al.: Prevalence, Predictors, and Treatment of Impostor Syndrome: a Systematic Review. J Gen Intern Med. 2020; 35(4): 1252–1275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreeze M: Imposter Syndrome as a Public Feeling. Palgrave Studies in Gender and Education. 2018; 191–219. Publisher Full Text\n\nBurstein AG, Loucks S, Kobos J, et al.: A longitudinal study of personality characteristics of medical students. J Med Educ. 1980; 55(9): 786–787. PubMed Abstract \| Publisher Full Text\n\nClance PR, Imes SA: The imposter phenomenon in high achieving women: Dynamics and therapeutic intervention. Psychotherapy: Theory Res Practice. 1978; 15(3): 241–247. Publisher Full Text\n\nEva EO, Islam MZ, Mosaddek ASM, et al.: Prevalence of stress among medical students: A comparative study between public and private medical schools in Bangladesh. BMC Res Notes. 2015; 8(1): 327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarzana N, Shahjahan M, Nayan SK: Stress among Female Medical Students in selected Medical Colleges of Dhaka City in Bangladesh. Northern Int Med College J. 2017; 8(1): 181–184. Publisher Full Text\n\nGottlieb M, Chung A, Battaglioli N, et al.: Impostor syndrome among physicians and physicians in training: A scoping review. Med Educ. 2020; 54(2): 116–124. PubMed Abstract \| Publisher Full Text\n\nHameed R, Bhat A, Nowreen N: Prevalence of Overweight and Obesity among Medical Students and its Correlation with Sleep Pattern and Duration. Int J Contemporary Med Res [IJCMR]. 2019; 6(6). Publisher Full Text\n\nHolliday AM, Gheihman G, Cooper C, et al.: High Prevalence of Imposterism Among Female Harvard Medical and Dental Students. J Gen Internal Med. 2020; 35(8): 2499–2501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIkbaal MY, Salim Musa N. ’Aqilah: Prevalence of Impostor Phenomenon among Medical Students in a Malaysian Private Medical School. Int J Med Students. 2018; 6(2): 66–70. Publisher Full Text\n\nIsrael GD: Determining Sample Size 1 The Level of Precision.1992.Retrieved August 11, 2021.Reference Source\n\nLevant B, Villwock JA, Manzardo AM: Impostorism in third-year medical students: an item analysis using the Clance impostor phenomenon scale. Perspectives Med Educ. 2020; 9(2): 83–91. Publisher Full Text\n\nMak KKL, Kleitman S, Abbott MJ: Impostor phenomenon measurement scales: A systematic review. Front Psychol. 2019; 10(APR). Publisher Full Text\n\nMaqsood H, Shakeel HA, Hussain H, et al.: The descriptive study of imposter syndrome in medical students. Int J Res Med Sci. 2018; 6(10). Publisher Full Text\n\nMontazeri A, Harirchi AM, Shariati M, et al.: The 12-item General Health Questionnaire (GHQ-12): translation and validation study of the Iranian version. Health Qual Life Outcomes 2003 1:1. 2003; 1(1): 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiemi PM, Vainiomäki PT: Medical students’ distress - Quality, continuity and gender differences during a six-year medical programme. Med Teacher. 2006; 28(2): 136–141. PubMed Abstract \| Publisher Full Text\n\nNuttall FQ: Body Mass Index. Nutrition Today. 2015; 50(3). Publisher Full Text\n\nQureshi M, Taj J, Latif M, et al.: Imposter Syndrome among Pakistani Medical Students. Annals King Edward Medical University. 2017; 23(2). Publisher Full Text\n\nRosenstein A, Raghu A, Porter L: Identifying the prevalence of the impostor phenomenon among computer science students. Annual Conference Innovation Technology Computer Science Education. ITiCSE;2020; 30–36. Publisher Full Text\n\nShahjalal M, Khan MNA, Mohsin FM, et al.: Imposter Syndrome Data Set (Version v3.0) [Data set]. Mendeley. 2021a. Publisher Full Text\n\nShahjalal M, Khan MNA, Mohsin FM, et al.: Distribution of imposter syndrome (IS) among medical students of Bangladesh: A cross-sectional study: Copy of questionnaire (Version v3.0). Mendeley. 2021b. Publisher Full Text\n\nSullivan JB, Ryba NL: Prevalence of impostor phenomenon and assessment of well-being in pharmacy residents. Am J Health-System Pharmacy. 2020; 77(9): 690–696. Publisher Full Text\n\nVillwock JA, Sobin LB, Koester LA, et al.: Impostor syndrome and burnout among American medical students: a pilot study. Int J Med Educ. 2016; 7. Publisher Full Text\n\nWang KT, Sheveleva MS, Permyakova TM: Imposter syndrome among Russian students: The link between perfectionism and psychological distress. Personality Individual Differences. 2019; 143(February): 1–6. Publisher Full Text" }	[ { "id": "97308", "date": "21 Jan 2022", "name": "Md Zakirul Islam", "expertise": [ "Reviewer Expertise Pharmacology" ], "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\nA major health issue is surveyed in this study which was not identified or given importance before. The number of references should be more. The article needs some graphical representation also. A detailed comparison between private and government medical college and among different years of medical college is also required. So this article can be indexed after required update.\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": "160903", "date": "23 Jan 2023", "name": "Adam Neufeld", "expertise": [ "Reviewer Expertise Medical education", "self-determination", "well-being", "impostor phenomenon" ], "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 inviting me to review this study, which addresses a timely topic in medical education, in the impostor phenomenon. The authors used a cross-sectional survey-based approach to assess the prevalence of IP among a sample of year 1-5 medical students, and determined how various sociodemographic factors played a role. They found that 32% met criteria for IP and that gender, year of study, and several physical health factors (e.g., BMI, smoking) were predictive. The study has a reasonable methodological approach and findings contribute to the literature; however, the authors would need to do a better job of setting up their hypotheses, the introduction is lacking in updated research that has occurred on this topic in the last 2-3 years, there are statistical shortcomings and areas of unclarity about the survey and analysis, and the results are somewhat overstated and generic. I make my comments based on each section, to help them improve the manuscript.\nTitle: First, it is generally recommended to use terminology \"impostor phenomenon\" and not \"imposter syndrome\" based on Clance and Imes' specific recommendations in the literature (regardless of which IP scale is used). The word \"distribution\" might also be better replaced by prevalence, and there is no mention of demographic predictors in the title, which the study looks at.\nAbstract: The background mentions nothing in its aim about determining predictive nature of demographics, yet the Methods does. The Conclusion is quite generic, as it could be argued that senior medical students require support and guidance, without even conducting this study.\nIntroduction: The authors draw on systematic reviews and studies that lend support to their ideas, but there is little detail provided or rationale for measuring things like 'living at home', 'BMI', or 'smoking'. Why would smoking be considered a risk factor for IP? Why would living at home do this? There is no explanations or hypotheses laid out, which makes these research pursuits unclear. The authors also don't touch on private versus public medical schools, and this is covered more in Discussion. The other concern is what studying gender (which is measured dichotomously) does, since targeting females differently is likely to be stigmatizing. The authors would do well to more carefully consider why they are measuring what they are measuring, and what the implications of doing that are, for students in medical education. There is also a lot of studies on IP and aspects of motivation, academic performance, and well-being, that are not cited in this paper, which deserve citation (see works beyond 2020).\n\nMethods: Please provide ethics number. The number of participants should be in Results. Measures section is largely redundant. BMI is also considered, in medicine, to be a poor predictor, given it's strong reliance on height, which skews results (someone \"obese\" can be healthy and someone \"normal\" can be unhealthy). Reasons for studying medicine are not mentioned at all in Introduction. Survey procedures again repeats details from above and provides far more statistical jargon that is needed. Simply provide a reference and tell the reader what the method was.\nStatistical Analysis: What does \"incomplete\" mean in terms of survey? Also, how is reliability of the YIS determined? Generally, Cronbach's alpha reliability estimates are needed for survey-based items; othewise we don't know if they measured what they were supposed to measure, and thus, if results can be considered valid.\nResults: The authors confusingly now use \"YIS\" instead of \"IS\" which should really just be \"IP\" throughout. They also unpack results here, \"...this indicates that\" and \"In our study...\", which is normally done in Discussion. Results should also be reported in past-tense.\n\nDiscussion: An important pattern I noticed throughout the Discussion section is that the authors simply re-state their findings, compare them to others, and move on. There is no attempt to understand why or explain what might be going. Why was it that smoking, or living at home, or having different BMI, etc., contributed? What are the implications? Why does this even matter? How will it guide interventions? The Discussion section should not simply be a regurgitation of the Results section and a comparison to other studies, since studies often look at things that probably have little weight or bearing. Looking at prevalence and demographic predictors is fine, but you can't change someone's gender or year of study. Are we going to instigate exercise programs? I feel a lot more work is needed here, and the Intro and Discussion require better synthesis.\nThe Strengths and Limitations is also lacking. There is no attempt to assess the YIS psychometrically, which should be done, and the YIS is among the least commonly used measures of IP, compared to the CIPS or HIPS. This also represents a shortcoming, since results are inevitably harder to interpret and compare across studies, due to this. Gender was also measured as a binary construct, which we know it is not, and this automatically stigmatizes and excludes those who may be gender-neutral, transgender, etc. There is also nothing wrong with sampling in one city; it's more how the sampling was done and how the analyses hold up statistically.\n\nConclusions: This is not the place to discuss risk for suicide, which is better placed in Introduction. Recommendations are also overstated and generic, since this study did not assess reasons why they found what they found, and senior medical students are known to be at higher risk for stress.\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/10-1059
https://f1000research.com/articles/10-1058/v1	18 Oct 21	{ "type": "Data Note", "title": "The relationship between umbilical–portal–systemic venous shunt and outcome in 11 fetuses", "authors": [ "Liu Fang", "Wang Xueyan", "Xiao Yangxue", "Zhang Xiaohang", "Ran Suzhen", "Zhou Lan", "He Deying", "Wang Xueyan", "Xiao Yangxue", "Zhang Xiaohang", "Ran Suzhen", "Zhou Lan", "He Deying" ], "abstract": "Objective: To investigate the relationship between congenital umbilical–portal–systemic venous shunt (UPSVs) and fetal outcome. Methods: The ultrasonographic and genetic characteristics of 11 cases of UPSVS were retrospectively analyzed and followed up to postnatal. Results: Four cases of ductus venosus -- systemic shunt (DVSS), one case of extrahepatic portal -- systemic shunt (EHPSS), and one case of umbilical systemic shunt (USS) combined with intrahepatic portal-systemic shunt (IHPSS), six cases of intrahepatic portal-systemic shunt (IHPSS) were observed. chromosomal abnormalities were observed in 9.1% (1/11), other ultrasonic abnormalities in 54.5% (6/11), cardiothoracic ratio increase in 45.5% (5/11), fetal growth restriction in 36.4% (4/11), edema was in 9.1% (1/11) and live birth was in 72.7% (8/11). Conclusion: The incidence of IHPSS is the highest and the outcome is the best. Shunt of DVSS and IHPSS can be closed spontaneously after birth. When the prenatal diagnosis is congenital UPSVs, chromosomal abnormalities and other ultrasonic abnormalities should be required further examination, and the growth and development of the fetus should be closely monitored.", "keywords": [ "umbilical-portal-systemic venous shunt", "fetus", "the outcome" ], "content": "Introduction\n\nCongenital portosystemic shunt (CPSS) is associated with some complications, such as cholestasis, hyperammonemia, pulmonary arterial hypertension, hepatopulmonary syndrome, and benign and malignant liver tumors, in childhood. Some types of CPSS including extrahepatic, persistent intrahepatic shunt and ductus venosus must be inhibited by interventional radiology or surgery.1\n\nDue to the low incidence, there is limited prenatal information on the relationship between umbilical-porta-systemic shunt and its prognosis. In the past, portal systemic shunts were divided into extrahepatic and intrahepatic shunts. They have been used to study fetal umbilical-portal system shunt and pediatric congenital portal systemic shunt.2–6 The recent systematic reclassification of the term “umbilical-porta-systemic venous shunt (UPSVS)” has been considered as the best choice for fetal prognosis analysis. This classification is based on the theory of UV-PV-DV as an intact structure.\n\nAccording to Achiron and Kivilevitch,7 the UPSVs are divided into three types: Type I, umbilical–systemic shunt (USS), with the blood flow from the umbilical vein entered directly into the systemic veins; Type II, ductus venosus–systemic shunt (DVSS), with the DV blood flow shunted from its normal path into the systemic veins; Type III, portal–systemic shunt, which divided into two subgroups: Type IIIa, intrahepatic portal–systemic shunt (IHPSS), with an intrahepatic shunt between the IHPVS and the hepatic vein; and Type IIIb, extrahepatic portal–systemic shunt (EHPSS), with an extrahepatic shunt between the portal system and systemic veins (IVC, iliac vein, renal vein). According to previous studies, the incidence of trisomy 21 in UPSVS was 10.4%.8 Achiron R et al. observed that DVSS and IHPSS had the best prognosis, with spontaneous shunt closure after birth.7 Berg et al.9 observed that cases with extrahepatic shunt were more likely to develop into cardiac decompensation. Delle et al.10 observed a causal relationship between IHPSS and FGR.\n\nThe purpose of this study was to review our experience with fetal UPSVS and analyze its clinical and prognostic characteristics for better prenatal counseling with UPSVS.\n\n\nMethods\n\nRetrospective analysis was performed on 11 cases of UPSVSs admitted to the prenatal diagnosis center of our hospital from December 1, 2019, to December 1, 2020. According to the UPSVS classification criteria, four cases were DVSS, and six cases were IHPSS, including one case of USS combined with IHPSS and one case of EHPSS. All patients signed an informed consent form, performed routine ultrasound examinations of the fetus, placenta and amniotic fluid, and then performed detailed examinations and records of each fetus’s heart, celiac vessel and middle cerebral artery, focusing on observation of the umbilical vein and ductus venosus, main portal vein, left and right branch morphology, internal echo and surrounding structures, observed whether there are abnormal ducts between the umbilical vein, ductus venosus, portal vein, hepatic vein and other systemic veins, and follow up until the shunt is closed after birth. The karyotype and low-coverage massively parallel copy number variation sequencing (CNV-seq) of the fetus were further examined by amniocentesis. Statistical methods: Descriptive statistics were used to retrospectively analyze the clinical features and pregnancy outcomes of 11 cases. SPSS20.0 software was used for statistical analysis of data. Measurement data was expressed as mean (±SD), and the Bonferroni adjustment method in ANOVA was used for pairwise comparison. p < 0.05 indicates that the difference is statistically significant. Counting data are expressed as percentages.\n\n\nResults\n\nAccording to the UPSVS classification criteria, four cases of DVSS, six cases of IHPSS, one case of USS combined with IHPSS and one case of EHPSS were recorded.\n\nIn two cases, the ductus venosus entered the middle of the inferior vena cava (Cases 2 and 4) and was observed at the gestational age of 23 weeks and 24+4 weeks, respectively. In one case, observed at 18 weeks of gestation, the ductus venosus was inserted into the hepatic segment of the inferior vena cava (Case 1, Figure 1). In one case, observed at 23+4 weeks, the ductus venosus was inserted into the middle hepatic vein (Case 3). Three cases received amniocentesis to do karyotype and CNV-seq test, and trisomy 21 was observed in Case 1; the other two cases had normal results. Two cases terminated their pregnancies: Case 1 terminated pregnancy because of trisomy 21, Case 3 terminated because of other associated structural abnormalities, including multiple hemivertebrae with scoliosis, and the left and right branches of the portal vein are not detected on ultrasound. Two cases were delivered prematurely (Cases 2 and 4), of which Case 4 had fetal growth restriction. The ductus venosus of the two cases had closed one month after birth, and the growth and development were normal.\n\n• One case of EHPSS\n\n• In Case 5, at 24+4 weeks of gestation, ultrasound showed fetal edema including abdominal effusion and skin thickening, widened hepatic veins, and increased cardiothoracic ratio, suggesting congestive heart failure. The umbilical vein was directly connected to the ductus venosus, the left and right branches of the portal vein and the main portal vein are not shown. The splenic vein was thin, and did not appear to enter the portal vein, the superior mesenteric vein was unclear. The distal end of the hepatic vein was widened and tortuous, seeming to be connected to the distal end of the hepatic artery, suggesting hepatic arteriovenous fistula. The pregnant woman finally chose to terminate the pregnancy without a chromosome examination.\n\nCase 8 displayed USS combined with IHPSS, see IHPSS for details.\n\nThe location of shunt in six cases is shown in Table 1. Amniocentesis was performed in two cases. The results showed no abnormality in karyotype or gene copy number variation. Two cases had fetal growth restriction (Cases 7 and 10). Ultrasound revealed cardiothoracic ratio increased in three cases (Cases 7, 8 and 10), congestive heart failure with possible cerebral edema in one case (Case 10). In Case 10, MRI revealed: cerebral vein, superior sagittal sinus, right transverse sinus and enlarged sigmoid sinus. Finally, one case underwent full-term delivery, five cases underwent premature delivery, due to congestive heart failure with possible cerebral edema (Case 10), increased cardiothoracic ratio (Cases 7 and 8), breech presentation combined with premature rupture of membranes (Case 9), fetal growth restriction with oligohydramnios (Case 11). All six cases had live births. Follow-up of those six cases showed that the shunts were closed within half a year after birth, and blood ammonia, liver function, growth and development were normal.\n\nResults are shown in Tables 1 and 2.\n\n* Represents p-value: * p < 0.05.\n\n\n\n• What’s known/what’s new statements\n\n• What’s already known about this topic?\n\nUPSVSs are divided into three types in recent research. Limited prenatal information is available on the relationship between umbilical–portal–systemic venous shunt and outcome due to the low incidence.\n\nWe collected 11 cases of fetus from different UPVSV types, and analyze the clinical and prognostic characteristics in details to enable better prenatal counseling with UPSVS.\n\n\nEthics statement\n\nThe report was ethically approved by the institutional review board (Chongqing Health Center of Women and Children) and written informed consent was obtained from the mothers to publish this paper.\n\n\nData availability\n\nDryad: Underlying data for ‘The relationship between umbilical–portal–systemic venous shunt and outcome in 11 fetuses’.\n\nhttps://doi.org/10.5061/dryad.crjdfn34g\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\nBernard O, Franchi-Abella S, Branchereau S, et al.: Congenital portosystemic shunts in children: recognition, evaluation, and management. Semin Liver Dis. 2012; 32: 273–287. PubMed Abstract \| Publisher Full Text\n\nGorincour G, Droullé P, Guibaud L: CAJR Am J Roentgenol. 2005; 184: 163–168. Publisher Full Text\n\nDelle Chiaie L, Neuberger P, Von Kalle T: Congenital intrahepatic portosystemic shunt: prenatal diagnosis and possible influence on fetal growth. Ultrasound Obstet Gynecol .2008; 32: 233–235. PubMed Abstract \| Publisher Full Text\n\nBekdache GN, Hamdan MA, Begam MA, et al.: Prenatal diagnosis of extrahepatic umbilicoportosystemic shunt: impact on postnatal management. J Obstet Gynaecol .2011; 31: 542–543. PubMed Abstract \| Publisher Full Text\n\nStringer MD: The clinical anatomy of congenital portosystemic venous shunts. Clin Anat .2008; 21: 147–157. PubMed Abstract \| Publisher Full Text\n\nSokollik C, Bandsama RHJ, Gana JC, et al.: Congenital portosystemic shunt: characterization of a multisystem disease. J Pediatr Gastroenterol Nutr .2013; 56: 675–681. PubMed Abstract \| Publisher Full Text\n\nAchiron R, Kivilevitch Z: Fetal umbilical-portal-systemic venous shunt: in-utero classification and clinical significance. Ultrasound Obstet Gynecol .2016; 47: 739–747. PubMed Abstract \| Publisher Full Text\n\nDong X, Wu H, Zhu L, et al.: Prenatal Ultrasound Analysis of Umbilical-Portal-Systemic Venous Shunts Concurrent With Trisomy 21. J Ultrasound Med. 2020; 9999: 1–6. PubMed Abstract \| Publisher Full Text\n\nBerg C, Kamil D, Geipel A, et al.: Absence ¨ of ductus venosus – importance of umbilical venous drainage site. Ultrasound Obstet Gynecol .2006; 28: 275–281. PubMed Abstract \| Publisher Full Text\n\nDelle Chiaie L, Neuberger P, Von Kalle T: Congenital intrahepatic portosystemic shunt: prenatal diagnosis and possible influence on fetal growth. Ultrasound Obstet Gynecol. 2008; 32(2): 233–235. PubMed Abstract \| Publisher Full Text" }	[ { "id": "119590", "date": "03 Feb 2022", "name": "Bijoy K. Balakrishnan", "expertise": [ "Reviewer Expertise Fetal medicine specialist" ], "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 this article. The cases that have been discussed are quite interesting and will provide necessary insight for clinicians while counselling their patients with UPVSS. However, it would have been more interesting if more pictures were provided and video clips were added as supplementary material for the readers to access. I myself would be interested in viewing the case of EHPSS and the cases with multiple shunts between the LPV and the hepatic veins. Once they are submitted I will provide a detailed review of the portal system and an easy method to screen for portal system anomalies.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "8200", "date": "07 Oct 2022", "name": "Liu Fang", "role": "Author Response", "response": "Thank you for your review, we are still collecting cases. EHPSS cases are relatively rare, so it takes some time to summarize the cases. At present, A case of USS has given birth, the prognosis is good. We will collect more data for publication, and would like to discuss further with you in the field of fetal diseases." } ] }, { "id": "140424", "date": "27 Jun 2022", "name": "Johannes van der Merwe", "expertise": [ "Reviewer Expertise Fetal 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 manuscript describes the clinical outcomes of a small series of prenatal diagnosed UPSVS cases. The manuscript is overall sound and the topic is relevant. The biggest criticism of this manuscript is that it seems to add no new insights to this rare finding. Structured comments:\nGeneral:\nThe manuscript is sometimes difficult to read due to syntax issues and vocabulary choice. The manuscript could benefit from formal copyediting. Structurally some headings and subheadings have been omitted.\n\nIntroduction:\nAdequate although the authors don’t reflect on recent publications. A lot have been published in this field in the last 2-3 years.\n\nCase descriptions\nThe case descriptions could benefit from ultrasound images being added of the EHPSS case and at least one example case of the IHPSS case.\n\nDiscussion – what the study adds.\nThe authors provoke no new discussion on this topic. Nor is there any reflection on current literature or systematic reviews on this pathology. It is unclear to the reader what future direction should be taken with this pathology. Some examples of recent publications that could help:\n\nVan Houdt M, van der Merwe J, Gewillig M, De Catte L. Prenatal 3D-ultrasound diagnosis of isolated intrahepatic portal-systemic shunt with intact ductus venosus: A case report and literature review. Radiology Case Reports. 2021 May 1;16(5):1173-8.\nNagy RD, Cernea N, Dijmarescu AL, Manolea MM, Zorilă GL, Drăgușin RC, Vrabie SC, Dîră LM, Sîrbu OC, Novac MB, Drăgoescu NA. Ductus Venosus Agenesis and Portal System Anomalies—Association and Outcome. Biology. 2022 Apr 1;11(4):548.\nErenel H, Karsli MF, Ozel A, Korkmaz SO, Sen C. Ductus venosus-systemic shunt. Report of six cases and systematic review of the literature. The Journal of Maternal-Fetal & Neonatal Medicine. 2020 Mar 18;33(6):1015-23.\nKivilevitch Z, Kassif E, Gilboa Y, Weisbuch T, Achiron R. The intra‐hepatic umbilical‐Porto‐systemic venous shunt and fetal growth. Prenatal Diagnosis. 2021 Mar;41(4):457-64.\nPacheco D, Brandao O, Montenegro N, Matias A. Ductus venosus agenesis and fetal malformations: what can we expect?–a systematic review of the literature. Journal of Perinatal Medicine. 2018 Dec 1;47(1):1-1.\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? Partly", "responses": [ { "c_id": "8876", "date": "07 Oct 2022", "name": "Liu Fang", "role": "Author Response", "response": "It's my great honor to receive your review. Thank you for your detailed review and the added references. Because the manuscript was submitted as datasets, discussion on this topic was omitted. It took me some time to collect more interesting cases, and I will submit a new view soon. Hope to discuss further with you in the field of fetal diseases. Best regards." } ] } ]	1	https://f1000research.com/articles/10-1058
https://f1000research.com/articles/10-568/v1	14 Jul 21	{ "type": "Case Report", "title": "Case Report: severe hemoperitoneum due to spontaneous rupture of uterine vessels during the second trimester of pregnancy", "authors": [ "G.V. Ishara Geelaka Bandara Jayarathna", "G.K.C. Jayalath", "Ramya Pathiraja", "G.V. Ishara Geelaka Bandara Jayarathna", "Ramya Pathiraja" ], "abstract": "Spontaneous rupture of uterine vessels during pregnancy is a life-threatening condition though, it has a rare occurrence. This case report discusses about a 32-year-old lady at 16 weeks of gestation presented with spontaneous rupture of uterine artery and she was managed with emergency laparotomy with suturing of ruptured artery. She had delivered a healthy baby after 37 weeks of gestation by a caesarean section due to pregnancy induced hypertension at 36 weeks of gestation.", "keywords": [ "Rare complications in Pregnancy", "spontaneous uterine vessel rupture", "Second-trimester abdominal pain", "decidualization of endometriotic tissue", "Spontaneous Hemoperitoneum" ], "content": "Introduction\n\nSpontaneous hemoperitoneum in the second trimester of pregnancy is a rare complication that gives potentially high mortality for both the mother and foetus.1 This may result from ruptured ectopic, ruptured uterine or ovarian vessels, ruptured spleen, or rupture of the pelvic vessel due to decidualization of the endometriotic deposits. Clinical features include acute abdominal pain, hypovolemic shock, and features of hemoperitoneum. Prompt clinical suspicion and diagnosis, timely intervention with volume replacement and emergency surgical approach will provide the best outcome.\n\nWe report this case of spontaneous hemoperitoneum in the second trimester due to rupture of uterine vessels, that had a good maternal and foetal outcome, with the objective of sharing our experience of diagnosis and management of rare obstetric complication. Obstetricians may find this article helpful as it is essential to be keen on this kind of rare condition that can endanger both the maternal and foetal lives.\n\n\nCase report\n\nA 32-year-old Sri Lankan woman in her third pregnancy at 16 weeks of gestation was admitted to the ward at midnight with a history of acute abdominal pain lasting for six hours. She had a previous history of primary subfertility for three years and underwent a laparotomic cystectomy and adhesiolysis four years back, where she was diagnosed with grade four endometriosis. Following that, she had one first trimester miscarriage and one normal vaginal delivery. Other than that, she had denied having any history of trauma to the abdomen, significant medical, family or psychosocial history.\n\nThe house officer had seen her on admission, pain killers were given, and urine investigations were sent, with suspicions of urinary tract infection. No scan or further investigations performed as the patient was haemodynamically stable other than having abdominal pain.\n\nThe following morning, the patient was in severe pain, she could not lie down on the bed and complained of fainting and shoulder tip pain. A senior registrar had seen her and on examination she was pale, her heart rate was 120/min (70/min), blood pressure was 90/60 mmHg (120/80 mmHg) and her abdomen was tense and tender. A clinical suspicion of an internal bleeding was made.\n\nBedside Ultrasound Scan (USS) was done and it had shown free fluid, probably blood in the peritoneal cavity with viable intrauterine pregnancy at 16 weeks of gestation. Her haemoglobin level found to be 7.4 g/dL (>11.0 g/dL).\n\nUterine rupture, abdominal, liver or splenic rupture, ruptured appendix and peptic ulcer perforation were suspected as differential diagnosis and immediate surgical intervention was needed.\n\nShe had an emergency laparotomy in which she was found to have hemoperitoneum with an estimated blood loss of one litre. Once the blood was removed, it was difficult to find the bleeding point. The gravid uterus was exteriorised and active bleeding from left uterine vessels noted (Figure 1). Significant decidualized endometriotic tissue patches around the ruptured vessels and the posterior aspect of the uterus were noted. Left uterine vessels were ligated above and below the bleeding points (Figure 2). The uterus was put back and the abdominal wall was closed following the insertion of a drain. She had been transfused two pints of blood intra-operatively. A specimen of suspected endometriotic patches was not sent for histological confirmation.\n\nHer recovery was normal. She was discharged on the third day of post-op after confirmation of the foetal wellbeing by a USS.\n\nShe was followed up at the clinic regularly with routine USS. Her pregnancy went uncomplicated until 36 weeks where she developed pregnancy-induced hypertension. She underwent caesarean section at 37 weeks by the same surgeon who performed the laparotomy. A healthy baby was delivered and her post-partum period was uneventful.\n\n\nDiscussion\n\nSpontaneous rupture of uterine vessels remains a rare condition that causes abdominal pain during pregnancy and it can be a life-threatening complication.2 Several studies highlight that the maternal mortality rate is 3.6% with a perinatal mortality rate of 30%.3 Though this condition is common during the 3rd trimester,4 there has been evidence to prove that this can occur during other trimesters and puerperium.5 This case report contains a second-trimester spontaneous uterine vessel rupture which is a rarer condition.\n\nThe patient presented with severe abdominal pain that was later followed by hypovolemia, low haemoglobin level and a positive USS for fluid in the abdominal cavity. Usually, these patients present with acute onset severe abdominal pain and hypovolemic shock with decreased haemoglobin levels, but there is usually no trauma or bleeding history. An ultrasound scan can be used to detect free fluid in the abdomen and paracentesis and has been done in hemodynamically stable patients to identify the fluid type if the patient is stable.3 It would be immensely helpful if the bedside USS could be done immediately after admission in pregnant women presenting with severe abdominal pain. However, the diagnosis was difficult as several causes can give rise to similar presentation such as uterine rupture, abdominal, liver or splenic rupture, appendicitis, peptic ulcer perforation, urinary tract infections, HELLP syndrome intestinal obstruction and rarely ectopic pregnancies.6\n\nHigh utero-ovarian venous tension in combination with conditions causing high intra-abdominal pressure such as defecation, pushing in the second stage of pregnancy, and inflamed vessels and adhesions associated with endometrial deposits have been reported as potential mechanisms.6 In this case report, decidualization of endometrial deposits over the uterine vessels causing rupture of the vessels can be considered as the mechanism.\n\nThe following paragraphs describe two cases that have been published regarding spontaneous rupture of uterine vessels before the third trimester with different presentation, management and outcomes.\n\nOne case study described a 30-year-old pregnant woman (G2P0) presenting with a history of acute abdominal pain that lasted 12 hours duration during the 20 weeks of gestation. This patient was haemodynamically unstable with a blood pressure of 65/30 mmHg and had a tender uterus. Ultrasound scan showed an absent foetal heartbeat and placental abruption was taken as a provisional diagnosis. Following resuscitation with colloids, she had undergone a laparotomy and found out there was a three liter hemoperitoneum with a normal uterus, liver, and spleen. The foetus and placenta were delivered with a small anterior hysterotomy and there was no evidence for placental abruption. After lifting the uterus there was a fresh venous blood collection at the left side uterine artery with a purplish mass attached in between the cervix and pelvic sidewall. The tissue sample sent for histopathology confirmed the presence of endometrial tissue. The patient with a four liter blood loss and transfusion of 10 units of blood, recovered well following 24-hour ICU care and discharged on day seven.3 Unlike our patient this patient already had lost the foetus without the option to continue the pregnancy till term.\n\nIn another case study, a 23-year-old pregnant woman in her 22 weeks of gestation presented with sudden abdominal pain and hemodynamically unstable condition with hypovolemic shock without a trauma history. She had undergone an exploratory laparotomy and found a laceration in the right-side uterine artery and the bleeding vessel ligated with sutures. However, the patient continued her pregnancy for 38 weeks of gestation.6 This is a similar case to our case though the presentation is more acute.\n\nThe treatment is immediate surgical intervention following maintenance of the balance of intravascular circulation. In this case, the patient underwent an immediate laparotomy and found to have ruptured uterine vessels due to decidualization of endometrial deposits. Though it is essential to confirm endometrial tissue with histology, the sample had not been sent during the surgical intervention.\n\nIn the above case records, there was a similar case to our case report as the woman also ended up delivering a baby even though the foetus was at 20 weeks of gestation. In our patient, the ligation of uterine vessels might contribute to causing pregnancy-induced hypertension, but no association is discussed in this case report. This case report can be presented as a peculiar condition compared to most of the similar cases that had ended up with hysterotomy and uterine artery ligation with sutures to control bleeding.\n\n\nConclusion\n\nObstetricians should consider this rare and potentially life-threatening condition in pregnant women presenting with abdominal pain and hypovolemic shock. It requires a keen observation to identify this condition as it is usually masked by several other conditions as mentioned in the discussion. With maintaining intra-vascular circulation properly, the patient should undergo an urgent laparotomy to identify the cause. During the procedure, there is a chance of continuing the pregnancy if the foetus is viable and maternal stability could establish with surgery, so that, suturing of ruptured vessels can be done without a hysterotomy. If this condition occurred at term, both the mother and neonate should be treated with extra care. However, there is an open path to identify the prevalence of presentation and outcome of this kind of clinical condition.\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.\n\n\nAuthor contributions\n\nDr G.K.C. Jayalath – the surgeon who detected the case and performed the surgery, Conceptualization, Investigation, Resources, Supervision, Writing – Original Draft Preparation.\n\nProf. R. Pathiraja – Supervision.\n\nDr G.V.I.G.B. Jayarathna – Writing – Review & Editing.", "appendix": "Acknowledgements\n\nTo the patient who gave consent to publish this case report and the staff in the teaching hospital, Colombo-South.\n\n\nReferences\n\nSwaegers MCR, Hauspy JJP, Buytaert PMHG, et al.: Spontaneous rupture of the uterine artery in pregnancy. Eur. J. Obstet. Gynecol. Reprod. Biol. 1997; 75(2): 145–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Connell MP, Prendiville W: Spontaneous uterine artery rupture: a rare cause of abdominal pain in pregnancy. J. Obstet. Gynaecol. J. Inst. Obstet. Gynaecol . 2005; 25(5): 511–512. PubMed Abstract\n\nFoley MR, Sonek JD, Lavender LM, et al.: Spontaneous rupture of uteroovarian veins in pregnancy: two case reports. Am. J. Obstet. Gynecol. 1987; 156(4): 962–964. PubMed Abstract \| Publisher Full Text\n\nLiu C-M, Hsu J-J, Hsieh T-T, et al.: Postpartum hemorrhage of the uterine artery rupture. Acta Obstet. Gynecol. Scand. 1998; 77(6): 695–697.\n\nRosales RG, Saldaña MÁC, Leal IA, et al.: Rotura espontánea de los vasos uterinos durante el embarazo: Comunicación de un caso y revisión bibliográfica. Ginecol. Obstet. Mex. 2008; 76(4): 221–223.\n\nAziz U, Kulkarni A, Lazic D, et al.: Spontaneous rupture of the uterine vessels in pregnancy. Obstet. Gynecol. 2004; 103(5 Pt 2): 1089–1091. PubMed Abstract \| Publisher Full Text\n\nWilliamson H, Indusekhar R, Clark A, et al.: Spontaneous Severe Haemoperitoneum in the Third Trimester Leading to Intrauterine Death: Case Report. Case Rep. Obstet. Gynecol. 2011; 2011: 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGinsburg KA, Valdes C, Schnider G: Spontaneous utero-ovarian vessel rupture during pregnancy: three case reports and a review of the literature. Obstet. Gynecol. 1987; 69(3 Pt 2): 474–476. PubMed Abstract" }	[ { "id": "89676", "date": "31 Aug 2021", "name": "Augustus Keshala Prabhodana Ranaweera", "expertise": [ "Reviewer Expertise Maternal 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 case is a rare but interesting case. I would think it would benefit readers especially clinicians providing care for pregnant women. This will allow readers to keep an open mind and to be aware of life-threatening rare conditions otherwise missed and may cause maternal or neonatal mortality.\n\nMy main criticism is there are many grammatical errors that need attention before indexing. The content is enough to get a comprehensive picture and to be aware of the important aspects of the management.\nIf these mistakes are corrected this should be considered for indexing.\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/10-568
https://f1000research.com/articles/10-544/v1	07 Jul 21	{ "type": "Research Article", "title": "Knowledge, attitude, and behaviours on diet, physical activity, and tobacco use among school students: A cross-sectional study in two Indian states", "authors": [ "Shalini Bassi", "Deepika Bahl", "Melissa Blythe Harrell", "Neha Jain", "Arun Kandasamy", "Subhash R. Salunke", "Vinod Gajanan Shah", "Prema Raghunathan", "Selvarajan Markandan", "Pratima Murthy", "Monika Arora", "Deepika Bahl", "Melissa Blythe Harrell", "Neha Jain", "Arun Kandasamy", "Subhash R. Salunke", "Vinod Gajanan Shah", "Prema Raghunathan", "Selvarajan Markandan", "Pratima Murthy", "Monika Arora" ], "abstract": "Background: Non-communicable diseases (NCDs) are escalating in India and can be attributed to behavioural risk factors such as unhealthy diet, physical inactivity and tobacco use that began in early years. Understanding adolescents’ knowledge, attitudes and behaviours (KAB) related to NCD risk factors would inform the development of school-based health programmes to prevent NCDs. Methods: Sixth-grade students (n=1026) in 20 schools (10 private, 10 public) from two Indian cities (n=667 from Pune; n=359 from Bengaluru) participated in a KAB survey in 2019. Differences in KAB by gender, school type within cities were investigated. Results: Knowledge about the harms of tobacco use was higher than knowledge about a healthy diet and the importance of physical activity. Only a small proportion of students did not eat breakfast (8.7%) or fruits (11.3%) daily. Only 33.4% of students read nutrition labels before choosing their food. Moderate-to-vigorous physical activity of less than an hour per day was reported by 42.5% of students. Approximately one-third of students had ever tried smoking tobacco (30.1%), smokeless tobacco (30.5%), and e-cigarettes (32.4%). Differences in these behaviours by gender and school type showed that both boys, girls and students of private and public schools are vulnerable. Conclusions: The findings highlight that knowledge is low for thematic areas like diet and physical activity. Low knowledge can be attributed to unfavourable behaviours like lack of reading nutrition labels and indulgence in sedentary activities. To protect India’s young population (adolescents), there is a need to amplify health education activities and context-specific health intervention materials for them by engaging parents and communities. Thus, these programmes should be incorporated into the curriculum as part of the regular teaching, as they may induce positive changes in their knowledge and behaviours. In India, school health programmes should dedicate significant time to health promotion and NCD risk prevention.", "keywords": [ "Non-communicable diseases", "risk factors", "school", "children", "lifestyle related behaviours", "health promotion" ], "content": "Introduction\n\nThe rising epidemic of non-communicable diseases (NCDs), such as cardiovascular diseases, cancers, diabetes and chronic respiratory diseases, is recognised as a leading cause of mortality and morbidity across all age groups in India and globally.1 NCDs are responsible for more deaths in India each year than all other causes of death combined.2 In the year 2019, 9.3 million deaths in India were attributed to all cause mortality, of which 6.1 million (64.9%) were due to NCDs.3\n\nMost NCDs are attributable to potentially modifiable behavioural risk factors, including unhealthy diets, physical inactivity, tobacco and alcohol use.4 Synergistic effects of demographic transition, globalisation, and economic growth have resulted in an environment where more children and adolescents are exposed to and/or engage in these behaviours than ever before.5 It is a well-known fact that lifestyle-related behaviours are often formed early and persist throughout life.6,7 Evidence has shown that 70% of premature deaths in adults due to NCDs are associated with risky behaviours that began in childhood.8 Primordial prevention, including community-based intervention to promote healthier lifestyles, is extremely important for national development and to achieve the Sustainable Development Goals by 2030. Investment at an early age can yield a triple dividend of benefits, i.e., improving health later during adolescence, enhancing health throughout the course of life, and eventually contributing to the health of the next generation of children.9 By 2030, India is expected to be the first country in the world to become home to more than 1.5 billion people, and its population is on target to reach 1.7 billion by 2050.10 Thus, protecting and supporting child and adolescent health in this country is paramount not only to India’s overall well-being, but also to global health and economy.\n\nSchools have been widely recognised as an important location for promoting healthy behaviours among children and adolescents.11,12 Incorporating health-based programmes into the school curriculum can substantially influence both health promoting behaviours and educational achievements. The above outcomes can be achieved by providing children and adolescents with adequate knowledge and skills, allowing them to establish healthy attitudes and social norms through such a curriculum.13 Understanding their current knowledge, attitudes and lifestyle-related behaviours would immensely help both the health and education sectors in developing and implementing effective and efficient school-based health lifestyle programmes.\n\nWe have developed Project PaTHWay, a three-year, school-based programme (2018-2021), to promote healthier lifestyle practices among children.14,15 The overall objective of the programme is to prevent and control the risk factors of NCDs by delivering a school-based health lifestyle programmes to students from two cities in India. Specifically, the programme aims to addresses key behavioural NCD risk factors, such as unhealthy diet, physical inactivity, and tobacco use among school children. The students followed for three years in the project PaTHWay. The first year of the programme included, baseline knowledge, attitude, and behaviour (KAB) assessment and development of the health education programme based on the KAB assessment. Subsequently, school-based health lifestyle programme is being implemented for two years, followed by endline evaluation. In this paper, we present the baseline KAB of students relevant to these NCD behavioural risk factors, which have helped us develop Project PaTHWay school intervention.\n\n\nMethods\n\nA cross-sectional study was conducted during January- March 2019 in schools of Pune and Bengaluru to understand the KAB of school students related to diet, physical activity and tobacco use.\n\nThe programme is being carried out in two cities of India, namely Pune (Maharashtra) and Bengaluru (Karnataka). Pune is situated in Western India, while Bengaluru is situated in Southern India. The number of NCD related deaths in both Maharashtra (70.6%) and Karnataka (72.4%) is higher than the national average of all Indian states (64.9%), across all age groups and both sexes.16 Both Pune and Bengaluru are densely populated and highly urbanised cities. The total population of Pune is 9,429,408, of which there are 4,924,105 males and 4,505,303 females. The average literacy rate is 86.2%.17 Similarly, Bengaluru is home to 9,621,551 individuals, of which there are 5,022,661 males and 4,598,890 females, and the average literacy rate is 87.6%.18\n\nTwenty schools (n = 10 public and n = 10 private) in Pune and Bengaluru were selected to implement Project PaTHWay. These schools were purposely selected to represent different socioeconomic strata (private schools: middle to higher socioeconomic status; public schools: lower socioeconomic status) within the two cities. The selected schools had common characteristics, such as each school having a division of the sixth grade into not more than one to two sub-sections, availability of playgrounds and equipment for physical activity, and provision of a school meal programme (public schools only).\n\nStudents from these 20 schools, enrolled in the sixth grade (n = 1238; Pune: n = 806; Bengaluru: n = 432) were eligible and invited to participate in the baseline KAB questionnaire administered in 2019. Total sample of 1238 students (Pune: n = 806; Bengaluru: n = 432) were included in the present study.\n\nA self-administered, baseline questionnaire was implemented, to assess the students’ KAB related to NCD behavioural risk factors (unhealthy diet, physical inactivity, and tobacco use). The questionnaire included a section on socio-demographic profile of the student on aspects such as age, gender as well as education and occupation of their parents and other section on KAB. The diet related knowledge was measured with six questions, each related to elements of a balanced diet (e.g., How many times per week should one eat breakfast?). Knowledge about physical activity was measured with five questions (e.g., How many minutes of physical activity should people of your age have daily?); and tobacco use knowledge was assessed with 16 questions (e.g., Does using chewing tobacco cause oral cancer?).\n\nAttitudes towards diet (e.g., It is important for me to eat breakfast daily), physical activity (e.g., Taking part in physical activities can help me get better marks at school) and tobacco use (e.g., Using tobacco makes a person appear to be braver and more grown-up) were measured using a 5-point Likert scale - strongly agree, agree, not sure, disagree and strongly disagree. Cronbach alpha was calculated for this construct as the mean score was calculated and presented for attitude towards diet, physical activity, and tobacco use. A higher diet and physical activity score denotes a positive attitude towards a healthy diet and physical activity, indicating lower risk of indulging in risky behaviours. A higher score for tobacco indicates that the adolescents have a less favourable attitude towards tobacco use, indicating a lower risk of tobacco consumption.\n\nThe questionnaire also included the behaviours specific to diet (e.g. In a week, on how many days do you eat breakfast?), physical activity (e.g., How many hours do you spend doing moderate to vigorous physical activities?), and tobacco use (e.g., Have you ever tried a cigarette/beedi/hookah - even once or twice?). Susceptibility to tobacco use, for both smoking tobacco and smokeless tobacco was also assessed using eight items (e.g. Do you think you will smoke any type of tobacco when you enter college?).\n\nThis questionnaire was developed based on the socio-ecological model19 and adapting measures from reliable instruments that have been validated with adolescents in India. Surveys that we referred were, Global Youth Tobacco Survey (GYTS-2010),20 Global Adult Tobacco Survey (GATS-2016-17),21 Project EAT,22 SPAN survey23 and surveys used by the author of this study in previous studies conducted in India.24–26 An English version of the questionnaire was administered in private schools, and Kannada and Marathi versions were administered in public schools in Bengaluru and Pune, respectively. The questionnaire was pre-tested to assess its validity (face and content) and reliability (internal reliability of attitude construct).\n\nFor content validity, feedback was obtained from experts (n = 5) from multi-disciplinary field. These national and international experts have more than two decades of experience in psychology, epidemiology, public health and nutrition. Based on their feedback, the sequence of the questions was modified, more relevant questions were incorporated and language edits were made to simplify the questionnaire for students for better comprehension. To assess face validity, focus group discussions were conducted with students (n = 80) of grade six from both cities. These 80 students were selected from two schools in each city (one public and one private). To assess reliability, the questionnaire was administered with another 177 students from both cities. These schools or the students who were involved in face validity and internal reliability were not part of the main study. Based on students' feedback and internal reliability findings, language of the questionnaire was simplified, repeated and offending questions were deleted, options for questions were changed and added, detailed marking instructions were added to the questionnaire. Revised questionnaire was administered by a trained research team of the project. The questionnaire was administered during school hours, using a standardised protocol. Students were given unique identification codes to ensure confidentiality. Copies of the questionnaire in all three languages (English, Marathi, and Kannada) are available in Extended data.65\n\nPermissions for implementation of the study were obtained from the Maharashtra State Board of Secondary and Higher Education and Karnataka Secondary Education Examination Board. We also received permission from authorities at schools, written active informed parental consent, and student assent, indicated by a signature on the consent form. Information sheets were sent from the schools to the parents of all eligible students, wherein details of the study and questionnaire were outlined. The consent stated the permission for data collection, scientific publications, dissemination in conferences by maintaining the confidentiality of the study participants and anonymity of the collected data. Ethical approval for this study was obtained from the Institutional Ethics Committee of both PHFI (TRC-IEC-373/18) and NIMHANS [NIMHANS/EC (BEH.SC.DIV.)].\n\nStatistical analyses were performed using STATA Version.13.1 software.27 The descriptive data were expressed as mean with standard deviation (SD) or proportions (%). A chi-square test was performed to examine differences in the categorical variables by socio-demographic factors (gender, school type). The summary scores for all attitude scales by gender and school type within each city were compared using the t-test. All statistics were analysed through a two-sided test; p-value less than or equal to 0.05 was considered statistically significant. Participants with missing information, parent refusal, student refusal or absenteeism on the day of questionnaire administration were excluded.\n\n\nResults\n\n82.8% of the recruited sample participated in the baseline questionnaire (n = 1026 out of 1238). The reasons for non-participation included absenteeism (n = 112; boys: 67, girls: 45) and parent refusal (n = 100, boys: 54, girls: 46). The final sample size was 1026 (Private: 518 and Public: 508) consisting of 61.1% boys and 55.1% of the sample was from private schools. The mean age of the students recruited for the study was 12.5 ± 0.75 years. The full, de-identified dataset of student responses is available in Underlying data.64\n\nDiet\n\nOverall, students’ knowledge about healthy dietary practices was low (Table 1). Many students (67.5%) knew that breakfast was the most important meal of the day. Only a few students (6.8%) knew they should eat at least five servings of fruits and/or vegetables a day. Knowledge about healthy dietary practices was generally highest among girls and private school students. In Pune, more girls (68%) knew breakfast was the most important meal of the day compared to boys (59.4%) (p = 0.02). Similarly, more girls (15.1%) were able to identify iron rich foods than boys (7.7%) (p = 0.03). In Bengaluru, more girls (42.2%) knew about the importance of a balanced diet than boys (22.2%) (p = 0.000). Knowledge on the importance of a balanced diet was more prevalent among private school students than public school students in both cities (p < 0.01). In Bengaluru, more private school students (49.2%) knew salty foods could lead to hypertension, compared to public school students (32.9%) (p = 0.002) (Table 1).\n\n* P < 0.05.\n\n** p < 0.01.\n\nPhysical activity\n\nCompared to knowledge about healthy dietary practices, students’ understanding of the benefits of physical activity was higher (Table 1). Around 66%-69% of students were aware of the positive impact physical activity can have on reducing the risk of NCDs such as diabetes, heart disease, and obesity. Difference in the knowledge level of boys and girls was seen in both Pune and Bengaluru but a significant difference was seen only in Bengaluru for variables linking physical activity with low risk of diabetes (boys: 84.8% vs girls: 73.9%, p = 0.01) and obesity (boys: 79.3% vs girls: 86.9%, p = 0.05). However, less than one-fifth of the students (18.9%, overall) knew that the level of recommended daily physical activity is 60 minutes or more. More students (59.2%, overall) knew screen time should be limited to two hours or less per day.\n\nTobacco use\n\nFew differences in tobacco use knowledge by gender were seen in Pune and Bengaluru. Where present, there was higher comprehension among boys in Pune for items like tobacco use is harmful to health (boys: 83.2% vs girls:76%, p = 0.025) and the legal age for buying or selling tobacco (boys: 61% vs girls: 52.1%, p = 0.025). In Bengaluru, girls had higher knowledge for smoking and its association with heart attack (boys: 84.3% vs girls: 95%, p = 0.001) and cancer (boys: 88.8% , girls: 95%, p = 0.03). In Bengaluru, significantly more public school students were knowledgeable about tobacco use related harms and policies in India than private school students (p < 0.01) (Table 2).\n\n* P < 0.05.\n\n** p < 0.01.\n\nDiet\n\nThe maximum score a participant could achieve for attitude towards diet was 40. The mean score of students was 28.4 ± 6.5, which is 70% of the maximum score. Within the two cities, students’ attitude about healthy dietary practices was similar for boys and girls (p > 0.05) and was similar in both school types (p > 0.05) (Table 3).\n\n* P < 0.05.\n\n** p < 0.01\n\nPhysical activity\n\nThe highest score a student could achieve for attitude towards physical activity was 45. The students’ mean score was 29.5 ±7.5, which is 65% of the maximum score. No significant differences were observed between boys and girls in both cities, highlighting that both have a positive attitude towards physical activity (p > 0.05). In Bengaluru, public school students had a more positive attitude towards physical activity in comparison to private school students (p = 0.001), whereas the opposite was observed in Pune (Table 3).\n\nTobacco use\n\nThe maximum score a student could achieve in this construct was 45. The students’ mean score was 31.3 ± 12.7, which is 68% of the maximum score. In Pune, mean scores were significantly higher among girls than boys (29.7 ± 13.3 in girls vs 26.8 ± 14.1 in boys; p = 0.01) and among public school (28.3 ± 12.5) students than private schools’ students (27.6 ± 4.8) (p = 0.01), suggesting girls and public school students had less favourable attitudes towards initiating a tobacco habit. In Bengaluru, no significant differences between gender were observed but a significant difference in scores by school type was observed (public school: 38.5 ± 5.8 vs private school: 36.4 ± 6.6, p = 0.002) (Table 3).\n\nDiet\n\nOverall, the vast majority of students (94.7%) were eating “outside food” (i.e. takeaway/street food) daily. Few students (8.7%) skipped breakfast daily. In Pune, fewer girls (8.8%) than boys (18.9%) reported that they had never eaten fruits (p = 0.001) or pulses (p = 0.004). In Bengaluru, fewer girls than boys reported that they had never eaten pulses (4.3% girls and 14.1% boys; p = 0.002) or salads (1.2% girls and 11.1% boys; p = 0.000) and more boys (51.8%) than girls (39.4%) reported that they had never read nutrition labels (p = 0.020). In Bengaluru, more girls (46.3%) than boys (35.2%) reported watching television while eating meals (p < 0.01), while the reverse was observed in Pune (boys: 20.2% vs girls 17.2%) (p < 0.01).\n\nWith respect to school type, in Pune skipping breakfast and never reading nutrition labels were more common among students in public schools (15.2%) compared to private school (9.7%)(p < 0.05). Nearly one-third of public school students (30%) and 22.7% of private school students never read a food label before buying food items (p < 0.005). Never reading nutrition labels was also very common among public school students in Bengaluru (64.8%) (Table 4).\n\n* P < 0.05.\n\n** p < 0.01.\n\nPhysical activity\n\nOverall, very few students (3.3%) watched screens (television, computer, video games etc.) for more than two hours a day. No significant differences were seen between girls and boys for screen time either in Pune or Bengaluru (p > 0.05). However, significantly more public-school students compared to private school students reported at least two hours of screen time a day in both Pune (4.3% in public school and 1.0% in private schools; p = 0.006) and Bengaluru (6.9% in public schools and 1.6% in private schools; p = 0.026).\n\nNo significant differences in moderate-to-vigorous physical activity were seen by gender or school type in both cities (p > 0.05). However, fewer students in Bengaluru (21.9%-29.9%) reported that they had less than adequate moderate-to-vigorous physical activity per day compared to students in Pune (50.3%-52.1%) (Table 4).\n\nTobacco\n\nOverall more students in Pune (50.7%) than Bengaluru (2.8%) reported having ever used tobacco. In both cities, no significant differences by gender were observed. However, the prevalence of ever smoking (65.4% vs. 33.0%, p = 0.001), ever use of smokeless tobacco (64.8% vs. 33.5%, p = 0.001), and ever e-cigarette use (69.2% vs. 36.7%, p = 0.001) was significantly higher among public school students in Pune than private school students (Table 4).\n\nThe boys from the participating schools of Pune were found to be more susceptible to tobacco use (23.3% smoking and 25.9% smokeless tobacco) in comparison to the girls (15.5% both smoking and smokeless tobacco use) (p < 0.05) (Table 5).\n\n* P < 0.05.\n\n** p < 0.01.\n\n\nDiscussion\n\nThis is one of the few studies on NCD risk factors that has comprehensively assessed KAB of school going children from two major metropolitan areas of India and the KAB findings helped to develop the school-based health lifestyle programme. During recruitment, it was ensured that the schools included in the study had similar characteristics, as differences in school characteristics might have influenced the results. Given the wide geographic variability in NCDs in this country,16,28,29 it is important to assess students’ KAB in a myriad of contexts.\n\nThe findings of our study revealed low dietary knowledge among students irrespective of gender and school type. Knowledge was better among girls and private school students. Similar differences were reported in school going students of Haryana (North India).30 The students’ knowledge of physical activity was better than their knowledge of diet and 66%-69% of students were aware of association between NCDs and physical inactivity. In contrast to our study findings, a better knowledge about physical activity (88%) was reported in students of Gujarat,31 and Delhi-India.32 Similarly, adolescents from the eighth and ninth grade in other low-and-middle-income countries (LMICs) such as Nepal, knew that physical activity is imperative for disease prevention and for staying healthy.33\n\nIn our study, students’ knowledge about the harms of tobacco use was much higher in comparison to their knowledge about diet and physical activity. This increase in knowledge could be attributed to the Government of India’s efforts to combat rising burden of tobacco use and its associate health impact, including comprehensive national tobacco control law i.e., COTPA-2003,34 National Tobacco Control Programme35 as well as tobacco-free educational institutional guidelines.36 Evidence from other LMICs, including Bangladesh, also reported high awareness of the harms of tobacco use among rural adolescents (13-18 years).37\n\nWith regard to behaviours, only 8.7% of students were missing their breakfast daily. This is similar to the findings of a study conducted among school students of Gujarat, India.38 Findings from a study conducted in Kenya also showed similar results, wherein 92.1% of the children and adolescents (8-11 years) consumed breakfast daily.39 In our study, more students from public schools reported missing their daily breakfast than students in private schools. This could be attributed to a low level of knowledge and the presence of students from lower middle-income families in these schools. Students from lower income families are more likely to experience scarcity of good quality food, meaning they may lack a wholesome breakfast as well as other meals of the day.40 An additional factor could be the availability of a mid-day meal scheme (i.e., school meal) in public schools,41 which may preclude the importance of their first meal at home (i.e., breakfast).\n\nOther protective dietary factors include fruit consumption, and only 11.3% of students did not eat fruits daily. The Comprehensive National Nutrition Survey (2019) showed that among adolescents (10-14 years), 59% in Maharashtra and 74% in Karnataka ate whole fruits only once a week.28 Though fruits are “price elastic” (a term that describes the connection between income and the demand of a product),42,43 no difference in consumption was seen by school type. Results from other LMICs were poorer, with only 9% of adolescents (8-11 years) consuming fruits four to seven times a week.39 On other hand, in a cross-country comparison among 49 LMICs, India had highest percentage (29.5%) of adolescents (13-17 years old) who met WHO’s recommendation for fruit and vegetable consumption.44 All of the included 49 countries were selected due to the availability of the Global School Based Student Health Survey (GSHS) data from 2004-2013 for this ecological study and provided a data on a total of 164,771 adolescents.\n\nAnother worrisome behaviour observed among students was the habit of eating “outside food” (i.e., takeout) every day (95%). Contributing factors to its popularity could be ease of access, taste, parent’s occupation, and fast-food companies marketing strategies.45 Fast food consumption data from 153,496 young adolescents (12-15 years) from 54 LMICs showed that 55.2% (51.3–59.1%) of the adolescents consumed fast food at least once per week, and 10.3% (8.3–12.4%) consumed fast food between four to seven days per week. The prevalence of fast-food consumption for four to seven days per week was lowest in the Americas (8.3%; 95% CI: 6.7–9.9) and highest in Southeast Asia (17.7%; 95% CI: 2.3–33.2%).46 Our study found that students are not reading nutritional labels while making food choices, as only 33% of students read labels. Students in public schools were practicing this behaviour more often than students in private schools. In contrast to this trend, 88% of students in Kolkata reported reading nutritional labels, and this was most common among girls.47\n\nLack of physical activity among adolescents is a major NCD risk factor. More than half of the students (57.5%) in this study reported engaging in moderate-to-vigorous physical activity for at least 60 minutes, daily, with no significant differences by gender or school type. One recent publication showed that 73.9% of adolescents in India are insufficiently physically active.48 However, a global study of 49 LMICs with 164,771 adolescents found that India had the highest percentage (29.5%) of adolescents who met WHO’s recommendations for physical activity.44 On an encouraging note, in the same study, few adolescents (3%) reported screen time of more than two hours a day. This is in contrast to 52.5% of school going adolescents in Tamil Nadu (Southmost part of India) who had an average daily screen time of more than two hours a day.49\n\nOne third of adolescents had ever tried tobacco (smoke, smokeless and e-cigarette). Evidence from 68 LMICs showed that mean prevalence of tobacco use among adolescents (12-15 years) was 13.6%, ranging from 2·8% in Tajikistan to 44·7% in Samoa and in most countries prevalence among boys was higher compared to girls.50 However, no difference among boys and girls was observed in this study, indicating a narrowing of gender gap for tobacco use which is much wider among adults in India.51 In Pune, public school students used more tobacco (all forms) in comparison to private schools. Whereas in Bengaluru, smokeless tobacco was more commonly used among public school students in comparison to smoke forms. Tobacco use (all forms) was found to be more prevalent in public school boys (13.4%) in comparison to private school boys (11.7%).52 This could be due to the students’ lack of compliance with provisions of Indian tobacco control law as well as the availability and ease of access to tobacco products outside of their schools.53 Surprisingly, 32.4% of the students also reported that they have tried e-cigarettes in the past. To the best of our knowledge, no other study in India has yet reported the prevalence of e-cigarette use among school going children. The prevalence found in our study was much higher than that found in a study from Mexico.54\n\nResults suggest that tobacco use prevention will be especially important for schools in Pune, while promoting physical activity may be a key strategy to pursue in Bengaluru. Students in both cities would benefit from additional school-based intervention to support healthy dietary practices, including eating pulses and vegetables frequently; eating healthier, eating home-cooked meals; and reading nutrition labels.\n\nAs the 2030 deadline for Sustainable Development Goals are only a decade away, the low levels of knowledge, particularly about unhealthy diet and physical inactivity, are a matter of concern.55 Aiming to reduce the NCD burden, the budding role of education in primary prevention was emphasised at the 2011 United Nations’ Meeting on the Prevention and Control of NCDs.56 Lack of healthy behaviours among adolescents has been linked with lack of sufficient knowledge, and research has also indicated a positive relationship between information level and overt behaviour.57 Sufficiently designed health promotion programmes58 may provide the much needed knowledge that will help to reduce risk behaviours such as unhealthy diet, physical inactivity, and tobacco use, hence providing a pathway for behaviour change. Health promotion strategies in schools provide an avenue to reach school children at a time coinciding with their cognitive development. Such an approach can impart information effectively, especially with respect to lifestyle choices.59 Studies have shown that if implemented efficiently, health promoting schools can enhance decision-making skills among students, too.60\n\nIn India, various education programmes have been designed for school going adolescents but there are only few comprehensive education programs addressing diet, physical activity and tobacco.61 In 2018, the Government of India launched the National School Health Programme under Ayushman Bharat or Healthy India. The programme comprehensively addresses the above risk factors, and is delivered by health ambassadors (two teachers per school) only in government and government aided schools across country.62\n\nThe results of the present study are being used to inform the development of a two-year intervention programme in these cities which will target students in the sixth to eighth grades, their parents, and community members. This intervention programme is being implemented from 2019-2021 by trained teachers and is facilitated by trained peer leaders in both public and private schools. This model of delivery has proven successful in India and elsewhere globally for tobacco63 and nutrition12 education.\n\nOne of the limitations of this study was that the sample of schools was not randomly selected from the population but was representative of the mix of types of schools in these two cities. This limits generalisability of the findings. The self-reported method utilised (i.e., survey) also may have led to skewed estimates of dietary intake, physical activity and tobacco use patterns among the students due to the fact that adolescents would have wanted to report positive health behaviours (social desirability - reporting bias). Behaviour for every type of tobacco use was evaluated without further investigating the number of times the tobacco was used by the students. Given budgetary constraints, no anthropometric or biochemical (e.g., salivary cotinine) measurements were completed, which may be a drawback of this study.\n\n\nConclusion\n\nThere is a need to amplify health education activities in schools in India, and school health interventions need to be adequately designed to ensure that they do not further widen existing socio-economic inequalities. This study highlights the need to augment school health programmes in India with a differential approach based on the issues which are specific to each school type (public and private) and city (Pune and Bengaluru). School health programmes should be structured with a dedicated focus on health promotion and NCD prevention. Developed intervention materials should be incorporated in the curriculum to become part of the regular teaching.\n\n\nData availability\n\nFigshare: Knowledge, Attitude and Behaviours (KAB) on diet, physical activity, and tobacco use among school students: Survey Dataset. https://doi.org/10.6084/m9.figshare.14760147.v4.64\n\nThis project contains the following underlying data:\n\n• KAB Survey Dataset.xlsx (student knowledge, attitude and behaviour responses regarding their diet, physical activity and tobacco consumption).\n\nFigshare: Knowledge, Attitude and Behaviours (KAB) on diet, physical activity, and tobacco use among school students: Survey Tool. https://doi.org/10.6084/m9.figshare.14760480.v2.65\n\nThis project contains the following extended data:\n\n• KAB Questionnaire_English.pdf\n\n• KAB Questionnaire_Marathi.pdf\n\n• KAB Questionnaire_Kannada.pdf\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 acknowledge the contribution of Dr. Nivedita Mishra, Ms. Rashmi Sangoram, Ms. Hema Vathi and Dr. Rayeesa Zainab for their contribution to this study and for aiding in data collection. This study would not have been possible without the participation of students and the permission of the authorities from the selected schools of Pune and Bengaluru (India).\n\n\nReferences\n\nWorld Health Organization: Non-communicable diseases: progress monitor 2020.Geneva: World Health; 2020.\n\nWorld Health Organisation: Global Status Report on Non-Communicable Diseases.2011. Reference Source\n\nInstitute for Health Metrics and Evaluation: Global Health Data. GBD Results Tool\|GHDx.Reference Source\n\nWHO: Global action plan for the prevention and control of noncommunicable diseases 2013-2020. World Heal Organisation. 2013; 102. Reference Source\n\nWatkins D, Hale J, Hutchinson B, et al.: Investing in non-communicable disease risk factor control among adolescents worldwide: A modelling study. BMJ Glob Heal. 2019 Apr 1; 4(2). 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKigaru DMD, Loechl C, Moleah T, et al.: Nutrition knowledge, attitude and practices among urban primary school children in Nairobi City. Kenya: a KAP study. BMC Nutr. 2015 Dec 29; 1(1): 1–8. Publisher Full Text\n\nArora M, Nazar GP, Gupta VK, et al.: Association of breakfast intake with obesity, dietary and physical activity behavior among urban school-aged adolescents in Delhi, India: Results of a cross-sectional study. BMC Public Health. 2012; 12(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Human Resource Development, Government of India: Mid Day Meal Scheme.Reference Source\n\nAndreyeva T, Long MW, Brownell KD: The impact of food prices on consumption: A systematic review of research on the price elasticity of demand for food. Am J Public Health. American Public Health Association; 2010; 100: p. 216–22. 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Drug Alcohol Depend. 2017 Nov 1; 180: 427–430. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnited Nations: Sustainable Development Goals.2015. Reference Source\n\nBay JL, Hipkins R, Siddiqi K, et al.: School-based primary NCD risk reduction: education and public health perspectives. Health Promot Int. 2017; 32: 369–379. PubMed Abstract \| Publisher Full Text Reference Source\n\nFleary SA, Joseph P, Pappagianopoulos JE: Adolescent health literacy and health behaviors: A systematic review. J Adolesc. Academic Press; 2018; 62: p. 116–127. PubMed Abstract \| Publisher Full Text\n\nMagarey AM, Pettman TL, Wilson A, et al.: Changes in Primary School Children’s Behaviour, Knowledge, Attitudes, and Environments Related to Nutrition and Physical Activity. ISRN Obes. 2013; 2013: 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDay RE, Sahota P, Christian MS: Effective implementation of primary school-based healthy lifestyle programmes: A qualitative study of views of school staff. BMC Public Health. 2019 Sep 9; 19(1): 1239. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nKarkada S, Pai MS: Concept of Health Promoting School. Manipal J Nurs Heal Sci. 2016; 2(2): 65–8. Reference Source\n\nSaraf DS, Gupta SK, Pandav CS, et al.: Effectiveness of a School Based Intervention for Prevention of Non-communicable Diseases in Middle School Children of Rural North India: A Randomized Controlled Trial. Indian J Pediatr. 2015; 82(4): 354–362PubMed Abstract \| Publisher Full Text\n\nMinistry of Health and Family Welfare and Ministry of Human Resource Development: School Health Programme under Ayushman Bharat, Operational Guidelines.New Delhi; 2018. Reference Source\n\nArora M, Stigler MH, Reddy KS: Effectiveness of health promotion in preventing tobacco use among adolescents in India research evidence informs the national tobacco control programme in India. Glob Health Promot. 2011; 18(1): 09–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBassi S: Knowledge, Attitude and Behaviours (KAB) on diet, physical activity, and tobacco use among school students: Survey Dataset. figshare. Dataset. 2021. Publisher Full Text\n\nBassi S: Knowledge, Attitude and Behaviours (KAB) on diet, physical activity, and tobacco use among school students: Survey Tool. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "89265", "date": "17 Aug 2021", "name": "Subhash Pokhrel", "expertise": [ "Reviewer Expertise Public health", "behaviour change", "health economics" ], "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 well-written paper that provides data on the knowledge, attitude and behaviours of school students in two Indian states. Such data will provide a baseline against which the impact of any behaviour change intervention can be measured in the future. As such, this is a timely and valuable paper.\nA few areas where the authors may want to improve on:\nAbstract\nConclusions: The last few sentences are policy speculations, not study conclusions, so they may be better placed in the Discussion section instead (of course with some backup references there).\nMethods\nSetting: Purposive sampling technique - explain a bit further to explain how exactly the 20 schools were selected. What was the sampling frame (total number of schools) and how exactly did you balance various characteristics mentioned here.\n\nParticipants: The number given for the final sample size (n=1238) does not match the number given in the Results section. Need to rephrase this sentence.\n\nStatistical analysis: A commentary needs to be added in the limitation section later saying that statistical tests are indicative only, as the sampling design was purposive (not random). Also, need justification for a t-test in the light of Likert-scale data.\nResults\nThe presentation looks fine as this is largely descriptive data. However, I wonder whether additional analysis examining if the reported KAB differed by parental education and occupation may provide more helpful insights. In other words, would observed gender differences in KAB disappear (or the other way around) once you have controlled for parental education and/or parental occupation? Note that in the Discussion section (p.13, para 6), you hypothesise the habit of eating outside food may have been influenced by parental occupation, among other things. Why not test these when you have collected your own data?\nDiscussion\nMost space in the Discussion section has been devoted to compare and contrast study findings with wider literature, which is important but doing so has limited the ability for the study to provide valuable insights for policymakers in relation to answering “what next”. So, you may choose to summarise the comparison of your findings with the study results in addition to providing a succinct discussion as to how Indian (and wider South Asian) policymakers can go from here. For example, for the sake of the argument, if we followed COM-B model of behaviour change to design and implement interventions for this age group, how can the study results help us identify where capabilities, opportunities and motivations lie and how can adolescents be supported in their lacking space to change their behaviour. I think the rich discussion in this area makes this paper more useful.\n\nA confusion/error, p.13, para 5: Seems to be a confusion between “price elasticity” and “income elasticity”. Please revise the sentence.\n\nLimitations: Acknowledge that statistical tests are indicative only as the whole sample was purposive.\nConclusion\nStrongly recommend revising the conclusion section. The sentences here must be backed up by the study findings and leave any speculative policies to the Discussion section above. An example conclusion may be: “In the two states of India, a higher proportion of school students know the harm of tobacco use than the benefits of a healthy diet and physical activity. Both gender of the student and which type of the school that the student went to influenced specific KABs but both the magnitude and direction of such influence were variable. The data suggests that there is an urgent need for designing and implementing interventions that can improve school students’ capability and motivation as well as create opportunities to change their unhealthy behaviours.\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": "7189", "date": "15 Oct 2021", "name": "Shalini Bassi", "role": "Author Response", "response": "Abstract Conclusions: The last few sentences are policy speculations, not study conclusions, so they may be better placed in the Discussion section instead (of course with some backup references there). Response: This has been revised as suggested. The conclusion highlighted the key study findings. Methods Setting: Purposive sampling technique - explain a bit further to explain how exactly the 20 schools were selected. What was the sampling frame (total number of schools) and how exactly did you balance various characteristics mentioned here. Response: Schools and colleges were selected based on the existing network of collaboration to represent different socio-economic statuses. This information has been incorporated under the setting section to make information comprehendible. Participants: The number given for the final sample size (n=1238) does not match the number given in the Results section. Need to rephrase this sentence. Response: The line “Total sample of 1238 students (Pune: n = 806; Bengaluru: n = 432) were included in the present study” has been deleted. The existing lines under this section emphasized that 1238 students were eligible and invited to participate in the study. Out of 1238 eligible students, 1026 students participated, which is already highlighted in the result section. Statistical analysis: A commentary needs to be added in the limitation section later saying that statistical tests are indicative only, as the sampling design was purposive (not random). Also, need justification for a t-test in the light of Likert-scale data. Response: Suggested accepted, added a line in the limitation section regarding purposive sampling. Each attitude statement was given a score and a mean score was calculated for this construct. This has been explained under the instrument and measure section. Thus, a t-test was used for this construct to do gender and school type comparisons within each city. Results The presentation looks fine as this is largely descriptive data. However, I wonder whether additional analysis examining if the reported KAB differed by parental education and occupation may provide more helpful insights. In other words, would observed gender differences in KAB disappear (or the other way around) once you have controlled for parental education and/or parental occupation? Note that in the Discussion section (p.13, para 6), you hypothesise the habit of eating outside food may have been influenced by parental occupation, among other things. Why not test these when you have collected your own data? Response: We anticipated doing this analysis, however, many of the students did not attempt the questions (missing response) related to their parental education and occupation. Discussion Most space in the Discussion section has been devoted to compare and contrast study findings with wider literature, which is important but doing so has limited the ability for the study to provide valuable insights for policymakers in relation to answering “what next”. So, you may choose to summarise the comparison of your findings with the study results in addition to providing a succinct discussion as to how Indian (and wider South Asian) policymakers can go from here. For example, for the sake of the argument, if we followed COM-B model of behaviour change to design and implement interventions for this age group, how can the study results help us identify where capabilities, opportunities and motivations lie and how can adolescents be supported in their lacking space to change their behaviour. I think the rich discussion in this area makes this paper more useful. Response: Some information on the applicability of the research findings exist in the discussion and is linked to the implications for Sustainable Development Goals and School Health Programmes by the Government of India. Further information has been added on the underlying theory of change models and linked with recommendations for the Government. Few comparative literatures has also been deleted. A confusion/error, p.13, para 5: Seems to be a confusion between “price elasticity” and “income elasticity”. Please revise the sentence. Response: As suggested, revised. Limitations: Acknowledge that statistical tests are indicative only as the whole sample was purposive. Response: Suggestion accepted and this has been incorporated in the limitation section. Conclusion Strongly recommend revising the conclusion section. The sentences here must be backed up by the study findings and leave any speculative policies to the Discussion section above. An example conclusion may be: “In the two states of India, a higher proportion of school students know the harm of tobacco use than the benefits of a healthy diet and physical activity. Both gender of the student and which type of the school that the student went to influenced specific KABs but both the magnitude and direction of such influence were variable. The data suggests that there is an urgent need for designing and implementing interventions that can improve school students’ capability and motivation as well as create opportunities to change their unhealthy behaviours. Response: Suggestion accepted and the conclusion has been revised." } ] }, { "id": "91108", "date": "23 Aug 2021", "name": "Nishibha Thapliyal", "expertise": [ "Reviewer Expertise Public health nutrition", "physical activity", "NCD", "obesity" ], "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 “Knowledge, attitude and behaviours on diet, physical activity and tobacco use among school students: A cross-sectional study in two Indian states by Shalini Bassi et.al. looks at measuring the knowledge, attitudes and behaviours (KAB) of school children (grade 6) in relation to NCD behavioural risk factors, primarily unhealthy dietary intake, physical inactivity and tobacco use. This information gathered is the baseline evaluation of Project PaTHway (a 3-year school-based healthy lifestyle programme).\n\nOverall, an interesting and impressive read. The strength of the paper lies in its massive contribution (KAB findings) towards limited literature of school-going children about NCDs awareness and also in providing emphasis to support the development of school-based health education programmes (policy) in India. However, since the subject population from rural areas (villages) could not be captured, it is suggested to be added as a limitation.\nAbstract: It is an extremely well structured abstract with reasonable focus on the key areas.\nIntroduction: The introduction is sound and clearly demarcates the problem that will be addressed in the paper. The area of focus and the aims are easily identified. However, a little more on the prospective role of KAB as a tool to develop any kind of intervention programme can be added. A little on the current behavioural habits of school children can also be mentioned, including the reason for the selection of school children (specifically 6th grade) as the study population.\nMethods: It is a well planned and thoroughly designed methodology, keeping in mind all permissions, clearance and consents requirement of a good study design. The credibility and validity of the tools have also been established. However, certain factors that could not be controlled (home environment) should be mentioned as a limitation. Also, the use of an additional supportive tool (like a food frequency or anthropometric) to decrease the reporting bias of a ‘self-reporting survey’ (also highlighted by the author) should be mentioned as a suggestive approach for future researches.\nResults & Discussion: The results are extensive and logically sequenced with a simple and easy to understand presentation. All areas of interest (KAP of dietary intake, tobacco use and physical inactivity) have been clearly depicted. However, there are certain findings (results of fruit intake, screen time and consumption of outside food) that challenge the traditional findings/beliefs of people’s behaviours that vary due to socioeconomic inequality. This could be due to the bias (subjects giving out socially approved answers) in self-reporting surveys (also listed as a limitation by the author) and can be added as a limitation in the study.\nConclusion: The findings of this project correctly identify the need for school-based lifestyle intervention programmes in terms of national policy planning for the prevention of NCD’s. The conclusion is precise and 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?\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": "7190", "date": "15 Oct 2021", "name": "Shalini Bassi", "role": "Author Response", "response": "Overall, an interesting and impressive read. The strength of the paper lies in its massive contribution (KAB findings) towards limited literature of school-going children about NCDs awareness and also in providing emphasis to support the development of school-based health education programmes (policy) in India. However, since the subject population from rural areas (villages) could not be captured, it is suggested to be added as a limitation. Response: Suggestion accepted. Added a line in the limitation section. Abstract: It is an extremely well structured abstract with reasonable focus on the key areas. Introduction: The introduction is sound and clearly demarcates the problem that will be addressed in the paper. The area of focus and the aims are easily identified. However, a little more on the prospective role of KAB as a tool to develop any kind of intervention programme can be added. A little on the current behavioural habits of school children can also be mentioned, including the reason for the selection of school children (specifically 6th grade) as the study population. Response: Suggestion accepted, we have incorporated a justification for assessing a KAB at baseline. “The baseline analysis of the KAB of the target population would provide better insight on the ‘on-ground’ situation and can guide the development of the intervention programme”. We have included a justification of recruiting class 6th students under the Introduction section: “Recruitment of sixth graders was done as the World Health Organization defines that adolescent age begins at 10 years. When a child enters adolescence, there is a change in explorative and emotive behaviors due to the shift from parental dependence to independence. Adolescents are vulnerable as they gain more control of what, when, how, and where they eat; moreover, their preferences and personal choices may gain priority over eating habits that are nurtured within the family setting”. Methods: It is a well-planned and thoroughly designed methodology, keeping in mind all permissions, clearance and consents requirement of a good study design. The credibility and validity of the tools have also been established. However, certain factors that could not be controlled (home environment) should be mentioned as a limitation. Also, the use of an additional supportive tool (like a food frequency or anthropometric) to decrease the reporting bias of a ‘self-reporting survey’ (also highlighted by the author) should be mentioned as a suggestive approach for future researches. Response: With context to the role of home environment in shaping the individual behavior has been added in the limitation section. We have also added the use of a food frequency questionnaire in the limitation section. Results & Discussion: The results are extensive and logically sequenced with a simple and easy to understand presentation. All areas of interest (KAP of dietary intake, tobacco use and physical inactivity) have been clearly depicted. However, there are certain findings (results of fruit intake, screen time and consumption of outside food) that challenge the traditional findings/beliefs of people’s behaviours that vary due to socioeconomic inequality. This could be due to the bias (subjects giving out socially approved answers) in self-reporting surveys (also listed as a limitation by the author) and can be added as a limitation in the study. Response: This information already exists in the limitation section: “The self-reported method utilised (i.e., survey) also may have led to skewed estimates of dietary intake, physical activity and tobacco use patterns among the students due to the fact that adolescents would have wanted to report positive health behaviours (social desirability - reporting bias).”. Conclusion: The findings of this project correctly identify the need for school-based lifestyle intervention programmes in terms of national policy planning for the prevention of NCD’s. The conclusion is precise and clear. Response: Thanks." } ] } ]	1	https://f1000research.com/articles/10-544
https://f1000research.com/articles/10-1051/v1	15 Oct 21	{ "type": "Research Article", "title": "Enhancement of digitized X-ray films using Contrast-Limited Adaptive Histogram Equalization (CLAHE)", "authors": [ "Wan-Noorshahida Mohd-Isa", "Joel Joseph", "Noramiza Hashim", "Nbhan Salih", "Joel Joseph", "Noramiza Hashim", "Nbhan Salih" ], "abstract": "Background: Rural clinics still have X-ray facilities that produce physical films, which are sent to the nearest hospital for evaluation. Purchasing digitalization facilities is costly, thus, sending digitized films to the radiologist may be a solution. This can be achieved via digital photo capture. However, there can be different output resolutions that may not be optimized for online diagnosis. This paper investigates if digitized X-ray films can be enhanced using image processing techniques of Contrast-Limited Adaptive Histogram Equalization (CLAHE), Normalized-CLAHE (N-CLAHE) and Min-Max Normalized-CLAHE (MMCLAHE).\n\nMethods: We collected and digitized 21 X-ray films with low, medium, and high resolutions and implemented the CLAHE, N-CLAHE and MMCLAHE image enhancement. These methods introduced a limit to clip the histogram of image intensities so as to reduce any noise amplification before file compression with the Fast Fourier Transform (FFT) and Discrete Cosine Transform (DCT). Quantitative metrics of the Peak Signal-to-Noise Ratio (PSNR) and Mean-Squared Error (MSE) were used to compare the accuracies between digitized and processed X-ray films. A qualitative evaluation was performed by a medical practitioner to validate the accuracy of enhanced digitized X-ray. Results: It had been found that both CLAHE and MMCLAHE provided good average PSNR values of 31dB - 32dB and produced low MSE values compared to N-CLAHE. The results of qualitative evaluation attained 89.9% correct diagnosis on nine randomly selected images. Generally, the evaluation indicated that the results fulfill the acceptable criteria for further evaluation and there seemed to be no pathological differences observed. Conclusion: This paper presented a proof of concept on an implementation of the CLAHE technique and its variations on digitized X-ray films. This paper had shown potential improvements with the proposed enhancement methods that may contribute to an increase efficiency in healthcare processes at rural clinics.", "keywords": [ "digital imaging", "image enhancement", "image compression", "histogram equalization", "telemedicine" ], "content": "Introduction\n\nThe pandemic situation has accelerated digitalization to many countries. However, people living in rural areas are having an inconvenience to access medical technologies due to unavailability of specialists and also shortage of medical equipments.1 In Malaysia rural clinics, for example, there are still X-ray facilities that produce physical X-ray films. The films are then sent using courier services to the nearest available hospital for diagnosis by radiologists, making the whole process becomes inefficient. Moreover, purchasing new digital X-ray facilities for rural clinics may not be cost beneficial since the population numbers are low.\n\nDigital technology has seen growth that leads to telemedicine.2–4 Telemedicine makes use of communication technology in healthcare and can be cost effective. However, telemedicine is not commonly practiced in Malaysia5 despite its potential to address healthcare shortcomings in the rural areas. In this paper, we propose a proof of concept work to digitalize physical X-ray films via digital photo capture so that their digital versions can be sent via email or cloud for evaluation by a radiologist elsewhere. This may improve efficiency in remote diagnosis as well as reducing physical storage.6\n\nA study by1 had described a simple implementation of 70 digitized chest X-ray films using a digital camera with an application of lossy compression technique. The digitized images had been shown in random order to two radiologists for diagnosis at 8 weeks after image capture. Then, lossy compression of different percentages were applied. The compressed images were again shown to the same radiologists in a random order for diagnosis at 8 weeks after the compression. The study achieved a mean percentage of 90% correct diagnosis with compression levels up to 20%. But with a compression of 40% and 50%, the correct percentages were 84% and 80%, respectively.\n\nA similar study by7 used an 8-megapixel smartphone on 44 X-ray films consisting of 16 chest and 28 musculo-skeletal. The digitize X-ray films were shown in random order to two radiologists for diagnosis through a LCD cellphone. The accuracy of diagnosis was reported as high. Also it was reported that a diagnosis was difficult when involving uncommon and difficult pathology cases.\n\nThese studies purely implemented digital photo capture of X-ray films and performed diagnosis without requiring online transmission. For sending images over the network, small file size with good resolution is best. However, there can be different resolutions due to differences in digital imaging software and hardware, hence, producing different qualities of digital X-ray films.8 These variations open an opportunity to explore image enhancement techniques on images of digitized X-ray films.9–15 In this paper, we investigate the use of CLAHE image processing techniques as a proof of concept and we validate the output via quantitative metrics and qualitative evaluation by a medical practitioner.\n\n\nMethods\n\nFigure 1 shows the flow of our proposed methodology, which includes, data collection and digitalization, image enhancement and compression, and evaluation. These would be discussed in the preceding sections.\n\nThis research project had received approval from the Multimedia University Research Ethics Committee (EA2242021).\n\nWe selected 21 upright chest X-ray images consisting of multiple abnormal diagnoses from multiple online databases.16 Then, we printed these online sources to obtain the physical chest X-ray films (CXR). After that, the CXR were captured using an Apple iPhone XS Max. To alter the image resolutions, the images were taken using a third-party application, DSLR Camera.17 An X-ray illuminator (Victor Steel VS148-A) was used as shown in Figure 2.\n\nThe setup in Figure 2 was done in a well-lit surrounding. A view box was placed on a flat surface approximately 30 cm away from a tripod mounted with a smartphone. A CXR was clipped to the view box in a side-view position with the illuminated area placed facing the smartphone. The position of the tripod head was adjusted to ensure the CXR filled the phone display and the personal details at the corner side were not in view. The flashlight setting was turned off to avoid reflections. The ISO speed was fixed to ISO-24 with an exposure time fixed to 1/100 seconds. Images were taken at High, Medium, and Low resolutions for a total of 63 digitized CXR. These images were stored as a JPEG format and unnecessary details were cropped using Windows 10 Photos application.\n\nHistogram Equalization (HE) is the commonly used method for image enhancement. In HE the contrast of an image is enhanced by modifying the distribution of pixel intensities. By distributing pixel intensities equally to each histogram, a global equalization is achieved. Thus, HE tends to over-enhance parts of an image that adds unnecessary artifacts and may increase image noise.11\n\nAdaptive Histogram Equalization (AHE) is an improvement of HE. AHE improves local contrast by dividing an image into blocks and applying computations to every block.11 Bilinear interpolation is used to combine all blocks into one image. A study conducted by10 applied AHE on 50 microfocus X-ray images. It showed that 80% of these had significant increase in contrast and detailing. However, AHE had a slow processing speed and enhanced image noise.\n\nContrast-Limited Adaptive Histogram Equalization (CLAHE) is an extension of AHE. Similar to AHE, CLAHE uses blocks in an image. The only difference is the addition of a contrast limit to reduce the noise amplification by clipping the histograms.14 The limit is a predefined value prior to calculation of a Cumulative Distributive Function (CDF). Histograms that exceed the clipped value are redistributed evenly to other histograms instead of removal. A study by9 found that images produced from CLAHE had higher pixel values than that of original images and had brought out more details and structure to the images. Both9 and14 had agreed that CLAHE was good at maintaining image brightness level.\n\nA study by18 proposed Normalized-CLAHE (N-CLAHE) that involved both Normalization function and CLAHE. In this method, a Logarithmic-Normalization function calculated output pixel intensity values by applying a Logarithmic function to input pixel intensity values. Normalization corrected the global intensity value before CLAHE computed the local contrast enhancement value.\n\nThe Min-Max Normalized-CLAHE (MMCLAHE) is a method that uses Min-Max values for the pixel intensity values. The pixel intensity is calculated using a ratio between new maximum (newMax) and new minimum (newMin) intensities to previous maximum (Max) and previous minimum (Min) intensity values.\n\nImage compression is a process to reduce large-sized images into smaller-sized images without degrading the image quality. In this work, it is required to compress the digitized CXR since these need to be sent to the specialists using a low or moderate network bandwidth. Compression works by reducing the number of bits to represent an image. It has three types, lossless, lossy and hybrid.19 A hybrid compression combines both lossless and lossy based on region of interest (ROI) and non-ROI of images.20 However, in the absence of subject experts, the ROI could not be determined21–23 as in our work.\n\nA lossless compression produces high-fidelity reproduction of images but may tend to have large file sizes. On the other hand, lossy compression produces good compression for use in telemedicine that suits rural areas with limited internet connectivity. Thus, in this research, standard lossy image compression techniques such as the Fast Fourier Transform (FFT) and Discrete Cosine Transform (DCT) are used.24\n\nThe Peak Signal-to-Noise Ratio (PSNR) and Mean-Squared Error (MSE) are our quality metrics for comparing the before and after processing of the digitized CXR. A higher PSNR value means better quality of the compressed image. A lower MSE value means less errors contain in the image.\n\nTo validate the diagnosis of the digitized CXR, we invited a medical practitioner to perform a blind evaluation. A randomly selected processed CXR with specific diagnosis were sent to the evaluator. The evaluator was asked to confirm the diagnosis by answering a series of “yes/no” questions and the responses were recorded.\n\n\nResults and discussion\n\nTable 1 shows the average PSNR and MSE values for each image enhancement and image compression pairs. From this table the results show that CLAHE-DCT gave the lowest average MSE of 35.59 and the highest average PSNR values of 32.85dB compared to other methods. Nevertheless, CLAHE-FFT, MMCLAHE-DCT and MMCLAHE-FFT also attained comparable good values of PSNR at 32.67dB, 31.98dB, and 31.80dB, respectively. Also CLAHE-FFT produced comparable low average MSE results at 36.96. Both MMCLAHE-DCT and MMCLAHE-FFT attained MSE values of 57.16 and 58.71, respectively.\n\nFigure 3 shows samples of processed CXR with a pneumothorax at High resolution level. They are labelled as A, B, C, D, E, F from top-left to bottom-right. Samples of processed CXR for CLAHE and MMCLAHE are in Figure 3 (A, B, C, and D).\n\nOn the contrary, both N-CLAHE FFT and N-CLAHE DCT attained the highest MSE and the lowest PSNR values at about 6533 and 10dB, respectively. It can be seen in Figure 3 (E and F) that the N-CLAHE method had produced an overexposure on the digitized CXR.\n\nFor qualitative evaluation, samples of Figure 3 were presented to the medical practitioner. Generally, the practitioner was extremely satisfied with the quality of the digitized and processed CXR. Images C and D (using MMCLAHE) were said to be better than Images A and B (using CLAHE). Images E and F (using N-CLAHE) were the worst among all due to overexposure.\n\nTable 2 shows the results of blind qualitative evaluation where nine images were randomly selected for diagnosis. All diagnosis of the presented digitized CXR had managed to be identified without any difficulties except for Image 7. The true diagnosis for Image 7 was Normal. However, the diagnosis reported that there might be some abnormalities present even though Image 7 was a High resolution MMCLAHE-FFT enhanced image. Thus, a simple accuracy attained by the qualitative evaluation is 89.9%, which is comparable to the work of Refs. 1 and 7.\n\nAlso, for Image 6, which was a Medium resolution CLAHE-FFT, it was highlighted that further testing would be required to confirm the diagnosis of Infiltration. Nonetheless it was generally concluded that no pathological differences were observed between all processed CXR and all digitalized CXR.\n\n\nConclusions\n\nIn this paper, we presented the results of image processing techniques using CLAHE and its variations to enhance digitized X-ray films. Twenty-one physical X-ray films had been digitized via mobile phone capture at Low, Medium, and High resolutions. Three different image enhancement methods with two different image compression techniques had been compared. Results of quantitative evaluation indicated that N-CLAHE may not be a suitable method due to producing an overexposure. Also, the performance of DCT or FFT did not affect the quality of output.\n\nResults of qualitative evaluation further validated the accuracy of the digitized X-ray with a medical practitioner. It had been found that the accuracy of correct diagnosis is close to the work by others in literatures. The overall presentation of the digitized X-ray had been found to be acceptable though some images might require further testing to confirm a diagnosis. Nevertheless, this paper had shown potential improvements with the proposed methods of enhancement that in turn may contribute to an increase efficiency in healthcare processes at rural clinics.\n\n\nAuthor contributions\n\nMohd-Isa W: Conceptualization, Formal Analysis, Project Administration, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Joseph J: Data Curation, Investigation, Methodology, Investigation; Hashim N: Conceptualization, Project Administration; Salih N: Formal Analysis, Validation.\n\n\nData availability\n\nFigshare: CLAHE-Enhanced X-ray Images DOI: https://doi.org/10.6084/m9.figshare.c.5490822.25\n\nThis project contains the following data:\n\n- A collection of processed chest X-ray digitized images with CLAHE, MMCLAHE, N-CLAHE techniques and FFT and DCT compression.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\nFigshare. CLAHE-Enhanced X-ray Images\n\nThis project contains the following data:\n\n- Processed images after digitization of physical X-ray films using CLAHE, MMCLAHE, N-CLAHE, and DCT and FFT compression.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank the medical practitioner involved for assisting with evaluation of the results.\n\n\nReferences\n\nKhodaie M, Askari A, Bahaadinbeigy K: Evaluation of a very low-cost and simple teleradiology technique. J. Digit. Imaging. 2015; 28(3): 295–301. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThati J, Sravanthi SB, Narayana YV: Optimized compression techniques for telemedicine therapeutic images. Proceedings of 2016 International Conference on Advanced Communication Control and Computing Technologies, ICACCCT 2016. 2016; 978: 7–11. Publisher Full Text\n\nNasifoglu H: Medical Image Compression for Telemedicine Applications. Archives in Biomedical Eng. & Biotechn. 2019; 2(1): 2–4. Publisher Full Text\n\nConway S: Article - Teleradiology and telemedicine: They are not equal.2016. Reference Source\n\nSu-Lyn B: Allow Telemedicine, Malaysia Urged, After Study Finds Higher Covid-19 Risk For Cancer Patients\|CodeBlue.2020. Reference Source\n\nSackey AH: A Low-Cost High-Quality Mobile X-ray Film Digitiser with Storage Facilities. SN Comprehensive Clin. Med. 2019; 1(1): 1–3. Publisher Full Text\n\nLicurse MY, Kim SH, Kim W, et al.: Comparison of diagnostic accuracy of plain film radiographs between original film and smartphone capture: a pilot study. J. Digit. Imaging. Dec 2015; 28(6): 646–653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWestberg M, Vasko T, Owen LS, et al.: Personal smartphones for neonatal diagnostic imaging: A prospective crossover study. J. Paediatr. Child Health. 2017; 53(4): 343–347. PubMed Abstract \| Publisher Full Text\n\nSaenpaen J, Arwatchananukul S, Aunsri N: A comparison of image enhancement methods for lumbar spine X-ray image. ECTI-CON 2018-15th International Conference on Electrical Engineering/Electronics, Computer, Telecommunications and Information Technology. 2019; 798–801. Publisher Full Text\n\nStaroverov NE, Gryaznov AY, Kholopova ED: Digital X-ray image processing with using the adaptive histogram equalization and adaptive background correction. 13th Russian-German Conference on Biomedical Engineering, XIII RGC, 23-25 May 2018, Aachen, Germany. Publisher Full Text\n\nKaur A, Singh C: Contrast enhancement for cephalometric images using wavelet-based modified adaptive histogram equalization. Appl. Soft Comput. 2017; 51: 180–191. Publisher Full Text\n\nMagudeeswaran V, Singh JF: Contrast limited fuzzy adaptive histogram equalization for enhancement of brain images. Int. J. Imaging Syst. Technol. 2017; 27(1): 98–103. Publisher Full Text\n\nQiu J, Harold Li H, Zhang T, et al.: Automatic x-ray image contrast enhancement based on parameter auto-optimization. J. Appl. Clin. Med. Phys. 2017; 18(6): 218–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh RP, Dixit M: Histogram equalization: a strong technique for image enhancement. Int. J. Sig. Process. Image Process. Pattern Recogn. 2015; 8(8): 345–352. Publisher Full Text\n\nAl Wadud MA, Kabir MH, Dewan MAA, et al.: A dynamic histogram equalization for image contrast enhancement. IEEE Trans. Consum. Electron. 2017; 53(2): 593–600. Publisher Full Text\n\nWang X, Peng Y, Lu L, et al.: ChestX-Ray8: Hospital-Scale Chest X-Ray Database and Benchmarks on Weakly-Supervised Classification and Localization of Common Thorax Diseases. 2017 IEEE Conference on Computer Vision and Pattern Recognition (CVPR). 2017; 3462–3471. Publisher Full Text\n\nScichilone F: DSLR Camera App.2014.Reference Source\n\nKoonsanit K, Thongvigitmanee S, Pongnapang N, et al.: Image enhancement on digital X-ray images using N-CLAHE. 10th Biomedical Engineering International Conference 2017. January 2017; 1–4. Publisher Full Text\n\nSatone K, Deshmukh A, Ulhe P: A review of image compression techniques. Proceedings of the International Conference on Electronics, Communication and Aerospace Technology, ICECA 2017. Jan 2017; 97–101. Publisher Full Text\n\nAli AH: Medical Image Data Compression using Hybrid Methods. ARPN J. Eng. App. Sci. 2018; 13(5): 1877–1886.Reference Source\n\nAziz SA, Sam SM, Mohamed N, et al.: A review on region of interest-based hybrid medical image compression algorithms. Telkomnika Telecommunication Computing Electronics and Control. 2020; 18(3): 1650–1657. Publisher Full Text\n\nKamargaonkar C, Sharma M: Hybrid medical image compression method using SPIHT algorithm and Haar wavelet transform. International Conference on Electrical, Electronics, and Optimization Techniques, ICEEOT 2016. 2016; 897–900.Publisher Full Text\n\nKaur M, Wasson V: ROI Based Medical Image Compression for Telemedicine Application. Procedia Computer Science. 2015; 70: 579–585. Publisher Full Text\n\nHussain AJ, Al-Fayadh A, Radi N: Image compression techniques: A survey in lossless and lossy algorithms. Neurocomputing. 2018; 300: 44–69. Publisher Full Text\n\nMohd-Isa: CLAHE-Enhanced X-ray Images. figshare. Collection. 2021. Publisher Full Text" }	[ { "id": "97129", "date": "15 Nov 2021", "name": "Rubita Sudirman", "expertise": [ "Reviewer Expertise signal processing", "image processing", "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\n1. The methods provides some overview on how the digital x-ray images are processed, however, this section is missing the important equations to the algorithm employed to perform the processing.\n2. Statistical analysis and its interpretation are not cited, to show the performance of CLACHE designed.\n3. In the conclusion, some brief numerical results should also be included to strengthen the finding from the study.\n\n4. There is no comparison made to past research, whether in the results or discussion section or in the conclusion. Adding some comparisons would enhance the quality of the paper.\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? Partly", "responses": [] }, { "id": "150520", "date": "20 Sep 2022", "name": "Himanshu Singh", "expertise": [ "Reviewer Expertise signal and image processing", "machine learning" ], "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\nResults of qualitative evaluation further validated the accuracy of the digitized X-ray with a medical practitioner. It had been found that the accuracy of correct diagnosis is close to the work by others in the literature. The overall presentation of the digitized X-ray had been found to be acceptable though some images might require further testing to confirm a diagnosis. Nevertheless, this paper had shown potential improvements with the proposed methods of enhancement that in turn may contribute to an efficiency increase in healthcare processes at rural clinics. This manuscript can be accepted.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-1051
https://f1000research.com/articles/10-1050/v1	15 Oct 21	{ "type": "Case Report", "title": "Case Report: Intracranial hypertension in an adult-onset Still’s disease patient initially presented with prolonged fever", "authors": [ "Gerasimos Eleftheriotis", "Elias Skopelitis", "Elias Skopelitis" ], "abstract": "This article describes the case of a 19-year-old woman who presented with prolonged fever, positive antinuclear antibodies (ANA) and splenomegaly. Pulmonary infiltrates were discovered and the patient was treated for community-acquired pneumonia, with no clinical amelioration. A more thorough evaluation was subsequently made, revealing elevated serum IgE and IgG4 levels and negative ANA tested by the hospital’s laboratory with two methods. During hospitalization thrombocytopenia, liver function test impairment, and evanescent rash during some febrile episodes developed. Vomiting also presented without any concomitant symptoms or signs; a funduscopic examination was consequently ordered, showing bilateral papilledema. Brain imaging was totally normal but a lumbar puncture revealed elevated opening pressure and lymphocytic pleocytosis along with low cerebrospinal fluid lactate dehydrogenase (CSF LDH). The patient was empirically treated with antimicrobials, dexamethasone, and acetazolamide and had immediate clinical and laboratory improvement. Diagnostic workup, however, was negative for an infectious agent; antimicrobials were ceased but the patient continued to improve. Adult-onset Still’s disease (AOSD) was considered as the working diagnosis because the patient fulfilled Yamaguchi criteria, responded to corticosteroids, and an alternative diagnosis was lacking. Nevertheless, because of the patient’s atypical features a trial to discontinue dexamethasone was undertaken, leading to immediate recurrence; the possibility of a self-limiting viral illness was excluded. Thrombocytopenia was attributed to hemophagocytic lymphohistiocytosis (HLH) that complicated AOSD. Corticosteroid reinitiation combined with methotrexate fully controlled all clinical and laboratory parameters. One month later papilledema had disappeared and the patient remained symptom-free even without acetazolamide. To our knowledge, this is the first report in the literature of an AOSD case presenting intracranial hypertension without cerebral imaging abnormalities and neurological or meningeal symptoms and signs, as well as with the initial observation of serum IgG4 elevation. A classic regimen combined with acetazolamide led to a positive outcome.", "keywords": [ "Adult onset Still’s disease", "intracranial hypertension", "papilledema", "cerebrospinal fluid pleocytosis", "IgE", "IgG4", "hemophagocytic lymphohistiocytosis" ], "content": "Introduction\n\nAOSD is an autoinflammatory, multisystemic disease that mainly presents in one of two distinctive clinical patterns, one with prominent systemic manifestations and another manifested predominantly with articular symptoms. These two patterns can overlap and either of these can be monocyclic (lasting from weeks to months), polycyclic, or chronic. Nervous system involvement has been reported in 7-12% of cases, manifesting with seizures, cranial nerve palsies, encephalitis, meningitis, stroke, demyelinating encephalopathy, peripheral neuropathy, and Miller Fisher-like syndrome.1-8\n\nOne of the most serious AOSD complications is HLH, also termed as macrophage activation syndrome in patients with autoimmune or autoinflammatory disorders. Approximately 14% of AOSD patients present that manifestation according to two retrospective studies including 176 patients, either at initial diagnosis or during follow-up, which significantly decreases their survival with a hazard ratio of 12.71, compared with non-HLH AOSD patients.9,10 Notably, AOSD was complicated by HLH in eight out of 20 patients in a French case series of AOSD patients requiring intensive care unit admission.11\n\nOn the other hand, intracranial hypertension can be associated with intracranial lesion, hydrocephalus, meningitis, encephalitis, encephalopathy, or with none of these. The rest of the cases are termed as idiopathic (pseudotumor cerebri) and can be caused by obstruction of cerebral venous drainage, medications and drugs used for other indications (e.g. insecticides) or other hematologic, neurological, endocrine or systemic disorders. Symptoms and signs attributed to intracranial hypertension can be bilateral papilledema, headache, vomiting, dizziness, abducens nerve palsy, tinnitus, visual or cognitive impairment (episodic or constant) and neck or back pain.\n\n\nCase presentation\n\nA 19-year-old Caucasian woman presented to the emergency department due to multiple febrile episodes the previous two weeks, along with loss of appetite and serious fatigue (she was able to walk only with assistance). Fever responded to paracetamol.\n\nThe patient was a computer engineering student. Her medical history, as well as her travel and sexual history were unremarkable. Her father had arterial hypertension and her mother had depression; no other significant information from the family history could be extracted. She denied special alimentary habits or close contact with people having fever. Some days ago, the patient had visited an Internal Medicine clinic where cefuroxime axetil 500 mg bid for 10 days was prescribed, but symptoms didn’t ameliorate. Blood tests had been ordered (Table 1), with the only notable result being the positive ANA test on a titer of 1/160, performed with enzyme-linked immunosorbent assay (ELISA). An upper abdominal ultrasound had been also performed showing splenomegaly, with maximum craniocaudal spleen diameter of 15 cm.\n\nVital signs on admission were as follows: 94 heart beats per minute, axillary temperature 39.2oC, blood pressure 110/70 mm Hg, oxygen saturation 100% and 24 breaths per minute while breathing ambient air and being totally alert. Heart auscultation revealed a mild systolic murmur, equally audible over the entire precordium. Searching for palpable lymph nodes revealed two non-tender, mobile right axillary nodes, sized approximately 1 cm2. Joint examination showed that all big joints were slightly warm with no other abnormal signs, while the patient was free of articular symptoms.\n\nA 12-lead electrocardiogram, upright chest radiograph and urinalysis were normal. From a complete blood count, hypochromic and microcytic anemia was found along with low red blood cells, high erythrocyte sedimentation rate, and C-reactive protein (CRP) (Table 2). The findings above were considered as manifestations of anemia due to inflammation because they were accompanied by low serum iron and normal serum ferritin. A transthoracic cardiac ultrasound was ordered for heart murmur evaluation; it was totally normal and the murmur was subsequently attributed to hyperdynamic circulation in the context of febrile illness.\n\nThe 3rd hospital day fever continued and the patient developed thrombocytopenia and liver function test impairment (Table 2). The day after, she underwent an abdominal and chest computed tomography (CT), which revealed the already known splenomegaly and mild axillary lymphadenopathy, as well as bilateral pulmonary infiltrates, especially on the upper lung fields (Figure 1). With these findings doxycycline 100 mg bid and ceftriaxone 2 g qd were initiated with a working diagnosis of community-acquired pneumonia.\n\nApproximately 24 hours after the first dose of antibiotics a mild, asymptomatic malar-papular reddish rash was discovered on clinical examination during a febrile episode and totally subsided when body temperature returned to normal range (Figures 2 and 3). This sign was also occurred in three other febrile episodes the next six days. Because fever persisted after four days of antibiotic treatment, despite a significant reduction of CRP values at the eighth hospital day (Table 2), the diagnostic workup was expanded to a thorough investigation for infectious and autoimmune diseases. All tests were normal, including ANA (performed twice with ELISA and indirect immunofluorescence by the hospital’s Laboratory of Immunology), except of an elevation of serum IgE and IgG4 immunoglobulins (Table 3).\n\nDue to the aforementioned inconclusive results, the patient was scheduled to undergo a bronchoscopy in order to obtain a bronchoalveolar lavage for further evaluation of pulmonary infiltrates. Then, she had two episodes of vomiting without any concomitant symptoms or signs. Given the lack of any other obvious explanation, it was supposed to be a sign of intracranial hypertension. For that reason, an ophthalmologic consultation was obtained in order to perform a funduscopic examination. Bilateral papilledema was found; the rest of the examination was normal. With this finding the patient urgently underwent brain CT scan and brain CT venography in order to exclude venous sinus thrombosis, with no abnormal findings. A lumbar puncture was performed afterwards, revealing a very high CSF opening pressure (51 cm H2O), low CSF LDH (20 IU/L) and lymphocytic pleocytosis (Table 4).\n\nDoxycycline was discontinued because tetracycline antibiotics can rarely elevate intracranial pressure, although more prolonged administration is usually a prerequisite.12,13 Ceftriaxone dosage was doubled to achieve better CSF concentrations and the following drug regimen was empirically added: IV ampicillin 2 g q4h for Listeria coverage, IV moxifloxacin 400 mg qd replacing doxyxycline against Staphylococcus aureus and atypical pathogens like Coxiella burnetii that can cause fever along with pulmonary infiltrates and CSF pleocytosis, IV ganciclovir for herpes virus family, IV dexamethasone 8 mg bid to cover immune-mediated disorders and acetazolamide tablets 250 mg qid, following the standard of care for idiopathic intracranial hypertension.\n\nFrom the first day with that new regimen vomiting and fever subsided and the patient felt less fatigued. The next laboratory tests were improved except of a first appearing leucocytosis with neutrophil predominance, which was attributed to dexamethasone use (Table 2). CSF culture was sterile and multiplex CSF PCR (Table 4), as well as CSF cytology and serum West Nile virus antibodies were negative. For that reason blood PCR for Epstein–Barr virus, cytomegalovirus, human herpes virus 6 and 7, adenovirus and enterovirus was ordered, with also negative results.\n\nIn summary, the patient had rapid improvement after empirical treatment initiation, but a specific diagnosis was lacking. Because the workup for infectious causes was negative antimicrobial agents were ceased. Dexamethasone and acetazolamide dosage was decreased because of rapid improvement (4 mg bid and 250 mg tid respectively), with the patient continuing to be free of febrile episodes and in even better general condition. Meanwhile, the patient underwent a brain MRI with five days of delay until she got her dental braces off, with no abnormal findings.\n\nIn order to differentiate a self-limited viral infection from a non-infectious inflammatory process dexamethasone was discontinued. Two days after corticosteroid cessation fever reappeared and laboratory parameters deteriorated (Table 2). The aforementioned clinical course advocated that the diagnosis was an autoinflammatory or autoimmune disorder, with AOSD being first on the differential diagnosis because of the evanescent rash. Yamaguchi criteria were applied due to their highest sensitivity; the patient fulfilled six criteria, with two of them being major (fever above 39.2oC for >1 week and typical rash the two major criteria and enlarged lymph nodes, splenomegaly, abnormal liver function tests, negative ANA and rheumatoid factor the four minor), thus establishing AOSD diagnosis, which requires at least five criteria, including at least two major. Dexamethasone was subsequently reinitiated along with methotrexate 10 mg per week, folic acid 5 mg per week, calcium carbonate/cholecalcipherol 1.000 mg/800 iu fixed combination qd and trimethoprim/sulfamethoxazole (800 + 160) mg thrice weekly for Pneumocystis jirovecii prophylaxis. Influenza and Streptococcus pneumoniae vaccination was performed, too. Fever subsided again from the next day and CRP values declined (Table 2). Meanwhile, a gastrocnemius muscle biopsy was performed to exclude sarcoidosis more strongly based on studies from Andonopoulos et al and Yanardag et al; the biopsy was normal.14,15\n\nThe patient was discharged and had been followed-up closely at the outpatient clinic. These visits included frequent funduscopic examinations from the department of ophthalmology. She remained asymptomatic and one month after diagnosis of intracranial hypertension papilledema had disappeared; acetazolamide was subsequently stopped. Papilledema or symptoms attributed to intracranial hypertension never presented thereafter. A repeated chest CT was performed 20 days after the first, showing complete resolution of pulmonary infiltrates (Figure 4).\n\n\nDiscussion\n\nThe case of AOSD analyzed herein combined ordinary AOSD manifestations, such as evanescent rash and splenomegaly, with rare or unique manifestations. It should be emphasized that AOSD is a diagnosis of exclusion, even if the patient fulfills the applied criteria.\n\nPulmonary infiltrates is a relatively rare AOSD manifestation, usually resolving after successful treatment. It is found in 12.25% of the AOSD patients at the time of diagnosis according to a recent cohort from Italy.16 Besides that, the patient had normal white blood cell count and low platelets, with the exact opposite being a common finding in the majority of AOSD cases. That differentiation is usually found when AOSD gets complicated by reactive HLH, a serious and often detrimental complication. Further investigation of the patient included a bone marrow aspiration and biopsy, which revealed toxic bone marrow alterations reactive to systemic disease along with megakaryocytic and erythroid lineage suppression. Taking all parameters into account, HLH-score was calculated, resulting in a probability of 58% for HLH diagnosis.17 A high level of alertness for HLH recognition is needed, as it significantly increases mortality regardless of the precipitating disorder.\n\nA classic AOSD regimen (corticosteroids plus methotrexate) was opted for without adding anakinra or other biologics because of rapid response, choosing dexamethasone over other corticosteroids because it was the drug of choice in many studies for HLH and the HLH-2004 protocol, too.18 Corticosteroids can be also used as monotherapy in AOSD, although an initial drug combination strategy is usually followed when risk factors for relapse after corticosteroid tapering exist like young age, splenomegaly, marked erythrocyte sedimentation rate elevation and very low glycosylated ferritin.\n\nConcerning IgE elevation, its real prevalence and clinical significance is still unknown in AOSD. This finding has been anecdotally reported in the clinical course of AOSD, suggesting that it is either is pretty rare between AOSD patients or that IgE levels are not measured in a lot of AOSD cases, or both.19,20 Yokoi et al in their article had speculated that IgE levels in AOSD are dependent on disease activity.19\n\nElevated serum IgG4, on the other hand, has been primarily associated with IgG4-related disease, which is characterized by tissue infiltration from IgG4-producing B and plasma cells with concomitant serum IgG4 elevation in the majority of cases. However, recent data indicates that serum IgG4 levels may be also elevated in a variety of other diseases like helminthic infections, asthma and other eosinophilic disorders, primary sclerosing cholangitis, chronic hepatitis and liver cirrhosis, systemic vasculitides and other connective tissue diseases, cholangiocarcinoma, pancreatic cancer, lymphoma, plasma cell disorders, and multicentric Castleman disease, but has never been reported in AOSD before.21-24 Future research could better investigate this potential association.\n\nThe patient also presented some biochemical parameters found usually at IgG4-related disease except of merely having elevated serum IgG4. Specifically, IgG4/IgG ratio was above 0.114, element having great value for IgG4-related disease diagnosis according to Xia, Fan and Liu and IgG4/IgG1 ratio was above 0.24, a criterion used from Boonstra et al as a tool to discriminate IgG4-associated cholangitis from primary sclerosing cholangitis in patients with mild serum IgG4 elevation.23,24 However, IgG4-related disease was excluded based on clinical and laboratory grounds. These included high fever along with a rash and marked elevation of CRP values, the latter largely excluding IgG4-related disease.25 In addition, hypothesizing this was an IgG4-related disease case affecting central nervous system among others, the lack of characteristic pachymeningitis signs on brain MRI suggests an alternative diagnosis.\n\nThis case is the first AOSD case in the literature presenting intracranial hypertension with normal cerebral imaging and without neurological deficits or symptoms and signs of meningeal irritation. The possibility of idiopathic intracranial hypertension associated with doxycycline was excluded because of CSF findings. CSF pleocytosis in AOSD is neutrophilic in the majority of the cases, although lymphocytic pleocytosis has also been reported.6 We believe this case indicates that intracranial hypertension is a rare manifestation of AOSD in the context of subclinical meningitis. This is the reason why acetazolamide was added, extrapolating data from acetazolamide administration in cases of intracranial hypertension associated with systemic lupus erythematosus.26\n\nAnother interesting finding was the patient’s low CSF LDH levels (20 IU/L), as data from a 2009 study by Vázquez et al suggest that even in critically ill patients without brain trauma and decreased level of consciousness CSF LDH levels ≤40 IU/L are associated with a non-structural etiology of their symptoms (i.e., underlying diagnosis other than stroke, intracranial space-occupying lesion, encephalitis or meningitis).27 In addition, combining data from Quaglia et al and Lee et al who analyzed specimens from 157 patients with bacterial, viral or tuberculous meningitis, no sample had CSF LDH levels lower than 33 IU/L.28,29 Further studies should assess the prevalence and prognostic role of intracranial hypertension in adult-onset Still’s disease patients and if acute non-infectious, non-neoplastic meningitis is also accompanied with elevated CSF LDH.\n\n\nConclusion\n\nThe patient fulfilled the diagnostic criteria for AOSD. Treatment response to dexamethasone was dramatic, both initially and after the (almost immediate) recurrence of symptoms following the early trial to stop corticosteroids. This case report highlights that symptoms of intracranial hypertension may be the only ones when central nervous system is affected in AOSD. Even if acetazolamide was added to immunosuppressants, following dosing strategies for the management of idiopathic intracranial hypertension, it is not known if that medication contributed to positive outcome; future data should assess its role. Physicians taking care of patients with AOSD should be aware of that potential complication and thus promptly perform a funduscopic examination and a lumbar puncture if clinical suspicion exists.\n\n\nAuthor roles\n\nEleftheriotis G: Conceptualization, Methodology, Validation, Writing – Original Draft; Skopelitis E: Validation, Supervision, Writing – Review & Editing\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.\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\nOhta A, Yamaguchi M, Kaneoka H, et al.: Adult Still’s disease: review of 228 cases from the literature. J Rheumatol. 1987 Dec; 14(6): 1139–46. PubMed Abstract\n\nOhta A, Yamaguchi M, Tsunematsu T, et al.: Adult Still’s disease: a multicenter survey of Japanese patients. J Rheumatol. 1990 Aug; 17(8): 1058–63. PubMed Abstract\n\nPouchot J, Sampalis JS, Beaudet F, et al.: Adult Still’s disease: manifestations, disease course, and outcome in 62 patients. Medicine (Baltimore). 1991 Mar; 70(2): 118–36. PubMed Abstract \| Publisher Full Text\n\nMarie Ι, Levesque H, Perraudin N, et al.: Aseptic meningitis and cranial nerve palsy revealing adult-onset Still’s disease. Clin Infect Dis. 1999 Jul; 29(1): 220–1. PubMed Abstract \| Publisher Full Text\n\nDesai SS, Allen E, Deodhar A: Miller Fisher syndrome in adult onset Still’s disease: case report and review of the literature of other neurological manifestations. Rheumatology (Oxford). 2002; 41(2): 216–22. PubMed Abstract \| Publisher Full Text\n\nAkkaraVeetil BM, Yee AH, Warrington KJ, et al.: Aseptic meningitis in adult onset Still’s disease. Rheumatol Int. 2012 Dec; 32(12): 4031–4. PubMed Abstract \| Publisher Full Text\n\nJie W, Miao L, Yankun S, et al.: Demyelinating encephalopathy in adult onset Still’s disease: case report and review of the literatures. Clin Neurol Neurosurg. 2013; 115(10): 2213–16. PubMed Abstract \| Publisher Full Text\n\nGoh Y, Wong VY, Tan WL, et al.: An unusual cause of acute ischemic stroke: adult onset Still’s disease. J Thromb Thrombolysis. 2020; 49(1): 141–144. PubMed Abstract \| Publisher Full Text\n\nGerfaud-Valentin M, Maucort-Boulch D, Hot A, et al.: Adult-onset still disease: manifestations, treatment, outcome, and prognostic factors in 57 patients. Medicine (Baltimore). 2014; 93(2): 91–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuscitti P, Iacono D, Ciccia F, et al.: Macrophage Activation Syndrome in Patients Affected by Adult-onset Still Disease: Analysis of Survival Rates and Predictive Factors in the Gruppo Italiano di Ricerca in Reumatologia Clinica e Sperimentale Cohort. J Rheumatol. 2018 Jun; 45(6): 864–872. PubMed Abstract \| Publisher Full Text\n\nNéel A, Wahbi A, Tessoulin B, et al.: Diagnostic and management of life-threatening Adult-Onset Still Disease: a French nationwide multicenter study and systematic literature review. Crit Care. 2018 Apr 11; 22(1): 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLochhead J, Elston JS: Doxycycline induced intracranial hypertension. BMJ. 2003 Mar 22; 326(7390): 641–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedman DI, Gordon LK, Egan RA, et al.: Doxycycline and intracranial hypertension. Neurology. 2004 Jun 22; 62(12): 2297–9. PubMed Abstract \| Publisher Full Text\n\nAndonopoulos AP, Papadimitriou C, Melachrinou M, et al.: Asymptomatic gastrocnemius muscle biopsy: an extremely sensitive and specific test in the pathologic confirmation of sarcoidosis presenting with hilar adenopathy. Clin Exp Rheumatol Sep-Oct. 2001; 19(5): 569–72. PubMed Abstract\n\nYanardag H, Tetikkurt C, Bilir M: Clinical and prognostic significance of muscle biopsy in sarcoidosis. Monaldi Arch Chest Dis. 2018 Apr 30; 88(1): 910. PubMed Abstract \| Publisher Full Text\n\nRuscitti P, Berardicurti O, Iacono D, et al.: Parenchymal lung disease in adult onset Still’s disease: an emergent marker of disease severity - characterisation and predictive factors from Gruppo Italiano di Ricerca in Reumatologia Clinica e Sperimentale (GIRRCS) cohort of patients. Arthritis Res Ther. 2020 Jun 22; 22(1): 151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFardet L, Galicier L, Lambotte O, et al.: Development and Validation of the HScore, a Score for the Diagnosis of Reactive Hemophagocytic Syndrome. Arthritis Rheumatol. 2014 Sep; 66(9): 2613–20. PubMed Abstract \| Publisher Full Text\n\nHenter JI, Horne A, Aricó M, et al.: HLH-2004: Diagnostic and Therapeutic Guidelines for Hemophagocytic Lymphohistiocytosis. Pediatr Blood Cancer. 2007 Feb; 48(2): 124–31. PubMed Abstract \| Publisher Full Text\n\nYokoi K, Hosoi E, Nakanishi M, et al.: Increased serum IgE level and interleukin-4 release from cultured lymphocytes from a patient with adult onset Still’s disease. Ann Rheum Dis. 1995 Sep; 54(9): 752–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkkurt ZM, Bozkurt M, Uçmak D, et al.: Atypical cutaneous features in adult onset Still’s disease. Indian J Dermatol Venereol Leprol May-Jun. 2014; 80(3): 250–3. Publisher Full Text\n\nYamamoto M, Tabeya T, Naishiro Y, et al.: Value of serum IgG4 in the diagnosis of IgG4-related disease and in differentiation from rheumatic diseases and other diseases. Mod Rheumatol. 2012 Jun; 22(3): 419–25. PubMed Abstract \| Publisher Full Text\n\nOhara H, Nakazawa T, Kawa S, et al.: Establishment of a serum IgG4 cut-off value for the differential diagnosis of IgG4-related sclerosing cholangitis: a Japanese cohort. J Gastroenterol Hepatol. 2013 Jul; 28(7): 1247–51. PubMed Abstract \| Publisher Full Text\n\nBoonstra K, Culver EL, Maillette de Buy Wenniger L, et al.: Serum immunoglobulin G4 and immunoglobulin G1 for distinguishing immunoglobulin G4-associated cholangitis from primary sclerosing cholangitis. Hepatology. 2014 May; 59(5): 1954–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXia CS, Fan CH, Liu YY: Diagnostic performances of serum IgG4 concentration and IgG4/IgG ratio in IgG4-related disease. Clin Rheumatol. 2017 Dec; 36(12): 2769–74. PubMed Abstract \| Publisher Full Text\n\nAbraham M, Khosroshahi A: Diagnostic and treatment workup for IgG4-related disease. Expert Rev Clin Immunol. 2017 Sep; 13(9): 867–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHershko AY, Berkun Y, Mevorach D, et al.: Increased intracranial pressure related to systemic lupus erythematosus: a 26-year experience. Semin Arthritis Rheum. 2008 Oct; 38(2): 110–5. PubMed Abstract \| Publisher Full Text\n\nVázquez JA, Adducci MDC, Monzón DG, et al.: Lactic dehydrogenase in cerebrospinal fluid may differentiate between structural and non-structural central nervous system lesions in patients with diminished levels of consciousness. J Emerg Med. 2009 Jul; 37(1): 93–7. PubMed Abstract \| Publisher Full Text\n\nQuaglia A, Karlsson M, Larsson M, et al.: Total lactate dehydrogenase in cerebrospinal fluid for identification of bacterial meningitis. J Med Microbiol. 2013 Nov; 62(Pt 11): 1772–3. Publisher Full Text\n\nLee SA, Kim SW, Chang HH, et al.: A New Scoring System for the Differential Diagnosis between Tuberculous Meningitis and Viral Meningitis. J Korean Med Sci. 2018 Jun 14; 33(31): e201. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "118883", "date": "13 Jan 2022", "name": "Luke Y. C. Chen", "expertise": [ "Reviewer Expertise HLH", "cytokine storm" ], "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 interesting case of AOSD with intracranial hypertension from Greece by Dr.’s Eleftheriotis and Skopelitis.\nThe case is of interest but I have some suggestions for revision:\nAbstract - currently too long and detailed:\nTry and trim about 300 words or so and keep only essential details.\n\nThe serum ferritin should be reported (currently the text just says it was normal). It is extremely unusual for the serum ferritin to remain normal through the course of AOSD.\n\nWas sIL-2r checked? If so, please report it. Likewise, if other cytokine such as IL-6 were checked, please report them.\n\nDiscussion - Concerning the overlap with between HLH/MAS and AOSD, a few important points are:\nThere is quite a lot of literature on the fact that many patients with AOSD may present with an MAS type of picture. The authors cite the HScore, which is reasonable, but more recent research shows that IL-18 may be an important biomarker to distinguish between AOSD with MAS vs. without.1\n\nAnecdotally, I have not found etoposide-based therapy to be of much value in severe or catastrophic AOSD with MAS. JAK inhibition such as ruxolitinib or tofacitinib may hold more promise in this particular cytokine storm syndrome.2,3\n\nIt should be noted that in cases of catastrophic or life-threatening AOSD, there is emerging evidence that IL-1 inhibition such as anakinra can induce rapid responses.4\nDiscussion - Concerning the mildly elevated serum IgG4 level of of 1.78 g/L, a few points should be made:\nMildly elevated serum IgG4 < 5 g/L is very non specific and is seen in many inflammatory, neoplastic and infectious conditions other than IgG4-RD.5\n\nElevated IgG4 in Still’s disease has been reported elsewhere.6\n\nPlease report the serum protein electrophoresis, if it was done. Polyclonal hypergammaglobulinemia (PHGG), including elevation in IgG4, is common in inflammatory disorders such as AOSD. This is primarily driven by IL-6, which leads to the PHGG.7\nReferences:\nShiga T, Nozaki Y, Tomita D, et al. Usefulness of Interleukin-18 as a Diagnostic Biomarker to Differentiate Adult-Onset Still’s Disease With/Without Macrophage Activation Syndrome From Other Secondary Hemophagocytic Lymphohistiocytosis in Adults. Frontiers in Immunology 2021; 12(4245). Hu Q, Wang M, Jia J, et al. Tofacitinib in refractory adult-onset Still's disease: 14 cases from a single centre in China. Annals of the rheumatic diseases 2020; 79(6): 842-4. Hansen S, Alduaij W, Biggs CM, et al. Ruxolitinib as adjunctive therapy for secondary hemophagocytic lymphohistiocytosis: A case series. Eur J Haematol 2021. Néel A, Wahbi A, Tessoulin B, et al. Diagnostic and management of life-threatening Adult-Onset Still Disease: a French nationwide multicenter study and systematic literature review. Crit Care 2018; 22(1): 88. Varghese JL, Fung AWS, Mattman A, et al. Clinical utility of serum IgG4 measurement. Clin Chim Acta 2020; 506: 228-35. Zhao EJ, Carruthers MN, Li CH, Mattman A, Chen LYC. Conditions associated with polyclonal hypergammaglobulinemia in the IgG4-related disease era: a retrospective study from a hematology tertiary care center. Haematologica 2020; 105(3): e121-e3. Zhao EJ, Cheng CV, Mattman A, Chen LYC. Polyclonal hypergammaglobulinaemia: assessment, clinical interpretation, and management. The Lancet Haematology 2021; 8(5): e365-e75.\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/10-1050
https://f1000research.com/articles/10-303/v1	19 Apr 21	{ "type": "Research Article", "title": "Study of Antibodies to Cytolethal Distending Toxin B (CdtB) and Antibodies to Vinculin in Patients with Irritable Bowel Syndrome", "authors": [ "Maysaa El Sayed Zaki", "Dina Elhammady", "Mona Foda Salama", "Mostafa Abdelsalam", "Asmaa Osama Bakr Osman", "Dina Elhammady", "Mona Foda Salama", "Mostafa Abdelsalam", "Asmaa Osama Bakr Osman" ], "abstract": "Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, categorized into various subtypes. Post-infection IBS may be attributed to the release of cytolethal distending toxin B (CdtB), which cross-reacts with the adhesion protein vinculin responsible for normal intestinal contractility. Objective: This study aims to identify anti-CdtB and anti-vinculin levels in IBS patients compared to healthy control. Subjects and methods: This retrospective case-control study was conducted on 100 patients with IBS, as determined by a questionnaire based on Rome IV criteria, recruited from the outpatient clinics of the Tropical Medicine at Mansoura University Hospital from January 2019 to January 2020. Results: Anti-vinculin and anti-CdtB levels were significantly elevated in patients with IBS (1.58±0.496ng/ml, 2.47±0.60ng/ml) when compared to control subjects (1.13±0.249ng/ml, 2.1±0.24 ng/ml), respectively with P=0.001 for both. Anti-vinculin level was significantly higher in the IBS-D subtype than the other subtypes (P=0.001) while, Anti-CdtB was significantly elevated in IBS-C, IBS-D subgroups compared to control subjects (P=0.001). Conclusion: Findings of the present study support the hypothesis that IBS results from post-infectious disorders initiated by bacterial enteritis. A hypothesis could be applied to all IBS subgroups. On the other hand. These biomarkers might reflect the post-infectious state's severity. These findings need further extensive longitudinal studies in patients with IBS.", "keywords": [ "irritable bowel syndrome", "anti-vinculin", "anti-CdtB", "Rome IV" ], "content": "Introduction\n\nIrritable bowel syndrome (IBS) is a common gut disorder that affects approximately 11% of the global population. 1,2 IBS mainly manifests in abdominal pain with bowel habit changes in the absence of either radiological evidence of associated pathological conditions or detectable chemical and physiological abnormalities. The diagnosis of this clinical condition relies upon Rome criteria. 3–7\n\nTo understand the pathogenesis of irritable bowel syndrome (IBS), previous studies have developed a rat model utilizing infection with Campylobacter jejuni in order to elicit a post-infection phenotype resembling human post-infection IBS (PI-IBS) characterized by apparent changes in the composition of small intestinal microbiota. 8,9 In these studies, progression to IBS was accompanied by the detection of a specific bacterial toxin named cytolethal distending toxin B (CdtB), a potential factor attributing to the pathogenesis of PI-IBS. This was supported by the low incidence of IBS in patients infected with a mutant strain of C. jejuni that lacks CdtB. 8,10\n\nFurthermore, the development of antibodies to CdtB was associated with altering gut microbiota associated with reducing specific interstitial cells of Cajal (ICC). 11,12 These findings are possibly due to the ability of anti-CdtB to cross-react with the host cell adhesion protein vinculin present in interstitial cells of Cajal and the myenteric ganglia that control the normal activity of the intestinal tract, including phase III of inter-digestive motor activity. 13 Absence or decrease in phase III contractions results in small intestinal bacterial overgrowth in animal models and human patients with IBS. 14,15 In this sense, autoimmunity may profoundly affect the host immune response to infections with C. jejuni, subsequently leading to IBS. 16,17 Based on these data, it has been suggested that loss of vinculin in the neuromuscular system of the GIT may be associated with the affection of the gut in animal models of post-infection C. jejuni. Detection of circulating levels of anti-CdtB and anti-vinculin by enzyme-linked immunosorbent assay (ELISA) has been used to identify patients with IBS-D, 18 and to differentiate it from other IBS subtypes. 19\n\nThe present study aims to detect anti-CdtB and anti-vinculin levels in patients with IBS and their possible role in the diagnosis of different IBS subtypes.\n\n\nMethods\n\nThis was a prospective case-control study comprising 100 adult patients aged >18 years with IBS, recruited from the Tropical Medicine Department's outpatient clinics at Mansoura University Hospital from January 2019 to January 2020, in addition to 100 healthy individuals as a control group. According to the actual calculated sample size, we needed to enroll 385 with IBS and 193 for control group at a power of 80% and type I error = 0.05, while at a power of 99% and type I error = 0.01, the minimum number of the patients was 16585 cirrhotic patients and 830 for the control group. This is very hard to achieve in lower economy countries like Egypt. We cannot afford to measure all the parameters in these patients. We had to design the study depending on self-funding without any further support.\n\nPatients were recruited and determined by a questionnaire-based upon the Rome IV criteria, then classified according to their predominant stool composition over 25% of the time: into IBS-C (hard or lumpy stools), IBS-D (loose and watery stools), or IBS-M (a mix of both types). 19 Exclusion criteria included patients with hepatic, renal, or autoimmune diseases, those with history of inflammatory bowel disease, gastrointestinal surgeries, thyroid disorders, diabetes mellitus, and patients with a history of taking antibiotics in the last 30 days.\n\nA 10 ml blood sample was obtained from each subject, which was then divided into three aliquots. Two aliquots were used to determine complete blood counts, and one aliquot was utilized for serum separation to assess complete liver function tests, including alanine transaminase, aspartate transaminase, total bilirubin, total albumin, and the kidney function test creatinine. The third aliquot was overlaid on heparin for plasma separation, and the remaining sera were stored at -20°C to be used for evaluation of anti-vinculin antibodies by laboratory prepared ELISA and anti-CdtB antibodies by commercial ELISA (Creative Diagnostics. 45-16 Ramsey Road Shirley, NY 11967, USA).\n\nAnti-vinculin levels were measured in separated plasma using human vinculin protein in a concentration of 1.21.2 μg/ml (Novoprotein Scientific, Summit, New Jersey, USA) as an antigen. The vinculin was used to coat wells of the plate following overnight incubation in the wells at 4°C with 100 mmol/l borate buffered saline AQ4 at a pH of 8.2 (Sigma-Aldrich). The reaction was blocked by using BSA 3% and incubating for one hour at room temperature, then washing three times with 0.05% PBS and Tween 20 (pH 7.4). Plasma was added after a 1:32 dilution in saline, then antibodies for vinculin (R and D Systems Cat# MAB6896, RRID:AB_10992930), were added as positive control and incubated for one hour at room temperature followed by washing three times with 0.05% PBS and Tween 20 (pH 7.4). Horseradish peroxidase-conjugated secondary antibodies (Millipore–Merck) were added and incubated for one hour at room temperature. After washing, a tetramethylbenzidine substrate solution (BioRad) was used for detection using a micro-plate reader (stat Fax-1200; Awareness Technology, Florida, USA). Optical densities (ODs) were read at 370nm, and the results were interpreted as OD. 12\n\nThe kit applies the double-sandwich technique using a pre-coated microplate with anti-CDTB antigen. Samples and antigen were added into ELISA plate wells and washed out with PBS, after which Avidin-peroxidase conjugates were added to ELISA wells in order. Tetramethyl benzidine (TMB) substrate was used for coloring after PBS thoroughly washed out the reactant. TMB turns blue in peroxidase reaction and finally turns yellow under the action of acid. Optical densities (ODs) were read at 450, and the results were interpreted as ng/ml.\n\n\nStatistical analysis\n\nData are reported as means and standard deviation (SD) or counts and percentages when appropriate. Comparisons between groups were made using t-tests, Mann-Whitney tests, Chi-square, or Fisher exact tests dictated by data type and distribution.\n\nOne-way analysis of variance (ANOVA) was used to test differences between more than two groups. P-value < 0.05 was considered significant for all statistical analyses in this study. All analyses were performed using the Statistical Package of Social Sciences (SPSS) version 22 for Windows (SPSS, Inc., Chicago, IL, USA).\n\n\nResults\n\nThis study included 100 patients with IBS (49 males and 51 females) aged 46.6 ± 6.8 years and 100 healthy controls with a statistically insignificant difference between patients and control regarding age and sex (P = 0.8 and P = 0.6, respectively). Patients were classified according to Rome III criteria into 40 patients with IBS-C, 26 patients with IBS-D, and 34 patients with IBS-M (Figure 1). Laboratory investigations, including ALT, AST, albumin, total bilirubin, hemoglobin, total leucocytes count, platelets, and creatinine, showed non-significant differences between patients and control subjects (P = 0.6, P = 0.5, P = 0.7, P = 0.6, P = 0.99, P = 0.99, and P = 0.58) respectively ( Table 1).\n\n\n\n• Male (No/%)\n\n• Female (No/%)\n\nAnti-vinculin and anti-CdtB levels were significantly elevated in IBS patients (1.58 ± 0.496ng/ml and 2.47 ± 0.60 ng/ml) respectively compared to the control subjects (1.13 ± 0.249 ng/ml and 2.1 ± 0.24 ng/ml) respectively with P = 0.001 for both ( Table 2).\n\nAnti-vinculin levels were also significantly higher in different IBS subgroups compared to control subjects, with the anti-vinculin level being significantly elevated in the IBS-D subtype when compared to the other subtypes with P = 0.001. Similarly, anti-CdtB showed significant elevation in IBS-C and IBS-D compared to control subjects (P = 0.001), with a significantly higher level detected in IBS-D than IBS-C (P = 0.001). However, the level of anti-CdtB in IBS-M was detected at a non-significant lower level compared to control subjects (P = 0.2), but at a significantly lower level when compared to IBS-C and IBS-D (P = 0.001) ( Table 3).\n\n\nDiscussion\n\nThere is an extreme necessity for the utilization of accessible and reliable, low-cost biomarkers in identifying IBS for the low-risk population to reduce the use of colonoscopy. 20 Previous studies have recognized anti-CdtB and anti-vinculin for use as valuable biomarkers in different IBS patients from healthy controls 21,22 in different populations. However, these biomarkers have not been sufficiently evaluated in Egyptian patients.\n\nIn the current study, both anti-vinculin and anti-CdtB demonstrated significantly elevated levels in IBS patients when compared to the control subjects, a finding that mirrors those from a previous study by Talley et al. 23 However, data reported by Rezaie et al. 16 depicts significant elevation in levels of both biomarkers only in IBS-M and IBS-D, but not IBS-C. This discrepancy in findings may be attributed to the difference in etiology of different IBS subtypes, 4 as it is hypothesized that most cases of post-infectious IBS manifest as IBS-D or IBS-M, with a minority of patients manifesting as IBS-C. 9 Another factor may make the microbiome profile difference between IBS patient subgroups; bacterial species producing methane are decreased in IBS-D and IBS-M 23 and increased in IBS-C. 24 Patients included in the present study, particularly those in the IBS-C subgroup, may represent patients who develop IBS following infections associated with their microbiota profile changes. These findings need extensive longitudinal studies to be confirmed.\n\nAnti-vinculin and anti-CdtB levels in this study were significantly elevated in patients with IBS-D, a concordance finding with Pimentel et al., 21 who reported that anti-CdtB and anti-vinculin distinguished IBS-D from IBD or other organic GI disease and healthy control. On the other hand, Bayoumy et al. 25 reported that anti-vinculin could be an important biomarker for IBS-D diagnosis among Egyptian patients. Cytolethal distending toxin represents a virulence factor for bacterial pathogens such as Escherichia coli, Salmonella, Shigella, and Campylobacter jejuni, by causing epithelial barrier breakdown and suppression of the acquired immune response to invading pathogens, resulting in an amplified pro-inflammatory response with consequent persistence of bacterial infection. 16 Development of anti-CdtB antibodies occurs in response to secretion of cytolethal distending toxin following infection with bacterial pathogens. Molecular mimicry accounts for the potential cross-reaction between anti-CdtB and vinculin with resultant anti-vinculin autoantibody production leading to injury to interstitial cells of Cajal (ICC) with the development of IBS. 12 Based on the suggestion of an association between the metabolic syndrome and liver affection and IBS, this study group performed liver function tests as a simple evaluation of liver affection. However, liver enzymes were normal in IBS patients' studied group, in contrast to reports by Lee et al. 26\n\nThe principal limitation of the present study was the small sample size. This necessitates the extension of the study to include large sample size. The findings of the present study are supported by previous studies (18, 19).\n\n\nConclusion\n\nThe present study’s findings support the hypothesis that IBS results from post-infectious disorders initiated by bacterial enteritis. Moreover, this hypothesis can be applied to all IBS subgroups as both anti-CdtB and anti-vinculin biomarkers were significantly elevated in IBS-C and IBS-D subgroups, with only anti-vinculin being elevated in IBS-M when compared to healthy control. These biomarkers were significantly elevated in IBS-D compared to IBS-C and IBS-M, possibly reflecting the post-infectious state's severity. These findings need further extensive longitudinal studies in patients with IBS.\n\n\nConsent\n\nAll participants provided written informed consent and the study was conducted according to the principles outlined in the Declaration of Helsinki. Confidentiality and privacy were considered regarding personal, clinical and laboratory data.\n\n\nEthical approval\n\nMansoura Faculty of Medicine Institutional Research Board approved the research (R.21.01.1141).\n\n\nData availability\n\nFigshare: “Study of antibodies to cytolethal distending toxin B (CdtB) and antibodies to vinculin in patients with irritable bowel syndrome” https://doi.org/10.6084/m9.figshare.14178908.v1. 27\n\nData are available under the terms of the Creative Commons CC BY 4.0", "appendix": "Acknowledgements\n\nThe authors are thankful for Mansoura Faculty of Medicine for providing laboratory to perform this work.\n\n\nReferences\n\nLacy BE, Patel NK: Rome Criteria and a Diagnostic Approach to Irritable Bowel Syndrome. J Clin Med. 2017; 6(11): 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanavan C, West J, Card T: The epidemiology of irritable bowel syndrome. Clin Epidemiol. 2014; 6: 71–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLongstreth GF, Thompson WG, Chey WD, et al.: Functional bowel disorders. Gastroenterology. 2006; 130(5): 1480–1491. PubMed Abstract \| Publisher Full Text\n\nTibble JA, Sigthorsson G, Foster R, et al.: Use of surrogate markers of inflammation and Rome criteria to distinguish organic from nonorganic intestinal disease. Gastroenterology. 2002; 123: 450–460. PubMed Abstract \| Publisher Full Text\n\nLongstreth GF, Thompson WG, Chey WD, et al.: Functional bowel disorders. Gastroenterology. 2006; 130: 1480–1491. PubMed Abstract \| Publisher Full Text\n\nSchmulson MJ, Drossman DA: What is new in Rome IV. J Neurogastroenterol Motil. 2017 Apr 30; 23(2): 151–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFord AC, Bercik P, Morgan DG, et al.: Validation of the Rome III criteria for the diagnosis of irritable bowel syndrome in secondary care. Gastroenterology. 2013; 145: 1262–1270. PubMed Abstract \| Publisher Full Text\n\nJee SR, Morales W, Low K, et al.: ICC density predicts bacterial overgrowth in a rat model of post-infectious IBS. World J Gastroenterol. 2010; 16: 3680–3686. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorales W, Pimentel M, Hwang L, et al.: Acute and chronic histological changes of the small bowel secondary to C. jejuni infection in a rat model for post-infectious IBS. Dig Dis Sci. 2011; 56: 2575–2584. PubMed Abstract \| Publisher Full Text\n\nPokkunuri V, Pimentel M, Morales W, et al.: Role of Cytolethal Distending Toxin in Altered Stool Form and Bowel Phenotypes in a Rat Model of Post-infectious Irritable Bowel Syndrome. J Neurogastroenterol Motil. 2012; 18: 434–442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorales W, Weitsman S, Kim G, et al.: Circulating antibodies to cytolethal distending toxin B correlate with the development of small intestinal bacterial overgrowth in a rat model of post-infectious IBS. Gastroenterology. 2013; 144: S-931–932.\n\nPimentel M, Morales W, Pokkunuri V, et al.: Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model. Dig Dis Sci. 2015 May; 60(5): 1195–205. PubMed Abstract \| Publisher Full Text\n\nVanderwinden JM, Liu H, De Laet MH, et al.: Study of the interstitial cells of Cajal in infantile hypertrophic pyloricstenosis. Gastroenterology. 1996; 111: 279–288. PubMed Abstract \| Publisher Full Text\n\nNieuwenhuijs VB, Verheem A, van Duijvenbode-Beumer H, et al.: The role of interdigestive small bowel motility in the regulation of gut microflora, bacterial overgrowth, and bacterial translocation in rats. Ann Surg. 1998; 228: 188–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVantrappen G, Janssens J, Hellemans J, et al.: The interdigestivemotor complex of normal subjects and patients with bacterial overgrowth of the small intestine. J Clin Invest. 1977; 59: 1158–1166. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRezaie A, Park SC, Morales W, et al.: Assessment of anti-vinculin and anti-cytolethal distending toxin B antibodies in subtypes of irritable bowel syndrome. Dig Dis Sci. 2017; 62: 1480–1485. PubMed Abstract \| Publisher Full Text\n\nLombardero M, Heymann PW, Platts-Mills TA, et al.: Conformational stability of B cell epitopes on group I and group II Dermatophagoides spp. allergens. Effect of thermal and chemical denaturation on the binding of murine IgG and humanIgE antibodies. J Immunol. 1990; 144: 1353–1360. PubMed Abstract\n\nMorales W, Rezaie A, Barlow G, et al.: Second-Generation Biomarker Testing for Irritable Bowel Syndrome Using Plasma Anti-CdtB and Anti-Vinculin Levels. Dig Dis Sci. 2019 Nov; 64(11): 3115–3121. PubMed Abstract \| Publisher Full Text\n\nRezaie A, Park SC, Morales W, et al.: Assessment of Anti-vinculin and Anti-cytolethal Distending Toxin B Antibodies in Subtypes of Irritable Bowel Syndrome. Dig Dis Sci. 2017 Jun; 62(6): 1480–1485. PubMed Abstract \| Publisher Full Text\n\nBurbige EJ: Irritable bowel syndrome: Diagnostic approaches in clinical practice. Clin Exp Gastroenterol. 2010; 3: 127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPimentel M, Morales W, Rezaie A, et al.: Development and validation of a biomarker for diarrhea-predominant irritable bowel syndrome in human subjects. PLoS One. 2015; 10(5): e0126438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTalley NJ, Holtmann G, Walker MM, et al.: Circulating Anti-cytolethal Distending Toxin B and Anti-vinculin Antibodies as Biomarkers in Community and Healthcare Populations With Functional Dyspepsia and Irritable Bowel Syndrome. Clin Transl Gastroenterol. 2019; 10(7): e00064. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPozuelo M, Panda S, Santiago A, et al.: Reduction of butyrate- and methane-producing microorganisms in patients with Irritable Bowel Syndrome. Sci Rep. 2015; 5: 12693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim G, Deepinder F, Morales W, et al.: Methanobrevibacter smithii is the predominant methanogen in patients with constipation-predominant IBS and methane on breath. Dig Dis Sci. 2012; 57: 3213–3218. PubMed Abstract \| Publisher Full Text\n\nBayoumi E, Sabrya M, Soliman NRA: Antivinculin antibodies as a marker of irritable bowel syndrome–diarrhea in Egyptian patients. Egyp Liver J. 2018; 8(4): 132–135.\n\nLee SH, Kim KN, Kim KM, et al.: Irritable Bowel Syndrome May Be Associated with Elevated Alanine Aminotransferase and Metabolic Syndrome. Yonsei Med J. 2016; 57(1): 146–152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaki EM, Elhammady D, Abdelsalam M, et al.: Study of antibodies to cytolethal distending toxin B (CdtB) and antibodies to vinculin in patients with irritable bowel syndrome. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "86122", "date": "27 May 2021", "name": "Gabriela Leite", "expertise": [ "Reviewer Expertise Drug development", "test development for diagnosis of antibodies and antigens associated with bacterial diseases." ], "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, Zaki and collaborators observed increased levels of plasma CdtB and vinculin antibodies in IBS subjects when compared to controls (non-IBS subjects) and analysis also revealed differences between IBS subtypes. The study has an important clinical relevance, but to be published, several corrections and clarifications should be addressed. The major concern is about the cdtB antibody test. It is not clear which kit was used for this test, authors need to clarify and double-check if the clostridium difficile toxin B (CDTB) kit was used. CdtB from C. difficile is a binary toxin termed the C. difficile toxin (CDT) and it has pore-forming or delivery subunit termed CDTb. This toxin is not the same of that observed in Campylobacter jejuni, the cytolethal distending toxin B. These are two completely different toxins. All publications with cdtB in IBS subjects were based on cdtB from C. jejuni. I couldn’t find the ELISA kit the authors mentioned on Creative Diagnostics webpage.\nSuggestions of writing are marked in italic.\nAbstract\nAuthors need to restructure the abstract considering suggestions on the manuscript.\n\nOverall suggestion: the word “patients” should be replaced by “subjects”.\n\nIntroduction\nParagraph 01:\n“IBS mainly manifests in subjects with abdominal pain and bowel habit changes in the absence…”\n\nIn between Paragraph 01 and 02, authors should present info about IBS subtypes.\n\nParagraph 02:\n“Irritable bowel syndrome” needs to be substituted by IBS.\n\n“In these studies, progression to IBS was accompanied by the detection of circulating levels of a specific bacterial toxin named cytolethal distending toxin B (CdtB), a potential factor attributing to the pathogenesis of PI-IBS.”\n\n“This was supported by the low incidence of IBS in patients infected with a mutant strain of C. jejuni that lacks CdtB.” These studies were based on results in rats, not patients.\n\nParagraph 03:\n“These findings were linked to the ability of anti-CdtB to cross-react with vinculin, a host cell adhesion protein present in interstitial cells of Cajal and the myenteric ganglia that control the normal activity of the intestinal tract, including phase III of inter-digestive motor activity.”\n\n“Based on these data, it has been suggested that loss of vinculin in the neuromuscular system of the GIT may be associated with the affection of the gut in animal models of post-infection C. jejuni.” This statement needs a reference. GIT needs to be spelled out.\n\nParagraph 04”\n“The present study aims to detect and to quantify anti-CdtB and anti-vinculin levels in subjects with IBS and their possible role in the diagnosis of different IBS subtypes.”\n\nMethods\nIn the abstract, authors mentioned this study was based in a retrospective study, but in Methods, a prospective study was mentioned\n\nThe authors provided information about samples size calculation (which in my opinion is not needed for this study), but authors mentioned the need of 16585 cirrhotic patients (Paragraph 01 in Methods). Not sure why cirrhotic subjects.\n\n“According to the actual calculated sample size, we needed to enroll 385 with IBS and 193 for control group at a power of 80% and type I error = 0.05, while at a power of 99% and type I error = 0.01, the minimum number of the patients was 16585 cirrhotic patients and 830 for the control group. This is very hard to achieve in lower economy countries like Egypt. We cannot afford to measure all the parameters in these patients. We had to design the study depending on self-funding without any further support.” I don’t think this info is needed. Since this is a retrospective study, 100 subjects with matched control group (gender and age) should give you enough power to check differences in quantities/levels. If you are planning to define a cutoff for clinical/diagnosis purpose, then you would need a sample size calculation.\n\n“Patients were recruited, and IBS was determined by a questionnaire-based upon the Rome IV criteria”\n\n“The third aliquot was overlaid on heparin for plasma separation, and the remaining sera were stored at -20°C to be used for evaluation of anti-vinculin antibodies by laboratory prepared ELISA and anti-CdtB antibodies by commercial ELISA (Creative Diagnostics. 45-16 Ramsey Road Shirley, NY 11967, USA).” If heparin was used on third aliquot, everything should be plasma, not serum. What was the kit catalog number used for this assay? I only could find the kit “Human Anti-Clostridium Difficile Toxin B (CDTB) ELISA” on Creative Diagnostics webpage. CdtB from this kit refers to clostridium difficile toxin B, but cdtB from IBS studies refers to cytolethal distending toxin B, mostly from C. jejuni. This information should be clarified as a major priority. The toxin B from Clostridium is not the same as cdtB from C. jejuni.\n\n“Anti-vinculin levels were measured in separated plasma using human vinculin protein in a concentration of 1.21.2 μg/ml (Novoprotein Scientific, Summit, New Jersey, USA) as an antigen.” Vinculin concentration should be fixed.\n\nAuthors said that “Optical densities (ODs) were read at 370nm, and the results were interpreted as OD”, but vinculin antibodies were reported as ng/mL. Please explain. If a calculation was performed, this need to be described.\n\nResults\n“This study included 100 patients with IBS (49 males and 51 females) aged 46.6 ± 6.8 years and 100 healthy controls (please add sex distribution and mean age/SD here). No differences were observed between IBS subjects and control regarding age and sex (P = 0.8 and P = 0.6, respectively).” I added a suggestion.\n\n“Patients were classified according to Rome III criteria into 40 patients with IBS-C, 26 patients with IBS-D, and 34 patients with IBS-M (Figure 1).” Authors mentioned Rome IV criteria in Methods. Figure 1 is not needed.\n\n“Anti-vinculin and anti-CdtB levels were significantly elevated in IBS patients (1.58 ± 0.496 ng/ml and 2.47 ± 0.60 ng/ml) respectively compared to the control subjects (1.13 ± 0.249 ng/ml and 2.1 ± 0.24 ng/ml) respectively with P = 0.001 for both (Table 2).” Authors need to double check vinculin antibodies unit.\n\nIn table 3, the first P-value of the anti-cdtB results was calculated based on what? All the other P-values (P1 to P6) are explained on the text below the table.\n\nDiscussion\nOverall suggestion: the discussion can be better expanded.\n\n“There is an extreme necessity for the utilization of accessible and reliable, low-cost biomarkers in identifying IBS for the low-risk population to reduce the use of colonoscopy”. Low-risk population? What do the authors mean with low risk? Please expand a little bit more why the need of development of a test for IBS.\n\n“Previous studies have recognized anti-CdtB and anti-vinculin for use as valuable biomarkers in different IBS patients from healthy controls”. Please rephrase it. It is confusing. I encourage the authors to perform a language review of the manuscript. Some suggestions were already included in my review, but I strongly recommend the authors to perform a in deep language review of the entire manuscript.\n\n“This discrepancy in findings may be attributed to the difference in etiology of different IBS subtypes, as it is hypothesized that most cases of post-infectious IBS manifest as IBS-D or IBS-M, with a minority of patients manifesting as IBS-C. Another factor may make the microbiome profile difference between IBS patient subgroups; bacterial species producing methane are decreased in IBS-D and IBS-M and increased in IBS-C. Patients included in the present study, particularly those in the IBS-C subgroup, may represent patients who develop IBS following infections associated with their microbiota profile changes. These findings need extensive longitudinal studies to be confirmed.” These statements are very confusing, not sure if that can be used to explain why IBS-C also had increased cdtB and vinculin antibodies. This needs to be better clarified. In addition, methane producers are mostly Archaea, not bacteria. Increases in methanogens also represents microbiome changes.\n\n“Anti-vinculin and anti-CdtB levels in this study were significantly elevated in patients with IBS-D, a concordance finding with Pimentel et al., who reported that anti-CdtB and anti-vinculin distinguished IBS-D from IBD, other organic GI diseases and healthy control. In addition, Bayoumy et al. reported that anti-vinculin could be an important biomarker for IBS-D diagnosis among Egyptian patients.”\n\n“Cytolethal distending toxin (use abbreviation) is a virulence factor for bacterial pathogens such as Escherichia coli, Salmonella, Shigella, and Campylobacter jejuni, by causing epithelial barrier breakdown and suppression of the acquired immune response to invading pathogens, resulting in an amplified pro-inflammatory response with consequent persistence of bacterial infection. Development of anti-CdtB antibodies occurs in response to secretion of cytolethal distending toxin (use abbreviation) following infection with bacterial pathogens. Molecular mimicry accounts for the potential cross-reaction between anti-CdtB and vinculin with resultant anti-vinculin autoantibody production leading to injury to interstitial cells of Cajal (ICC) with the development of IBS. Based on the suggestion of an association between the metabolic syndrome and liver affection and IBS, this study group performed liver function tests as a simple evaluation of liver affection. However, liver enzymes were normal in IBS patients' studied group, in contrast to reports by Lee et al.” This entire paragraph should be rewritten, it is confusing and not well constructed.\n\nConclusion\n“The present findings support the hypothesis that IBS may results from post-infectious bacterial gastroenteritis. Moreover, this hypothesis can be applied to all IBS subgroups as both anti-CdtB and anti-vinculin biomarkers were significantly elevated in IBS-C and IBS-D subgroups, with only anti-vinculin being elevated in IBS-M when compared to healthy control.”\n\n“These biomarkers were significantly elevated in IBS-D compared to IBS-C and IBS-M, possibly reflecting the post-infectious state's severity. These findings need further extensive longitudinal studies in patients with IBS.” How was severity of the disease accessed? There is no mention of that in any part of the manuscript, so no conclusion can be made based on severity.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6737", "date": "30 Jun 2021", "name": "Maysaa El Zaki", "role": "Author Response", "response": "Response to reviewers: We would like to thank the reviewer the insightful comments on the paper, as these comments led us to an improvement of the work. Our revisions reflect all reviewers suggestions. Detailed responses to the reviewers are given below. In this study, Zaki and collaborators observed increased levels of plasma CdtB and vinculin antibodies in IBS subjects when compared to controls (non-IBS subjects) and analysis also revealed differences between IBS subtypes. The study has an important clinical relevance, but to be published, several corrections and clarifications should be addressed. Response: Many thanks, we really appreciate that. The major concern is about the cdtB antibody test. It is not clear which kit was used for this test, authors need to clarify and double-check if the clostridium difficile toxin B (CDTB) kit was used. CdtB from C. difficile is a binary toxin termed the C. difficile toxin (CDT) and it has pore-forming or delivery subunit termed CDTb. This toxin is not the same of that observed in Campylobacter jejuni, the cytolethal distending toxin B. These are two completely different toxins. All publications with cdtB in IBS subjects were based on cdtB from C. jejuni. I couldn’t find the ELISA kit the authors mentioned on Creative Diagnostics webpage. Response: Thanks for your meticulous comment and observation and we already clarify this point in the methodology as the measured was anticdtB from C.jejuni. Abstract Authors need to restructure the abstract considering suggestions on the manuscript. Overall suggestion: the word “patients” should be replaced by “subjects”. Response: Thanks, we corrected it. Introduction Paragraph 01: “IBS mainly manifests in subjects with abdominal pain and bowel habit changes in the absence…” Response: Thanks, corrected. In between Paragraph 01 and 02, authors should present info about IBS subtypes. Response: Thanks for your suggestion, we added it. Paragraph 02: “Irritable bowel syndrome” needs to be substituted by IBS. Response: Thanks, corrected. “In these studies, progression to IBS was accompanied by the detection of circulating levels of a specific bacterial toxin named cytolethal distending toxin B (CdtB), a potential factor attributing to the pathogenesis of PI-IBS.” Response: Thanks for your advise, corrected. “This was supported by the low incidence of IBS in patients infected with a mutant strain of C. jejuni that lacks CdtB.” These studies were based on results in rats, not patients. Response: Thanks for meticulous observation, modified. Paragraph 03: “These findings were linked to the ability of anti-CdtB to cross-react with vinculin, a host cell adhesion protein present in interstitial cells of Cajal and the myenteric ganglia that control the normal activity of the intestinal tract, including phase III of inter-digestive motor activity.” Response: Thanks, corrected. “Based on these data, it has been suggested that loss of vinculin in the neuromuscular system of the GIT may be associated with the affection of the gut in animal models of post-infection C. jejuni.” This statement needs a reference. GIT needs to be spelled out. Response: Thanks, corrected. Paragraph 04: “The present study aims to detect and to quantify anti-CdtB and anti-vinculin levels in subjects with IBS and their possible role in the diagnosis of different IBS subtypes.” Response: Thanks, corrected. Methods In the abstract, authors mentioned this study was based in a retrospective study, but in Methods, a prospective study was mentioned Response: Thanks for your comment, corrected The authors provided information about samples size calculation (which in my opinion is not needed for this study), but authors mentioned the need of 16585 cirrhotic patients (Paragraph 01 in Methods). Not sure why cirrhotic subjects. “According to the actual calculated sample size, we needed to enroll 385 with IBS and 193 for control group at a power of 80% and type I error = 0.05, while at a power of 99% and type I error = 0.01, the minimum number of the patients was 16585 cirrhotic patients and 830 for the control group. This is very hard to achieve in lower economy countries like Egypt. We cannot afford to measure all the parameters in these patients. We had to design the study depending on self-funding without any further support.” I don’t think this info is needed. Since this is a retrospective study, 100 subjects with matched control group (gender and age) should give you enough power to check differences in quantities/levels. If you are planning to define a cutoff for clinical/diagnosis purpose, then you would need a sample size calculation Response: Thanks for your advise and suggestion. This calculation was added as a response to the editor of the journal and now it is removed as your suggestion. “Patients were recruited, and IBS was determined by a questionnaire-based upon the Rome IV criteria” Response: Thanks, corrected. “The third aliquot was overlaid on heparin for plasma separation, and the remaining sera were stored at -20°C to be used for evaluation of anti-vinculin antibodies by laboratory prepared ELISA and anti-CdtB antibodies by commercial ELISA (Creative Diagnostics. 45-16 Ramsey Road Shirley, NY 11967, USA).” If heparin was used on third aliquot, everything should be plasma, not serum. What was the kit catalog number used for this assay? I only could find the kit “Human Anti-Clostridium Difficile Toxin B (CDTB) ELISA” on Creative Diagnostics webpage. CdtB from this kit refers to clostridium difficile toxin B, but cdtB from IBS studies refers to cytolethal distending toxin B, mostly from C. jejuni. This information should be clarified as a major priority. The toxin B from Clostridium is not the same as cdtB from C. jejuni. Response: Thanks for your comments, the study of antibodies for anti cdtB was performed on serum samples as clarified in the methods and the commercial refers to the used components of the ELISA method used. The plasma was used for anti-vinculin antibodies. “Anti-vinculin levels were measured in separated plasma using human vinculin protein in a concentration of 1.21.2 μg/ml (Novoprotein Scientific, Summit, New Jersey, USA) as an antigen.” Vinculin concentration should be fixed. Response: Thanks for your observation, corrected. Authors said that “Optical densities (ODs) were read at 370nm, and the results were interpreted as OD”, but vinculin antibodies were reported as ng/mL. Please explain. If a calculation was performed, this need to be described. Response: Thanks for your insightful advise, we corrected it. “This study included 100 patients with IBS (49 males and 51 females) aged 46.6 ± 6.8 years and 100 healthy controls (please add sex distribution and mean age/SD here). No differences were observed between IBS subjects and control regarding age and sex (P = 0.8 and P = 0.6, respectively).” I added a suggestion. Response: Thanks, corrected. “Patients were classified according to Rome III criteria into 40 patients with IBS-C, 26 patients with IBS-D, and 34 patients with IBS-M (Figure 1).” Authors mentioned Rome IV criteria in Methods. Figure 1 is not needed. Response: Thanks for your suggestion, we removed it. “Anti-vinculin and anti-CdtB levels were significantly elevated in IBS patients (1.58 ± 0.496 ng/ml and 2.47 ± 0.60 ng/ml) respectively compared to the control subjects (1.13 ± 0.249 ng/ml and 2.1 ± 0.24 ng/ml) respectively with P = 0.001 for both (Table 2).” Authors need to double check vinculin antibodies unit. Response: Thanks for your comments, we check it and methodology was modified. In table 3, the first P-value of the anti-cdtB results was calculated based on what? All the other P-values (P1 to P6) are explained on the text below the table. Response: Thanks for your comments, we added it and it was already present in the primary submission. Discussion Overall suggestion: the discussion can be better expanded. Response: Thanks, done. “There is an extreme necessity for the utilization of accessible and reliable, low-cost biomarkers in identifying IBS for the low-risk population to reduce the use of colonoscopy”. Low-risk population? What do the authors mean with low risk? Please expand a little bit more why the need of development of a test for IBS. Response: Thanks for your comment, we clarified this point. “Previous studies have recognized anti-CdtB and anti-vinculin for use as valuable biomarkers in different IBS patients from healthy controls”. Please rephrase it. It is confusing. I encourage the authors to perform a language review of the manuscript. Some suggestions were already included in my review, but I strongly recommend the authors to perform a in deep language review of the entire manuscript. Response: Thanks, Paraphrasing was done “Anti-vinculin and anti-CdtB levels in this study were significantly elevated in patients with IBS-D, a concordance finding with Pimentel et al., who reported that anti-CdtB and anti-vinculin distinguished IBS-D from IBD, other organic GI diseases and healthy control. In addition, Bayoumy et al. reported that anti-vinculin could be an important biomarker for IBS-D diagnosis among Egyptian patients.” Response: Thanks, corrected “Cytolethal distending toxin (use abbreviation) is a virulence factor for bacterial pathogens such as Escherichia coli, Salmonella, Shigella, and Campylobacter jejuni, by causing epithelial barrier breakdown and suppression of the acquired immune response to invading pathogens, resulting in an amplified pro-inflammatory response with consequent persistence of bacterial infection. Development of anti-CdtB antibodies occurs in response to secretion of cytolethal distending toxin (use abbreviation) following infection with bacterial pathogens. Molecular mimicry accounts for the potential cross-reaction between anti-CdtB and vinculin with resultant anti-vinculin autoantibody production leading to injury to interstitial cells of Cajal (ICC) with the development of IBS. Based on the suggestion of an association between the metabolic syndrome and liver affection and IBS, this study group performed liver function tests as a simple evaluation of liver affection. However, liver enzymes were normal in IBS patients' studied group, in contrast to reports by Lee et al.” This entire paragraph should be rewritten, it is confusing and not well constructed. Response: Thanks, paraphrasing was done The present findings support the hypothesis that IBS may results from post-infectious bacterial gastroenteritis. Moreover, this hypothesis can be applied to all IBS subgroups as both anti-CdtB and anti-vinculin biomarkers were significantly elevated in IBS-C and IBS-D subgroups, with only anti-vinculin being elevated in IBS-M when compared to healthy control Response: Many thanks, corrected “This discrepancy in findings may be attributed to the difference in etiology of different IBS subtypes, as it is hypothesized that most cases of post-infectious IBS manifest as IBS-D or IBS-M, with a minority of patients manifesting as IBS-C. Another factor may make the microbiome profile difference between IBS patient subgroups; bacterial species producing methane are decreased in IBS-D and IBS-M and increased in IBS-C. Patients included in the present study, particularly those in the IBS-C subgroup, may represent patients who develop IBS following infections associated with their microbiota profile changes. These findings need extensive longitudinal studies to be confirmed.” These statements are very confusing, not sure if that can be used to explain why IBS-C also had increased cdtB and vinculin antibodies. This needs to be better clarified. In addition, methane producers are mostly Archaea, not bacteria. Increases in methanogens also represents microbiome changes. Response: Many thanks, deleted." } ] }, { "id": "85381", "date": "11 Jun 2021", "name": "Simon Keely", "expertise": [ "Reviewer Expertise Gastroenterology", "mucosal inflammation", "immunology", "microbiome", "functional GI 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\nThis is an interesting study validating previous work on the utility of CdtB and vinculin as potential biomarkers for functional GI disorders, specifically IBS.\nThe study is clearly reported although there are a number of inconsistencies that need to be addressed.\nThere are also a number of statements that would benefit from revision with the more current literature in mind.\nThe introduction speculates on microbiome alterations in IBS however it should be noted that the idea of a specific IBS microbiome is someone controversial with larger studies analysing mucosal microbiomes showing now distinct signature (Hugerth et al. (20211)).\n\nThe introduction discusses the association between CdtB and vinculin antibodies and post-infectious IBS and C. jejuni, however there is no mention of whether the patients have a post infectious history (reading the manuscript, one assumes they are idiopathic IBS cases) and the increases in these antibodies in a potentially non PI cohort is not addressed in the discussion. This warrants revision.\n\nMethodology - the abstract and methods state that these are Rome IV diagnosed patients but the results state that they are Rome III. Please explain this discrepancy.\n\nThe power calculation is very concerning. The line \" the minimum number of the patients was 16585 cirrhotic patients and 830 for the control group\" appears to be copied from a different power calculation for a different study. There should be no reason to do this as the power calculation should be independent. Please show the detailed power calculation for this study, including the data used to estimate the sample size.\n\nWere any additional data taken on patients that may impact results, BMI or anxiety/depression, for instance? There are previous studies that suggest that HADS can improve the sensitivity of blood based biomarkers for FGIDs (Jones et al. (20142)).\n\nThe conclusion is that a larger sample cohort is warranted, but this study validates previous, larger 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? 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": "6789", "date": "14 Jun 2021", "name": "Maysaa El Zaki", "role": "Author Response", "response": "Thanks for reviewing the article. The response will be followed." }, { "c_id": "6804", "date": "30 Jun 2021", "name": "Maysaa El Zaki", "role": "Author Response", "response": "Response to reviewers: We would like to thank the reviewer the insightful comments on the paper, as these comments led us to an improvement of the work. Our revisions reflect all reviewers suggestions. Detailed responses to the reviewers are given below. This is an interesting study validating previous work on the utility of CdtB and vinculin as potential biomarkers for functional GI disorders, specifically IBS. The study is clearly reported although there are a number of inconsistencies that need to be addressed. There are also a number of statements that would benefit from revision with the more current literature in mind. Response: Thanks, we really appreciate that. The introduction speculates on microbiome alterations in IBS however it should be noted that the idea of a specific IBS microbiome is someone controversial with larger studies analysing mucosal microbiomes showing now distinct signature (Hugerth et al. (2021)). Response: Thanks for your nice comment, we mentioned this observation in the introduction. The introduction discusses the association between CdtB and vinculin antibodies and post-infectious IBS and C. jejuni, however there is no mention of whether the patients have a post infectious history (reading the manuscript, one assumes they are idiopathic IBS cases) and the increases in these antibodies in a potentially non PI cohort is not addressed in the discussion. This warrants revision. Response: The following was added: 'In the present study there was no history of previous infection with C. jejuni. However, the elevated levels of antiCdtB and antivinculin can be used as biomarkers for diagnosis of IBS either post infections or without previous infection'. Methodology - the abstract and methods state that these are Rome IV diagnosed patients but the results state that they are Rome III. Please explain this discrepancy. Response: Thanks for your meticulous observation but we depend on Rome IV and we divided patients according to the results of the questionnaire into three groups as there were no patients unclassified. The power calculation is very concerning. The line \" the minimum number of the patients was 16585 cirrhotic patients and 830 for the control group\" appears to be copied from a different power calculation for a different study. There should be no reason to do this as the power calculation should be independent. Please show the detailed power calculation for this study, including the data used to estimate the sample size. Response: Thanks for your comments, it was corrected. Were any additional data taken on patients that may impact results, BMI or anxiety/depression, for instance? There are previous studies that suggest that HADS can improve the sensitivity of blood based biomarkers for FGIDs (Jones et al. (2014)). Response: Thanks for your observation but actually we did not have full data about these points. The conclusion is that a larger sample cohort is warranted, but this study validates previous, larger studies. Response: Thanks, we agree with you our study validates previous larger studies but to the best of our knowledge this was the first study which discussed this point in Egypt. Unfortunately, due to lack of financial support it was difficult to do the study on large scale of patients." } ] } ]	1	https://f1000research.com/articles/10-303
https://f1000research.com/articles/10-314/v1	23 Apr 21	{ "type": "Systematic Review", "title": "Cost-effectiveness of patient navigation for lung cancer – a systematic review", "authors": [ "Benjamin Kass", "Christina Dornquast", "Nina Rieckmann", "Ute Goerling", "Christine Holmberg", "Thomas Reinhold", "Christina Dornquast", "Nina Rieckmann", "Ute Goerling", "Christine Holmberg", "Thomas Reinhold" ], "abstract": "Background: Patient navigation (PN) programs have been shown to increase patient satisfaction and quality of life among patients with lung cancer and to decrease time to treatment. However, the general cost-effectiveness of such programs in the context of lung cancer remains unknown. Hence, the aim of the present systematic review was to analyze the scientific literature and quantitatively assess the level of evidence on the cost-effectiveness of PN programs for patients diagnosed with lung cancer. Methods: A systematic literature search was carried out in PubMed, EMBASE, CENTRAL, CINAHL and PsycINFO databases without time limitations. Randomized controlled trials written in English or German were eligible for inclusion if any results regarding the cost-effectiveness of personally delivered PN programs for patients after lung cancer diagnosis were reported. A manual search was carried out to supplement the systematic search. Additionally, the authors of ongoing or unpublished relevant research were contacted. The titles, abstracts and full texts of relevant citations were screened independently by two reviewers.\n\nResults: The initial search yielded 814 articles, including four papers identified manually. Twenty-one articles were included in the full text screening. However, no study met the inclusion criteria. Contacting the authors of ongoing or unpublished research and cross-cancer studies did not yield any studies that met the inclusion criteria.\n\nConclusion: Since no study met the inclusion criteria, this study reveals a research gap in this area. Furthermore, no conclusive statement regarding the cost-effectiveness of patient navigation programs for patients diagnosed with lung cancer can be made. Since the implementation of new healthcare models such as PN at least partially depends on their cost-effectiveness, future attempts to evaluate PN programs for lung cancer patients should consider examining outcomes related to cost-effectiveness to overcome the identified research gap.", "keywords": [ "Patient Navigation", "Cost-Benefit Analysis", "Lung Cancer", "Systematic Review", "Empty Review", "Randomized Controlled Trials" ], "content": "Introduction\n\nLung cancer remains the most commonly diagnosed cancer type worldwide, with 2.1 million new cases annually. It accounts for an estimated 1.8 million deaths per year and is thus the most common cause of cancer-related deaths in men and the second most common cause in women.1 Steady progress in the detection, treatment and prevention of lung cancer has led to increased survival as well as decreased incidence.2 However, more than 50 percent of lung cancer patients die within a year after diagnosis, and the 5-year survival rate is still lower than 21 percent for men and women combined.2,3 In addition to the poor prognosis, lung cancer patients often experience severe symptom burdens, such as fatigue, shortness of breath and pain. These symptoms, along with anxiety and depression, are associated with reduced quality of life in lung cancer patients.4–7\n\nDepending on patients’ overall health and their lung cancer stage, treatment patterns can be very complex and overwhelming for patients. Chemotherapy, immunotherapy, radiotherapy, surgery, or therapy combinations are possible treatment options in curative or palliative settings. Furthermore, targeted therapy can be applied. These care options are provided by numerous different health care professionals within and across health care sectors.8 Patients’ complex needs, the large number of involved caregivers and expensive treatment patterns result in a high economic burden of lung cancer, varying across disease stages, treatment patterns and health care systems.9–12\n\nA promising approach to improve patient-oriented outcomes (e.g., health-related quality of life) and to reduce health care costs for patients with cancer and chronic diseases is patient navigation (PN).13–16 PN was first introduced by Harold Freeman to address barriers to timely care among African-American women with breast cancer in Harlem, New York.17 While PN programs initially focused on barriers within breast cancer care, there is currently a wide range of different programs across the cancer care continuum and beyond, including programs for other chronic diseases.15,18 Typically, PN programs focus on the elimination of one or multiple barriers to appropriate health care services, such as financial and access barriers, information barriers, medical system barriers and emotional barriers.17 Programs are situated across the care continuum, including screening, diagnosis, treatment and surveillance.17,18 PN programs differ based on the targeted barriers, diseases and program components, and the programs are conducted by a variety of usually specifically trained health care professionals or lay health workers.13,14 Components of PN programs can be very heterogeneous, including aiding in care coordination, transportation services, educational components, emotional support and assistance with financial barriers such as insurance coverage.16\n\nWithin the context of lung cancer, the introduction of PN delivered by nurses reduced the time to treatment initiation after suspicion of a lung cancer diagnosis in different settings.19–21 PN carried out by lay health workers has been shown to improve lung cancer screening rates in current smokers who receive care in community health centers.22 In one study, newly diagnosed lung cancer patients highly appraised the provision of information, emotional support and care coordination delivered by trained volunteer lay navigators.23 Compared to a control group receiving enhanced standard care over a 12-month follow-up, patients in a 16-week PN support program who were newly diagnosed with lung, breast or colon cancer reported comparable cancer-related quality of life and cumulative direct costs as well as higher patient satisfaction.24 The authors found lower cumulative direct costs in the small subsample of 30 lung cancer patients; however, effectiveness measures were not reported separately for this diagnosis. However, the general cost-effectiveness of such PN programs in the context of patients diagnosed with lung cancer has not yet been evaluated. Therefore, to date, no systematic and quantitative assessment of the cost-effectiveness of PN programs for patients diagnosed with lung cancer is available. Hence, the aim of this review was to analyze the scientific literature to quantitatively assess the level of evidence on the cost-effectiveness of PN programs for patients diagnosed with lung cancer.\n\n\nMethods\n\nSystematic and comprehensive literature searches in the PubMed, EMBASE, CENTRAL, CINAHL and PsycINFO databases were conducted to retrieve studies published prior to January 15, 2020. Medical subject headings (MeSH) terms and keywords were used to identify cost-effectiveness analyses of PN focusing on lung cancer patients. Broad search terms for patient navigation, including potential synonyms (e.g., case management, case coordination, and community health workers), cost-effectiveness and lung cancer, were applied. The PubMed search strategy (Table 1) was adapted to the other electronic databases. An extensive manual search, including screening of study registers, and reference lists of studies and reviews in the context of patient navigation and lung cancer was carried out to identify additional studies. The authors of study protocols and cross-cancer studies were contacted up to two times to retrieve unpublished lung cancer-specific results.\n\nRetrieved citations from all databases and manual searches were merged using Endnote Version X9.2 for Windows (Clarivate Analytics, Boston, MA, USA). After the review and removal of duplicates, study titles and abstracts were screened for inclusion. The full texts of the remaining articles were assessed for eligibility. Each step was independently performed by two review authors (CD, BK) to verify the retrieved results. Studies were eligible for inclusion if the following criteria were met:\n\n1) the study was a randomized controlled trial (RCT);\n\n2) the study was published in English or German; and\n\n3) the study reported results regarding the cost-effectiveness of PN for lung cancer patients after the diagnosis was confirmed.\n\nBased on a modification of the definition by Wells et al. (2008), PN was defined as an intervention that is carried out personally (e.g., by nurses, care managers, lay health workers) for a defined episode of individual lung cancer-related care and focuses on the identification and resolution of individual barriers to receive appropriate healthcare services.13 To reduce the heterogeneity of the included programs and effectiveness outcomes, PN programs focusing on the screening and early detection of lung cancer were a priori excluded. No further restrictions regarding the interventions were made. Studies describing all possible PN program components applied after diagnosis were eligible. No restrictions regarding the publication date, age of participants, lung cancer classification and stage, follow-up lengths, evaluation perspectives or reported cost types were made. Studies were excluded if they did not meet all eligibility criteria.\n\nData extraction and quality assessment of the included studies were planned to be carried out independently by two review authors. Data extraction using the 3rd version of the Cochrane data collection form for intervention reviews (Cochrane, London, UK) or an adaption of this form as well as the Revised Cochrane tool for assessing risk of bias in randomized trials tool for quality assessment were considered.25,26 Disagreements of any kind regarding data extraction and quality assessment were resolved by consensus. The two review authors worked closely together with an experienced senior author (TR) through the entire process. The systematic selection and screening of studies followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines.27\n\n\nResults\n\nA total of 810 citations were identified through database searches (PubMed n = 447, EMBASE n = 122, CENTRAL n = 72, CINAHL n = 165, PsycINFO n = 4), and an additional four articles were found manually. The removal of duplicate citations resulted in the inclusion of 741 titles and abstracts for eligibility screening. In this first-stage screening, 720 articles did not meet the inclusion criteria and were excluded. The remaining 21 articles proceeded to the full text assessment. Eight articles did not investigate patient navigation programs28–35; six articles did not focus on lung cancer or did not present lung cancer-specific results36–41; three articles did not include an economic evaluation8,24,42; three articles were study protocols43–45; and one article focused on promoting lung cancer screening prior to the diagnosis.22 During the full text assessment, the interrater reliability between CD and BK was 100%. Figure 1 illustrates the described study identification and selection process according to the PRISMA guidelines.\n\nAdditional attempts have been made to contact the authors of eight study protocols or cross-cancer studies.24,33,39–42,44,46 The authors of four studies could not be reached or did not reply. One research group did not collect cost-related data in their study, and the remaining three groups did not perform lung cancer-specific analyses. Furthermore, the screening of reference lists of approximately matching reviews and studies did not yield any relevant record either. Since no eligible studies on the cost-effectiveness of PN in lung cancer patients were found, this review reveals a research gap in this area.\n\n\nDiscussion\n\nThe systematic review did not identify any RCTs examining the cost-effectiveness of PN programs for patients diagnosed with lung cancer, revealing a research gap. Hence, no conclusion about the cost-effectiveness of such programs can be drawn.\n\nA Markov model by Shih et al. from 2016 is the only analysis in this field.47 These authors offer early evidence that PN might be cost-effective for lung cancer patients. However, since the study did not meet the inclusion criteria for the current review and in line with recommendations for empty reviews, its results will not be further discussed as they are not able to answer the research question at hand.48\n\nDuring the review process, one RCT focusing on the cost-effectiveness of a lung cancer PN screening program was detected.22 A priori, heterogeneity concerns led to the exclusion of programs focusing on the time before lung cancer diagnosis. The personal circumstances prior to and after such a diagnosis and thus the needs of potential patients seemed too different for a systematic comparison of respective PN programs. Other systematic reviews regarding the effectiveness or cost-effectiveness of PN did not distinguish between programs that were conducted prior to diagnosis and those that were conducted after diagnosis.13,14,16,18,49 However, heterogeneity was an issue regarding the generalizability of the results.\n\nThe specificity of the investigated research question to overcome heterogeneity concerns and the comprehensive attempts to gather unpublished information were considered strengths of the present review. Nevertheless, heterogeneity concerns are irrelevant in the absence of studies meeting the inclusion criteria, and the contact attempts were unsuccessful since no findings could be included and only half of the contacted authors replied.\n\nConsidering the high mortality and particularly severe disease burden of lung cancer, as well as the potentially beneficial effects of PN in other clinical fields, we encourage further research regarding the cost-effectiveness of PN programs for lung cancer patients. The low survival rates of lung cancer patients indicate the need for individually tailored treatment and support (e.g., PN programs) but might also be a cause for reduced study participation. Additionally, such studies need short follow-up periods, which in turn requires a high compliance among patients. The disease severity might also result in small subsample sizes in studies examining different cancer types, which would lead to a main analysis among all cancer types rather than subsample analyses. This assumption was substantiated by the answers from the contacted authors. Moreover, patients in advanced stages of the disease might be less likely to participate in an RCT, in which the kind of intervention will be decided by chance. Nonetheless, we focused on RCTs to build on the best available evidence. Since no eligible studies were identified, the focus on RCTs could be seen as a limitation. However, our broad search strategies did not include a restriction by study type, and no further thematically relevant nonrandomized studies except for the above-mentioned Markov model were identified during the selection process. Therefore, even if we had loosened the inclusion criteria, e.g. to non-RCTs with a sufficiently high number of participants, no further studies would have been identified. Recently published systematic reviews and study protocols related to the cost effectiveness of PN in the context of other cancer types and chronic diseases, might be a good starting point to develop and conduct similar studies in the context of lung cancer and thereby help overcome the research gap.18,49–52\n\nFuture review attempts could consider an a priori broader research question and less restrictive inclusion criteria. Hence, all attempts should weigh heterogeneity concerns and its attached problems of comparability against further potential inclusions.\n\n\nConclusions\n\nNo evidence meeting the inclusion criteria was detected. Hence, this systematic review reveals a research gap regarding the cost-effectiveness of PN programs for patients diagnosed with lung cancer. Furthermore, no conclusive statement regarding the cost-effectiveness of such PN programs can be made. Since the implementation of new healthcare models such as PN at least partially depends on their cost-effectiveness, future evaluation attempts of PN programs in the field should consider examining outcomes related to cost-effectiveness analyses to overcome this research gap and thus help stakeholders make informed decisions regarding the implementation of PN programs among lung cancer patients in routine care.\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\nReporting guidelines\n\nRepository: PRISMA checklist and flow chart for Cost-effectiveness of patient navigation for lung cancer – a systematic review. http://dx.doi.org/10.17169/refubium-29583", "appendix": "Acknowledgements\n\nWe thank American Journal Experts (AJE) for English language editing.\n\n\nReferences\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Zwieten A, Caldwell P, Howard K, et al.: NAV-KIDS(2) trial: protocol for a multi-centre, staggered randomised controlled trial of a patient navigator intervention in children with chronic kidney disease. BMC nephrol. 2019; 20(1): 134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGervès-Pinquié C, Daumas-Yatim F, Lalloué B, et al.: Impacts of a navigation program based on health information technology for patients receiving oral anticancer therapy: the CAPRI randomized controlled trial. BMC Health Serv Res. 2017; 17(1): 133. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "94423", "date": "29 Sep 2021", "name": "Derek Falk", "expertise": [ "Reviewer Expertise Patient navigation and health equity" ], "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\nIntroduction, 3rd Paragraph. How exactly would PN reduce costs for patients? Generally, it is accepted that PN improves timely screening and care.\n\nIntroduction, 3rd Paragraph. The last sentence seems repetitive compared to the barriers presented. Also, it can be simply stated that navigators include both trained health care professionals and lay health workers.\n\nMethods. What was the start date of the literature review?\n\nWhy did the authors only include RCTs in the inclusion criteria? This would severely limit the available literature in this area.\n\nI wonder if the definition of PN should be included in the introduction rather than Methods. Also, are case management, care coordination, and community health working interchangeable concepts?\n\nDiscussion. Could the authors explain how exactly the Shih et al. 2016 study did not meet inclusion criteria without extensive discussion?\n\nDiscussion. Perhaps explaining the design of an ideal study would help the reader to understand the type of study and rigor required for inclusion. Also, do the authors intend to perform such a study as a next step since the literature does not address it?\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": [ { "c_id": "7294", "date": "08 Oct 2021", "name": "Benjamin Kass", "role": "Author Response", "response": "Comment 1: Introduction, 3rd Paragraph. How exactly would PN reduce costs for patients? Generally, it is accepted that PN improves timely screening and care. Response 1: Thank you for this helpful comment. PN generally aims to reduce barriers to appropriate care and has thereby the potential to reduce treatment costs e.g. for people who receive treatment at an earlier (more appropriate) stage of disease. As pointed out by Wells et al. 2008, PN has the potential to move a person to an earlier stage of tumor diagnosis with a potentially less severe degree of cancer and thereby potentially reduce future treatment costs. A sentence has been rewritten for clarification (Introduction, 3rd Paragraph). Comment 2: Introduction, 3rd Paragraph. The last sentence seems repetitive compared to the barriers presented. Also, it can be simply stated that navigators include both trained health care professionals and lay health workers. Response 2: Thank you for pointing this out. We have rewritten the last part of the paragraph (Introduction, 3rd Paragraph). Comment 3: Methods. What was the start date of the literature review? Response 3: The literature review started with the systematic search at January 15th 2020. The first sentence of the methods section has been clarified (Methods, first sentence). Comment 4: Why did the authors only include RCTs in the inclusion criteria? This would severely limit the available literature in this area. Response 4: Thank you for this comment. We see the potential controversy of limiting our review to RCTs. In the process of planning this review, we a priori focused on RCTs, since it was our aim as authors to present the best available evidence. After revealing no relevant RCTs during the review process, this can be seen as a weakness of our review. However, our broad search strategy, which was not restricted to RCTs, could only identify one nonrandomized thematically relevant study, the briefly discussed Markov model by Shih et al. 2016. We tried to elaborate on this topic at the 5th paragraph of the discussion section. Please let us know, if we should include further aspects to the discussion of the study selection. Comment 5: I wonder if the definition of PN should be included in the introduction rather than Methods. Response 5: Thank you very much for your comment. The topic was discussed prior to submission and we considered this point in detail once more. Against the background of our rewritten general description of PN in the introduction, we would prefer to leave our specific PN definition in the Methods to give the audience a general overview about inclusion and exclusion criteria at one place. Comment 6: Also, are case management, care coordination, and community health working interchangeable concepts? Response 6: Our research has shown that these concepts are inconsistently labeled in the literature. Against this background, it was our aim to include all potentially relevant concepts to the search strategy and thereafter to check potentially relevant programs against the background of our PN definition. Comment 7: Discussion. Could the authors explain how exactly the Shih et al. 2016 study did not meet inclusion criteria without extensive discussion? Response 7: The Shih et al. publication is a Markow model based calculation without an underlying RCT. Hence, it did not meet the a priori defined suitable study type for inclusion. A sentence has been rewritten for clarification (Discussion, 2nd Paragraph). Comment 8: Discussion. Perhaps explaining the design of an ideal study would help the reader to understand the type of study and rigor required for inclusion. Response 8: Thank you for your suggestion. We included a sentence to the penultimate paragraph of the Discussion. Comment 9: Also, do the authors intend to perform such a study as a next step since the literature does not address it? Response 9: Yes, it is intended and has already started. As part of the NAVICARE project, some of the authors are working on a project that aims to evaluate a patient navigation program for stroke and lung cancer patients in Germany, which would fit the inclusion criteria for patient navigation of this review in the future. The study is called CoreNAVI - Feasability of a patient navigation programme, and is registered at the German registry for clinical studies (https://www.drks.de/drks_web/setLocale_EN.do). The study will also address the cost-effectiveness issues of patient navigation based on cost data provided by a cooperating statutory health insurance and effectiveness data collected during the trial." } ] } ]	1	https://f1000research.com/articles/10-314
https://f1000research.com/articles/10-719/v1	02 Aug 21	{ "type": "Research Article", "title": "Presentation of cytokine profile in relation to oxidative stress parameters in patients with severe COVID-19: an observational pilot study", "authors": [ "Marija Petrushevska", "Dragica Zendelovska", "Emilija Atanasovska", "Aleksandar Eftimov", "Katerina Spasovska", "Dragica Zendelovska", "Emilija Atanasovska", "Aleksandar Eftimov", "Katerina Spasovska" ], "abstract": "Introduction: COVID-19 can be worsened by hyper-production of cytokines accompanied by increased level of oxidative stress. The aim of this study was to investigate the correlation between a set of cytokines and the markers of the oxidative stress. Methods: The levels of cytokines IL-2, IL-4, IL-6, IL8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, MCP-1 and EGF were determined by using High Sensitivity Evidence Investigator™ Biochip Array technology. The oxidative stress parameters (d-ROM, PAT, OS index) were measured in serum on FRAS5 analytical photometric system. Results: IL-6, IL-8, IL-10, VEGF, MCP-1 and EGF were significantly higher (p<0.05) in the patients with severe COVID-19 with increased levels of IL-2, IFN-g, TNF-a and IL-1α. The d-ROM, OS index, and PAT were significantly higher (p<0.05) in severe COVID-19 patients. IL-6 demonstrated the strongest correlation with all of the markers of the oxidative stress, d-ROM (r=0.9725, p=0.0001), PAT (r=0.5000, p=0.0001) and OS index (r=0.9593, p=0.012). Similar behavior was evidenced between IFN-g and d-ROM (r=0.4006, p=0.0001), PAT (r=0.6030, p=0.0001) and OS index (r=0.4298, p=0.012). Conclusion: The oxidative stress markers show good correlation with the tested cytokines which can be measured at the beginning of the disease in a primary care setting to predict the course of COVID-19.", "keywords": [ "oxidative stress", "COVID-19", "cytokines" ], "content": "1. Introduction\n\nCytokine storm syndrome has been widely discussed and proposed as one of the underlying aetiologies of respiratory failure in patients infected with SARS-CoV-2. Pro-inflammatory cytokines play a key role in large number of respiratory viral infections by activation of the adaptive immune response and, when this response is not controlled, it can lead to involvement of the lung tissue in the course of ARDS or can result in severe damages of multiple organs. For example, following influenza viral infection, an excessive amount of reactive oxygen species (ROS) is produced in several tissues including alveolar epithelium and endothelium1 for which induced expression of cytokines through activation of Toll-like receptors (TLR3, TLR7 and TLR8, retinoic acid inducible gene I and members of NOD-like receptor family) stand in the background of the pathogenesis.2,3 Oxidative stress is typical for infection of human respiratory syncytial virus,4 rhinoviruses,5 and many other viruses. This has been discussed in previously published reviews7-12 and as well, several experimental studies suggest that cytokine storm correlated with direct tissue injury and lead to unfavourable prognosis of severe form of the COVID-19 disease.7 Briefly, particularly high levels of IL-6, IL-10, IL-2R and TNF-α have been reported in patients with severe form of the disease13,14 although other authors suggest that more cytokines, such IL-1β, IL-1RA, IL-8, IL-18 are included in the COVID-19 pathogenesis.7,13,14\n\nAuthors have suggested that the innate immune response follows same pathway for SARS-CoV-2 infection.1,8 Namely, ROS is a strong ligand and a direct mediator in the NLPR3 (inflammasome) trigger. Moreover, NF-κB, which is activated by ROS, triggers transcriptional levels of NLPR3 are enhanced by TLR and NLR ligands. This means that the inflammasome is increased by ROS either directly or indirectly.8,12 To the addition of ROS, H2O2 activates NF-κB to produce inflammatory cytokines.15 Hyperproduction of IL-6, TNF-α, IL-1β, IP-10, GCSF, MCP-1, MIP1-α/CCL3 and elevated blood ferritin are also observed in patients infected with SARS-CoV-2.7,16\n\nFor this purpose, and in the light to share more experimental data as evidence to the suggested pathogenesis of COVID-19 with the scientific community, we have utilized a highly standardized cytokine assay to measure plasma levels of 11 inflammatory cytokines potentially associated as key factors with the cytokine storm syndrome. Afterwards, we have investigated which of these cytokines involved in the cytokine storm of COVID-19 show good association/correlation with the oxidative stress markers determined with fast and inexpensive photometric analytical method. Moreover, the relation between the cytokines, oxidative stress markers and the most commonly used inflammation-related biomarkers (CRP, D-dimers, PLR, NLR and LDH) in severe form of the disease was investigated.\n\n\n2. Methods\n\n52 patients with COVID-19 were hospitalized at the University Clinic for Infectious Diseases and Febrile Conditions, Skopje, Republic of North Macedonia at the beginning of the pandemic within a period of 1 month. 14 patients classified with severe COVID-19 (nine males and five females) with a mean age of 58.36 years (range from 36 to 71 years) were included in this study. The diagnosis and classification of COVID-19 were based on the Interim Guidance for Clinical Management of COVID-19 issued by WHO. Severe cases in addition to severe pneumonia met at least one of the following conditions: SpO2 <90% on room air, respiratory rate >30 breaths/minute or presence of severe respiratory distress. All patients were confirmed to have SARS-CoV-2 infection by real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR). Severe form of COVID-19 as primary exposure variable, demographic characteristics, medical history, clinical symptoms and signs, concomitant medication, outcome data, as well as laboratory analyzes were obtained from the patients’ medical records were other predictor variables. The study flow chart is shown in Figure 1. The study was approved by the local ethics committee (Ethics Committee of the Faculty of Medicine, University of Ss Cyril and Methodius, Skopje, Republic of North Macedonia, No #03-366/7) and complies with the STrengthening the Reporting of Observational studies in Epidemiology (STROBE) statements for reporting of observational trials.17\n\nPAT (total antioxidant power, iron reducing) and d-ROMs (plasma peroxides) were measured on a FRAS5 analytical photometric system (H&D, Italy). Samples were collected and analyzed immediately after hospital admission. The instructions of the manufacturer were followed for the both tests. The d-ROM and PAT are reported in equivalents of H2O2 and ascorbic acid, respectively. Oxidative stress index (OSI) presents information obtained from d-ROMs Fast test and the PAT test that is automatically calculated by the manufacturer’s software (OB manager, FRAS5, H&D, Italy) with normal reference values less than 40.\n\nThe High Sensitivity Evidence Investigator™ Biochip Array technology (Randox Laboratories, GB) was used to perform simultaneous quantitative detection of multiple analytes from a single patient sample (14 SARS-CoV-2 infected and 20 non-infected individuals).\n\n100 μL of plasma was used in biochip carriers, following by incubation on thermo-shaker for 1 hour at 37°C and 370 rpm and 16–20 hours incubation at 4°C. Afterwards, carry out of two wash cycles and 300 μL conjugate was added into each well followed by another incubation of 1 hour at 37°C and 370 rpm. At the final step after twice washing the carriers, fluorescent dye was added to carriers according to protocol and carriers were captured by Evidence Investigator Array. Results were processed automatically using EvInvest software and levels of cytokines IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α, MCP-1 and EGF were calculated as pg/mL.\n\nExposure variables were summarized using descriptive statistics. Data were described as number and/or percentage, or median and range or mean and standard deviation (SD) or standard error of mean (SEM), where appropriate. Differences between groups were explored using the t-test followed by Mann–Whitney where appropriate. A p-value less than 0.05 was considered significant. For purpose of control and comparison between groups, we have analyzed samples of 20 healthy individuals with negative RT-PCR test for SARS-CoV-2 (12 males and eight females, mean age 54). Spearman r coefficient of correlation was performed. All analyses were made using the statistical program GraphPad Prism 9 (USA) (RRID:SCR_000306); an open-access alternative is JASP (RRID:SCR_015823).\n\n\n3. Results\n\nAll 14 patients with a mean age of 58.36 years had severe form of the disease. The average time from onset of symptoms to hospital admission was 10.52±2.33 days (range 7–16 days). All of them had underlying medical conditions at admission. The most frequently reported comorbidities were hypertension, diabetes and chronic cardiac disease. The most prominent and disturbing symptoms reported by the patients on admission were high body temperature (80%), dyspnea (64%), malaise (62%) and cough (56%). The mean value of all clinical laboratory parameters upon hospitalization are presented in Table 1. Abnormal values for CRP, LDH, PLR, D-dimer and NLR were observed. The mean ± SEM value for CRP was 144.7 ± 21.37 mg/L, LDH was 823.4 ± 80.02 IU/L, PLR was 538.2 ± 85.09, NLR was 17.08 ± 2.058, and D-dimer was 2688 ± 499.1 ng/mL. All 14 patients had increased values for ALT, AST and WBC in comparison to the individuals not infected with SARS-CoV-2. The observed statistically difference between the two groups was significant in all cases (p < 0.05).\n\nAs presented in Table 1, 11 cytokines (including chemokines and growth factors) were analyzed in 14 patients infected with SARS-CoV-2 with severe form of the disease and these values were compared with individuals without SARS-CoV-2 infection. In this comparison, statistically significant increase (p < 0.05, t-test) was observed for IL-6, IL-8, IL-10, VEGF, MCP-1 and EGF in the SARS-CoV-2 patients, while IL-2, IFN-γ, TNF-α and IL-1α were increased but this difference was not significant when compared to the individuals without SARS-CoV-2 infection (p < 0.05, t-test). Important finding of this pilot study is that the parameters of the oxidative stress, d-ROM (448.8 ± 30.37 U.Carr), OS index (107.7 ± 14.38) and PAT (3048 ± 100.1 U.Carr) were significantly higher (p < 0.05, t-test) in severe COVID-19 patients when compared to the not infected individuals (Table 1). Moreover, we have investigated the correlation among the investigated cytokines, the oxidative stress parameters and CRP, LDH, PLR, D-dimer and NLR. The Spearman r coefficient of correlation between all these parameters is presented as a heat-map on Figure 2. The heat-map confirmed a positive and significant correlation between all cytokines and the parameters of the oxidative stress (d-ROM, PAT and OSI), except a negative correlation between IL-10 and the total antioxidant capacity, PAT. The correlation was not considered to be significant between OS index and the IL-8 (r = 0.3762, p = 0.8552) and between d-ROM and VEGF (r = 0.2156, p = 0.999). IL-6 demonstrated strongest correlation with all of the markers of the oxidative stress, d-ROM (r = 0.9725, p = 0.0001), PAT (r = 0.5000, p = 0.0001) and OS index (r = 0.9593, p = 0.012). Alongside, similar behavior was evidenced between IFN-γ and d-ROM (r = 0.4006, p = 0.0001), PAT (r = 0.6030, p = 0.0001) and OS index (r = 0.4298, p = 0.012). We further investigated the correlation between the cytokines and CRP as one of the most commonly used biomarkers, where the strongest one was observed with IL-6, IL-8, MCP-1 and IFN-γ. Moreover, in terms of correlation, investigated inflammatory cytokines IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, IFN-γ, TNF-α, IL-1α and MCP-1 showed a strong positive correlation between each other, except between IL-6 and EGF (Figure 2).\n\n\n4. Discussion\n\nCytokines including chemokines and growth factors together with lipid metabolites are among the main factors of immune cell function and their differentiation, hence upon their dysregulation various diseases can arise.7-9,12 Herein, we share our results to give an add-on to the clinical evidences that oxidative stress is increased in patients with severe form of COVID-19 and that the measured oxidative stress parameters had shown a good correlation with the cytokines and the commonly used laboratory biomarkers. This pilot study focused on the possibility to utilize the oxidative stress parameters (d-ROM, PAT and OS index) as a fast and inexpensive prognostic tool for disease progression and potentially predict the outcome of COVID-19 in patients. Several retrospective studies and reviews have been published where abnormal levels of cytokines involved in the adaptive immunity (IL-2, IL-4) or pro-inflammatory cytokines and interleukins (IFNs, IL-1, IL-6, IL-10 IL-17 and TNF-α) were reported.7,13,18,19\n\nOur study revealed that several cytokines and biomarkers were significantly increased in infected SARS-CoV-2 patients with severe form of the disease in comparison to those who were not, which was accompanied with coagulopathy as determined by deterioration of the platelet related parameters (PLR, D-dimer, IL-6) and MCP-1 as thrombosis related indicator. Huang et al. (2020) reported that MCP-1 levels were much higher in critical ICU patients and additionally that the platelet count was lower in those patients that do not survive.19 Patients from our study were all with severe form of COVID-19 and all of them had died during hospitalization. Moreover, in our patients several of the cytokines had been increased more than 10-fold above the levels of the non-infected that we considered as a baseline. It is worth noting, the statistically significant increase of the VEGF levels more than 10-fold that can be related to the essential role of VEGF in endothelial cell activation by binding to cell surface VEGF receptors. VEGF up-regulation was observed in several viral infections and it has been investigated as a target for potential therapy development.20 In addition, Huang et al., report higher levels of VEGF in hospitalized COVID-19 patients.19\n\nThe strong correlation between the investigated cytokines (including chemokines and growth factors), the oxidative stress parameters and some of the commonly used biomarkers (CRP, D-dimers, NLR, PLR) are in line with the proposed cytokine storm as underlying mechanism of the infection. The cytokine storm syndrome occurs when large numbers of leukocytes are activated and release a high concentration of proinflammatory cytokines, with IL-6, IL-10, IFN, MPC-1, IL-1, IL-2 and IL-8 being the foremost. Generally, SARS-CoV-2 infection is associated with oxidative stress, the proinflammatory state, cytokine production, and cell death demonstrated by increase in ROS levels and an alteration of antioxidant defense during the infection.11,21\n\nEven though limited published data are available, we believe that SARS-CoV-2 in line with other RNA viruses triggers oxidative stress by disturbing the pro-antioxidant–antioxidant balance.8,22,23 We have demonstrated the significantly higher level of the d-ROM and OS index values in the infected patients with SARS-CoV-2 when compared with those who were not infected, supporting the hypothesis that viral infection will increase the oxidative stress and complicate the course of the disease. Whilst we consider that the OS index value presents an important parameter that we can have an impact on against COVID-19, by supplementation with antioxidants especially when there is applicable knowledge for several nutraceuticals/vitamins (vitamin C, vitamin D, curcumin, selenium, quercetin and other polyphenols) with proven anti-inflammatory, antioxidant and antiviral capacity.24,25\n\nThere are several limitations of the study besides being a single-center experience and a pilot study with only severe and critically ill patients. The herein presented patients were hospitalized at the beginning of the global pandemic when no specific and official guidelines were issued and available to assist the need for hospitalization. They had symptoms developed several days prior being hospitalized, however we believe that these symptoms were not life threatening and the hyper-inflammatory phase was at its beginning stage which is deemed by the obtained levels of the cytokines and the oxidative stress index. Nevertheless, further studies concerning COVID-19 patients with high levels of d-ROMs and OS index are warranted to determine whether supporting antioxidant therapy can reduce the possibility for the fatal outcome of the critically ill COVID-19 patients.\n\n\n5. Conclusion\n\nThis observational pilot study demonstrates a good correlation between the panel of tested cytokines and the parameters of the oxidative stress measured by a fast photometric method that could be used at the beginning of the disease to predict whether COVID-19 will develop in severe form. The presented results will contribute to support the evidences that the cytokine storm syndrome lies as an immunopathogenesis during SARS-CoV-2 infection and by using the oxidative stress parameters (d-ROM, PAT, OS index) physicians can provide timely and early interventions in COVID-19 patients.\n\n\nAuthor contributions\n\nMP, DZ, EA contributed to the conception and design of the study. MP and DZ contributed to the oxidative stress parameters analyses, collated the data for the study, and completed all statistical analysis of data. MP wrote the first draft of the manuscript. AE performed the cytokine assay. KS and EA contributed to the clinical evaluation and medical data collection from the COVID-19 patients. All authors read and approved the final version of the manuscript.\n\n\nData availability\n\nDataDryad: Underlying data for ‘Presentation of cytokine profile in relation to oxidative stress parameters in patients with severe COVID-19: an observational pilot study’. https://doi.org/10.5061/dryad.gf1vhhmqg.\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\nChernyak BV, Popova EN, Prikhodko AS, et al.: COVID-19 and Oxidative Stress. Biochemistry (Mosc). 2020; 85(12):1543–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIwasaki A, Pillai PS: Innate immunity to influenza virus infection. Nat Rev Immunol. 2014; 14:315–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHui DSC, Zumla A: Severe Acute Respiratory Syndrome: Historical, Epidemiologic, and Clinical Features. Infect Dis Clin North Am. 2019; 33(4):869–89. 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PubMed Abstract \| Publisher Full Text\n\nNtyonga-Pono M: COVID-19 infection and oxidative stress: an under-explored approach for prevention and treatment? Pan Afr Med J. 2020; 35(Suppl 2):12. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "94168", "date": "21 Sep 2021", "name": "Atakan Tanacan", "expertise": [ "Reviewer Expertise Perinatology", "maternal-fetal medicine", "prenatal diagnosis" ], "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 have read the manuscript with great interest. The authors have made a comprehensive study on an important topic. I have some minor recommendations for the authors:\nThe inflammatory background of COVID-19 should be discussed.\n\nTreatment options and targeted therapies should be discussed in more detail.\n\nSevere COVID-19 complications should be discussed in more detail.\n\nThe clinical implication of findings obtained from the present study should be underlined.\n\nThe following literature by Tanacan et al. 20211 may be useful and in my opinion, the mechanisms behind the possible immunologic processes behind COVID-19 may help the authors to improve their 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? 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": "7213", "date": "14 Oct 2021", "name": "Marija Petrushevska", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your contribution to the increase in the quality of the manuscript. Below I attend to all of your comments. The changes will be included in the 2nd version of the manuscript after we receive an additional report from another reviewer. Comment 1, Comment 2, and Comment 5: The authors have discussed the inflammatory background of the disease in the Section Discussion on page 6 of 10 (first paragraph). A revision will be inserted where the reference Tanacan et al. 2021 will be included, additionally, in this section, we will discuss treatment options and targeted therapies. Comment 3: COVID-19 complications are also presented and well referenced in the manuscript. We believe that this has been a subject of numerous publications in the past year. Also, we discussed the most prominent and disturbing symptoms and their onset followed by the clinical biochemical parameters. Comment 4: The clinical implication of findings has been pointed out in the Section Conclusion. However, we will elaborate in more detail as stated below: \"The presented results will contribute to supporting the evidence that the cytokine storm syndrome lies as an immunopathogenesis during SARS-CoV-2 infection. By using the oxidative stress parameters (d-ROM, PAT, OS index) physicians can provide timely and early interventions in COVID-19 patients. Namely, we consider that the investigated parameters can be used as a tool at the beginning of COVID-19 disease for the general assessment of oxidative stress and hence enabling a better triage of the patients in terms of disease severity.\"" } ] }, { "id": "94846", "date": "07 Oct 2021", "name": "Samuel Asamoah Sakyi", "expertise": [ "Reviewer Expertise Infectious and non-communicable diseases", "biomarkers discovery", "(diabetes", "hypertension", "kidney disorders", "cytokines)" ], "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 current study examined the levels of cytokines and oxidative stress markers among COVID-19 patients and correlated them with disease severity.\nThe manuscript is well written except that in the Methods section the authors failed to state the study design. I inferred from the statistical section that the study design was a case-control study. The authors should describe the control very well - how they were recruited to ensure they were devoid of confounding factors, more especially since cytokines are involved. Cytokines are affected by many factors. Additionally, conclusion statements should explain the statistical terms correlation.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7293", "date": "14 Oct 2021", "name": "Marija Petrushevska", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your contribution to increasing the quality of our manuscript. We have accepted all your remarks, and we made changes in the material section in order to describe the control (recruitment, risk factors). All changes are marked in red and with track changes. Additionally, in the Section Conclusion - we have elaborated in detail on the statistical correlation." } ] } ]	1	https://f1000research.com/articles/10-719
https://f1000research.com/articles/10-550/v1	08 Jul 21	{ "type": "Research Article", "title": "Toxin-like peptides in plasma, urine and faecal samples from COVID-19 patients", "authors": [ "Carlo Brogna", "Simone Cristoni", "Mauro Petrillo", "Maddalena Querci", "Ornella Piazza", "Guy Van den Eede", "Carlo Brogna", "Maddalena Querci", "Ornella Piazza", "Guy Van den Eede" ], "abstract": "Background: SARS-CoV-2 that causes COVID-19 disease and led to the pandemic currently affecting the world has been broadly investigated. Different studies have been performed to understand the infection mechanism, and the involved human genes, transcripts and proteins. In parallel, numerous clinical extra-pulmonary manifestations co-occurring with COVID-19 disease have been reported and evidence of their severity and persistence is increasing. Whether these manifestations are linked to other disorders co-occurring with SARS-CoV-2 infection, is under discussion. In this work, we report the identification of toxin-like peptides in COVID-19 patients by application of the Liquid Chromatography Surface-Activated Chemical Ionization – Cloud Ion Mobility Mass Spectrometry.\n\nMethods: Plasma, urine and faecal samples from COVID-19 patients and control individuals were analysed to study peptidomic toxins’ profiles. Protein precipitation preparation procedure was used for plasma, to remove high molecular weight proteins and efficiently solubilize the peptide fraction; in the case of faeces and urine, direct peptide solubilization was employed.\n\nResults: Toxin-like peptides, almost identical to toxic components of venoms from animals, like conotoxins, phospholipases, phosphodiesterases, zinc metal proteinases, and bradykinins, were identified in samples from COVID-19 patients, but not in control samples. Conclusions: The presence of toxin-like peptides could potentially be connected to SARS-CoV-2 infection. Their presence suggests a possible association between COVID-19 disease and the release in the body of (oligo-)peptides almost identical to toxic components of venoms from animals. Their involvement in a large set of heterogeneous extra-pulmonary COVID-19 clinical manifestations, like neurological ones, cannot be excluded. Although the presence of each individual symptom is not selective of the disease, their combination might be related to COVID-19 by the coexistence of the panel of the here detected toxin-like peptides. The presence of these peptides opens new scenarios on the aetiology of the COVID-19 clinical symptoms observed up to now, including neurological manifestations.", "keywords": [ "SARS-CoV-2", "COVID-19", "toxin-like peptides" ], "content": "Introduction\n\nNumerous clinical extra-pulmonary manifestations co-occurring with COVID-19 disease have been reported (e.g. neurological, haemorrhagic, and thrombotic) and evidence of their severity and persistence is increasing. Gupta et al. reviewed the extrapulmonary organ-specific pathophysiology of patients with COVID-19, 'to aid clinicians and scientists in recognizing and monitoring the spectrum of manifestations, and in developing research priorities and therapeutic strategies for all organ systems involved'1. Liotta et al. characterized the incidence of neurological manifestations in a cohort of hospitalised patients with confirmed COVID-19: the most frequent were myalgia, headache, encephalopathy, dizziness, dysgeusia, and anosmia; encephalopathy was found to be 'associated with increased morbidity and mortality, independent of respiratory disease severity'2. Whether these manifestations are linked to disorders co-occurring with SARS-CoV-2 infection is under discussion, including their concomitant occurrence, which could be strongly related COVID-19 disease. Frontera et al., by conducting a prospective, multi-centre, observational study of hospitalised adults with laboratory-confirmed SARS-CoV-2 infection, concluded that 'neurologic disorders were detected in 13.5% of COVID-19 patients during the study timeframe. Many of these neurologic disorders occur commonly among patients with critical illness. Encephalitis, meningitis or myelitis referable to SARS-CoV-2 infection did not occur, though post-infectious Guillain-Barre syndrome was identified. Overall, neurologic disorders in the context of SARS-CoV-2 infection confer a higher risk of in-hospital mortality and reduced likelihood of discharge home'3.\n\nStudies on the use of mass spectrometry in COVID-19 context focus on the search for augmented human inflammatory molecules to be used as biomarkers to assess the severity status of COVID-19 (see for example the work4 of Messner and colleagues). Different studies report the use of proteomic approaches to characterise SARS-CoV-2 proteins5–7. Other studies highlight challenges in their use due to the need of enriching the protein fraction to be analysed for maximizing the technology sensitivity8.\n\nLiquid Chromatography Surface-Activated Chemical Ionization – Cloud Ion Mobility Mass Spectrometry (LC-SACI-CIMS) is reported as a high sensitivity mass spectrometry technique able to maximize the peptide signal intensity9–12. We used LC-SACI-CIMS to reveal the presence of metabolites that could explain the clinical descriptions of neurological, coagulation and inflammatory symptoms, and here we present the results of our analyses. We found toxin-like peptides in plasma, urine, and faecal samples from COVID-19 patients, but not in control samples. As our findings do not correspond with current thinking of the aetiology related to the observed clinical manifestations in COVID-19 patients, we feel their immediate sharing with the scientific community is critical.\n\n\nMethods\n\nLiquid Chromatography-Surface Activated Chemical Ionization – Cloud Ion Mobility Mass Spectrometry (LC-SACI-CIMS) exhibits a high selectivity in peptide detection thanks to its ability to selectively isolate peptide ions through an in-source ion mobility (IM) effect. In fact, it allows a selective regulation of the potential difference between the low voltage of the SACI surface (47 V) and the entrance lens (-50 / -600 V), and a selective focalization on solvent ion cloud containing species at low or high m/z ratio. By switching the entrance voltage lens between -50 and -600 V during the analysis, it is possible to separate the low m/z from the high m/z potential signal, to avoid ion trap saturation, and to maximize the number of detected compounds. The mass spectra chemical noise is also strongly reduced due to the lower amounts of solvent cluster ions that are produced in low voltage ionization conditions. Thus, the peptide detection efficiency is strongly increased by the IM selectivity and lower chemical noise with respect to the classical high voltage ionization approaches. Thanks to the specificity of the SACI-CIMS technology in focalizing the solvent ion clouds containing the high m/z (oligo-)peptide species, it was possible to increase the detection efficiency.\n\nIn the use of LC-SACI-CIMS, the following strategies have been adopted:\n\nTo reduce the presence of contamination as much as possible and to avoid the formation of acetonitrile polymers occurring in acid conditions (as reported by Eizo et al.13), formic acid was not added to the CH3CN chromatographic phase.\n\nTo separate low from high m/z solvent ion clusters by reducing the ion trap saturation, the space/charge effect, and by increasing the detected compounds recovery, LC-SACI–CIMS entrance lens voltage was switched between -50 and -600 V every 10 ms during the analysis.\n\nTo enhance the SACI ionization efficiency, NH4HCO3 was added to the samples. As reported in the literature14,15, the peptide ionization efficiency (and consequently the sensitivity) is enhanced in SACI conditions when ionic salts are present in the sample, due to peptide ion specific coordination.\n\nTo decrease the total run time, a shot gun chromatographic gradient was used to desalt the sample.\n\nTo avoid sample molecular profile alteration, and to evaluate the potential biological activities of the circulating species, no enzymatic digestion was applied to samples.\n\nTo normalize the m/z signal intensity, 5 µL of standard ESI tune mix (Agilent, USA) were added to each sample extract.\n\nNH4HCO3, methanol, acetonitrile and formic acid were purchased from Sigma-Aldrich (Milan, Italy). Bi-distilled water was purchased from VWR (Milan, Italy).\n\nSamples used in the present study: plasma samples collected from 15 COVID-19 patients from different cities of Italy and from five control individuals (i.e. negative to SARS-CoV-2 tests and not affected by cancer or autoimmune diseases); urine samples collected from two additional COVID-19 patients and from two control individuals; stool samples from three COVID-19 patients and from three control individuals. The human biological samples used in the experimentation were collected and used with the expressed free and informed written consent, of the person from whom the material was taken, according to current legislation. The study received approval from “Comitato Etico Campania Sud” (n.36/2021, request submitted on 06-05-2020).\n\nPlasma. Each plasma sample was treated as follows: 5 µL of CH3CN were added to 50 µL of plasma and vortexed for one minute. The procedure was repeated 10 times. Then the sample was centrifuged at 1,500 g for 10 minutes and two 100 µL aliquots of supernatant were dried and resuspended in 70 µL of NH4HCO3 50 mmol. The solution was analysed by LC-SACI-CIMS (see Rationale).\n\nUrine. Each urine sample was treated as follows: an equivalent volume of bi-distilled water was added, followed by centrifugation at 1,500 g for 10 minutes. 100 µL were dried and resuspended in 70 µL of NH4HCO3 50 mmol. The sample was analysed by LC-SACI-CIMS (see Rationale).\n\nStool. Each stool sample was treated as described by Cristoni et al.11 and analysed by LC-SACI-CIMS (see Rationale).\n\nThe Ultimate 3000 LC (by ThermoFisher) was used to achieve separation of analytes for each sample prior to mass spectrometry (MS) analysis. A reversed phase Kinetex C-18 LC column (50 × 2.1 mm; particle size, 5 µm; pore size, 100 Å, by Phenomenex, USA) was used. The eluent flow was 0.25 mL/min and the injection volume was 15 µL. The mobile phases were:\n\nA. 0.2% (v/v) formic acid (HCOOH)\n\nB. acetonitrile (CH3CN)\n\nThe elution gradient was: 2% (v/v) of B between 0 and 2 min; 2 to 30% between 2 and 7 min; 30 to 80% between 7 and 9 min; 80% between 9 and 12 min; 80-2% between 12 and 12.1 min. The column was rebalanced with 2% of B between 12.1 and 17 min.\n\nAll samples were analysed for the presence of proteins with potential toxic effect by using the LC-SACI-CIMS as already described in the literature9–12. Samples were analysed with an ORBITRAP mass spectrometer (Breme, Germany) coupled to a surface-activated chemical ionization (SACI) source and operated in positive ion mode.\n\nThe surface voltage was 47 V and the entrance lens was switched between -50 and -600 V each 10 ms. Auxiliary gas: 2 L / min; Nebulizer gas: 80 psi; Temperature: 40 °C. Full scan spectra were acquired in the 40–3,500 m/z range for non-targeted metabolomics/proteomics analyses to detect analytes. The same m/z range was used for both discovery and selective biomarker identification, and to standardize (primarily in terms of scan rate) the instrument. The software used for data elaboration is SANIST, a modified version of the Global Proteome Machine (GPM, https://www.thegpm.org/GPM/), implanted as described in 9–12. SANIST output files are available as supplementary material16 (see section Data availability).\n\nSANIST software here used is freely available, upon email request to CranioMed group (dir.brogna@craniomed.it).\n\nMass spectrometry on samples was performed with collision-induced dissociation using data dependent scan and helium as the collision gas. The ion trap was applied to isolate and fragment the precursor ions (windows of isolation, ± 0.3 m/z; collision energy, 30% of its maximum value, which was 5V peak to peak), and the ORBITRAP mass analyser was used to obtain fragments with an extremely accurate m/z ratio (resolution 15,000; m/z error <10 ppm).\n\nDetected high m/z peptides were used to identify toxins thanks due to the selectivity given by their long chain.\n\nThe complete UniprotKB set of manually reviewed venom proteins and toxins (UniprotKB, Animal toxin annotation project. https://www.uniprot.org/program/Toxins, Accessed October 4, 2020), mixed with a subset of non-venom proteins and toxins from UniprotKB database17 was used as reference protein dataset in order to give statistical significance to the results.\n\nTBLASTN18 was run at the National Center for Biotechnology Information (NCBI) website19 with default options and parameters, with the exception of the following ones: max target sequences = 1,000; expect threshold = 100; word size = 3; gap cost existence = 9; gap cost extension = 1; filter of low complexity regions = No. Searches have been performed versus: Nucleotide collection (nr/nt); Reference RNA sequences (refseq_rna); RefSeq Genome Database (refseq_genomes); Whole-genome shotgun contigs (wgs) from metagenomic experiments; Sequence Read Archive (SRA) sequences from metagenomic experiments; Transcriptome Shotgun Assembly (TSA); Patent sequences (pat); Human RefSeqGene sequences (RefSeq_Gene); Betacoronavirus Genbank sequence dataset.\n\nThe information reported in Table 1 has been retrieved from the UniprotKB database and from the NCBI Taxonomy database20, after confirmation by BLAST sequence comparison analysis18.\n\nThirty-six candidate protein sequences on which the identified toxin-like peptides have been mapped are here reported, together with information retrieved from UniprotKB and NCBI Taxonomy databases. The table is split in three sections according to the phylum of the reported species: Chordata (green), Echinodermata (pink), Mollusca (azure).\n\nSANIST was set to perform the database search considering all potential protein points and post-translational modifications, and to consider proton rearrangements. No enzyme cutting rules were specified, but all the protein subsequence combinations were considered. Database search calculation was performed by means of General Processing Graphic Processing Units (GPGPU).\n\nThe MS data are available on the ZENODO platform16 (see section Data availability).\n\n\nResults and discussion\n\nThe presence of (oligo-)peptides characterised as toxic components of animal venoms was observed in plasma and urine samples from SARS-CoV-2 infected patients and never in plasma, urine and faecal samples from control individuals. Examples of SACI-CIMS chromatograms are reported in Figure 1 and Figure 2 (panels a and b), showing the spectra acquired by means of the LC-SACI-CIMS technology. Figure 2c and d show the spectra obtained using ESI extracted at the same retention time. SACI-CIMS give rise to higher signal intensities probably due to the low ion trap saturation.\n\n(a) Base peak LC Full Scan (MS), tandem mass (MS/MS) chromatogram of an extracted plasma sample of a patient and a control subject and (b) a blow-up of a specific chromatogram region (5.713–5.719 min). The blow-up shows the four regions of data acquisition: 1) Full scan mass spectrum originated by the cloud containing low m/z ratio molecular species; 2) Tandem mass spectra (MS/MS) mass spectrum originated by the cloud containing low m/z ratio molecular species; 3) Full scan mass spectrum originated by the cloud containing medium-high (MedHigh) m/z ratio molecular species; 4) Tandem mass spectra (MS/MS) mass spectrum originated by the cloud containing medium-high (MedHigh) m/z ratio molecular species.\n\nExamples of full scan mass spectra, obtained by analysing a COVID-19 positive urine sample and acquired focalizing solvent ion cloud species containing a) low, b) high m/z species extracted in the 5.713–5.719 min chromatographic region and ESI full scan mass spectrum obtained analysing the same sample and extracting the signal at the same retention time extracting c) low and d) high m/z ratio.\n\nSeveral (oligo-)peptides (between 70 and 115, depending on the analysed sample) matched to different animal venom proteins and toxins like conotoxins, phospholipases A2, metalloproteinases (86% of assignments have a -log(e) higher than 25). An overview of 36 proteins covered by the toxin-like peptides found is reported in Table 1; details of -log(e) and false discovery rates are reported in Table 2. Examples of mass spectra peptide characterization together with the peptide ion fragmentation pathways are shown in Figure 3a. All the MS/MS signal were assigned to the different N-terminal y,z (blue and purple colour) and c-terminal b,c (red and yellow colour) fragmentation series (see Figure 3b for fragmentation series details). In the defined SACI-CIMS conditions, doubly charged m/z ion of medium-high molecular weight peptide species are produced, allowing high identification accuracy, in line with what is already described in the literature that high identification statistical rates are achieved analysing peptide doubly charged species with medium high molecular weight. Different fragmentation anomalies with proton rearrangements have also been detected and considered in phase of data elaboration. Only mass spectra exhibiting a statistical -log(e) score higher that 10 and a false discovery rate lower than 0.05 were considered for the identification (see Figure 3c). False discovery rate and statistical score were estimated by means of reverse sequence approach.\n\nSome of the toxin-like peptides found mapped on the same reference protein (UniprotKB: D2DGD8), are reported in Figure 4: these peptides were found in the five plasma samples and in the three faecal samples.\n\nConotoxin Pu6.1 from Conus pulicarius (UniprotKB:D2DGD8) is aligned with the toxin-like peptides identified in four out of five plasma samples. Being the protein secreted and cleaved, leader-region pro-peptide and mature cysteine rich domains are highlighted in green, yellow and red, respectively. Each identified toxin-like peptide is named according to the sample of origin and its uniqueness. For each of them, the number reported in square brackets indicates the number of identical toxin-like peptides identified in the same sample.\n\nThe types of toxic-like peptides found resemble known conotoxins, phospholipases A2, metalloproteinases, prothrombin activators, coagulation factors, usually present in animal venoms, which are known to have high specificity and affinity towards human ion channels, receptors, and transporters of the nervous system, like the nicotinic acetylcholine receptor.\n\nThe same results have been observed in an additional set of 10 plasma samples from 10 different patients.\n\nWhat follows is our attempt to elaborate a potential relation between their presence and extra-pulmonary COVID-19 symptomatology.\n\nConotoxins are neurotoxic peptides isolated from the venom of marine (genus Conus) cone snails. In their mature form, they consist of 10 to 30 amino acid residues, with often one or more disulphide bonds, which are used to classify them in structural classes (μ-conotoxins, ω-conotoxins, and α-conotoxins are the major classes). The mechanism of action of conotoxins is not yet fully understood21. Studies have found that they are able to modulate the activity of several receptors, including ion channels, nicotinic acetylcholine receptors (nAChRs) and acetylcholine-degrading enzymes (acetylcholinesterases), thus resulting in the alteration of acetylcholine levels and of cholinergic transmission22–24. Regarding cholinesterases, a potential association between cholinesterase levels and severity of pneumonia in COVID-19 patients has been proposed25.\n\nThe presence of conotoxin peptides might explain the occurrence of many symptoms (like hyposmia, hypogeusia and the signs typical of Guillain-Barre syndrome) observed in some COVID-19 patients. Their presence can alter normal functioning of ion channels, nicotinic acetylcholine receptors and of acetylcholine levels.\n\nPhospholipases A2 (PLA2, E.C. 3.1.1.4) hydrolyse phospholipids and lead to release of lysophosphatidic acid and arachidonic acid26. Arachidonic acid is a major precursor of many pro-inflammatory mediators like leukotriene, thromboxane and prostaglandin; as a consequence, abnormal presence of active PLA2 can induce severe inflammation27. In animal venoms, PLA2 act as neurotoxic proteins: they hydrolyse membrane phospholipids of the motor nerve terminal, and the plasma membrane of skeletal muscle, thus triggering a severe inflammatory degenerative response, which in turn leads to degeneration of the nerve terminal and skeletal muscle26. The drug dexamethasone can inhibit prostaglandins synthesis and leukotriene formation28. As dexamethasone is still the only therapeutic shown to be effective against the novel coronavirus in patients29 with severe symptoms, it can be that the positive effect of this drug on COVID-19 patients is also due to the reduction of the here identified PLA2-like peptides.\n\nThe last example of identified toxin-like peptides is those recognised as metalloproteinases present in animal venoms, zinc-dependent enzymes of varying molecular weight having multidomain organization. These toxic enzymes cause haemorrhage, local myonecrosis, skin damage, and inflammatory reaction30. It has been reported that symptomatic COVID-19 patients have significantly lower zinc levels in comparison to controls and that zinc deficient patients develop more complications31. The presence of this specific group of toxin-like peptides, which capture zinc, can be one of the reasons for such significantly low zinc levels in symptomatic COVID-19 patients.\n\nSimilarity searches by TBLASTN14 with relaxed parameters at the National Center for Biotechnology Information (NCBI) website (see Methods) revealed (in addition to mRNA sequences from the animal species reported in Table 1) almost identical short stretches (up to 10 amino acids) of these peptides in potential coding regions of many bacterial and viral sequences, but no long potential coding frame entirely covering any of them was found. Consequently, at the time of writing we have not yet identified the \"genetic source\" of these peptides, which could be:\n\nThe SARS-CoV-2 RNA genome with its protein reading set, as proposed by Brogna32, who reported the identification in SARS-CoV-2 RNA of many regions encoding for oligopeptides (four–five amino acids long) identical to neurotoxin peptides typical of animal venoms.\n\nThe SARS-CoV-2 genome directly read by bacteria, assuming that the SARS-CoV-2 genome, or parts thereof, is capable of replicating with a possible ‘bacteriophage-like’ mode of action, as previously described33.\n\nGenomes of bacteria, which, as a reaction to the presence of the virus, secrete these peptides. This could happen by using still not well known and debated mechanisms, like alternative reading due to rRNA sequence heterogeneity (as described in 34,35), or the involvement of small bacterial ncRNA (sRNAs), known to be key players of gene regulation under conditions like stress response, quorum sensing, or virulence (in this context, in 1984 Coleman et al. reported the micF non-coding RNA as a functional bacterial sRNA36).\n\nA combination of the above e.g. the ‘toxin’ genetic code is present in the bacteria and expression may be triggered by SARS-CoV-2, acting like temperate bacteriophages, which are known to interact with bacteria so that they express (or not) certain genes, as described by Carey et al.37.\n\nA detailed 3D structural similarity analysis between the toxin-like peptides found and reference proteins has not yet been conducted. Accordingly, at the time of writing, we can only speculate that these toxin-like peptides are involved in the clinical extra-pulmonary manifestations in symptomatic COVID-19 patients. According to our knowledge, these toxin-like peptides have never been searched in animals considered reservoirs of SARS-CoVs.\n\n\nConclusions\n\nThe presence of (oligo-)peptides almost identical to toxic components of venoms from animals has been observed. Data and results reported here suggest an association between COVID-19 disease and the release in the body of these, and raise a series of questions:\n\nAre these findings in line with what was proposed by Tizabi et al.38, i.e. a potential therapeutic role for nicotine, nicotinic agonists, or positive allosteric modulators of nicotinic cholinergic receptors in COVID-19?\n\nIf induced by SARS-CoV-2, can the production of toxin-like peptides be involved in the neurological disorders and injuries observed in hospitalized COVID-19 patients?\n\nIf induced by SARS-CoV-2, can the production of toxin-like peptides influence complex diseases apparently triggered or enhanced by COVID-19, like e.g. Guillain-Barré Syndrome39 or Parkinson's disease40?\n\nAre toxin-like peptides associated with SARS-CoV-2 infection or to other viral infections or, more in general, is their presence related to sickness condition?\n\nAre our findings supporting the suggestion made by the iVAMP Consortium41 on the relationships between animal venom glands and microorganisms' microenvironments?\n\nWe consider that the immediate sharing of these results can contribute to the untangling of the multifaceted set of clinical manifestations in symptomatic COVID-19 patients, and to the further understanding of the mechanisms involved.\n\n\nData availability\n\nUniprot: Kunitz-type serine protease inhibitor homolog beta-bungarotoxin B1 chain [Bungarus candidus (Malayan krait)]. Accession number Q8AY46, https://identifiers.org/uniprot:Q8AY46\n\nUniprot: Basic phospholipase A2 BFPA, svPLA2, EC 3.1.1.4 (Antimicrobial phospholipase A2) (Phosphatidylcholine 2-acylhydrolase) [Bungarus fasciatus (Banded krait) (Pseudoboa fasciata)]. Accession number A6MEY4, https://identifiers.org/Uniprot:A6MEY4\n\nUniprot: Phospholipase A2 MALT0035C, svPLA2, EC 3.1.1.4 [Micrurus altirostris (Uruguayan coral snake) (Elaps altirostris)]. Accession number F5CPF1, https://identifiers.org/Uniprot:F5CPF1\n\nUniprot: Zinc metalloproteinase-disintegrin-like NaMP, EC 3.4.24.- (Snake venom metalloproteinase, SVMP) [Naja atra (Chinese cobra)]. Accession number A8QL59, https://identifiers.org/Uniprot:A8QL59\n\nUniprot: Acidic phospholipase A2 D, svPLA2, EC 3.1.1.4 (APLA) (Phosphatidylcholine 2-acylhydrolase) [Naja sputatrix (Malayan spitting cobra) (Naja naja sputatrix)]. Accession number Q9I900, https://identifiers.org/Uniprot:A9I900\n\nUniprot: Venom prothrombin activator omicarin-C non-catalytic subunit, vPA (Venom coagulation factor Va-like protein) [Cleaved into: Omicarin-C non-catalytic subunit heavy chain; Omicarin-C non-catalytic subunit light chain] [Oxyuranus microlepidotus (Inland taipan) (Diemenia microlepidota)]. Accession number A58L90, https://identifiers.org/Uniprot:Q58L90\n\nUniprot: Venom prothrombin activator oscutarin-C non-catalytic subunit, vPA (Venom coagulation factor Va-like protein) [Cleaved into: Oscutarin-C non-catalytic subunit heavy chain; Oscutarin-C non-catalytic subunit light chain] [Oxyuranus scutellatus (Coastal taipan)]. Accession number Q58L91, https://identifiers.org/Uniprot:Q58L91\n\nUniprot: Short neurotoxin 4, SNTX4 (Alpha-neurotoxin 4) [Pseudonaja textilis (Eastern brown snake)]. Accession number Q9W7J9, https://identifiers.org/Uniprot:Q9W7J9\n\nUniprot: Acidic phospholipase A2 homolog textilotoxin D chain, svPLA2 homolog [Pseudonaja textilis (Eastern brown snake)]. Accession number P23028, https://identifiers.org/Uniprot:P23028\n\nUniprot: Coagulation factor V [Cleaved into: Coagulation factor V heavy chain; Coagulation factor V light chain] [Pseudonaja textilis (Eastern brown snake)]. Accession number Q593B6, https://identifiers.org/Uniprot:Q593B6\n\nUniprot: Venom prothrombin activator pseutarin-C non-catalytic subunit, PCNS, vPA (Venom coagulation factor Va-like protein) [Cleaved into: Pseutarin-C non-catalytic subunit heavy chain; Pseutarin-C non-catalytic subunit light chain] [Pseudonaja textilis (Eastern brown snake)]. Accession number Q7SZN0, https://identifiers.org/Uniprot:Q7SZN0\n\nUniprot: Cysteine-rich venom protein ENH1, CRVP (Cysteine-rich secretory protein ENH1, CRISP-ENH1) [Pseudoferania polylepis (Macleay's water snake) (Enhydris polylepis)]. Accession number Q2XXQ3, https://identifiers.org/Uniprot:Q2XXQ3\n\nUniprot: Bradykinin-potentiating and C-type natriuretic peptides (Brain BPP-CNP, bBPP-CNP) (Evasin-CNP) [Cleaved into 12 chains] [Bothrops jararaca (Jararaca)]. Accession number Q9PW56, https://identifiers.org/Uniprot:Q9PW56\n\nUniprot: Snake venom metalloprotease inhibitor 02D01 (02E11) (10F07) (Svmpi-Eoc7) [Cleaved into 15 chains] [Echis ocellatus (Ocellated saw-scaled viper)]. Accession number A8YPR6, https://identifiers.org/Uniprot:A8YPR6\n\nUniprot: Zinc metalloproteinase/disintegrin [Cleaved into: Snake venom metalloproteinase brevilysin L4, SVMP (Snake venom metalloproteinase hxl-1, EC 3.4.24.-) ; Disintegrin brevicaudin-1a; Disintegrin brevicaudin-1b (Disintegrin adinbitor) (Disintegrin halystatin)] [Gloydius brevicaudus (Korean slamosa snake) (Agkistrodon halys brevicaudus)]. Accession number Q698K8, https://identifiers.org/Uniprot:Q698K8\n\nUniprot: Zinc metalloproteinase-disintegrin-like halysase, EC 3.4.24.- (Snake venom metalloproteinase, SVMP) (Vascular apoptosis-inducing protein, VAP) [Gloydius halys (Chinese water mocassin) (Agkistrodon halys)]. Accession number Q8AWI5, https://identifiers.org/Uniprot:Q8AWI5\n\nUniprot: Alpha-elapitoxin-Oh2b, Alpha-EPTX-Oh2b (Alpha-neurotoxin) (LNTX3) (Long neurotoxin OH-6A/OH-6B) (OH-3) [Ophiophagus hannah (King cobra) (Naja hannah)]. Accession number P82662, https://identifiers.org/Uniprot:P82662\n\nUniprot: Acidic phospholipase A2 PePLA2, svPLA2, EC 3.1.1.4 (Phosphatidylcholine 2-acylhydrolase) [Protobothrops elegans (Elegant pitviper) (Trimeresurus elegans)]. Accession number Q2PG83, https://identifiers.org/Uniprot:Q2PG83\n\nUniprot: Basic phospholipase A2 PL-X, svPLA2, EC 3.1.1.4 (Phosphatidylcholine 2-acylhydrolase) [Protobothrops elegans (Elegant pitviper) (Trimeresurus elegans)]. Accession number P06860, https://identifiers.org/Uniprot:P06860\n\nUniprot: Bradykinin-potentiating and C-type natriuretic peptides (BPP-CNP) [Cleaved into six chains] [Protobothrops flavoviridis (Habu) (Trimeresurus flavoviridis)]. Accession number P0C7P5, https://identifiers.org/Uniprot:P0C7P5\n\nUniprot: Phospholipase A2 AP-PLA2-I, PLA2, EC 3.1.1.4 (Phosphatidylcholine 2-acylhydrolase 2) [Acanthaster planci (Crown-of-thorns starfish)]. Accession number Q2C2C2, https://identifiers.org/Uniprot:Q3C2C2\n\nUniprot: Conotoxin Cl9.6 [Californiconus californicus (California cone) (Conus californicus)]. Accession number D6C4M3, https://identifiers.org/Uniprot:D6C4M3\n\nUniprot: Kunitz-type serine protease inhibitor conotoxin Cal9.1a [Californiconus californicus (California cone) (Conus californicus)]. Accession number D2Y488, https://identifiers.org/Uniprot:D2Y488\n\nUniprot: Conotoxin Cl14.9 [Californiconus californicus (California cone) (Conus californicus)]. Accession number D6C4J8, https://identifiers.org/Uniprot:D6C4J8\n\nUniprot: Alpha-conotoxin CIB (C1.2) [Conus catus (Cat cone)]. Accession number P0DPT2, https://identifiers.org/Uniprot:P0DPT2\n\nUniprot: Conotoxin Fla16d (Conotoxin Fla16.1) [Cleaved into: Conotoxin fla16a; Conotoxin fla16b; Conotoxin fla16c] [Conus flavidus (Yellow Pacific cone)], Accession number V5V893, https://identifiers.org/Uniprot:V5V893\n\nUniprot: Sigma-conotoxin GVIIIA [Conus geographus (Geography cone) (Nubecula geographus)]. Accession number P58924, https://identifiers.org/Uniprot:P58924\n\nUniprot: Conotoxin Mr15.2 (Mr094) [Conus marmoreus (Marble cone)]. Accession number P0DM19, https://identifiers.org/Uniprot:P0DM19\n\nUniprot: Conotoxin mr3g (Mr3.6) [Conus marmoreus (Marble cone)]. Accession number P0C1N5, https://identifiers.org/Uniprot: P0C1N5\n\nUniprot: Conotoxin Pu6.1 [Conus pulicarius (Flea-bitten cone)]. Accession number D2DGD8, https://identifiers.org/Uniprot:D2DGD8\n\nUniprot: Alpha-conotoxin-like Pu1.5 [Conus pulicarius (Flea-bitten cone)]. Accession number P0C8U9, https://identifiers.org/Uniprot:P0C8U9\n\nUniprot: Putative alpha-conotoxin Qc alphaL-1, QcaL-1 [Conus quercinus (Oak cone)]. Accession number A1X8B8, https://identifiers.org/Uniprot:A1X8B8\n\nUniprot: Contryphan-R (Bromocontryphan) [Cleaved into: [Des-Gly1]-contryphan-R] [Conus radiatus (Rayed cone)]. Accession number P58786, https://identifiers.org/Uniprot:P58786\n\nUniprot: Rho-conotoxin TIA, Rho-TIA [Conus tulipa (Fish-hunting cone snail) (Tulip cone)]. Accession number P58811, https://identifiers.org/Uniprot:P58811\n\nUniprot: Conotoxin 10 [Conus virgo (Virgin cone)]. Accession number Q5K0C5, https://identifiers.org/Uniprot:Q5K0C5\n\nUniprot: Conotoxin Vi15a (Vi15.1) [Conus virgo (Virgin cone)]. Accession number B3FIA5, https://identifiers.org/Uniprot:B3FIA5\n\nZenodo: Underlying data for ‘Toxin-like peptides in plasma, urine and faecal samples from COVID-19 patients’, https://doi.org/10.5281/zenodo.490315416\n\nThis project contains the following underlying data:\n\nData file 1: Toxins.fasta\n\nData file 2: Toxins.mgf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY4.0)\n\n\nConsent\n\nThe human biological samples used in the experimentation were collected and used with the expressed free and informed written consent of the person from whom the material was taken, according to current legislation.", "appendix": "Acknowledgements\n\nThe authors thank Martina Larini and Simone Madama for paper revision.\n\n\nDeclarations\n\nThe scientific output expressed does not imply a policy position of the European Commission. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "90696", "date": "09 Aug 2021", "name": "Paolo Grumati", "expertise": [ "Reviewer Expertise 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\n12th August 2021: This peer review report was originally published with a Not Approved status, but the reviewer has since notified the Editorial team that this was not intended and it should be an Approved with Reservations. The report approval status has been updated to reflect this.\nIn the present manuscript the authors proposed an interesting consequence of COVID 19 infection. The idea is that COVID infection induces the production of toxins that are responsible for the specific clinical manifestations. Despite the fact that the origin of the toxins is not clear. The authors identified via mass spectrometry peptides that match the sequences of toxin components of venoms from animals. These observations are surprising and provocative. However, there are several points that the authors should consider:\nCOVID-19 outbreak is a pandemic therefore the number of affected people is extremely high. The number of samples analysed should be much higher. Authors should consider to have at least three different groups. Non-infected control, infected without clinical symptoms, affected with severe symptoms. For each group at least 10 samples for each analysis (blood, urine) should be analysed in order to have a more reliable statistic analysis. Moreover, does the amount of toxins correlate with the severity of the phenotype?\n\nAuthors should provide some biological data that the toxins they identified are responsible for the clinical phenotype. They should perform some in vitro experiments infecting cells (CALU are the most used) with COVID or treating them with toxins. The outcome should be similar.\n\nIt is difficult to access the original data. It would be interesting to see the peptide sequences identified from the mass spectrometry. In Fig.4 the authors reported an example but it is unlike that a secreted toxin contains the leader region pro-peptide.\n\nThe text needs some editing. Citation of other research papers should be conform to the standard.\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? No", "responses": [ { "c_id": "7028", "date": "13 Aug 2021", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Grumati, Thanks a lot for your valuable comments and suggestions that you have provided in the report. We will address all of them, and wait for those of other reviewers, in order to provide a fully revised version of the manuscript. Best regards, Mauro Petrillo, on behalf of the authors." }, { "c_id": "7296", "date": "12 Jan 2022", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Grumati, Thanks a lot for your valuable comments and suggestions that you have provided in the report. As anticipated, we have addressed all your points, and provided a new version of the manuscript: COVID-19 outbreak is a pandemic therefore the number of affected people is extremely high. The number of samples analysed should be much higher. Authors should consider to have at least three different groups. Non-infected control, infected without clinical symptoms, affected with severe symptoms. For each group at least 10 samples for each analysis (blood, urine) should be analysed in order to have a more reliable statistic analysis. Moreover, does the amount of toxins correlate with the severity of the phenotype? Response: We thank the Dr. Grumati for the comment. We increased the number of analysed plasma samples to address this point: in addition to the 5 control cases and 15 hospitalised cases (already mentioned in the section “Methods: Cohort”), we have analysed 5 plasma samples of infected individuals with mild or no symptoms, together with 5 additional controls. Thus, for plasma, there are now 30 cases (20 positive to SARS-CoV-2 tests plus 10 controls). In this new added group, we observe the presence of toxin-like peptides, apparently in lower amounts (in terms of -loge) with respect to the samples from hospitalised individuals. However, as we have no additional information about the grade of severity of the hospitalised subjects, we prefer to not infer any correlation between the amount of identified peptides and the severity of the phenotype. To better clarify this point, we have added the following sentence in the section “Methods:Cohort”: Apart from positivity to SARS-CoV-2, no additional information (i.e. age, sex, blood serotype, severity of the disease, time of the collection, fatality, etc.) was provided. Authors should provide some biological data that the toxins they identified are responsible for the clinical phenotype. They should perform some in vitro experiments infecting cells (CALU are the most used) with COVID or treating them with toxins. The outcome should be similar. Response: We thank the Dr. Grumati for the comment. Experiments to assess neurotoxicity of these peptides on 3D neuronal/glial model (“neurospheres”) obtained from human induced Pluripotent Stem Derived Neural Stem Cells (iPS-NSCs) are ongoing and will be part of an additional publication. We added a sentence in the section “Results and discussion” to address this point: Experiments to assess neurotoxicity of these peptides on 3D neuronal/glial model (“neurospheres”) obtained from human induced Pluripotent Stem Derived Neural Stem Cells (iPS-NSCs) are ongoing. It is difficult to access the original data. It would be interesting to see the peptide sequences identified from the mass spectrometry. In Fig.4 the authors reported an example but it is unlike that a secreted toxin contains the leader region pro-peptide. Response: We thank the Dr. Grumati for these comments. Regarding the data, we followed the journal policy: all data produced and here presented are freely and publicly available on the Zenodo platform (http://www.doi.org/10.5281/zenodo.4903154, as reported in the section “Data availability” and in Reference 16, including MS files in MGF format and sequence files in FastA format). Regarding the alignment in Fig.4, the shown peptides correspond to the longest peptides we observed (LC-SACI-CIMS is particularly able in detect long peptides), aligned with respect to the precursor of the protein. We did not make any specific selection for secreted proteins; thus, precursors are expected to be present in our samples. To address this point, we modified the legend of Fig.4 accordingly. The text needs some editing. Citation of other research papers should be conform to the standard. Response: Thanks a lot for spotting these inconsistencies. We revised citations according to the journal specifications. We hope that the quality of the manuscript, thanks to your comments, has been improved and you consider it suitable for publication. Best regards, Mauro Petrillo, on behalf of the authors." } ] }, { "id": "93677", "date": "05 Oct 2021", "name": "Moshe Arditi", "expertise": [ "Reviewer Expertise Immunology", "Infectious Diseases", "Innate Immune Responses" ], "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 report the identification of toxin-like peptides in COVID-19 patients samples (Plasma, urine and stool samples) by using Liquid Chromatography Surface-Activated Chemical Ionization-Cloud Ion Mobility Mass Spectrometry. The investigators used a study cohort (for plasma) of 15 COVID 19 patients from different cities of Italy and from 5 control uninfected individuals. They collected urine samples from 2 COVID19 patients and 2 controls, and stool samples from 3 COVID19 patients and 3 controls. They report that toxin-like peptides, almost identical to toxic components such as conotoxins, phospholipases, phosphodiesterases etc. were identified from COVID19 patients, but not in any control samples.\n\nThey report an overview of 36 proteins covered by the toxin-like peptides they have found in plasma of COVID1- patients. These toxin-like peptides they discovered are very much like various neurotoxins, such as alpha Conotoxins, alpha Cobratoxins or similar to Bungarotoxins, all known to be neurotoxins.\nThese are very important observations, the authors are asking the question is the COVID-19 infection is somehow inducing these toxin-like peptides in the host and if so, if these neurotoxin- like peptides maybe playing a functional role of the neurologic findings that are frequently associated with COVID-19 infection. One very important and potentially critical paper that must be mentioned in the discussion and that authors have missed was recently published by Mary Hongying Cheng et al.(20201) where the investigators discovered a Superantigen-like motif in the S1 Spike protein, as well as two other neurotoxins that have peptide similarities to alpha cobratoxin and alpha bungarotoxin, alpha cobratoxin etc. Given this PNAS paper, it is now clear that the SARS -CoV2 virus contains neurotoxin-like peptides already. It would be incredibly interesting to see if the neurotoxin like peptides described and discovered in this PNAS paper are present in the toxin-like peptides described in this specific study. It will make this paper and its discussion much more impactful. At a minimum the PNAS paper should be discussed and cited.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7279", "date": "20 Jan 2022", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Arditi, thanks a lot for the valuable comments and suggestions that you have provided in the report. We will address all of them in order to provide a fully revised version of the manuscript. Best regards, Mauro Petrillo, on behalf of the authors." }, { "c_id": "7297", "date": "20 Jan 2022", "name": "Mauro Petrillo", "role": "Author Response", "response": "Dear Dr Arditi, Thanks a lot for your valuable comments and suggestions that you have provided in the report. As anticipated, we have addressed all your points, and provided a new version of the manuscript: One very important and potentially critical paper that must be mentioned in the discussion and that authors have missed was recently published by Mary Hongying Cheng et al.(2020) where the investigators discovered a Superantigen-like motif in the S1 Spike protein, as well as two other neurotoxins that have peptide similarities to alpha cobratoxin and alpha bungarotoxin, alpha cobratoxin etc. Given this PNAS paper, it is now clear that the SARS -CoV2 virus contains neurotoxin-like peptides already. It would be incredibly interesting to see if the neurotoxin like peptides described and discovered in this PNAS paper are present in the toxin-like peptides described in this specific study. It will make this paper and its discussion much more impactful. At a minimum the PNAS paper should be discussed and cited. Response: We thank the Dr. Arditi for the comment and we fully agree with his suggestion to cite the Mary Hongying Cheng et al. (2020) PNAS paper. With respect to this point, we would like to highlight that the peptides reported in the paper as examples are a subset of more than 100 peptides identified by MS. We checked in the full set of peptides, and we can confirm that we identified, in plasma and faecal samples, toxin-like peptides mapping on kappa 1a-bungarotoxin, Kappa 1b-bungarotoxin from Malayan krait, kappa-2-bungarotoxin and alpha-bungarotoxin from many-banded krait (Uniprot Accession Numbers Q8AY56, Q8AY55, P15816, and P60615, respectively). To address this point, we added the following paragraph in the section “Results and discussion”: Cheng et al. [REF] reported the discovery of a superantigen-like motif in the S1 Spike protein, as well as two other neurotoxin-like motifs that have peptide similarities to neurotoxins from Ophiophagus (cobra) and Bungarus genera. They conclude that neurotoxin-like motifs are present in SARS-CoV-2 protein products, acting as neurotoxin-like peptides. We checked in the full set of peptides we got (here we report only 36 examples), and we identified, in plasma and faecal samples, toxin-like peptides mapping on kappa 1a-bungarotoxin, Kappa 1b-bungarotoxin from Malayan krait, kappa-2-bungarotoxin and alpha-bungarotoxin from many-banded krait (Uniprot Accession Numbers Q8AY56, Q8AY55, P15816, and P60615, respectively), which were reported by Cheng and colleagues. Furthermore, we looked at the amino acid changes currently reported in GISAID data [REF], analysed by CoV-GLUE-Viz (update 15/09/2021) [REF], and occurring in the Y674QTQTNSPRRAR685 motif identified by these authors as homologous to neurotoxin motifs of animal venom proteins. We observed the existence of amino acid variations which makes this motif even more similar to the neurotoxin motifs of animal venom proteins (like variations Q677S and T676A observed in sequences assigned to PANGO Lineage B.1.596). Experiments to assess neurotoxicity of these peptides and of spike protein on 3D neuronal/glial model (“neurospheres”) obtained from human induced Pluripotent Stem Derived Neural Stem Cells (iPS-NSCs) are currently ongoing. What follows is our attempt to elaborate a potential relation between their presence and extra-pulmonary COVID-19 symptomatology. We hope that the quality of the manuscript, thanks to your comments, has been improved and you consider it suitable for indexing. Best regards, Mauro Petrillo, on behalf of the authors." } ] } ]	1	https://f1000research.com/articles/10-550
https://f1000research.com/articles/10-183/v1	05 Mar 21	{ "type": "Systematic Review", "title": "The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis", "authors": [ "Osfar Sjofjan", "Danung Nur Adli", "Rakhmad Perkasa Harahap", "Anuraga Jayanegara", "Dicky Tri Utama", "Ainun Pizar Seruni", "Danung Nur Adli", "Rakhmad Perkasa Harahap", "Anuraga Jayanegara", "Dicky Tri Utama", "Ainun Pizar Seruni" ], "abstract": "Introduction: The number of publications in Scopus on this topic increased from less than 50 in 1995 to more than 250 in 2015. In other hand, Inconsistency in results about the correlation between yeast and lactic acid bacteria as probiotics has been evident since the early publications on use in broilers. Methods: A meta-analysis was conducted to determine relationship between lactic acid bacteria and yeast as probiotics to broiler diets on the growth performance, relative organ weight, blood parameters, and immune response of the broiler. A database was designed based on published data that reported the use of probiotics on the broiler. The method used for selecting articles was based on the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) method. Articles selected were taken from PubMed, Web of science, Scopus, Google Scholar, and Science direct databases as well as individual. Results: The final database consists of 49 in vivo articles, 93 studies, and 225 treatments. The analysis statement in the system was a PROC MIXED procedure of SAS software. The level of probiotic increased (p <0.001) body weight, body weight gain, and feed intake of broiler. There was a reduction (p <0.01) on feed conversion ratio and mortality on the level probiotic given to broiler. Supplementation of probiotics in broiler diet increased (p <0.001) the weight of liver, spleen, gizzard, bursa of fabricius and carcass yield, while reduced (p<0.001) abdominal fat weight. The probiotic given increased the total of red and white blood cells (both at p < 0.001) but did not affect lymphocyte. Discussion: It can be concluded the yeast act as supporting agent that serves lactic acid bacteria as probiotic increases the growth performance, relative organ weight, blood parameters, and immune response of the broiler.", "keywords": [ "broiler", "lactic acid bacteria", "meta-analysis", "probiotic", "yeast." ], "content": "Introduction\n\nIn 1997, the use of antibiotics in livestock was first addressed in Denmark with avoparcin as an antibiotic growth promoter (AGP). The trend continued and a European Union (EU)-wide ban on AGPs in animal feed (poultry) took effect in 2006 (EC Regulation No 1831/2003)1. Since then, this type of regulation has spread to developing countries, including Indonesia, which has been banning antibiotics and imported poultry feed products since the most recent regulation, PERMENTAN/14/16/2017, was put in place2. The EU introduced probiotics as an alternative to antibiotics and this has subsequently become an area of great interest for researchers worldwide3. Probiotics are living microorganisms that when ingested in sufficient amounts, may positively improve growth, intestinal health and animal productivity. Probiotics are commonly sourced from lactic acid bacteria, namely, Lactobacillus and Bifidobacterium, which are usually found in the intestine4.\n\nEarlier studies have reported an active role for probiotics in reducing or eliminating the pathogen bacteria in the intestine. In recent research5,6 probiotic mixtures have also been found to have beneficial effects against a wide range of disorders, although evidence that mixtures are more effective than their component strains is more limited. Nevertheless, in the future, a further potential advantage of multi-strain probiotics, in addition to exerting additive or synergistic effects, is that the strain-specific effects of individual probiotic components could together exert a broader spectrum of activity5,6. Probiotics can be given in both powder and liquid form and positively modulate the composition of broiler intestinal microflora via the stimulation of potentially beneficial bacterial populations and the reduction of pathogenic bacteria1–4. The interaction between probiotics and micro biota added to diet influences the microbial population’s stability and the health of the host. The gut micro biota plays a crucial role in host metabolism and fundamentally influences physiology, health and well-being, functionality and performance5.\n\nYeasts have been reported to act as supporting agents for lactic acid bacteria but also as having the potential to reduce avian bacterial in the gut micro biota of poultry1,4. Inconsistency in results about the correlation between yeast and lactic acid bacteria as probiotics has been evident since the early publications on use in broilers4. Accordingly, the current study aims to determine the relationship between lactic acid bacteria and yeasts as probiotics in broiler diets on growth performance, meat quality, blood parameters, and immune responses, through a meta-analysis using data from published articles.\n\n\nMethods\n\nA database was constructed based on peer-reviewed and published research articles which reported the use of probiotics in the broiler diet. Articles were selected based on the Systematic Review Center for Laboratory Animal Experimentation (SYRCLE') method7 and Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA)8. Articles selected were taken from PubMed, Web of science, Scopus, Google Scholar, and Science direct databases as well as individual journals such as World Poultry Journal Science, British Poultry Science Journal, and International Journal of Poultry Science using the keywords ‘probiotic’, ‘broiler’, ‘performance’, ‘organ weight’, ‘carcass’, and ‘blood serum’. In each article evaluated, the reference list was also searched for relevant articles. The raw database information from articles, authors, year of study, broiler (strain and sex), diet used in trial, length of trial, level of treatment, form and dosage of probiotic contained in the study was recorded in a spreadsheet following the referenced method7. The parameters included were growth performance, relative organ weight, carcass quality, blood parameters, and immune responses. The strains recorded on the raw database were Ross308, which dominated at 63.26%; Arbor Acres at 32.65%; and others at 4.09%.\n\nCriteria for an article to be included in database were as follows: (a) article was published in a peer-reviewed with range 2008–2020, this paper length was chosen as related to journals aged last 10–12 years are often good9 (b) the broiler were modern-controlled-trial environment and management, (c) performed directly on broiler in vivo as the experimental animals, (d) The log concentration of lactic acid bacteria and yeast both powder and liquid form on the trial was transformation into 1010 in the database development, (e) non-probiotic treatment excluded from the database, (e) the articles written consistently in English were considered in studies, (f) the average duration of the study was minimum 0–21 days and the maximum at 0–53 days, (g) dosages given at the range 0–10 g/kg from total feed formulation. Moreover, the dependent and independent variables were selected with the aim of lactic acid bacteria and yeast related as probiotic on the broiler. Likewise, data extraction was completed in accordance with the task analysis to obtain the exact values from graphical data, the relevant figure from the papers were subjected to an online tool, WebplotDigitizer 4.4 (https://automeris.io/WebPlotDigitizer/), following the method10.\n\nThe final database consisted of 49 in vivo articles, 93 studies, and 225 n-total (3,375 n-total of total in this experiment). The details for the study selection included in this meta-analysis are provided in Figure 1. The search strategy is presented in Table 1. The summary of the final database is presented in Table 2, and PICOS criteria presented in Table 6.\n\nStatistical dataset analysis using a mixed-model approach was applied11–14 with statement analysis in the system using the MIXED procedure of SAS (version 9.1, SAS Institute Inc., 2008), the following model was applied: The findings of a study was then taken as a random effect, while the supplementation concentration was taken as the fixed effect as follows15–17:\n\n\n\nwhere Yij = the expected output for dependent variable Y at level j from the variable X as a continuous\n\nvariable in the study i,, B0 = overall intercept across all studies (fixed effect), B1 = linear regression coefficient of Y on X (fixed effect), B2 = quadratic regression coefficient of Y on X (fixed effect), Xij = value of the continuous predictor variable (probiotic supplementation level), si = random effect of study i, bi = random effect of study i on the regression coefficient of Y on X in study i and eij = unexplained residual error. In the statement CLASS, the “study” variable was declared. Data were weighted by the number of replicates in each study. Additionally, an unstructured variance – covariance matrix (type = un) was performed at the random effect part of the model to avoid a positive correlation between intercepts and slopes. Significance of an effect was stated at the probability level of p < .05, and p < .1 was considered as a tendency of significance. In case that the quadratic model above was not significant, the model was changed into its corresponding linear model. The variable of the study was declared in the class statement as it did not contain any quantitative information. The regression equations are also presented with p-value, and root mean square error (RMSE).\n\nFurthermore, to determine (1) interaction between lactic acid bacteria and yeast; (2) interaction of type probiotic (powder and liquid) according to the following model16:\n\n\n\nWhere Yij = the expected output for dependent variable Y, μ = overall mean, Si = random effect of I study, τj = fixed effect of the j level, Sτij = random interaction between i study and the j level, and eij =residual error. A significant effect was declared at p<0.05 or there is a tendency when the p-value was between 0.05 and 0.10.\n\n\nResults\n\nTable 3 presents the effects of probiotics on broiler performance. The meta-analysis results show the level of probiotic (p<0.001) body weight, body weight gain, and feed intake of broilers. In contrast, there was a reduction (p <0.01) on feed conversion ratio (FCR) and mortality on the level probiotic given to broiler. Furthermore, the analysis also shows that the form of probiotic in the feed does not create any significant difference in broiler performance.\n\nNote: P = powder; L=liquid; B=LAB; Y=yeast; Slope: The respond when the probiotic at the zero level, SE intercept: standard error intercept; BW: body weight; BWG; body weight gain; FCR: feed conversion ratio; FI; feed intake; root mean square error (RMSE)\n\nThe weight of abdominal organs and carcass yield of broilers were affected by the supplementation of probiotics in the diet (Table 4). Supplementation of probiotics in broiler diet increased (p <0.001) the weight of liver, spleen, gizzard, bursa of Fabricius and carcass yield, while reduced (p<0.001) abdominal fat weight. Different types of probiotic, i.e., powder or liquid, influenced the weight of liver (p =0.001), spleen (p <0.005), gizzard (p =0.045) and bursa of Fabricius, (p <0.001). In contrast, abdominal fat and carcass yield were not affected by the type of probiotics supplemented in the diet. Further, different culture type, i.e., lactic acid bacteria or yeast, had no significant effect on the abdominal organs weight and carcass yield of broilers.\n\nNote: P= powder; L=liquid; B=LAB; Y=yeast; Slope: The respond when the probiotic at the zero level, SE intercept: standard error intercept; root mean square error (RMSE)\n\nThe effects on blood parameters of lactic acid bacteria and yeasts as probiotics are presented in Table 5. The probiotic given increased the total of red and white blood cells (both at p < 0.001) but did not affect lymphocyte. Furthermore, the immune response hemoglobin results were not significantly influenced by the delivery of different lactic acid bacteria and yeast forms such as powder or liquid.\n\nNote: RBC: red blood cell; WBC: white blood cell; P= powder; L=liquid; B=LAB; Y=yeast; Slope: The respond when the probiotic at the zero level, SE intercept: standard error intercept; root mean square error (RMSE)\n\nP = powder; L=liquid; B=LAB; Y=yeast; Slope: The respond when the probiotic at the zero level, SE intercept: standard error intercept; BW: body weight; BWG; body weight gain; FCR: feed conversion ratio; FI; feed intake; RBC: red blood cell; WBC: white blood cell root mean square error (RMSE); PICOS: population, intervention, comparison, outcomes and study\n\n\nDiscussion\n\nOur meta-analysis shows that probiotics positively affect growth performance. In terms of growth performance, we suggest that this finding is related to the ability of probiotics to induce intestinal mechanisms, resulting in a reduction in pathogenic bacteria. In the digestive system, intestinal pH, intestinal bacteria composition, and digestive activity are improved when probiotics are present in diets18. Some probiotics are known to produce enzymes, amylase, protease, and lipase to optimize nutrients’ breakdown19,20. They can also increase specific enzymes in the host digestive tract to enhance nutrient absorption in the diet. In the poultry industry, probiotics are supplemented into the diet to maintain health by enhancing gut health, modulating the immune system, lowering glycaemic response, and improving various performances parameters21–23. Moreover, the administering of probiotics has several ways in practice. The administering of probiotics can be included in the basal diet or combined with raw materials that contain prebiotics to enhance its effect20,30,50–54,67. The probiotic can be given alone or with another additive without any negative effect such as acidifiers and phytogenics25,26,32,42,44. Furthermore, probiotics can contain one or multiple microorganism strains that can be added to animals' diets24,28,37.\n\nThere are previous studies that report that cell-wall components of yeast in dietary supplementation to lactic acid bacteria improve the growth rate, feed consumption, and feed efficiency in broilers51,55. These positive and consistent results were due to yeast activating spores to reduce and remove potential pathogens in the gut which possibly increases body weight44. Linearly, the factors related to synergism between yeast and lactic acid bacteria reported in the research could be related to environmental conditions in various experiments45,46.\n\nOne study from 46 showed that rearing the broiler with a heat-stress environment at 350c was more low weight than heat stress exposed. Thus, yeast failed to alleviate heat-stress on the performance of broiler46. The lower temperature in the chicken house may help increase feed intake to eat more of the experimental diets57. In addition, other factors related due to being reared under a stocking density stress of 43 kg live weight per m2 floor space57. Another result45 explains in more detail that yeasts in powder form significantly increase the body weight of broilers starting at 21 days old, with an increase in line with increasing feed intake and reducing FCR. These consistent results57–59 explain that the use of lactic acid bacteria as probiotics in powder form help to increase the body weight of broilers, as a result of digestibility and metabolic process improvement caused by the bacteria, affecting energy partition and putting more energy into growth than maintenance46 .\n\nMoreover, the positive use of probiotics both as powders and liquids was in line with the increasing level of treatment in broiler57. The probiotics enhance liquid lactic acid bacteria synergism with yeast in the feed but suggested at an optimized level57 of 0.8%. However, the dose-response relationship of probiotics in animal trials is rarely studied1. At low doses a probiotic may be specific, for example bifidobacteria, due to the high specificity of bifidobacteria for that particular probiotic1. In other hand, if the dose increased, this would leave some substrate for other probiotic strains able to ferment it. The outcome of high dose would show less specificity that that of the low dose1. Treatment with both powder and liquid forms increases body weight and feed intake and reduces feed conversion31,57,61. Moreover, probiotics for farm animals have positive effects on growth, efficiency of feed utilization68. In addition, the consistent result in studies45vs 50–52 show the relationship between both yeast and lactic acid bacteria working together to reduce potential pathogens in the gut of broilers but dose is dependent.\n\nThe meta-analysis results show limited effects on the carcass and organ weight of broilers. In agreement from 52,62 the carcass quality shows no significant difference after administering probiotics of both lactic acid bacteria and yeast. The one factor can be caused reduced of percentage carcass are heat-stress environmental. The carcass heat-stress was associated with the reduced of carcass quality52. Apart from physiological adjustment derived from depressed feed intake, the increased Corticosterone level may be responsible lower percentage of carcass52. Carcass percentage was reported to increase by one study45, with the saleable product in terms of edible portions. Reported45 the carcass quality can be affected from physiological and genetic potential, feed formulated, strain of the broiler rearing. The excess fat deposition in carcass of broiler is undesirable to producers because of reduced carcass yield and to consumers that prefer a leaner product1.\n\nLikewise, one study62 the use of yeast as probiotic reduced abdominal fat of broiler. Reported62 yeast help to reduce fat deposition because, modern-broiler-farming were intensive feeding (ad-libitum), fatter caused limb defects, and sudden death syndrome. The probiotics reported reduced fat of broiler compared without probiotic65. The mode of action was that probiotics decreased the activity of acetyl-CoA carboxylase66. Acetyl-CoA carboxylase has been widely suggested as the rate-limiting enzyme in fatty acid synthesis66. The decline in the synthesis of fatty acids, in turn, would decrease their availability for esterification to triglycerides for deposed in the adipose tissue66. Furthermore, the minimum dose of probiotic to stimulate fatty acids are currently unknown63,64,66. Differences in the broiler line/breed and conditions, as well as microorganism strains (highly species-and strain specific), origin species, concentrations, and methods of administration of the probiotic bacteria, may explain these results65. However, our study can’t exactly suggest the dose optimum for using this probiotic.\n\nIn one study42, the effects of probiotics on relative weights of liver and spleen were not significant (P > 0.05), while bursa of Fabricius relative weight increased (p < 0.05). Supplementing diets with probiotics could help to prevent necrotic enteritis which associated with degeneration of hepatocytes and immune system of the broiler69. The smaller liver in broiler may indicate a higher resistance to pathogen microorganism such as Clostridium perfrigens70. The IGF-1 can produce short-fatty acids (SCFAs), which act either directly or indirectly on the liver and adipose tissue to promote growth of organ and skeletal development65. The report from71at the end of the feeding trial showed that the development of gizzard weight was decreased, dateable irregularities in the gizzard are a sensitive index to reduce anti-nutritional factors in the basal diet after exposure to toxic substance not to amount of lactic bacteria71. Moreover, increased weight of this lymphoid organ may indicate a higher immunity achieved in treated broiler, which could be explained by probiotic anti-microbial activity18,42. The factor affected by significant differences in the relative organ weight is the ability to absorb substances from probiotics18,43,44. 18 stated that variances between the broilers result from impacts on absorption and other capacities of the relative organ weight. The growth factors correlate with age, while the broiler's uses in the relative age cause the same internal organ’s growth. In instances, an increased relative organ weight may be in line with an increase in lymphocyte concentration18.\n\nThe meta-analysis of different probiotic levels on some blood parameters showed red and white blood cell concentration increased (p <0.05) with increasing probiotic supplementation levels in the feed. The increased of the white blood cells had correlated with yeast reduce the uric acid (UA) content in the blood72. The uric acid is a metabolite of protein that has an antioxidant function, but is converted to a pro-oxidant in the cell or cytoplasm72. In contrast, lymphocyte, and hemoglobin were not significantly different (p >0.05)15. Linearly, blood serum rose in line with probiotic increase. Additionally, one18 shows that, the lactic acid bacteria that help reduce avian-pathogenic bacteria were Escherichia coli and Clostridium perfrigens. The beneficial action indicates that lactic acid bacteria produced extracellular enzymes to enhance the nutrient digestibility of feed and synthesize immune function using endogenous anti-microbial20. In terms of negative linear response in studies21 there was no positive result on red and white blood cells.\n\nMoreover, probiotics could be related to a lowered recycling of bile salts in the gut or inhibited hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity21. The mechanism operating in lactic acid bacteria, as probiotics to elicit their hypocholesterolemic effect is interference with intestinal bile acid transport and absorption, leading to an increase in bile acid excretion21. The potential pathogens reduce but are not eliminated, thus, probiotics balance the intestinal environment to enhance the broiler’s immune systems45,50–52. Although, the lactic acid bacteria do not produce butyric acid themselves, they stimulate the proliferation of butyric acid and cell-wall of yeast in the blood circulation by the mechanism that is called cross-feeding65. Continued research42 shows probiotics help to increase the white blood cell count as level of probiotic is increased. One study42 stated that the increase of white blood cells and immune response was due to the level increase of B and T lymphocyte production. In line with the 41 studies the amount of red blood cells, hemoglobin, and white blood cells consistently tends to increase compared to controls. The positive effect from yeast as a probiotic could derive from its outer cell wall components namely: chitin, mannan, and glucan which have an immunostimulant effect. Moreover, these outer wall components promote lactic acid bacteria activity, which is activated by producing enzymes that cause disintegration of bile salts, making them unconjugated68. The yeast can enhance the immune response by promoting growth of lactic acid bacteria and thus simultaneously producing antibacterial substances and stimulating the production of immunoglobulin33. Thus, yeast acts as a supporting agent of lactic acid bacteria, which adhere to the endogenous epithelial cells to initiate colonization33.\n\n\nConclusions\n\nThe results provided by this meta-analysis demonstrates the enhancement of overall performance of broilers supplemented with lactic acid bacteria and yeast as probiotics. Effects of the probiotics on blood parameters are dose dependent, where areas, the additives have limited effects on organ weight and carcass percentage. Both powder and liquid forms of probiotics do not affect the results differently. The future research trends are to determine the dose optimum of probiotic for broiler.\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 for ‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’. https://doi.org/10.6084/m9.figshare.1406041473.\n\nThis project contains extracted data of outcome measures (BW: body weight; BWG; body weight gain; FCR: feed conversion ratio; FI; feed intake; RBC: red blood cell; WBC: white blood cell).\n\nFigshare: PRISMA checklist for ‘‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’’. https://doi.org/10.6084/m9.figshare.1406050174.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "Acknowledgements\n\nThanks to Animal Feed and Nutrition Modelling (AFENUE) Research Group, Faculty of Animal Science, IPB University, Bogor 16680, Indonesia.\n\n\nReferences\n\nVenema K, do Carmo AP: Probiotics and prebiotics: current research and future trends. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen W, Wang JP, Yan L, et al.: Evaluation of probiotics in diets with different nutrient densities on growth performance, blood characteristics, relative organ weight and breast meat characteristics in broilers. Br Poult Sci. 2013; 54(5): 635–641. PubMed Abstract \| Publisher Full Text\n\nPark JH, Kim IH: Supplemental effect of probiotic Bacillus subtilis B2A on productivity, organ weight, intestinal Salmonella microflora, and breast meat quality of growing broiler chicks. Poult Sci. 2014; 93(8): 2054–2059. PubMed Abstract \| Publisher Full Text\n\nZhang ZF, Zhou TX, Ao X, et al.: Effects of β-glucan and Bacillus subtilis on growth performance, blood profiles, relative organ weight and meat quality in broilers fed maize–soybean meal based diets. Livest Sci. 2012; 150(1–3): 419–424. Publisher Full Text\n\nJamshidparvar A, Javandel F, Seidavi A, et al.: Effects of golpar (Heracleum persicum Desf.) and probiotics in drinking water on performance, carcass characteristics, organ weights, blood plasma constituents, and immunity of broilers. Environ Sci Pollut Res Int. 2017; 24(30): 23571–23577. PubMed Abstract \| Publisher Full Text\n\nKhan SH, Yousaf B, Mian AA, et al.: Assessing the effect of administering different probiotics in drinking water supplement on broiler performance, blood biochemistry and immune response. J Appl Anim Res. 2011; 39(4): 418–428. Publisher Full Text\n\nGheisar MM, Hosseindoust A, Kim IH: Effects of dietary Enterococcus faecium on growth performance, carcass characteristics, faecal microbiota, and blood profile in broilers. Vet Med. 2016; 61(1): 28–34. Publisher Full Text\n\nHussein E, Selim S: Efficacy of yeast and multi-strain probiotic alone or in combination on growth performance, carcass traits, blood biochemical constituents, and meat quality of broiler chickens. Livest Sci. 2018; 216: 153–159. Publisher Full Text\n\nNosrati M, Javandel F, Camacho LM, et al.: The effects of antibiotic, probiotic, organic acid, vitamin C, and Echinacea purpurea extract on performance, carcass characteristics, blood chemistry, microbiota, and immunity of broiler chickens. J Appl Poult Res. 2017; 26(2): 295–306. Publisher Full Text\n\nJavandel F, Nosrati M, van den Hoven R, et al.: Effects of Hogweed ( Heracleum persicum) Powder, Flavophospholipol, and Probiotics as Feed Supplements on the Performance, Carcass and Blood Characteristics, Intestinal Microflora, and Immune Response in Broilers. J Poult Sci. 2019; 56(4): 262–269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun HY, Kim IH: Dietary Supplementation of Mixed Yeast Culture Derived from Saccharomyces cerevisiae and Kluyveromyces maxianus: Effects on Growth Performance, Nutrient Digestibility, Meat Quality, Blood Parameters, and Gut Health in Broilers. J Poult Sci. 2019; 56(2): 140–147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSugiharto S, Isroli I, Yudiarti T, et al.: The effect of supplementation of multistrain probiotic preparation in combination with vitamins and minerals to the basal diet on the growth performance, carcass traits, and physiological response of broilers. Vet World. 2018; 11(2): 240–247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGhasemi-Sadabadi M, Ebrahimnezhad Y, Shaddel-Tili A, et al.: The effects of fermented milk products (kefir and yogurt) and probiotic on performance, carcass characteristics, blood parameters, and gut microbial population in broiler chickens. Arch Anim Breed. 2019; 62(1): 361–374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdel-Hafeez HM, Saleh ESE, Tawfeek SS, et al.: Effects of probiotic, prebiotic, and synbiotic with and without feed restriction on performance, hematological indices and carcass characteristics of broiler chickens. Asian-Australas J Anim Sci. 2017; 30(5): 672–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToghyani M, Mosavi SK, Modaresi M, et al.: Evaluation of kefir as a potential probiotic on growth performance, serum biochemistry and immune responses in broiler chicks. Anim Nutr. 2015; 1(4): 305–309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalah AS, El-Tarabany MS, Ali MA: Impact of dietary supplementation with a synbiotic, organic acids or their combination on growth performance, carcass traits, economic efficiency, jejunum histomorphometry and some blood indices of broiler chickens. Anim Prod Sci. 2018; 59(7): 1318–1326. Publisher Full Text\n\nParyad A, Mahmoudi M: Effect of different levels of supplemental yeast (Saccharomyces cerevisiae) on performance, blood constituents and carcass characteristics of broiler chicks. Afr J Agric Res. 2008; 3(12): 835–842. Reference Source\n\nKoc F, Samli H, Okur A, et al.: Effects of Saccharomyces cerevisiae and/or mannanoligosaccharide on performance, blood parameters and intestinal microbiota of broiler chicks. Bulgarian J Agr Sci. 2010; 16(5): 643–650. Reference Source\n\nZhou TX, Chen YJ, Yoo JS, et al.: Effects of chitooligosaccharide supplementation on performance, blood characteristics, relative organ weight, and meat quality in broiler chickens. Poult Sci. 2009; 88(3): 593–600. PubMed Abstract \| Publisher Full Text\n\nRezaeipour V, Fononi H, Irani M: Effects of dietary L-threonine and Saccharomyces cerevisiae on performance, intestinal morphology and immune response of broiler chickens. S Afr J Anim Sci. 2012; 42(3). Publisher Full Text\n\nCho JH, Zhang ZF, Kim IH: Effects of single or combined dietary supplementation of beta-glucan and kefir on growth performance, blood characteristics and meat quality in broilers. Br Poult Sci. 2013; 54(2): 216–221. PubMed Abstract \| Publisher Full Text\n\nPriya BS, Babu SS: Effect of different levels of supplemental probiotics (Saccharomyces cerevisiae) on performance, haematology, biochemistry, microbiology, histopathology, storage stability and carcass yield of broiler chicken. Int J Pharm Biol Arch. 2013; 4(1): 201–207. Reference Source\n\nLan RX, Lee SI, Kim IH: Effects of Enterococcus faecium SLB 120 on growth performance, blood parameters, relative organ weight, breast muscle meat quality, excreta microbiota shedding, and noxious gas emission in broilers. Poult Sci. 2017; 96(9): 3246–3253. PubMed Abstract \| Publisher Full Text\n\nSun HY, Kim IH: Dietary supplementation of mixed yeast culture derived from saccharomyces cerevisiae and Kluyveromyces maxianus: effects on growth performance, meat quality, blood parameters, and gut health in broilers. J Poult Sci. 2019; 56(2): 140–147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElnagar AS, Abdelkhalek E: Productive Performance, Blood Biochemical Traits And Immune Response of Sasso Chickens Supplemented With Saccharomyces Cerevisiae or Mannan Oligosaccharide As Natural Growth Additives. Egypt Poult Sci J. 2017; 37(2): 505–522.\n\nMashayekhi H, Mazhari M, Esmaeilipour O: Eucalyptus leaves powder, antibiotic and probiotic addition to broiler diets: effect on growth performance, immune response, blood components and carcass traits. Anim. 2018; 12(10): 2049–2055. PubMed Abstract \| Publisher Full Text\n\nAttia YA, Zeweil HS, Alsaffar AA, et al.: Effect of non-antibiotic feed additives as an alternative to flavomycin on productivity, meat quality and blood parameters in broilers. Arch Geflügelk. 2011; 75(1): 40–48. Reference Source\n\nPournazari M, AA-Qotbi A, Seidavi A, et al.: Prebiotics, probiotics and thyme (Thymus vulgaris) for broilers: performance, carcass traits and blood variables. Revista Colombiana de Ciencias Pecuarias. 2017; 30(1): 3–10. Publisher Full Text\n\nRiyazi SR, Ebrahimnezhad Y, Hosseini SA, et al.: Comparison of the effects of basil (Ocimum basilicum) essential oil avilamycin and protexin on broiler performance, blood biochemistry and carcass characteristics. Arch Anim Breed. 2015; 58(2): 425–432. Publisher Full Text\n\nSugiharto S, Yudiarti T, Isroli I, et al.: Effect of dietary supplementation with Rhizopus oryzae or Chrysonilia crassa on growth performance, blood profile, intestinal microbial population, and carcass traits in broilers exposed to heat stress. Arch Anim Breed. 2017; 60(3): 347–356. Publisher Full Text\n\nManafi M: Comparison study of a natural non-antibiotic growth promoter and a commercial probiotic on growth performance, immune response and biochemical parameters of broiler chicks. J of Poult Sci. 2015; 52(4): 274–281. Publisher Full Text\n\nAshayerizadeh O, Dastar B, Shargh MS, et al.: Influence of antibiotic, prebiotic and probiotic supplementation to diets on carcass characteristics, hematological indices and internal organ size of young broiler chickens. Journal of Animal and Veterinary Advances. 2009; 8(9): 1772–1776. Reference Source\n\nZahirian M, Seidavi A, Solka M, et al.: Dietary supplementation of Aspergillus oryzae meal and its effect on performance, carcass characteristics, blood variables, and immunity of broiler chickens. Trop Anim Healt Prod. 2019; 51(8): 2263–2268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSugiharto S, Yudiarti T, Isroli I: Growth performance, haematological parameters, intestinal microbiology, and carcass characteristics of broiler chickens fed two-stage fermented cassava pulp during finishing phase. Trop Anim Sci J. 2019; 42(2): 113–120. Publisher Full Text\n\nSugiharto S, Yudiarti T, Isroli I, et al.: Growth performance, haematological responses, intestinal microbiology and carcass traits of broiler chickens fed finisher diets containing two-stage fermented banana peel meal. Trop Anim Health Prod. 2019; 52(3): 1425–1433. PubMed Abstract \| Publisher Full Text\n\nSugiharto S, Yudiarti T, Isroli I, et al.: The effect of fungi-origin probiotic Chrysonilia crassa in comparison to selected commercially used feed additives on broiler chicken performance, intestinal microbiology, and blood indices. Journal of Advanced Veterinary and Animal Research. 2018; 5(3): 332–342. Publisher Full Text\n\nMiah MA, Sarker MMH, Das MR, et al.: The influence of probiotics and antibiotic growth promoter on growth performance and hemato-biochemical parameters in Broilers. Curr Res in Agr Sci. 2014; 1(4): 103–109. Reference Source\n\nSherief MAR, Sherief MSA: The Effect of single or combined dietary supplementation of mannan oligosacharide and probiotics on performance and slaughter characteristics of broilers. Int J of Poult Sci. 2011; 10(11): 854–862. Reference Source\n\nReisinger N, Ganner A, Masching S, et al.: Efficacy of a yeast derivative on broiler performance, intestinal morphology and blood profile. Livest Sci. 2012; 143(2–3): 195–200. Publisher Full Text\n\nAttia YA, Al-Hamid AEA, Ibrahim MS, et al.: Productive performance, biochemical and hematological traits of broiler chickens supplemented with propolis, bee pollen, and mannan oligosaccharides continuously or intermittently. Livest Sci. 2014; 164: 87–95. Publisher Full Text\n\nSjofjan O, Adli DN: Effect of dietary of supplementation mannan-riched fraction (mrf) and probiotic-enhanced liquid acidifier on the growth performance, serum blood biochemistry, and intestinal properties of broilers. IOP Conf Ser Earth and Environ Sci. 2020; 478(1): 12–66. Reference Source\n\nMakled MN, Abouelezz KFM, Gad-Elkareem AEG, et al.: Comparative influence of dietary probiotic, yoghurt, and sodium butyrate on growth performance, intestinal microbiota, blood hematology, and immune response of meat-type chickens. Trop Anim Health Prod. 2019; 51(8): 2333–2342. PubMed Abstract \| Publisher Full Text\n\nVase KK, Mortezavi SH, Rasouli B, et al.: The effect of three tropical medicinal plants and superzist probiotic on growth performance, carcass characteristics, blood constitutes, immune response, and gut microflora of broiler. Trop Anim Health Prod. 2019; 51(1): 33–42. PubMed Abstract \| Publisher Full Text\n\nWaqas M, Mehmood S, Mahmud A, et al.: Effect of yeast based mannan oligosaccharide (Actigen™) supplementation on growth, carcass characteristics and physiological response in broiler chickens. Indian J of Anim Res. 2018; 53(11): 1476–1479. Publisher Full Text\n\nKuprys-Caruk M, Michalczuk M, Chabłowska B, et al.: Comparison of the effect of lactic acid bacteria added to feed or water on growth performance, health status and gut microbiota of chickens broilers. Anim Sci. 2019; 58(1): 55–67. Publisher Full Text\n\nYalçin S, Yalçin S, Eser H, et al.: Effects of dietary yeast cell wall supplementation on performance, carcass characteristics, antibody production and histopathological changes in broilers. Kafkas Universitesi Veteriner Fakultesi Dergisi. 2014; 20(5): 757–764.Publisher Full Text\n\nYalçinkaya I, Çinar M, Yildirim E, et al.: The effect of prebiotic and organic zinc alone and in combination in broiler diets on the performance and some blood parameters. Ital J of Anim Sci. 2016; 11(3): e55. Publisher Full Text\n\nShokaiyan M, Ashayerizadeh O, Shargh MS, et al.: Algal crude fucoidan alone or with Bacillus subtilis DSM 17299 in broiler chickens diet: growth performance, carcass characteristics, blood metabolites, and morphology of intestine. Poult Sci J. 2019; 7(1): 87–94. Publisher Full Text\n\nSalehizadeh M, Modarressi MH, Mousavi SN, et al.: Effects of probiotic lactic acid bacteria on growth performance, carcass characteristics, hematological indices, humoral immunity, and IGF-I gene expression in broiler chicken. Trop Anim Health Prod. 2019; 51(8): 2279–2286. PubMed Abstract \| Publisher Full Text\n\nKhajeh BM, Afsharmanesh M, Ebrahimnejad H: Effect of dietary bacillus coagulans and different forms of zinc on performance, intestinal microbiota, Carcass and Meat Quality of Broiler Chickens. Probiotics Antimicrob Proteins. 2019; 12(2): 461–472. PubMed Abstract \| Publisher Full Text\n\nAdli DN, Sjofjan O, Natsir MH, et al.: The effect of replacing maize with fermented palm kernel meal (FPKM) on broiler performance. Livest Res Rural Dev. 2020; 32(7): 120. Reference Source\n\nMin Y, Choi Y, Choe J, et al.: Effects of dietary mixture of protease and probiotics on growth performance, blood constituents, and carcass characteristics of growing-finishing pigs. J Anim Sci Technol. 2019; 61(5): 272–277. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHussein EO, Ahmed SH, Abudabos AM, et al.: Effect of antibiotic, phytobiotic and probiotic supplementation on growth, blood indices and intestine health in broiler chicks challenged with clostridium perfringens. Animals (Basel). 2020; 10(3): 507. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamlucken U, Ramchuran SO, Moonsamy G, et al.: A novel Bacillus based multi-strain probiotic improves growth performance and intestinal properties of Clostridium perfringens challenged broilers. Poult sci. 2020; 99(1): 331–341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQorbanpour M, Fahim T, Javandel F, et al.: Effect of dietary ginger (Zingiber officinale Roscoe) and multi-strain probiotic on growth and carcass traits, blood biochemistry, immune responses and intestinal microflora in broiler chickens. Animals (Basel). 2018; 8(7): 117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChuang WY, Lin WC, Hsieh YC, et al.: Evaluation of the combined use of saccharomyces cerevisiae and aspergillus oryzae with phytase fermentation products on growth, inflammatory, and intestinal morphology in broilers. Animals (Basel). 2019; 9(12): 1051. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSjofjan O, Adli DN, Harahap RP, et al.: Extended data for ‘‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’. 2021. http://www.doi.org/10.6084/m9.figshare.14060414\n\nSjofjan O, Adli DN, Harahap RP, et al.: PRISMA checklist for ‘‘The effects of lactic acid bacteria and yeasts as probiotics on the growth performance, relative organ weight, blood parameters, and immune responses of broiler: A meta-analysis’. 2021. http://www.doi.org/10.6084/m9.figshare.14060501" }	[ { "id": "90146", "date": "26 Jul 2021", "name": "Hazem Mohammed Ebraheem Shaheen", "expertise": [ "Reviewer Expertise Veterinary Pharmacology." ], "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\nPlease specify what is the object of this study.\nYou could add an Economic interpretation of this study.\nData in the Results section were insufficient and need more clear for discussion.\nThis paragraph is not understandable:\n\"Criteria for an article to be included in database were as follows: (a) article was published in a peer-reviewed with range 2008–2020, this paper length was chosen as related to journals aged last 10–12 years are often good9 (b) the broiler were modern-controlled-trial environment and management, (c) performed directly on broiler in vivo as the experimental animals, (d) The log concentration of lactic acid bacteria and yeast both powder and liquid form on the trial was transformation into 1010 in the database development, (e) non-probiotic treatment excluded from the database, (e) the articles written consistently in English were considered in studies, (f) the average duration of the study was minimum 0–21 days and the maximum rom total feed formulation. Moreover, the dependent and independent variables were selected with the aim of lactic acid bacteria and yeast related as probiotic on the broiler. Likewise, data extraction was completed in accordance with the task analysis to obtain the exact values from graphical data, the relevant figure from the papers were subjected to an online tool, WebplotDigitizer 4.4 (https://automeris.io/WebPlotDigitizer/), following the method10\".\nI suggest an English language edit for the revision of the manuscript.\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? 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? Yes", "responses": [ { "c_id": "6965", "date": "19 Aug 2021", "name": "Danung Nur Adli", "role": "Author Response", "response": "We would like to express our sincere thanks and appreciation for comprehensively reviewed our manuscript that helped us to improve our manuscript. Regarding the reviewer suggestion and question 1. Please specify what is the object of this study. Answer: We already mentioned in the introduction that \"Accordingly, the current study aims to determine the relationship between lactic acid bacteria and yeasts as probiotics in broiler diets on growth performance, meat quality, blood parameters, and immune responses, through a meta-analysis using data from published articles\". 2.You could add an Economic interpretation of this study. Answer: Thank you for the suggestion. Regarding this suggestion the economic parameters, initially we input this parameter but in final paper we don't put down this parameter due to lack of data provided in the paper. 3. Data in the Results section were insufficient and need more clear for discussion. Answer: We mentioned clearly in the result section. 4. Answer: The statement that you are asking for are the requirements when we select the paper and criteria when we put down the information, to develop the data. 5. I suggest an English language edit for the revision of the manuscript. Answer: Authors would like say thank to second reviewer. The manuscript has been edited for English language usage by a native British proof-reader of Britannia Proofreading Service." } ] } ]	1	https://f1000research.com/articles/10-183
https://f1000research.com/articles/10-1042/v1	13 Oct 21	{ "type": "Research Article", "title": "Survival models for right censored breast cancer data: theory, application and comparison", "authors": [ "Madiha Liaqat", "Shahid Kamal", "Florian Fischer", "Waqas Fazil", "Madiha Liaqat", "Shahid Kamal", "Waqas Fazil" ], "abstract": "Background: Censoring frequently occurs in disease data analysis, which is a key characteristic of time to failure modeling. Typically, time to failure studies are conducted through non-parametric and semi-parametric modelling techniques. Parametric models provide more efficient estimates, but are seldomly used, because of some of the limitations and assumptions which need to be fulfilled to apply them. The aim of this study is to illustrate the theoretical and application limitations and performance of different flexible and standard parametric models to evaluate the prognostic value for mortality risk of breast cancer after recurrence among women. Methods: This article describes the theoretical properties of flexible parametric models and compares their performances to standard parametric models, by studying mortality in women diagnosed with breast cancer. We describe how time to failure data may be analyzed with nonlinear flexible models. In this regard, we apply fractional polynomials, spline models, piecewise exponential models, and piecewise exponential additive mixed models. We also illustrate properties of standard parametric models. All analyses have been conducted with multiple covariates to identify significant predictors. Information criteria have been used to evaluate performances of models. Results: Fractional polynomial and spline-based generalized additive models work well in capturing local fluctuations. Parameter estimation with a piecewise exponential additive mixed model (PAMM) as an extension of the piecewise exponential modelling (PEM) approach automatically penalizes model complexity, which is very helpful to avoid over fitting. Conclusions: Flexible parametric time to failure models are more efficient than standard parametric time to failure models. By incorporating time dependent covariates, PAMM is a good approach to perform in-depth studies of predictors over different finite intervals of follow-up time. Until now, this approach is rarely used in time to failure right censored studies.", "keywords": [ "censoring", "time to failure analysis", "non-proportionality", "splines", "piecewise exponential models", "piecewise exponential additive models", "accelerated failure time", "oncology" ], "content": "Background\n\nCancer causes a large disease burden worldwide, among which breast cancer is the most frequent cause of cancer deaths in women. Pakistan, being a lower-middle-income country, has a greater number of breast cancer patients compared to its neighboring countries. It is the country with the highest age-standardized death rate in 2019 globally. Risk of death increases after early breast cancer recurrence in the first three to five years of primary treatment. Time after recurrence to death is analyzed through time to failure techniques, incorporating recorded prognostic factors before recurrence such as age, tumor grade, molecular subtype and treatment.1 In previous research, age has not been proven to be a significant influence on breast cancer deaths.2,3 To further explore its role, age at diagnosis and age at recurrence are included in this study with other covariates.\n\nTime to failure data have incomplete information about exact event occurrence time, which is known as censoring. Three common types of censoring are encountered in time to failure studies: right, left and interval censoring. The most common is right censoring, which is classified into three types: fixed type 1, type 11 and random type 1. In fixed type 1, right censoring occurs for all understudy subjects, who do not observe the event of interest during the predefined study time. Type 11 censoring is named for all subjects who do not observe a specific event after a pre-specified number of events have occurred. In random type 1 right censoring, censored subjects have different censoring times, as not all have same entry time into the study.4 Non-parametric, semi-parametric and parametric modelling techniques are amenable to analyze such types of time to failure disease studies.5\n\nKaplan-Meier (KM) is the simplest method, used to estimate survival function by a non-parametric maximum likelihood estimator (NPMLE), which has the limitation of studying only one factor at a time. Therefore, it is not suitable for multivariate studies.6 The Cox proportional hazard (PH) models, a semi-parametric approach, does not assume the shape of the baseline hazard function, so distributions of regression parameters’ outcomes remain unknown.7 Cox PH models incorporate multivariate predictors by holding the PH assumption, which assumes a fixed proportion of hazard for individuals. In case of right censoring where upper bounds of event occurrences are not specified, regression parameters are estimated through dividing the likelihood function of the PH model into two parts: one comprises of the baseline hazard and unknown parameters, while the other has only unknown parameters to be estimated, which is called partial-likelihood. Breslow8 and Efron9 introduced approximations in partial-likelihood to handle ordered ties in uncensored event times, while exact and discrete methods are also available, in which non-ordered tied survival times are applied through a partial likelihood approach.5\n\nValidity of PH assumption can be checked through a standard global test suggested by Grambsch and Therneau.10 Furthermore, graphical ways of plotting residuals versus predictors are also discussed in their research.10 In case of non-proportionality, extended Cox PH models can apply, which account for the effects of time varying predictors on survival times.11 Spline-based methods are a good choice to estimate effects of unknown nonlinear predictors on continuous response through penalized partial likelihood, they also explore the functional form of non-proportional predictors.12 Piecewise models are a good choice for long length follow-up studies, where predictors’ effects are checked at different finite time intervals to obtain in-depth information about disease progression.13\n\nParametric models rely on a fully maximum likelihood approach, parametric estimates are more efficient and precise if conducted through correctly specified forms. In parametric modelling, time to failure is assumed to follow any distribution, such as exponential, Weibull, gamma, generalized gamma, log-normal, log-logistic, Gompertz and Generalized F.14 By building a linear relationship between the logarithm of failure time and predictors, data can be analyzed through the accelerated failure time (AFT) model. In AFT models one-unit changes in predictors explain a proportional change in survival time, as illustrated by Lee and Go,15 while in PH parametric form assumes a proportional change in hazard due to a one-unit change in predictors.\n\nThe aim of this paper is to review and apply the above stated modelling techniques to time to failure data, and to evaluate their performances through statistical measures, to investigate the best fitted one for right censored data, while fulfilling limitations and assumptions.\n\n\nMethods\n\nOur data consists of 1,028 women diagnosed with breast cancer in Lahore, Pakistan. All women observed recurrence between February 2011 and February 2018 after initial treatment. They were treated at the same hospital (Institute of Nuclear Medicine & Oncology Lahore, Pakistan). The primary endpoint of this study is death due to breast cancer. Exclusion criteria were: incomplete or missing information, women diagnosed with another disease or another cancer before breast cancer, and bilateral carcinomas. Women who were still alive (survived) at the end of the study, or died due to another reason than breast cancer, are considered right-censored. Age at diagnosis, age at recurrence, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2), tumor grade, radiotherapy and chemotherapy are the predictors included in this study, all were chosen with the help of clinicians and oncologists.\n\nIn the data, age at diagnosis and age at recurrence (in years) are continuous variables, estrogen receptor, progesterone receptor and Her2 are represented by binary variables (0 = Negative, 1 = Positive), tumor grade is represented categorically (I,II,III). In addition, chemotherapy and radiotherapy are indicated by dummy variables (0 = No, 1 = Yes), here 0 indicated the patients who did not receive the treatment, while1=Yes meant they received treatment. Survival time was considered from recurrence to death or drop out, and censoring status was coded with0 for censored, and 1 for death due to breast cancer. Along with the aim of this study, which is the comparison of different parametric models, it is also a major interest to find out how the two treatments (radiotherapy and chemotherapy), in combination with the other predictors, affect the survival of breast cancer patients after recurrence.\n\nProportional hazards have been checked by scaled Schoenfeld residuals (Extended data). Furthermore, the statistical tests revealed that not all covariates are statistically significant (p < 0.05), but the global test is statistically significant. Therefore, we can assume that the proportional hazard assumption holds.\n\nIn this work, right censored time-to-event data are considered. Survival time is denoted by T and censoring time by Cj, where j=1,2,3,…,m are women diagnosed with invasive breast cancer who observed first recurrence after primary treatment. The jth event time is defined by tj=minTjCj, while time for censored and uncensored events is denoted by δj=ITj≤Cj. The general relationship form, between Tand X is given as\n\nThe hazard rate represents a probability of an instantaneous failure per unit of time given that an individual patient has survival after time t.5 The main objective of time to failure studies is to estimate the hazard function accurately. For this purpose, different modelling approaches are applied.\n\nIn the multivariate approach, the semi-parametric Cox PH model is most popular for the analysis of time to failure data. The general form is written as\n\nFPs provide flexible parameterization of continuous predictors, were first used for modelling families of curves by Royson and Altman,17 with polynomial of degreem, it can be written as\n\nm is a positive integer and α0,α1,α2,…,αm are regression parameters, while fjx1 is divided into two parts: one consists of x1pj when pj≠0, and another of lnx1 for pj=0.17 FP models have a wide variety of shapes based on different transformations. The main issue with fractional polynomials is in choosing a suitable power for polynomials, as this has a direct positive relationship with flexibility. To increase flexibility, a greater power can be used, but with the major threat of non-locality. That means that the fitted function at a given point of x0 depends on data points which are very far from that reference point.18\n\nSpline regression is an improved technique, used to overcome the non-locality of fractional polynomials. In spline models, the dataset is divided into multiple parts and these parts are joined with knots. In time to failure regression analysis, spline modelling is extensively used to smooth non-linear effects of continuous predictors. Spline fX has a smooth function. The major problem occurs in choosing the number of knots, as no hard and fast rule is available to apply the suitable number of knots.19\n\nUnder RCS, the best way of choosing the number of knots is using the quotient of the difference between the largest and average uncensored log survival time and the largest and smallest uncensored log survival time.20 Royston suggested another method in this respect; according to him, a good way to choose the suitable number of knots is to randomly apply different number of knots every time, and select the best model with measure of information criteria.21 Flexible parametric models have scaling for proportional hazards or proportional odds, which are usually based on transformation of survival function by a link function\n\nS0t is the baseline survival function, αis the vector of unknown parameters for predictors x. Piecewise exponential models (PEMs) are also a reasonable approach to estimate hazard ratios more accurately.21 Under the PEM modelling approach, follow-up time is divided into iintervals, by assuming a constant baseline hazard in each interval, so thatπ0t=πi simplifies to,\n\nFor the cut points of follow-up times, minimum to maximum time is divided into finite intervals, 0=n0<n1<n2<n3<…<ni=tmax. Here, n0 to ni are time intervals, and the hazard rate of exponential distribution is constant over time intervals. Censoring and all unique event times can be used as time interval cut points, but no hard and fast rule is available to choose cut points, which is a point to ponder; too small or too large cut points may cause under- or overfitting.\n\nOne approach to deal with this cut point problem is an extension of PEM, in which a large number of cut points are used and the hazard is estimated semi-parametrically. This is called the Piecewise exponential additive mixed model (PAMM), in which a hazard is modeled through a smooth nonlinear function. In PAMM, predictors contribute to the hazard additively, imposing a quadratic penalty on the basis coefficients:\n\nIn fully parametric PH modelling, baseline hazard function is assumed to follow a specific distribution and coefficients are estimated via maximum likelihood. A number of different parametric PH models are derived by applying distributions, such as exponential, Weibull, and Gompertz.\n\nAn alternative to parametric PH is AFT models, the corresponding log-linear form of the AFT model with respect to time is given as\n\nThe likelihood estimates are maximized using the Newton Raphson procedure,15 which may be time consuming and tricky without computer programming. The freely available R software is used to implement all modelling techniques.\n\nComparison of fitting models is done via measures of fit, which describe accuracy of fitted models for a given data set, usually called goodness of fit measures. Model fitting accuracy has nothing to do with the predictive ability for external data prediction. The Akaike Information Criterion (AIC)25 and the Bayesian Information Criterion (BIC)26 are two of the most common measures which are used to compare models’ performances. For the PH model AIC and BIC are based on log-partial likelihood\n\nAccording to the Ethical Guidelines for Epidemiologists (IEF-EGE) and the regulations of the ethics committee located at the Advanced Studies and Review Board, University of the Punjab, Lahore (Pakistan), no ethics approval is needed, because the analysis is based on routine data. The study was critically cleared by the Advanced Studies and Review Board of Punjab University. The letter of support written by the departmental head was submitted to the selected hospital. Prior to data collection, written consent was obtained from the head of oncology department and confidentiality was maintained by coding from data collection to analysis.\n\n\nResults\n\nIn the present study, women’s age was collected for: diagnosis and recurrence time. The median age at diagnosis of breast cancer was 47 years (range: 18–59); while the median age at recurrence was 49 years (range: 21–62). Median survival time after recurrence was 3 years, and just half (54.1%) of cancer cases were ER-negative. The majority of patients were PR-positive (64.6%) and had a positive human epidermal growth factor receptor 2 (52.9%). Overall, 207 women (20.1%) had tumor grade 1, whereas 821 (79.9%) had a higher level of malignancy. Chemotherapy (36.4%) and radiotherapy (87.4%) were given as primary treatments (Table 1).\n\nThere were 447 deaths among the 1,028 women included in the study. As shown in Table 1, 78.5% of deaths occurred due to breast cancer within three years after recurrence, while 20.8% between 3 to 6 years, and 0.7% of patients died due to breast cancer after 6 years of its recurrence. The molecular markers among women who died due to breast cancer were distributed as follows: 59.3% ER-positive, 66.7% PR-negative, and 65.5% human epidermal growth factor receptor 2-positive. Breast cancer death was positively associated with higher tumor grade (11 and 111; 97.3%) and no chemotherapy (67.8%).\n\nTable 2 presents information measure results. Low values of AIC, AICc, BIC and BICc are considered good; if a model’s fitting values for AIC, AICc, BIC and BICc are smaller than others, that model is considered a good fitted one. To make results less lengthy and meaningful, we only discuss here AIC and AICc values of first three good fitted accelerated failure time distributional models. From the fully parametric models, Weibull is the best fitted one (AIC = 7269.5, AICc = 7271.9) among others, generalized gamma (AIC = 7269.8, AICc = 7272.1), gamma (AIC = 7270.2, AICc = 7272.5), and Generalized F (AIC = 7271.7, AICc = 7274.0) come next in terms of preferences, respectively. We also presented BIC and BICc in Table 2.\n\nMultivariate fractional polynomial (MFP) is the best fitted model incorporating time dependent covariates (AIC = 5167.5, AICc = 5163.6), while the generalized additive model (GAM) (AIC = 5171.3, AICc = 5173.8) is also a good choice for analyzing non-linear continuous predictors in a multivariate setting, having the advantage of small numbers of parameters in non-integer, which is due to shrinkage during parameter estimation. Royston and Palmar’s flexible parametric models have been applied with different scales: we considered flexible parametric models on hazard and odd scales, by including time-dependent effects for the age at diagnosis and age at recurrence covariates. Hazard generalized gamma (AIC = 7211.3, AICc = 7214.8) outperformed odd generalized gamma (AIC = 7212.5, AICc = 7216.0). Although the subjective approach of knot selection may be criticized, sensitivity analyses studies showed insignificant differences in results while changing positions of knots.29–31\n\nFigure 1 shows cumulative hazard graphs for all parametric and flexible parametric models with Nelson Aalen cumulative hazard as a reference. The Nelson Aalen estimator is represented by a step function, which starts at zero. It provides an estimate of the expected number of deaths observed for a given amount of time. Visually, all models provide fitting accuracy for the right censored breast cancer failure time understudy data, with some slight variations existing to capture the fluctuations. The wider lines show a greater confidence interval, which is indicative of a poor fit, while narrower lines show good model fitting.32\n\nTime dependent covariates, age at diagnosis and age at recurrence are modeled using 4 degrees of freedom for splines. PEM and PAMM are applied to the understudy data to get baseline hazard estimates, where finite time intervals are considered as factors to maintain constant hazard for each interval. The age at diagnosis and recurrence are estimated using P-splines with the same 4 degrees of freedom. PEM and PAMM results are compared with Nelson Aalen estimator, graphical displays showed close agreement of good model fitting in Figure 2.\n\n\nDiscussion\n\nIn this paper, different parametric models are compared in terms of theoretical aspects and application. Our findings suggested that progesterone receptor negative, human epidermal growth factor receptor 2 positive, higher tumor grade, and no chemotherapy increase the risk of death after recurrence. The most surprising result is regarding radiotherapy treatment, which depicted no reduction in breast cancer time to death. This might be due to a higher level of physical impairment of patients receiving radiotherapy treatment. However, patients treated by radiotherapy at an early stage have a larger survival time.33\n\nWe applied distributional parametric models which are known as standard parametric models, with a full maximum likelihood estimator to estimate unknown parameters. AFT models make practical sense to study the influence of covariates, which may accelerate breast cancer mortality. The AFT model is the best choice for the analysis of time to failure data when hazards are non-proportional, as it provides efficient estimates and an estimate of the median failure time.\n\nThe exponential distribution having one rate parameter is often used in experiments to account for the amount of time until an event occurs. The Weibull distribution is a special case of the exponential distribution with shape and scale parameters. It provides a better fit than exponential with one extra degree of freedom. The same is true for gamma distribution, which has two parameters and has close results to Weibull. Generalized gamma has mean, location and scale parameters. Log-normal is a probability distribution, with a normally distributed logarithm. It is widely used in lifetime data analysis. The two parameters of mean and standard deviation have a more stable behavior than log-logistic distribution. Generalized F distribution is a good alternative to generalized gamma with one extra parameter. From the interpretation point of view, the AFT model’s results are easy to interpret and help clinicians to make wise decisions related to the patients’ conditions.\n\nFlexible parametric models have advantage of using restricted cubic splines, which incorporates time dependent effects of predictors on the log hazard and reduces the bias of non-proportionality. The Royston and Parmar generalized gamma flexible parametric model under hazard scale outperformed the odd scale model. Of course, one should not ignore the threat of overfitting, by including greater number of internal knots. The functional polynomial model has the advantage of only considering significant factors, so it gives better results than other spline-based models. GAM under spline basis function has the potential to provide a better fit of data than generalized linear flexible parametric models.34,35\n\nThe main strength of this study is that we described and applied different time to failure models, to right censored breast cancer data. In a piecewise exponential model the baseline hazard is modeled by step function with different intervals, estimation is done by including dummy variables for each interval. The major disadvantage of this technique is that data becomes too long, and parameter estimation becomes unstable. The piecewise additive mixed model overcomes this drawback. By adding a large number of basis functions and using P-splines between neighboring basis coefficients, parameters are estimated through restricted maximum likelihood (REML).13\n\nThere are several limitations to our study. First, the use of a single case study may be viewed as a limitation. However, a simulation study can also be designed to validate results. Second, the model comparisons are based on within sample information measures (AIC, AICc, BIC, BICc), while predictive performances of models can also be checked via different measures. Third, sensitivity analyses of choosing different numbers of knots in spline-based models can be performed to make firm conclusions.\n\n\nConclusion\n\nFlexible parametric modelling of the hazard function is more efficient than standard parametric models, incorporating the complex patterns of the observed failure data. Generalized additive models provide more accurate estimates under spline-basis function, with time dependent covariates. For long follow-up studies and multiple time dependent covariates, which may have effects on hazard, penalized models are more suitable.\n\n\nData availability\n\nData is available from the corresponding author, Dr. Florian Fischer (florian.fischer1@charite.de), upon reasonable request. Data can be used for research purposes, but cannot be published because it is taken from a hospital.", "appendix": "Acknowledgements\n\nWe thank the staff of the Institute of Nuclear Medicine & Oncology Lahore (INMOL), who supported in data collection. We also wish to thank Dr. Rab Nawaz Maken from INMOL cancer hospital, Lahore, Pakistan, for providing full support to conduct this research.\n\n\nReferences\n\nMcPherson K, Steel CM, Dixon JM: ABC of breast diseases. breast cancer-epidemiology, risk factors, and genetics. BMJ. 2000; 321: 624–628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarchielli A, Balzi D: Age at diagnosis, extent of disease and breast cancer survival: a population-based study in Florence, Italy. Tumori. 2000; 86: 119–123. PubMed Abstract \| Publisher Full Text\n\nCrowe JP Jr, Gordon NH, Shenk RR, et al.: Age does not predict breast cancer outcome. Arch. Surg. 1994; 129: 483–487. 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Publisher Full Text\n\nRoyston P, Sauerbrei W: Multivariable model-building: a pragmatic approach to regression analysis based on fractional polynomials for modelling continuous variables. Wiley;2008. Publisher Full Text\n\nDurrleman S, Simon R: Flexible regression-models with cubic-splines. Stat. Med. 1989; 8: 551–561. PubMed Abstract \| Publisher Full Text\n\nRoyston P: Flexible alternatives to the Cox model, and more. The State Journal. 2001; 1: 1–28. Publisher Full Text\n\nFriedman M: Piecewise exponential models for survival data with covariates. Ann. Stat. 1982; 10: 101–113. Publisher Full Text\n\nBender A, Andreas G, Fabian S: A generalized additive model approach to time-to-event analysis. Stat. Model. 2018; 18: 299–321. Publisher Full Text\n\nWei LJ: The accelerated failure time model: A useful alternative to the Cox regression model in survival analysis. Stat. Med. 1992; 11: 1871–1879. PubMed Abstract \| Publisher Full Text\n\nVilijandas B, Mikhail N: Accelerated Life Models; Modeling and Statistical Analysis. Chapman&Hall/CRC;2002.\n\nAkaike H: A new look at the statistical model identification. IEEE Trans. Autom. Control. 1974; 19: 716–723. Publisher Full Text\n\nSchwarz G: Estimating the dimension of a model. Ann. Stat. 1978; 6: 461–464. Publisher Full Text\n\nHurvich CM, Tsai CL: Regression and time series model selection in small samples. Biometrika. 1989; 76: 297–307. Publisher Full Text\n\nVolinsky CT, Raftery AE: Bayesian information criterion for censored survival models. Biometrics. 2000; 56: 256–262. PubMed Abstract \| Publisher Full Text\n\nLambert PC, Dickman PW, Nelson CP, et al.: Estimating the crude probability of death due to cancer and other causes using relative survival models. Stat. Med. 2010; 29: 885–895. PubMed Abstract \| Publisher Full Text\n\nRoyston P, Lambert PC: Flexible parametric survival analysis using Stata: beyond the Cox model. 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New York:Wiley Online Library;1990." }	[ { "id": "118775", "date": "19 Jan 2022", "name": "Benjamin Kearns", "expertise": [ "Reviewer Expertise Methodological research", "health economics", "survival analysis", "time-series analysis." ], "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 an interesting manuscript, but it feels very unfocused. For example, the first paragraph mentions the importance of age, and evaluating the effect of treatment is also mentioned, along with discussion of PH and AFT. This suggests that identifying and quantifying treatment effects is the main objective. But there are no model-based estimates of covariate effects provided in the results section.\nThere is also an extensive discussion and comparison of different model types (which is stated to be the main focus). This includes non and semi parametric approaches, but they are not presented in the results section. Parametric models are included, but it is unclear how the choice between these should be made (noting that there is no way to compare information criteria for statistical significance), and it is unclear how generalisable the results of this study are beyond the case-study provided. The abstract concludes \"PAMM is a good approach to perform in-depth studies of predictors over different finite intervals of follow-up time.\" This is not supported by the results presented (and this conclusion is missing from the main text).\nThere are some important omissions, such as technical details for the GAM, and model specifications that were used for analysis (such as which degrees of FPs, basis functions for GAMs) with justification.\nThere are some places where detailed information is provided which does not really contribute to the manuscript. Examples include a discussion of the types of right censoring in the introduction (this distinction is never used elsewhere in the manuscript), and discussion of both non- and semi- parametric methods, when these do not appear in the results. It is also unclear why Figures 1 and 2 are separate (and graphical results for the RP GG models look wrong, they have the lowest IC of the models presented in Fig 1, but the worst visual fit).\nOverall, the manuscript requires substantial additional restructuring to make it suitable for publication. I would recommend making the focus a tutorial-style paper to demonstrate how flexible models may be used to estimate time-varying treatment effects, and how this compares with the treatment-effects obtained from standard approaches. This could focus on the impact of treatment. To support this, the R code used should be made available (even if the data cannot be), to enhance reproducibility. The authors could also consider replicating the analysis on a publicly available dataset (see for example Kearns et al. 20191)\nSome additional feedback is provided below:\nBackground: first paragraph needs more references to support the statements made.\n\nBackground: first paragraph needs more justification for why the role of age is being explored when it was previously not found to be significant. What makes the authors think they will find a different association?\n\nAs noted, most of the flexible models (such as GAMs and FPs) were originally developed for non-survival data, so information is required as to how they can be applied to survival data.\n\nMethods, study design: it is unclear what the \"Extended data\" is.\n\nMethods: pi0 (for the Cox PH) needs defining.\n\nUse of information criteria will be limited as non- and semi-parametric models cannot then be compared. It is unclear why AIC(c) were used in preference to BIC(c) when presenting results. It is also unclear what would happen if the four IC measures gave conflicting results (which one would be used to select the best model)?\n\nResults, Table 2: it is unclear why IC are so much lower for FPs and GAMs - this suggests that the likelihood for these two models is defined differently.\n\nDiscussion: the benefits of AFT models will only hold if the aft assumption holes this is an important caveat that should be mentioned. Also, as the PH assumption is earlier stated to hold for this analysis, the relevance of the discussion of AFT models is unclear.\n\nDiscussion: \"The Weibull distribution is a special case of the exponential distribution\" - it is the other way around. This paragraph is on the whole too general.\n\nDiscussion: \"Flexible parametric models have advantage of using restricted cubic splines\" - there are a large number of flexible models that use other basis functions (or alternative approaches to induce flexibility).\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?\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": [] }, { "id": "149019", "date": "27 Sep 2022", "name": "Federico Ambrogi", "expertise": [ "Reviewer Expertise Multivariate analysis", "Survival analysis", "Study design", "high-dimensional data." ], "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\nAfter reading the title, I expected to read a tutorial or review paper for survival analysis for breast cancer patients. The paper is in part a review but I discovered that the focus is on parametric models and has some more general aspects scattered throughout the text.\nThe main critique has to do with the endpoint chosen by the authors. As death from breast cancer is used, some considerations about competing risks are necessary and nothing is said in the paper. The analysis performed using regression model is that of cause-specific hazards and generally must also take into account the other causes of death. There are plenty of tutorial papers on competing risks both in the methodological and applied medical literature. Moreover, the study population is described approximately. The type of recurrence, for example, is not specified: is it the same for all 1028 women? What is the time scale for the analysis? Is the time interval starting from the time of recurrence until eventual death? One option is to use age as time scale and consider late entry.\nThe Results section is also questionable, having to deal with time to event data. How was median survival time calculated? In general, we have to take into account censored observations. The cumulative incidence curve (accounting for competing events) must be used to calculate the median event time! The same applies to the percentages of women with the events within 3 years and so on (Results section): this must be calculated using cumulative incidence curves, not just calculating the percentages as censoring makes percentages meaningless.\nGeneral conclusions about models cannot be drawn from this study and the sentence about simulation is too vague to be of any utility. In my opinion, this could be a tutorial paper about regression models in survival analysis but the data used are really too complicated for a tutorial!\nSpecific comments:\nThe explanation of the different censoring type is not of interest here. Instead, late entry and time scales are of interest considering the data.\n\nMultivariate must be changed to multivariable.\n\nKM can be used with more than one variable. Obviously if using many variables, an excessive stratification may prevent any meaningful result.\n\nThe sentence is not clear \"...so distributions of regression parameters’ outcomes remain unknown.\" Regression parameters in the Cox model have clear statistical properties.\n\nWhat are \"unknown nonlinear predictors\" and how can splines model them?\n\nPH assumption \"assumes a fixed proportion of hazard for individuals\"? Must be better explained!\n\nWhat is the distinction between binary and dummy variables?\n\nModel (1) is the \"general relationship form\" or something very special?\n\n\"...the hazard rate of exponential distribution is constant over time intervals\", probably the hazard is constant in each interval.\n\n\"...too small or too large cut points may cause under- or overfitting\", probably the time intervals are too large or too small.\n\n\"The likelihood estimates are maximized using the Newton Raphson procedure,15 which may be time consuming and tricky without computer programming.\" This is a sentence from the fifties...\n\n\"...if a model’s fitting values for AIC, AICc, BIC and BICc are smaller than others, that model is considered a good fitted one\", it is considered better than the others...\n\nMFP and GAM information criteria are probably not on the same scale as the others and comparison cannot be direct.\n\nExplanation of the MFP is lacking...\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? 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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-1042
https://f1000research.com/articles/9-173/v1	10 Mar 20	{ "type": "Method Article", "title": "Applying machine learning EEG signal classification to emotion‑related brain anticipatory activity", "authors": [ "Marco Bilucaglia", "Gian Marco Duma", "Giovanni Mento", "Luca Semenzato", "Patrizio E. Tressoldi", "Marco Bilucaglia", "Gian Marco Duma", "Giovanni Mento", "Luca Semenzato" ], "abstract": "Machine learning approaches have been fruitfully applied to several neurophysiological signal classification problems. Considering the relevance of emotion in human cognition and behaviour, an important application of machine learning has been found in the field of emotion identification based on neurophysiological activity. Nonetheless, there is high variability in results in the literature depending on the neuronal activity measurement, the signal features and the classifier type. The present work aims to provide new methodological insight into machine learning applied to emotion identification based on electrophysiological brain activity. For this reason, we analysed previously recorded EEG activity measured while emotional stimuli, high and low arousal (auditory and visual) were provided to a group of healthy participants. Our target signal to classify was the pre-stimulus onset brain activity. Classification performance of three different classifiers (linear discriminant analysis, support vector machine and k-nearest neighbour) was compared using both spectral and temporal features. Furthermore, we also contrasted the classifiers’ performance with static and dynamic (time evolving) features. The results show a clear increase in classification accuracy with temporal dynamic features. In particular, the support vector machine classifiers with temporal features showed the best accuracy (63.8 %) in classifying high vs low arousal auditory stimuli.", "keywords": [ "EEG", "brain anticipatory activity", "machine learning", "emotion" ], "content": "Introduction\n\nIn last decades, the vision of the brain has moved from a passive stimuli elaborator to an active reality builder. In other words, the brain is able to extract information from the environment, building up inner models of external reality. These models are used to optimize the behavioural outcome when reacting to upcoming stimuli1–4.\n\nOne of the main theoretical models assumes that the brain, in order to regulate body reaction, runs an internal model of the body in the world, as described by embodied simulation framework5. A much investigated hypothesis is that the brain functions as a Bayesian filter for incoming sensory input; that is, it activates a sort of prediction based on previous experiences about what to expect from the interaction with the social and natural environment, including emotion6. In light of this, it is possible to consider emotions, not only as a reaction to the external world, but also as partially shaped by our internal representation of the environment, which help us to anticipate possible scenarios and therefore to regulate our behaviour.\n\nThe construction model of emotion7 argues that the human being actively builds-up his/her emotions in relation to the everyday life and social context in which they are placed. We actively generate a familiar range of emotions in our reality, based on their usefulness and relevance in our environment. In this scenario, in a familiar context we are able to anticipate which emotions will be probably elicited, depending on our model. As a consequence, the study of the anticipation of/preparation for forthcoming stimuli may represent a precious window for understanding the individual internal model and emotion construction process, resulting in a better understanding of human behaviour.\n\nA strategy to study preparatory activity could be related to the experimental paradigm in which cues are provided regarding the forthcoming stimuli, allowing the investigation of the brain activity dedicated to the elaboration of incoming stimuli8,9. A cue experiment to predict the emotional valence of the forthcoming stimuli showed that the brain’s anticipatory activation facilitates, for example, successful reappraisal via reduced anticipatory prefrontal cognitive elaboration and better integration of affective information in the paralimbic and subcortical systems10. Furthermore, preparation for forthcoming emotional stimuli also has relevant implications for clinical psychological conditions, such as mood disorders or anxiety11,12.\n\nRecently, the study of brain anticipatory activity has been extended to statistically unpredictable stimuli13–15, providing experimental hints of specific anticipatory activity before stimuli are randomly presented. Starting from the abovementioned studies, we focused on the extension of brain anticipatory activity to statistically unpredictable emotional stimuli.\n\nAccording to the so called dimensional model, emotion can be defined in terms of three different attributes (or dimensions): valence, arousal and dominance. Valence measures the positiveness (ranging from unpleasant to pleasant), arousal measures the activation level (ranging from boredom to frantic excitement) and dominance measures the controllability (i.e. the sense of control)16.\n\nEmotions can be estimated from various physiological signals17, such as via skin conductance, electrocardiogram (ECG) and electroencephalogram (EEG). The latter has received a considerable amount of attention in the last decade, introducing several machine learning and signal processing techniques, originally developed in other contexts, such as brain computer interfaces18. Emotion recognition has been re-drawn as a machine learning problem, where proper EEG related features are used as inputs to specific classifiers.\n\nThe most common features belong the spectral domain, in the form of spectral powers in delta, theta, alpha and gamma bands19, as well as power spectral density (PSD) bins20. The remaining belong to the time domain, in the form of event-related de/synchronizations (ERD/ERS) and event-related potentials (ERP)19, as well as shape related indices such as the Hjorth parameters and the fractal dimension20.\n\nThe most commonly used classifier is the support vector machine (SVM) with the radial basis function (RBF) kernel, followed by the k-nearest neighbour (kNN) and the linear discriminant analysis (LDA)19,20. Finally, most of the classifiers are implemented as non-adaptive (i.e. static)19, in contrast to the dynamic versions that take into account the temporal variability of the features21.\n\nThe classification performances are very variable because of the different features and classifiers adopted. The following examples are taken from 19 - in particular, from the subset (17 out of 63) of reviewed papers that focused on arousal classification. Using an SVM (RBF kernel) and spectral features (e.g. short-time Fourier transform), Lin and colleagues obtained 94.4% accuracy (i.e. percentage of corrected classification)22, while using similar spectral features (e.g. PSD) and classifier (SVM with no kernel), Koelstra and colleagues obtained an accuracy of 55.7%23. Liu and Sourina obtained an accuracy of 76.5% using temporal features (e.g. fractal dimension) with an SVM (no kernel)24, while Murugappan and Murugappan obtained a an accuracy of 63% using similar temporal features and an SVM with a polynomial kernel25. Finally, Thammasan and collegues obtained an accuracy of 85.3% using spectral features (e.g. PSD), but with a kNN (with k=3)26. All the classifiers were static.\n\nThe purpose of the present work is to provide new methodological advancements on the machine learning classification of emotions, based on the brain anticipatory activity. For this purpose, we compared the performances of tree different classifiers (namely LDA, SVM, kNN) trained using two types of EEG features (namely, spectral and temporal). In addition, each classifier was dynamically trained, to take into account the temporal variability of the features. The results provide useful insights regarding the best classifier-features configuration to better discriminate emotion-related brain anticipatory activity.\n\nA more detailed description of the machine learning algorithms is provided as Extended data27.\n\nIn introducing pattern recognition, we underlined that the classifiers are built using a set of previously annotated class-prototypical features for the training set. It is common practice to extract from the training set a subset of annotated features (the test set) and use it to evaluate the performances of the trained classifiers – but not to train it.\n\nSince the training set is limited, the specific train/test splitting introduce a bias in both the training and performance evaluation. This can be avoided following the so-called k -fold cross validation scheme. The original training set D is partitioned into k disjoint and equal sized sets, D=∪i=1kDk. The classifier is then trained k-times using, each time, as the test set a different partition Dj and as the training set the remaining ∪i≠jDi. Finally, the overall performance is computed as the average over the k single performances28 (pp. 483–485).\n\nWith the general term performance, we mostly refer to the classification accuracy ACC, defined as the ratio between the number of correctly classified features and the total number of features. Introducing the chance-level accuracy ACC0 as the ratio between the number of features for each class (i.e. how balanced is the training set), we can additionally define as performance the Kappa statistic: κ = (ACC – ACC0)/ACC029.\n\nCompared to ACC, κ is a more robust performance measure, since it is normalized by the class unbalances. Another solution to take into account the class unbalances, is to compare (using for example a t-test) the k cross-validated accuracies against k random accuracies, obtained from a random classifier29–35.\n\nTo classify a time-varying signal (i.e. to perform a dynamic classification), an ordered sequence of features {xi}i=1N (i.e. temporal features), corresponding to N adjacent temporal windows, is extracted. The temporal features are fed into either “dynamic” classifiers, such as the Hidden Markow Model (HMM)21, or an ordered sequence of “static” classifiers {fi}i=1N 36–39. The former fully takes into account the signal’s temporal variability, since it uses the entire sequence during the training phase. The latter train each static classifier fi, using only the corresponding features xi, but provides an ordered sequence of accuracies {ACCi}i=1N. where each ACCi corresponds to fi.\n\nAs stated in the previous sections, the curse of dimensionality arises when the number of available training features is small compared to the feature dimension m. In such situations, the parameter estimation becomes problematic (see for example the problem of the singularity of the estimated covariance matrix described in the LDA sub-section) and the trained classifier usually underperforms.\n\nAs a rule of thumb, the number of training features N should be an exponential function of the dimensionality (e.g. N = 10m), with the ratio growing with the complexity of the classifiers40. By fixing the feature dimension m, linear classifiers require, for example, a less numerous training set. Additionally, even with an adequate training set, feature dimensionality impacts on both the training and classification speed. In fact, as stated in the SVM sub-section, linear classification requires O(m) multiplications and sums to compute each scalar product. Reducing the feature dimensionality by means of so called feature selection algorithms, a classifier can be made more robust (i.e. less sensitive to the curse of dimensionality) and efficient (in terms of computational speed).\n\nFeature selection can be broadly described as a mapping function s:ℝm→ℝn such as:\n\n\n\nwhere n < m and {s1, s2, ... , sn} ⊂ {1, 2, ... , m}. In other words, a feature selection algorithm performs a projection of the original feature vector onto a lower dimensional subspace defined by a subset of scalar features. The best subspace, as selected among all the possible 2m, should not significantly decrease the classification performances, both globally (i.e. how features are classified overall) and locally (i.e. how the single feature is classified)41\n\nFeature selection algorithms can be broadly grouped according to the following criteria42:\n\n1. Label information. Supervised algorithms take into account the class information, while unsupervised algorithms do not, in assigning the training features as belonging to the same class.\n\n2. Search strategy. Filter algorithms (also known as classifier-independent) are based on a two-step “ranking and selecting” criterium: scalar features are first ranked according to a proper criterion; then only the “best” ones are selected. Wrapper methods (also known as classifier dependent methods) use the selected classifier, following an “ad hoc” approach: the selected scalar features are those that give the best classification performance\n\nAn example of a supervised filter algorithm is the biserial correlation coefficient. Given a training set D composed by N+ features belonging to the class +1 and N– features belonging to the class –1, the biserial correlation coefficient for the k-th scalar feature xk is given by 43:\n\n\n\nwhere m(·), s(·) are the sample mean and sample standard deviation operators, respectively, and xk+, xk- are the subset of xk belonging to the classes +1 and –1, respectively. The total feature score is obtained by summing the m coefficients of each scalar feature xk. Once the scores rk2 are sorted in descending order, the feature selection is made simply by selecting the first scalar features whose summing score get a percentage (e.g. 95%) of the total feature score.\n\n\nMethods\n\nThe data of the present study were obtained in the experiment described in 37, which was approved by the Ethical Committee of the Department of General Psychology, University of Padova (No. 2278). Before taking part in the experiment, each subject gave his/her informed consent in writing after having read a description of the experiment. In line with department policies, this re-analysis of an original study approved by the ethics committee did not require new ethical approval.\n\nIn the present study, we reanalysed the EEG data27 of the experiment described in 37, applying an original static and dynamic features selection and classification by using the three different algorithms explained above.\n\nA more detailed description of the experimental design is available in the original study. Here we describe only the main characteristics.\n\nTwo sensory categories of stimuli (i.e. visual and auditory), were extracted according to their arousal value from two standardized international archives. Visual stimuli consisted of pictures of 28 faces, 14 neutral faces and 14 fearful faces were extracted from the NIMSTIM archive44, whereas auditory stimuli consisted of 28 sounds, and 14 low- and 14 high-arousal sounds were chosen from the International Affective Digitized Sounds (IADS) archive45.\n\nTo all 28 adult healthy participants, two different experimental tasks, which were delivered in separate blocks were presented. The two tasks are described in Figure 1, which illustrates the sequence of events and the temporal trial structure relative to the passive (top) and the active (bottom) tasks. Within each task, the stimuli were randomly presented and equally distributed according to either sensory category (faces or sounds) and arousal level (high or low). Full details of these tasks have been described previously in 37.\n\nDuring the entire experiment, the EEG signal was continuously recorded using a Geodesic high density EEG system (EGI GES-300) through a pre-cabled 128-channel HydroCel Geodesic Sensor Net (HCGSN-128) referenced to the vertex (CZ), with a sampling rate of 500 Hz. The impedance was kept below 60kΩ for each sensor. To reduce the presence of EOG artefacts, subjects were instructed to limit both eye blinks and eye movements, as much as possible.\n\nThe continuous EEG signal was off-line band-pass filtered (0.1–45Hz) using a Hamming windowed sinc finite impulse response (FIR) filter (order = 16500) and then downsampled at 250 Hz. The EEG was epoched starting from 200 ms before the cue onset and ending at the stimulus onset. The initial epochs were 1300 ms long from the cue onset, including 300 ms of cue/fixation cross presentation and 1000 ms of interstimulus interval (ISI).\n\nAll epochs were visually inspected to remove bad channels and rare artefacts. Artefact-reduced data were then subjected to independent component analysis (ICA)45. All independent components were visually inspected, and those that related to eye blinks, eye movements, and muscle artefacts, according to their morphology and scalp distribution, were discarded. The remaining components were back-projected to the original electrode space to obtain cleaner EEG epochs.\n\nThe remaining ICA-cleaned epochs that still contained excessive noise or drift (±100 μV at any electrode) were rejected and the removed bad channels were reconstructed. Data were then re-referenced to the common average reference (CAR) and the epochs were baseline-corrected by subtracting the mean signal amplitude in the pre-stimulus interval. From the original 1300 ms long epochs, final epochs were obtained only from the 1000 ms long ISI.\n\nFrom each epoch and each channel k, the PSD was estimated by a Welch’s periodogram using 250 points long Hamming’s windows with 50% overlapping. PSD was first log transformed to compensate the skewness of power values46, then the spectral bins corresponding to alpha, beta and theta bands – defined as 13~30Hz, 6~13Hz and 4~6Hz, respectively47 – were summed together. Finally, alpha, beta and theta total powers were computed as:\n\n\n\n\n\n\n\nAs a measure of emotional arousal, we computed the ratio between beta and alpha total powers βtotk/αtotk48, while to measure cognitive arousal, we computed the ratio between beta and theta total powers θtotk/βtotk49.\n\nFor each epoch, the feature (with a dimensionality of 256) was obtained, concatenating the beta-over-alpha and beta-over-theta ratio of all the channels:\n\n\n\nIt has been previously shown that arousal level (high or low) can be estimated from the contingent negative variation potentials37. The feature extraction procedure, therefore, follows the classical approach for event-related potentials50. Each epoch from each channel was first band pass filtered (0.05~10Hz) using a zero-phase 2nd order Butterworth filter and decimated to a sample frequency of 20Hz. EEG signal was thus normalized (i.e. z-scored) according to the temporal mean and the temporal standard deviation:\n\n\n\nwhere x˜i(tk) is the raw signal from i-th channel at time point tk, and mi and si are, respectively, the temporal mean and the temporal standard deviation of the i-th channel. For each epoch, the feature (with a dimensionality of 2560) was obtained, concatenating all normalized signal from each channel:\n\n\n\nEach epoch was partitioned into 125 temporal segments, 500 ms long and shifted by 1/250 s (one sample). Within each time segment, we extracted the dynamic spectral and temporal features, following the same approaches described in Static spectral features and Static temporal features sub-sections, respectively. Dynamic temporal features had a dimensionality of 1280, corresponding to 0.5 × 20 = 10 samples per channel. Dynamic spectral features had the same dimensionality as their static counterparts (256), but the Welch’s periodogram was computed using a 16 points long Hamming’s window (zero-padded to 250 points) with 50% overlapping.\n\nThe extracted features (both static and dynamic) were grouped according to the stimulus type (sound or image) and the task (active or passive), in order to classify the group-related arousal level (high or low). A total of four binary classification problems (high arousal vs low arousal) were performed: active image (Ac_Im), active sound (Ac_So), passive image (Ps_Im) and passive sound (Ps_So).\n\nStatic features were reduced by means of the biserial correlation coefficient r2 with the threshold set at 90% of the total feature score. In order to identify the discriminative power of each EEG channel, a series of scalp plots (one for each feature type and each group) of the coefficients were drawn. Since each channel is associated with N > 1 features (as well as N r2 coefficients), the coefficients (one coefficient for each channel) are calculated as a mean value. In other words, spectral and temporal features had two and 20 scalar features, respectively, for each EEG channel. To compute their scalp plots, we averaged 2 and 20 r2 coefficients of each channel. To enhance the visualization of the plots, the coefficients were finally normalized to the total score and expressed as a percentage.\n\nEach classification problem was addressed by the mean of three classifiers: LDA with pseudo-inverse covariance matrix; soft-margin SVM with penalty parameter C = 1 and RBF kernel; and kNN with Euclidean distance and k=1. Additionally, a random classifier, giving a uniform pseudo-random class (Pr{HA} = Pr{LA} = 0.5), served as a benchmark29. The accuracy of the classifiers was measured, repeating 10 times for a 10-fold cross-validation scheme. The feature selection was computed within each cross-validation step, to avoid overfitting and reduce biased results43.\n\nFor each group (Ac_Im, Ac_So, Ps_Im, Ps_So) and each feature type (static spectral, static temporal), the classification produced a 10 × 4 matrix containing the mean accuracies (one for each of the 10-fold cross-validation repetitions) of each classifier.\n\nDynamic features were reduced and classified similarly to the static ones. For each temporal segment, the associated features were reduced by means of the biserial correlation coefficient (threshold at 90%) and the classifiers (SVM, kNN, LDA and random) were evaluated using a 10-fold cross-validation scheme – repeated 10 times.\n\nFor each group, each feature type (dynamic spectral, dynamic temporal), each temporal segment and each classifier, the classification produced 10 sequences of mean accuracies {ACCi}i=1125 – one for each repetition of the 10-fold cross-validation scheme.\n\nThe syntax in MATLAB used for all analyses is available on GitHub along with the instructions on how to use it (see Software availability)51. The software can also be used with the open source program Octave.\n\nThe results of the static classifications were compared against the benchmark classifier by means of a two-sample t-test (right tail).\n\nThe results of dynamic classifications (i.e. based on dynamic spectral or dynamic temporal features) were compared following a segment-by-segment approach. For each group, the accuracy sequences of the dynamic classifiers (SVM, kNN and LDA) were compared with the benchmark accuracy sequence. Each sample ACCik, with k = {SVM, kNN, LDA}, was tested against ACCiRandom by means of two-sample t-tests (right tail). The corresponding p-value sequences {pik}i=1125 were Bonferroni-Holm corrected for multiple comparisons. Finally, the best accuracy point was detected as the left extreme of the temporal window corresponding to the highest significant accuracy.\n\n\nResults\n\nIn Figure 2 and Figure 3, the scalp distributions of r2 coefficients for each binary static classification problem, grouped for feature (spectral, temporal) and groups (Ps_Im, Ps_So, Ac_Im, Ac_So), are shown.\n\nScalp distribution of the r2 coefficients (normalized to the total score and expressed as percentage) grouped for tasks and stimulus type. (a) Active task: left Image, right Sound; (b) Passive task: left Image, right Sound.\n\nScalp distribution of the r2 coefficients (normalized to the total score and expressed as percentage), grouped for tasks and stimulus type. (a) Active task: left Image, right Sound; (b) Passive task: left Image, right Sound.\n\nThe temporal feature gave the most consistent topographical pattern, showing that the regions that best differentiate between high vs low stimuli (auditory and visual) were located over the central-parietal electrodes, whereas a more diffuse pattern in the scalp topography emerged for the spectral features.\n\nIn Figure 4 and Figure 5, box plots of the accuracies of static temporal and spectral classifications, grouped for condition, are shown. Note that SVM accuracies (the 2nd boxplot from the left) are always shown as lines because the accuracies were constant within each cross-validation step (see also Table 1, Table 2 and Table 3).\n\nFrom left: Active Image (Ac_Im), Active Sound (Ac_So), Passive Image (Ps_Im) and Passive Sound (Ps_So).\n\nFrom left: Active Image (Ac_Im), Active Sound (Ac_So), Passive Image (Ps_Im) and Passive Sound (Ps_So).\n\nOrdered accuracies grouped for classifier, feature and group.\n\nSVM, support vector machine; LDA, linear discriminant analysis; kNN, k-nearest neighbour.\n\nActive Image (Ac_Im), Active Sound (Ac_So), Passive Image (Ps_Im) and Passive Sound (Ps_So).\n\nSVM, support vector machine; LDA, linear discriminant analysis; kNN, k-nearest neighbour.\n\nActive Image (Ac_Im), Active Sound (Ac_So), Passive Image (Ps_Im) and Passive Sound (Ps_So).\n\nSVM, support vector machine; LDA, linear discriminant analysis; kNN, k-nearest neighbour.\n\nNote that all the accuracies refer to the same static classification problem (high arousal vs low arousal), performed using different classifiers (SVM, LDA, kNN) and features (spectral, temporal), on different groups (Ps_Im, Ps_So, Ac_Im, Ac_So).\n\nUsing spectral features, in only two groups did some classifiers show an accuracy greater than the benchmark. In the Ac_So group, ACCSVM = 50.9% (t(18)=2.371, p=0.015) and ACCkNN = 50.9% (t(18)=1.828, p=0.042), while for Ps_Im, ACCLDA = 51.4% (t(18)=4.667, p<0.001) and ACCSVM = 51.8% (t(18)=9.513, p<0.001).\n\nUsing temporal features, in all the groups some classifiers showed an accuracy greater than the benchmark. In the Ac_So group, ACCSVM = 50.9% (t(18)=2.907, p=0.005) and ACCkNN = 51% (t(18)=2.793, p=0.006) and in the Ps_So group, AACSVM = 50.4% (t(18)=9.493, p<0.001).\n\nIn Figure 6–Figure 12, the results of the significant dynamic classifications are shown. In the upper section of the plots, the mean (bold line) and the standard deviation (shaded) of the accuracy sequence are shown. In the lower section of the plot (black dashed line), the Bonferroni-Holm corrected p-values sequence, discretized (as a stair graph) as significant (p<0.05) or non-significant (p>0.05) is shown.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ac_Im group for LDA (a) and SVM (b) classifiers.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ac_So group for LDA (a) and SVM (b) classifiers.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ps_Im group for LDA (a) and SVM (b) classifiers.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ac_So group for SVM (a) and kNN (b) classifiers.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ac_So group for LDA classifier.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ps_Im group for LDA (a), SVM (b) and kNN (c) classifiers.\n\nAccuracy (mean value, coloured line; standard deviation, shaded line) and p-values (black dotted line) in Ps_So group for LDA (a) and kNN (b) classifiers.\n\nNote that all the accuracy plots refer to the same dynamic classification problem (high arousal vs low arousal), performed using different classifiers (SVM, LDA, kNN) and features on different groups. Spectral: Ac_Im (Figure 6), Ac_So (Figure 7), Ps_Im (Figure 8) and Ps_So (Figure 9); temporal: Ac_So (Figure 10), Ps_Im (Figure 11) and Ps_So (Figure 12).\n\nUsing spectral features, in all the groups some classifiers showed an accuracy greater than the benchmark. In the Ac_Im group, ACCLDA = 51.97% @t = 0.080s (t(18)=6.291, p<0.001) and ACCSVM = 51.07% @t = 0.416s (t(18)=6.531, p<0.001). In the Ac_So group, ACCLDA = 53.04% @t = 0.332s (t(18)=8.583, p<0.001) and ACCSVM = 51.16% @t = 0.146s (t(18)=8.612, p<0.001). In the Ps_Im group, ACCLDA = 53.12% @t = 0.156s (t(18)=6.372, p=0.000) and ACCSVM = 51.83% @t = 0.140s (t(18)=6.668, p<0.001). In the Ps_So group, ACCSVM = 50.62% @t = 0.024s (t(18)=5.236, p=0.003) and ACCkNN = 51.41% @t = 0.476s (t(18)=4.307, p=0.026).\n\nUsing temporal features, in only three groups did some classifiers show an accuracy greater than the benchmark. In the Ac_So group, ACCSVM = 63.80% @t = 0.100s (t(18)=6.113, p=0.001). In the Ps_Im group, ACCLDA = 63.68% @t = 0.024s (t(18)=12.108, p<0.001) and ACCSVM = 51.43% @t = 0.084s (t(18)=4.881, p=0.008). In the Ps_So group, ACCLDA = 64.30% @t = 0.0276s (t(18)=11.092, p<0.001) and ACCkNN = 63.70% @t = 0.480s (t(18)=16.621, p<0.001).\n\nTable 4 reports the accuracies for dynamic features, ordered in descending order and grouped for classifier, feature group and time.\n\nOrdered accuracies grouped for classifier, feature and group.\n\nSVM, support vector machine; LDA, linear discriminant analysis; kNN, k-nearest neighbour.\n\n\nDiscussion\n\nThe aim of the study was to provide new methodological insights regarding machine learning approaches for the classification of anticipatory emotion-related EEG signals, by testing the performance of different classifiers on different features.\n\nFrom the ISIs (i.e. the 1000 ms long window preceding each stimulus onset), we extracted two kinds of “static” features, namely spectral and temporal, the most commonly used features in the field of emotion recognition19,20. As spectral features, we used the beta-over-alpha and the beta-over-theta ratio, whereas for the temporal feature we concatenated the decimated EEG values.\n\nAdditionally, we extracted the temporal sequences of both static spectral and temporal features, using a 500 ms long window moving along the ISI to build dynamic spectral and temporal features, respectively. This step is crucial for our work since, considering the temporal resolution of the EEG, an efficient classification should take into account the temporal dimension, to provide information about when the difference between two conditions are maximally expressed and therefore classified.\n\nWe trained and tested three different classifiers (LDA, SVM, kNN, the most commonly used in the field of emotion recognition19,20) using both static and dynamic features, comparing their accuracies against a random classifier that served as benchmark.\n\nOur goal was to identify the best classifier (static vs dynamic) and the best feature type (spectral vs temporal) to classify the arousal level (high vs low) of 56 auditory/visual stimuli. The stimuli, extracted from two standardized datasets (NIMSTIM52 and IADS44), for visual and auditory stimuli, respectively) were presented in a randomized order, triggered by a TrueRNG™ hardware random number generator.\n\nConsidering the number of groups (four), the number of classifiers (three) and the number of feature types (two), each classification (static or dynamic) produced a total of 24 accuracies, whose significances were statistically tested (using a two-sample t-test and the benchmark’s accuracies).\n\nWithin the nine significant accuracies obtained using static features, the classifier that obtained the highest number of accuracies was the SVM (six significant accuracies), followed by kNN (two significant accuracies) and LDA (one significant accuracy). The most frequent feature was the temporal (five significant accuracies). Finally, the best (static) feature-classifier combination was the SVM with spectral features (51.8%), followed by LDA with spectral features (51.4%) and kNN with temporal features (51%).\n\nWithin the 13 significant accuracies obtained using dynamic features, the classifier that obtained the highest number of accuracies was the SVM (six significant accuracies), followed by LDA (four significant accuracies) and kNN (three significant accuracies). The most frequent feature was the spectral (eight significant accuracies). Finally, the best (dynamic) feature-classifier combination was the SVM with temporal features (63.8%), followed by kNN with temporal features (63.70%) and LDA with temporal features (63.68%). Spectral features produced only the 5th highest accuracy (53.12% with LDA). The three best accuracies were all within the first 100ms of the ISI, although a non-significant Spearman’s correlation between accuracy and time was observed (r=-0.308, p=0.306).\n\nOur results show that globally the SVM presents the best accuracy, independent from feature type (temporal or spectral), but more importantly, the combination of SVM with the dynamic temporal feature produced the best classification performance. This finding is particularly relevant, considering the application of EEG in cognitive science. In fact, due to its high temporal resolution, EEG is often applied to investigate the timing of neural processes in relation to behavioural performance.\n\nOur results therefore suggest that, in order to best classify emotions based on electrophysiological brain activity, the temporal dynamic of the EEG signal should be taken into account with a dynamic feature and consequently with a dynamic classifier. In fact, by including also time evolution of the feature in the machine learning model, it is possible to infer when two different conditions maximally diverge, allowing possible interpretation of the timing of the cognitive processes and the behaviour of the underlying neural substrate.\n\nFinally, the main contribution of our results for the scientific community is that they provide a methodological advancement that is generally valid both for the investigation of emotion based on a machine learning approach with EEG signals and also for the investigation of preparatory brain activity.\n\n\nData availability\n\nFigshare: EEG anticipation of random high and low arousal faces and sounds. https://doi.org/10.6084/m9.figshare.6874871.v827\n\nThis project contains the following underlying data:\n\n- EEG metafile (DOCX)\n\n- EEG data related to the Passive, Active and Predictive conditions (CSV)\n\n- Video clips of the EEG activity before stimulus presentation (MPG)\n\nFigshare: EEG anticipation of random high and low arousal faces and sounds. https://doi.org/10.6084/m9.figshare.6874871.v827\n\nThis project contains the following extended data:\n\n- Detailed description of LDA, SVM and kNN machine learning algorithms (DOCX)\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/mbilucaglia/ML_BAA\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.366604551\n\nLicense: GPL-3.0", "appendix": "References\n\nFriston K: A theory of cortical responses. 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Nat Commun. 2019; 10(1): 5264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberts T, Cant JS, Nestor A: Elucidating the Neural Representation and the Processing Dynamics of Face Ensembles. J Neurosci. 2019; 39(39): 7737–7747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJain AK, Duin RP, Mao J: Statistical pattern recognition: A review. IEEE Trans Pattern Anal Mach Intell. 2000; 22(1): 4–37. Publisher Full Text\n\nTang J Alelyani S, Liu H: Feature selection for classification: A review. In Data Classification: Algorithms and Applications . Aggarwal, C.C.,Ed.; 2014; 37–64. Reference Source\n\nMiao J, Niu L: A Survey on Feature Selection. Procedia Comput Sci. 2016; 91: 919–926. Publisher Full Text\n\nMüller K, Krauledat M, Dornhege G, et al.: Machine learning techniques for brain-computer interfaces. Biomed Tech (Biomed Tech). 2004; 49: 11–22. Reference Source\n\nStevenson RA, James TW: Affective auditory stimuli: characterization of the International Affective Digitized Sounds (IADS) by discrete emotional categories. Behav Res Methods. 2008; 40(1): 315–21. PubMed Abstract \| Publisher Full Text\n\nStone JV: Independent component analysis: an introduction. Trends Cogn Sci. 2002; 6(2): 59–64. PubMed Abstract \| Publisher Full Text\n\nAllen JJ, Coan JA, Nazarian M: Issues and assumptions on the road from raw signals to metrics of frontal EEG asymmetry in emotion. Biol Psychol. 2004; 67(1–2): 183–218. PubMed Abstract \| Publisher Full Text\n\nBabiloni C, Stella G, Buffo P, et al.: Cortical sources of resting state EEG rhythms are abnormal in dyslexic children. Clin Neurophysiol. 2012; 123(12): 2384–2391. PubMed Abstract \| Publisher Full Text\n\nMert A, Akan A: Emotion recognition from EEG signals by using multivariate empirical mode decomposition. Pattern Anal Appl. 2018; 21: 81–89. Publisher Full Text\n\nClarke AR, Barry RJ, Karamacoska D, et al.: The EEG Theta/Beta Ratio: A marker of Arousal or Cognitive Processing Capacity? Appl Psychophysiol Biofeedback. 2019; 44(2): 123–129. PubMed Abstract \| Publisher Full Text\n\nBlankertz B, Lemm S, Treder M, et al.: Single-trial analysis and classification of ERP components - A tutorial. Neuroimage. 2011; 56(2): 814–825. PubMed Abstract \| Publisher Full Text\n\nMarco B: BAA - Matlab Code (Version 1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3666045\n\nTottenham N, Tanaka JW, Leon AC, et al.: The NimStim set of facial expressions: Judgments from untrained research participants. Psychiatry Res. 2009; 168(3): 242–249. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "92737", "date": "14 Sep 2021", "name": "Zahid Halim", "expertise": [ "Reviewer Expertise Machine learning" ], "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 presents an interesting approach using ML on EEG data for brain activity. Following issues need to be addressed.\nQuantify your results in the abstract.\n\nAdd a gap analysis section in your introduction and also mention the research questions.\n\nFig. 1 seems generic, please add steps specific to your solution. Just a suggestion.\n\nSummarize your key findings in a table in the discussion section.\n\nFollowing references are missing\nOn Identification of Driving-Induced Stress Using Electroencephalogram Signals: A Framework Based On Wearable Safety-Critical Scheme and Machine Learning A machine learning-based investigation utilizing the in-text features for the identification of dominant emotion in an email Imagined character recognition through EEG signals using deep convolutional neural network Investigating the use of pretrained convolutional neural network on cross-subject and cross-dataset EEG emotion recognition Cross-subject multimodal emotion recognition based on hybrid fusion A novel derivative-based classification method for hyperspectral data processing\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\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? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "7146", "date": "15 Sep 2021", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for your review. We will respond to your comments and suggestions after receiving at least a second review. Best wishes Patrizio" }, { "c_id": "7272", "date": "08 Oct 2021", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Quantify your results in the abstract. Reply: We added the accuracies of the 3 best classifier-feature combinations for both the static and dynamic classifiers in the abstract. Add a gap analysis section in your introduction and also mention the research questions. Reply: We split the past introduction into two different sections: introduction and state of the art. The introduction describes the theory of the emotion, the brain anticipatory activity, the physiological correlated of the emotions and introduce the emotion recognition as a ML approach. The state of the art summarizes previous works on the emotion recognition, underling their characteristics (in terms of chosen features/classifiers and obtained performances), as well as identify the limits (i.e. performances highly variable depending from the feature/classifier combination). Finally, it describes the aims of the present work. We hope now both the limits in the literature and the aim of the present study are clearer. Fig. 1 seems generic, please add steps specific to your solution. Just a suggestion. Reply: This paper re-analysed previously collected data, whose details can be found in the corresponding article (ref. 37). Fig. 1 graphically summarised the experimental task of (ref. 37). We agree that, probably, it is not such informative since interest readers can look at (ref. 37) for all the details. So, we removed it. Summarize your key findings in a table in the discussion section. Reply: We added the table 5 to summarise the best 3 classifier/features combinations for both the static and dynamic approaches. Following references are missing On Identification of Driving-Induced Stress Using Electroencephalogram Signals: A Framework Based on Wearable Safety-Critical Scheme and Machine Learning A machine learning-based investigation utilizing the in-text features for the identification of dominant emotion in an email Imagined character recognition through EEG signals using deep convolutional neural network Investigating the use of pretrained convolutional neural network on cross-subject and cross-dataset EEG emotion recognition Cross-subject multimodal emotion recognition based on hybrid fusion A novel derivative-based classification method for hyperspectral data processing Reply: We added the suggested references: (1), (2), (3), (5) and (6) in the introduction, while (4) in the discussion." } ] }, { "id": "92735", "date": "21 Sep 2021", "name": "Rajesh K Tripathy", "expertise": [ "Reviewer Expertise AI for healthcare", "deep learning", "Biomedical signal processing", "machine learning" ], "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, authors have applied machine learning to classify emotion‑related brain anticipatory activity using EEG signals. The paper has several flaws and it should be carefully revised before resubmission. My review comments are given as follows:\nWhy authors have considered linear discriminant analysis, support vector machine, and k-nearest neighbor classifiers for the classification of EEG signals? Why not RNNs and multilayer perceptron.\n\nCould you please add the arousal score? How you have divided into low-arousal and high-arousal. Why not three classes as low arousal vs. medium arousal vs. high arousal. Please clarify.\n\nThe cohen kappa score, Mathews correlation coefficients, F score values need to be evaluated.\n\nRecently, the rhythm-specific deep CNN is successful for the classification of emotions using EEG signals. Could you compare with CNN-based methods?\n\nThe advantages and disadvantages of your work must be written.\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": [ { "c_id": "7273", "date": "08 Oct 2021", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Why authors have considered linear discriminant analysis, support vector machine, and k-nearest neighbor classifiers for the classification of EEG signals? Why not RNNs and multilayer perceptron. Reply: According to the literature (ref. 19-20), LDA, SVM and kNN are the most commonly used (79.3% and 84% overall, respectively) classifiers in the Emotion recognition Field. A more recent review (ref. 53) partially confirmed this, showing that LDA, SVM and kNN are overall used 44.44% of the times. In contrast, neural‑network-based classifiers (e.g. CNN, MLP-BP, ANN) are used only 3.17% (ref. 19), 15.5% (ref. 20) and 5.56% (ref. 53) of the times. In addition to their popularity, LDA SVM and kNN were selected as representative of 3 different families, namely parametric, discriminative and non-parametric classifiers. Could you please add the arousal score? How you have divided into low-arousal and high-arousal. Why not three classes as low arousal vs. medium arousal vs. high arousal. Please clarify. Reply: Both the image and sounds were discretized into low and high arousal as those with a SAM score respectively lower and higher than 5. We chose a binary classification since it is associated to lower computational costs than the multiclass alternatives. Multiclass classification could produce different (and probably better) results, at the cost of lowering the number of available instances per class. We added this consideration in the study limitations. The cohen kappa score, Mathews correlation coefficients, F score values need to be evaluated. Reply: As evaluation metric, we chose the accuracy since the two classes were approximatively balanced (we reported in Table 1 the class distributions) and we were mainly interested in the overall correct classification (with equal weight on high and low arousal). We agree that additional metrics (that are mandatory for unbalanced dataset) would be very informative also for balanced datasets, providing, e.g., useful insights to which class is better classified. We added this consideration in the study limitations. Recently, the rhythm-specific deep CNN is successful for the classification of emotions using EEG signals. Could you compare with CNN-based methods? Reply: In the discussions we compared our results with two studies based on CNN classifiers and we identify that The advantages and disadvantages of your work must be written. Reply: In the discussions we added the paragraph “Study limitations”." } ] } ]	1	https://f1000research.com/articles/9-173
https://f1000research.com/articles/10-1039/v1	12 Oct 21	{ "type": "Study Protocol", "title": "Trajectories of somatic drug utilization patterns in Major Depressive Disorder: A Study protocol for a Danish nationwide register study using Latent Class Analysis", "authors": [ "Anne Marije Christina Overgaard Nielsen", "Kristoffer Jarlov Jensen", "Ramune Jacobsen", "Pernille Herold Jeberg", "Anna Birna Almarsdóttir", "Marie Kim Wium-Andersen", "Merete Osler", "Janne Petersen", "Kristoffer Jarlov Jensen", "Ramune Jacobsen", "Pernille Herold Jeberg", "Anna Birna Almarsdóttir", "Marie Kim Wium-Andersen", "Merete Osler", "Janne Petersen" ], "abstract": "Background: Major Depressive disorder (MDD) is a heterogeneous, multi-etiological disorder that is associated with chronic medical conditions and a high somatic treatment burden. A better understanding of the somatic diseases and treatment burden in MDD can be provided through a mapping of the somatic drug utilization patterns over time. The objective of this study is therefore to characterize the somatic drug profiles and their transitions over time (i.e. trajectories) among MDD patients.\n\nMethods: This descriptive study will be a nationwide register-based study including all Danish patients with an incident MDD diagnosis between 2011 and 2015. Using Latent Class Analysis, we will identify homogenous MDD patient subgroups according to somatic drug utilization (i.e. drug profiles). The development in somatic drug profiles will be depicted in four different time intervals from three years prior to the MDD diagnosis to three years after the diagnosis. Patients will be assigned to the latent class (drug profile) to which they have the highest probability of belonging using modal assignment. The treatment trajectories will be performed by cross tabulating these assignments. Discussion: Profiles and trajectories of somatic drug use will provide a new perspective on patterns of somatic drug burden in MDD patients. Moreover, identifying homogenous subgroups of MDD patients regarding somatic drug use can contribute to a deeper understanding of MDD etiology. In the future, this knowledge could help optimizing MDD treatment by studying if different antidepressants will show different efficacy and safety depending on the profiles and trajectories of somatic diseases.", "keywords": [ "Major depressive disorder", "prescription drugs", "drug utilization", "register study", "comorbidity", "latent class model" ], "content": "Introduction\n\nMajor Depressive disorder (MDD) is a heterogeneous, multi-etiological disorder that is considered a major global health burden with approximately 264 million people affected worldwide1 [WHO - depression]. The disorder is displayed with varying symptomology and treatment efficacy, and the full understanding of the pathophysiology of MDD remains incomplete. It is well-established that the prevalence of MDD is substantially increased in patients with somatic disorders including diabetes,2,3 cancer,4,5 cardiovascular diseases,6 chronic pain,7 migraine,8 and sleep disorders.9 Moreover, the co-occurrence of MDD and somatic diseases is associated with worsened prognostic outcomes.10 A high prevalence of somatic diseases in MDD patients indicates a high disease burden as well as a complex MDD etiology. Previous studies have mainly focused on one drug at a time, or one disease at a time. Investigating drugs or diseases related to MDD individually may be too simplistic an approach. Using Latent Class Analysis, we will map the overall use of drugs for somatic diseases in MDD patients which will reveal homogenous MDD patient subgroups with different somatic drug profiles. These homogenous patient subgroups can potentially provide an understanding of unique MDD etiology that requires unique treatment strategies and further, some treatment groups might have a higher risk of drug interactions.\n\nThe aim of our research is therefore to provide a full picture of the somatic pharmacological treatment burden in MDD patients. In this study, we will first characterize somatic drug profiles from three years prior to three years past index diagnosis of MDD, and secondly, depict the trajectories of patients from one drug profile to another over time. Finally, the drug profiles and trajectories will be characterized by age, gender, education, and MDD severity.\n\n\nMethods\n\nThe study will be a register-based drug utilization study assessing somatic pharmacological treatment profiles and their trajectories, or transitions over time, in patients with a first-time MDD diagnosis. The MDD patients are diagnosed at time 0 (index date) between 2011 and 2015 (the study inclusion period) and followed over the patient observation period defined as three years prior to the index MDD diagnosis until three years after the diagnosis (Figure 1A). Using Latent Class Analysis (LCA) the somatic drug profiles for the MDD patients will be determined at four seven-month intervals defined by the following time points: three years before the index date, index date, seven months after the index date, and three years after the index date (Figure 1B). The trajectories, i.e. drug profile changes throughout the four periods, will then be determined and characterized.\n\nInclusion criteria\n\nTo be eligible for the study, individuals must have met three inclusion criteria: 1) Patients have received an MDD diagnosis at a psychiatric unit between 1st of January 2011 till 31st of December 2015. 2) The MDD diagnosis must be a first-time (index) diagnosis. The diagnosis is defined as first-time if the patients do not have a history of MDD 15 years prior to the (index) diagnosis. 3) The MDD diagnosis must be the primary/action diagnosis (“A-diagnosis”).\n\nThe information regarding the MDD diagnosis, date of diagnosis, and diagnosis type is available through the Danish National Patient Register (DNPR).11 The International Classification of Diseases, Tenth Revision (ICD-10) will be used to identify the MDD diagnosis including either a single episode of depression (ICD-10 code F32) or recurrent depressive episode (ICD-10 code F33).\n\nExclusion criteria\n\nThe following individuals will be excluded from the study (Figure 2): 1) Patients with hospital contact up to 15 years prior to the first-time diagnosis with MDD due to the following A-diagnosis disorders: Manic episodes (F30), Bipolar affective disorder (F31), Schizophrenia (F20-F29), and Dementia (F00-F03), 2) Individuals with no available data on gender or date of birth from the Danish Civil Registration System (CRS)12 at any time during the study inclusion period from 20011-2015, 3) Patients that have migrated to or from Denmark within the period of 15 years prior to the first-time MDD diagnosis, and 4) MDD patients under the age of 10 at the index date.\n\nSample size calculation\n\nTo make the outcome significant, it is suggested to include at least 300 individuals in a Latent Class Analysis (LCA).13 We expect the number of included individuals in our study to exceed 300 considering an inclusion period of 5 years and a high prevalence of MDD.\n\nThe prescription of pharmacological treatment for somatic disorders will be available through the Danish National Prescription Register (NPR).14 The given somatic drug type is characterized by the Anatomical Therapeutic Chemical code (ATC code). All ATC codes except N05 and N06 (psychotropic drugs) will be defined as somatic drugs. For the purpose of not getting an excessive amount of observations, the drug types used in this study will be at the chemical subgroup level of the drugs (4th level). A prescription drug will be considered as a taken drug if a patient has redeemed the drug at the community pharmacy at least twice during any of the 7-month intervals. The chemical subgroup drug types for somatic diseases are chosen based on the frequency of prescription: For each of the four intervals, prescription drugs redeemed by at least 0.5% of the study population will be considered; the combination of these ATC codes across all intervals will comprise the final drug portfolio to be submitted to the LCA model (Figure 3).\n\nThe covariates used for this study are gender (male/female), age at index date (<18, 18-34, 35-64, and 65+), level of MDD severity, and education level. Gender and age are retrieved from the CRS. MDD symptom severity is categorized either as mild depression (F32.0, F33.0), moderate depression (F32.1, F33.1), severe depression (F32.2, F32.3, F33.2, F33.3), or unspecified (F32.4, F32.5, F33.4, F32.8, F32.9, F33.8, F33.9). This information is available through the DNPR. The educational level at index date will be available through the Danish Education Register (DER)15 and will be divided into four categories: ‘Primary lower secondary’, ‘Upper secondary’, ‘Bachelor, Masters, Doctoral’, or ‘Missing or not classified’.\n\nThis study is approved by the Regional Data Authorities (ref. P-2020-88). A Danish research study based on register data does not need ethical approval from the Danish Council on Ethics when the study does not involve biological material [The Danish National Committee On Health Research Ethics]. The data used for this study is obtained through Statistics Denmark (DST). All data is anonymized and solely aggregated data will be reported [DST Data for Research].\n\n\nStatistics\n\nAll statistical analyses will be performed using SAS Enterprise Guide 7.1 (Statistical Analysis System, RRID:SCR_008567). Alternatively, R (R Project for Statistical Computing, RRID:SCR_001905) could be used as an open-source software.\n\nLatent Class Analyses (LCA) are used to identify unobservable subgroups (classes or clusters) within a population. These unobservable subgroups, called latent classes, have similar patterns of their shared set of observable variables from a data set. In this study, LCA will be performed utilizing the drugs that the MDD patients have redeemed at the pharmacy as indicator variables. The variables will be dichotomized into two categories: prescription drugs redeemed twice during an interval (1) and redeemed once or less (0). The latent variable will represent the drug profiles for somatic diseases in patients with MDD (Figure 4A).\n\nFirst, the study population will be divided randomly into two equal groups: a development cohort and a validation cohort. For the four respective time intervals, the LCA model will be fitted with a different number of classes for the development and validation cohort. This will be done to confirm that the best model fit for the development cohort corresponds to the best model fit for the validation cohort. The optimum number of latent classes will be determined by the Bayesian Information Criterion (BIC),16,17 together with a clinical judgment of a psychiatrist. A decrease in BIC will indicate a better model fit, and the model that will yield the lowest BIC will be chosen.18–20 In a case where the model fit for the development- and validation cohorts lead to a different number of latent classes, the clinical judgment will define the final number of classes.\n\nAfter fitting the LCA model, the MDD patients will be assigned to a latent class (i.e. drug profile) where they have the highest probability of belonging compared to other classes (modal assignment). Similar analyses will be performed for each time interval. Each latent class/drug profile will have a unique pattern of redeemed prescription drugs based on the estimated item-response probabilities, i.e. the probabilities of receiving the chemical subgroup drugs given a particular class membership. All profiles will be characterized with the covariables gender, age, education level and MDD severity.\n\nThe treatment trajectories are the transitions in latent class membership (drug profiles) over time (Figure 4B). Through modal assignment, all patients will be assigned to a class at each time interval. The trajectories will be identified by cross tabulating these assignments in each time interval. Thereby, the frequencies of each treatment trajectory will be obtained and thereafter, covariables will be associated with each trajectory. The trajectories will be visualized through a Sankey Diagram.\n\nBased on this study and the obtained data we will generate a research article which will submitted for publication to a peer-reviewed journal.\n\n\nDiscussion\n\nDrug use in MDD with chronic comorbidities is exceedingly complex, and it is expected that MDD patients have a high pharmacological treatment burden. We have not been able to identify studies that have investigated this treatment burden through the characterization of multiple drug profiles. Drug use in a population is multiplicative, and it is therefore hoped that this study will provide new insights into MDD, comorbidities, treatment burden and patterns through the characterization of homogeneous MDD subgroups in respect to somatic drug use.\n\nSome limitations to the study have been identified. Our first issue, which is to only include patients with MDD as a primary diagnosis, can be considered both an advantage and a limitation. By exclusively involving patients with the primary diagnosis, we will get a more homogeneous group. Moreover, the primary diagnosis is usually given by a psychiatric specialist at the hospital, which is rarely the case with a secondary diagnosis. However, MDD can be given as a secondary diagnosis to patients after a chronic somatic disease, and it is, therefore, plausible that these patients have somatic drug profiles that might be overlooked in our study. Some important observations might be overlooked because the patient has been given a primary diagnosis of a chronic disease when going to the hospital and have been given MDD as a secondary diagnosis. Our second limitation is that the study solely includes prescription drugs redeemed at the community pharmacy. By using the Danish National Prescription Registry, it is not possible to retrieve over-the-counter drugs such as mild analgesics. The medication received at the hospital cannot be retrieved either. Therefore, the drug profiles may be more complex in reality than what will be obtained from the prescription drugs in this study. The third limitation is the neglect of drugs only redeemed once. It was decided that a prescription drug must be redeemed twice during a time interval of seven months. In Denmark, prescription drugs are typically redeemed in three-month supplies, and the intervals in our study are more than twice this duration. This design was chosen to ensure compliance in our population. However, by our approach, some somatic drugs that will only be redeemed once are not included, e.g. antibiotics treatment courses contained in a single package. Our fourth limitation is that we will include the most frequent drug types which can lead to a neglect of drugs for rare diseases. To minimize the running time of SAS, it was decided to not include the most rarely prescribed somatic drugs during each interval (drugs with a frequency less than 0.5%). This could potentially lead to a neglect of somatic drugs for rare chronic diseases, since they will not be as frequently prescribed, however, they could be equally valuable to analyze. These limitations will be considered when interpreting the results. Moreover, while interpreting the results we will consider that some prescription drugs can be given for several different indications (e.g. corticosteroids).\n\nThrough the identification of homogeneous MDD subgroups according to drug use, a better understanding of the somatic diseases and treatment burden in MDD can be provided. Identifying drug profiles in MDD will represent disease patterns which in turn can represent unique MDD etiology. In the future, it can therefore be investigated if the patient subgroups with unique somatic drug patterns will respond differently to various types of antidepressants. Moreover, specific patient subgroups and trajectories can be associated with treatment outcomes and thus become a novel way to identify long-term treatment strategies.\n\n\nData availability\n\nAll data sources used in this study are Danish registers that are linked to the unique personal identification number (CPR number) of the patients. The registers have been made available for us by Statistics Denmark (DST) [DST Registers in Statistics Denmark].\n\n\nReporting guidelines\n\nThe study will be conducted by following the Strengthening of Reporting of Observational Studies in Epidemiology (STROBE) guidelines [STROBE Statement].\n\n\nAuthor contributions\n\nConceptualization: All authors\n\nMethodology: Anne Marije Christina Overgaard Nielsen, Janne Petersen, Kristoffer Jarlov Jensen, Pernille Herold Jeberg\n\nSupervision: Kristoffer Jarlov Jensen, Janne Petersen, Ramune Jacobsen\n\nVisualization: Anne Marije Christina Overgaard Nielsen\n\nWriting – Original Draft Preparation: Anne Marije Christina Overgaard Nielsen\n\nWriting – Review & Editing: All authors", "appendix": "References\n\nKim Y-K: Major Depressive Disorder: Current Advances and Paradigm Shifts. Psychiatry Investig. 2020; 17(3): 179–180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerge LI, Riise T: Comorbidity between Type 2 Diabetes and Depression in the Adult Population: Directions of the Association and Its Possible Pathophysiological Mechanisms. Int. J. Endocrinol. 2015; 2015: 1–7. Publisher Full Text\n\nHolt RIG, de Groot M, Lucki I, et al.: NIDDK International Conference Report on Diabetes and Depression: Current Understanding and Future Directions. Diabetes Care. 2014; 37(8): 2067–2077. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrebber AMH, Buffart LM, Kleijn G, et al.: Prevalence of depression in cancer patients: a meta-analysis of diagnostic interviews and self-report instruments. Psychooncology. 2014; 23(2): 121–130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung K, Singh G: Biological Mechanisms of Cancer-Induced Depression. Front. Psych. 2018; 9. Publisher Full Text\n\nDhar AK, Barton DA: Depression and the Link with Cardiovascular Disease. Front. Psych. 2016; 7. Publisher Full Text\n\nSheng J, Liu S, Wang Y, et al.: The Link between Depression and Chronic Pain: Neural Mechanisms in the Brain. Neural Plast. 2017; 2017: 1–10. Publisher Full Text\n\nZhang Q, Shao A, Jiang Z, Tsai H, et al.: The exploration of mechanisms of comorbidity between migraine and depression. J. Cell. Mol. Med. 2019; 23(7): 4505–4513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartín-Merino E, Ruigómez A, Johansson S, et al.: Study of a Cohort of Patients Newly Diagnosed With Depression in General Practice: Prevalence, Incidence, Comorbidity, and Treatment Patterns. Prim Care Companion J Clin Psychiatry . 2010; 12(1): Publisher Full Text\n\nMoussavi S, Chatterji S, Verdes E, et al.: Depression, chronic diseases, and decrements in health: results from the World Health Surveys. Lancet. 2007; 370(9590): 851–858. Publisher Full Text\n\nSchmidt M, Schmidt SAJ, Sandegaard JL, et al.: The Danish National Patient Registry: a review of content, data quality, and research potential. CLEP. 2015; 7: 449–490. Publisher Full Text\n\nSchmidt M, Pedersen L, Sørensen HT: The Danish Civil Registration System as a tool in epidemiology. Eur. J. Epidemiol. 2014; 29(8): 541–549. PubMed Abstract \| Publisher Full Text\n\nWeller BE, Bowen NK, Faubert SJ: Latent Class Analysis: A Guide to Best Practice. J. Black Psychol. 2020; 46(4): 287–311. Publisher Full Text\n\nPottegård A, Schmidt SAJ, Wallach-Kildemoes H, Sørensen HT, et al.: Data Resource Profile: The Danish National Prescription Registry. Int. J. Epidemiol. 2017; 46(3): 798–798–798. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen VM, Rasmussen AW: Danish education registers. Scand. J. Public Health. 2011; 39(7_suppl): 91–94. PubMed Abstract \| Publisher Full Text\n\nDean N, Raftery AE: Latent Class Analysis Variable Selection. Ann. Inst. Stat. Math. 2010; 62(1): 11–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwarz G: Estimating the Dimension of a Model. Ann. Stat. 1978; 6(2): 461–464. Publisher Full Text\n\nKongsted A, Nielsen AM: Latent Class Analysis in health research. J. Physiother. 2017; 63(1): 55–58. PubMed Abstract \| Publisher Full Text\n\nPetersen KJ, Qualter P, Humphrey N: The Application of Latent Class Analysis for Investigating Population Child Mental Health: A Systematic Review. Front. Psychol. 2019; 10. Publisher Full Text\n\nSchreiber JB: Latent Class Analysis: An example for reporting results. Res. Soc. Adm. Pharm. 2017; 13(6): 1196–1201. PubMed Abstract \| Publisher Full Text" }	[ { "id": "96754", "date": "18 Nov 2021", "name": "Verónica Vitriol", "expertise": [ "Reviewer Expertise Dr Veronica Vitriol G is a Clinical psychiatrist and Magister in Psychology PHD Maria de la Luz Aylwin is Bachelor of Science", "mention in Biology Doctorate in Physiology." ], "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 trial, the authors propose to characterize the pattern of use of drugs for somatic disorders in patients with primary diagnoses of Major Depressive Disorder (MDD). They will characterise which pattern of somatic drugs and the changes in the trajectories of this pattern in a cohort of patients with first diagnosis of MDD using the Danish data of the use of somatic drugs and the MDD diagnosis by a psychiatrist. They will study the patients in 4 periods of 7 months, 3 years previous to the MDD diagnosis, 7 months previous to the diagnosis, at the time of diagnosis, and 3 years post diagnosis.\nThey will use Latent Class Analysis (LCA) to classify patients in groups the pattern of drug use at each period (7 months) and then characterize the trajectory of these LCA groups.\nI - The protocol rationale and objectives are described, however, it will improve the comprehension if:\n1) The LCA groups at the 4 time periods and the trajectories of these groups are defined in the abstract, and\n2) In the introduction, background information is provided regarding the prevalence of depression in the Danish population. Furthermore, please\nProvide a reference for the phrase “depression is heterogeneous and multi etiological disorder ()” and the next sentence “The disorder is displayed with varying symptomatology and treatment efficacy ()”.\n\nExplain further the sentence “somatic diseases in MDD patients indicates a high disease burden as well as a complex MDD etiology”. Why does somatic disease in MDD account for a complex etiology? Please be more explicit and provide a reference.\n\nThis protocol is a descriptive work, so it is clearly supposed to contribute towards understanding the different MDD clinical subtypes. However, the report could explain better why this work contributes to the understanding of the etiology of the different MDD types.\nII - The study design is appropriate for the research question:\n1) However, it is not coherent across all the manuscript the relationship between MDD and somatic drug use (as an indirect indication of a comorbid somatic disorder). A secondary MDD diagnosis after the diagnosis of a somatic disorder is clearly different that a primary MDD with a later use of somatic drugs. It is suggested that the authors explain across all the manuscript that the MDD diagnosis is the first psychiatrist diagnosis of the national data-base.\n2) The inclusion and exclusion criteria are well described\n3) Regarding the sample size, more information is needed about the universe and how it was calculated that 300 patients were needed.\n\n4) It is suggested to describe what is a “chemical subgroup level of the drugs (4th level)”, to facilitate its eventual replication\nThis work is a trial protocol so the datasets are not presented. The ethical considerations are described and the limitations are well described. However, it is suggested to explain how this work could contribute to improve the understanding of the etiology of depression by being descriptive. Furthermore, access to data through the Danish database is also suggested to include among the strengths of this protocol.", "responses": [] }, { "id": "246453", "date": "14 May 2024", "name": "Joëlle A Pasman", "expertise": [ "Reviewer Expertise psychiatric genetics", "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\nThe authors present a study protocol for investigating somatic drug use trajectories in MDD patients in the Danish population. They propose a latent class analysis to identify sub groups of patients based on their somatic drug use, and will assess demographic characteristics of the patients in each group. In addition, they will investigate transition trajectories from one group to another, and characterize these trajectories in the same manner. The proposed study sounds interesting and important, and the authors have selected appropriate methodology. I do have some questions and suggestions for improvement.\nWill this study rely on specialist care data only to identify MDD patients? This is suggested by the authors speaking of psychiatrist or hospital diagnoses. The prescription register presumably does include drugs prescribed in specialist or primary care? The implications of not including MDD patients from primary care should be discussed.\nThe authors need to explain better why they perform the specific analyses they propose and what specific questions they try to answer. The rationale is now very general and descriptive, while I think that the authors have more specific aims in mind. For instance, is the question about co-morbidity between somatic disease and MDD? But there are no base rates to compare to, as only patients are included. Also, looking at drug use to investigate comorbidity seems a bit roundabout if diagnostic information is available. If the purpose is to simply describe different drug use groups, the authors might want to think more on how to explain why this is interesting. They may want to give some examples for what kind of groups they expect to find (e.g., a low-drug use group, a highly-stable diabetes drug use group, a transient beta-blocker use group), and explain why it would be relevant to identify if individuals fall in this group (such as predicting transitions, development of disease).\n\nSome questions that the authors may want to consider:\nDo the authors have contingency plans for when they e.g. find an unwieldy amount of different classes, or none that fit the data well? Would they then e.g. increase the threshold for prevalence of prescription?\n\nWhy do the authors exclude patients with psychotic symptoms and dementia? If the aim is to capture idiosyncratic MDD, I think the choice to exclude dementia then is a bit random (e.g. why not other somatic 'causes' of MDD, such as stroke or epilepsy?). Also, dropping individuals with a psychotic may give a clearer but less realistic picture of patterns of somatic comorbidity. The problem with such exclusion criteria is that you have to explain well what you're aiming to do and why, and it's difficult to determine where to stop.\n\nCan any individual be a member of multiple classes?\n\nCan the authors elaborate on why clinical judgment is involved in selecting the optimal number of classes?\n\nFinally, the language in the paper may need editing. Formulations are often a bit awkward. As an example, what do the authors mean here: \"To make the outcome significant, it is suggested to include at least 300 individuals in a Latent Class Analysis (LCA).\"? The paper may benefit from input from a native speaker.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-1039
https://f1000research.com/articles/10-1032/v1	11 Oct 21	{ "type": "Method Article", "title": "Quantifying endothelial cell proliferation in the zebrafish embryo", "authors": [ "George Bowley", "Timothy JA Chico", "Jovana Serbanovic-Canic", "Paul C Evans", "Timothy JA Chico", "Jovana Serbanovic-Canic" ], "abstract": "Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.", "keywords": [ "Zebrafish", "Endothelial Cell", "Proliferation", "Microscopy" ], "content": "\n\n\n\nScientific benefit(s)\n\n• Endothelial cell (EC) proliferation can be visualised at the single cell level in live embryos.\n\n3Rs benefit(s)\n\n• Zebrafish embryos can be used to partially replace mouse models in early cardiovascular disease research.\n\n• This time-lapse zebrafish embryo proliferation assay is a non-terminal refinement ofimmunolabelling and cell labelling approaches, which require tissue fixation.\n\nPractical benefit(s)\n\n• Only one tank of adult zebrafish are required to produce transgenic embryos for this method (20-40).\n\n• Fewer than 30 embryos are required for time-lapse experiments, thus fewer adult pairs are required which reduces breeding stress.\n\n• Use of transgenic zebrafish lines allows for proliferating cells to be visualised in real-time without use of embryo fixation, compared to immunolabelling and cell labelling approaches which require embryo fixation.\n\n• The zebrafish EC proliferation assay allows simultaneous quantification of proliferation in up to 24 embryos. Assuming four embryos are used as controls, this makes it possible to compare four treatment groups where each group has n = 5 animals. A comparable experiment using 24 mice would require each mouse to be culled, which incurs significant additional cost and effort, and may be difficult to justify, particularly for screening experiments where the potential benefits of the research are unknown.\n\nCurrent applications\n\n• Screening for genes involved in EC proliferation in response to blood flow.\n\nPotential applications\n\n• As a screening assay to identify new drugs/agents that prevent pro-atherogenic changes in endothelial cell proliferation in response to flow.\n\n• Screening for genes involved in migration and apoptosis in response to flow by observing cell movement throughout the time-lapse, effects on apoptosis could be screened by counting the number of ECs undergoing apoptosis either by looking for blebbing of EC nuclei, or using a different transgenic.\n\n\nIntroduction\n\nEndothelial cell (EC) proliferation is vital for vascular development and homeostasis, but is also associated with increased vascular permeability which is a key driver of atherosclerosis.1 Cell culture and mouse models are widely used to study EC proliferation in atherosclerosis yet use of these models involves a trade-off between biological relevance, maintenance cost, and technical complexity depending on the model chosen. This paper explores the efficacy of immunostaining, molecular labelling, and transgenic approaches to study EC proliferation in zebrafish embryos.\n\nLike most forms of human disease research, cardiovascular disease (CVD) studies conventionally identify genes for in-depth research by first applying wider screening approaches. The models used in these types of research have several advantages and disadvantages. In vitro models can be used for high throughput analysis of the genes and pathways that control EC proliferation, but do not model the interaction of different tissues and are therefore reductionist. Conversely, rodents and other mammals have provided physiologically relevant models, such as the ApoE mutant mouse line which is the de facto model of plaque formation in CVD research. Whilst mice are an excellent model for study of genes involved in atherosclerosis (indeed, based on the number of mouse vascular mechanics papers on PubMed, 30,000 mice were used for this research in the past decade), they are unsuitable for screening multiple genes at once as generating mutants is costly, time consuming and may be difficult to justify due to concerns about the effect of mutations on animal welfare and the number of animals that would be required in such a study. Zebrafish embryos survive by oxygen and nutrient diffusion, thus mutations which are embryo lethal in mammals such as abolished development of ECs (cloche) or targeted knock-down of cardiac troponin (sih) are not lethal in zebrafish embryos. These characteristics mean zebrafish embryos are uniquely amenable to study of genes involved in ECs, and EC responses to flow.\n\nWhilst zebrafish embryos are not protected prior to 5 days post fertilization (dpf) in the UK, they are still live organisms and therefore present a partial replacement to use of other animals in CVD research. Nonetheless, the advantages of using zebrafish over in vitro models are multiple. One pair of adult zebrafish can produce hundreds of embryos in one day, and with just an incubator and E3 medium these embryos will each develop a complete cardiovascular system in two days,2 with zero human intervention. These characteristics give studies on zebrafish embryos simplicity comparable with that of two-dimensional in vitro models, far greater simplicity than comparable three-dimensional in vitro models, and comparable biological relevance to mammalian in vivo models but with reduced cost and reduced detriment to animal welfare.\n\nThe zebrafish embryo is ideal for studying vascular biology, it is small, optically transparent, and allows manipulation of gene expression using morpholino oligonucleotides3 (MOs), CRISPR-Cas94 and CRISPR-Interference.5 These characteristics allow rapid generation of genetically modified embryos and quantitative analysis of EC behaviour. With the exception of CRISPR-Cas9, these genetic approaches and quantitative methods are not viable for CVD research in mice. Whilst CRISPR-Cas9 is effective in mice, generation of a range of mutants for screening purposes remains infeasible for reasons described above.\n\nZebrafish have been used to model EC apoptosis and migration in response to blood flow.6–8 Whilst these events can be reliably observed using existing methods, EC proliferation is usually estimated by counting the total number of ECs. However, the number of ECs is determined by a complex interaction of multiple factors, including apoptosis, cell migration and differentiation/dedifferentiation, and therefore cell number is not a reliable metric for studying proliferation. It is therefore important to identify a reliable and direct assay of EC proliferation in zebrafish. Here we describe our attempts to directly quantify EC proliferation in zebrafish embryos using a combination of techniques currently used for in vivo and in vitro models; immunohistochemistry (a technique widely used to study protein expression in mouse endothelium using the en face technique), DNA labelling (used in cell culture and mice) and time-lapse imaging, to determine which method detected the greatest number of proliferating ECs. Indirect approaches for studying proliferation such as cell number counting, viability, and metabolic activity assays were not pursued as their results can be influenced by migration and apoptosis.\n\n\nMethods\n\nZebrafish care and experimental procedures were carried out under Project Licence 70/8588 issued by the UK Home Office and local ethical committee approval was obtained. Adult zebrafish were kept at a constant temperature of 28 ± 1°C and at pH 7.5 ± 0.5. They were subjected to a light/dark cycle of 14 hours of light and 10 hours of darkness. Adult zebrafish were fed a diet of artemia and dry Zebrafeed™. For mating, pairs of male and female zebrafish were placed into mating tanks and separated by a divider to allow accurate timing of fertilisation. For experiments where this was not required, a box for embryo collection with a mesh insert and marbles on top was placed in the fish tank to harvest progeny. The Nacre line9 and transgenic (Tg) (fli1a:nls-mCherry), (fli1a:LifeAct-mClover),10 and (fli1:EGFP) lines were used.11,12 Nacre zebrafish lack melanocytes and are thus more transparent than conventional wildtype lines, this makes Nacre highly suitable for light microscopy. Both transgenic lines use Nacre as background. Tg fli1a:nls-mCherry expresses a red fluorescent protein linked to a nuclear localisation sequence under the endothelial promoter, resulting in red fluorescence in EC nuclei. Tg fli1a:LifeAct-mClover expresses a f-actin:mClover fusion protein under the endothelial promoter, resulting in visualisation of the EC actin cytoskeleton. Initially, we intended to compare EC proliferation in embryos with and without blood flow, thus for early experiments (whole mount immunostaining) heart contraction was inhibited by silencing troponin T2A by injection of tnnt2a ATG morpholino at 3 ng final dose (sequence 5′-CATGTTTGCTCTGATCTGACACGCA-3′). As EC proliferation was not detected at this experimental stage, flow and no-flow comparison was stopped to minimise our use of embryos and only embryos with blood flow were used for subsequent experiments.\n\nAdults: For all experiments described, embryos were produced by pair mating eight male and eight female adults of the same transgenic line. This was done to minimize the chance that zero pairs produced embryos, and to minimise confounders by limiting the genetic diversity of the embryos. Adults do not suffer pain or distress from pair mating and were returned to their home tanks immediately after producing embryos, home tanks contain environmental enrichments such as fake jellyfish and fake seaweed. No adults suffered adverse events as a consequence of this work.\n\nEmbryos: No embryos used in this study exceeded 5.2 dpf. For all experiments described, embryo collection stopped after 60 embryos were obtained. Sex of embryos was not determined (as is standard practice in zebrafish research). For PCNA and EdU experiments, embryos were treated then screened for the endothelial marker (Tg fli1:EGFP for PCNA, and Tg fli1a:nls-mCherry for EdU) then three embryos were randomly selected for imaging. For time-lapse imaging experiments, embryos were screened for Tg flia:nls-mCherry and Tg fli1a:LifeAct-mClover, then three embryos were randomly selected for imaging. Embryos not selected for imaging were humanely destroyed using bleach.\n\nTg (fli1:EGFP) embryos were fixed in 4% paraformaldehyde (PFA) at 30 hpf to allow screening for proliferation in the main trunk vascular beds, then whole mount immunostaining was performed using anti-PCNA antibody (GeneTex, RRID: AB_11161916. Diluted 1:50) and AlexaFluor-568 mouse anti rabbit secondary antibody (ThermoFisher, RRID: AB_143165. Diluted 1:200). Colocalization of PCNA and fli1-EGFP was used to define proliferating ECs. Embryos not treated with PCNA antibody were imaged during protocol optimization to ensure the specificity of PCNA antibody. Detection of cell proliferation was defined as where a nucleus is both positive for PCNA and for fli1-EGFP.\n\nThe Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher) was used to label DNA of proliferating cells with AlexaFluor-488. The protocol was modified for zebrafish as follows:\n\nZebrafish larvae with Tg (fli1a:nls-mCherry) were collected at 30 hpf and incubated in EdU solution (EdU 500 uM, 15% DMSO, 85% E3) at 28°C for 1 h. Larvae were fixed in 4% PFA overnight then washed and transferred to permeabilization solution (supplied in kit) for 1 h. Permeabilised larvae were then treated according to manufacturer protocol. Proliferating ECs were defined as cells positive for fli1a:nls-mCherry and EdU.\n\nStandard light microscopy was used to screen out dead or undeveloped embryos, and to identify tnnt2a morphant embryos that lack a heartbeat. The morphant phenotype (no heartbeat) was observed in all (100%) of injected embryos, survival is indistinguishable from wildtype up to the time point tested (30 hpf).\n\nFluorescent microscopy was used to identify and select embryos containing fli1a:nls-mCherry or fli1:EGFP after they were non-terminally anaesthetised using E3 medium. Fluorescence screening was done using a ZEISS Axio Zoom V16 at 28 hpf. Zeiss filter set 63 (HE mRFP, cat no 489063-0000-000) was used. Camera mode was used and pixel binning set to 5 × 5 to maximise sensitivity to the fluorophore. Screened embryos were washed in E3 media without tricaine, then transferred into fresh E3 prior to further imaging.\n\nFor confocal imaging, embryos were non-terminally anaesthetised using tricaine and mounted in 1–2% agarose over which standard E3 medium was placed. Still images were taken using a Nikon A1 confocal microscope at 1024 × 1024 resolution (0.62 μm per pixel), z-stacks were taken in 8-μm increments. Time lapses were taken using a Zeiss LSM880 in Airyscan mode. Images were captured at 1848 × 1848 resolution (0.47 μm per pixel), z-stacks were taken in 3.43-μm increments. Images were analysed using NIS Elements or ZEN respectively. Composite images were generated by performing maximum intensity projection of the stack data, no other processing was performed. For both microscopes, excitation was done by a 488 nm and 568 nm laser.\n\nFor light sheet microscopy,8,10 non-terminally anaesthetised embryos were immobilised using 1% agarose and mounted in a glass capillary. Embryos were suspended in melted agarose (37°C, 0.8–1.2% w/v) then drawn up into a glass capillary using a plunger. Agarose suspended embryos were then pushed through the capillary and suspended into the imaging chamber containing E3 and tricaine. Images were captured at 1920 × 1920 resolution (0.23 μm per pixel), z-stacks were taken in 1-μm increments. Images were captured every 15 minutes for 4 h using ZEISS Z1 light sheet microscope and processed using ZEN software. Post-acquisition, maximum intensity projection was used to produce a single image for each time point. EC proliferation was defined as where fli1a:nls-mCherry nuclei visibly divide over the course of the time lapse. Experimental embryos were then euthanised using bleach.\n\nThe number of proliferation events detected using EdU labelling, time-lapse confocal microscopy and PCNA immunolabelling were compared using a two-way ANOVA (n = 3 embryos per each group). Each animal was treated as a single unit of analysis. Analysis was done using GraphPad PRISM (RRID:SCR_002798), an open-source alternative to this is JASP (RRID:SCR_015823).\n\n\nProtocols\n\nE3 medium: 5 mM NaCl, 0.17 mM KCL, 0.33 mM MgSO4 and 0.33 mM CaCl2 in dH2O. 4% PFA: paraformaldehyde diluted to 4% with E3 (One month shelf life when stored at 4°C). tnnt2a MO: diluted to a 100 μM stock with dH2O. Stock concentration should be determined using a Nanodrop and diluted to 3 ng/nL for microinjection. PBS: phosphate-buffered saline (ThermoFisher) diluted from 10× stock. PBST: phosphate-buffered saline with Tween-20. PBS made to 0.1% Tween-20. PDT: phosphate-buffered saline with 1% DMSO and 0.1% Triton X-100. TRIS buffer: tris base diluted in dH2O to 1 M stock. 150 mM solution was prepared from stock and pH adjusted to 8.0. Tricaine: 4 g tricaine methanesulfonate in 1 L dH2O pH 7–7.5. Blocking solution: 1% bovine serum albumin in PBS. Goat serum: goat serum stock (frozen) diluted to 10% in blocking solution. Bovine serum albumin (BSA): stock albumin powder should be added to dH20 to make 10% (w/v) BSA solution, this solution should be aliquoted into 1.5-mL Eppendorf tubes and frozen for use as needed. Click-IT EdU AlexaFluor-488 Flow Cytometry Assay Kit – ThermoFisher C10420.\n\n\n\n1) Collect embryos and transfer to a petri dish of E3 media.\n\n2) Incubate embryos at 28°C until at least 24 hpf.\n\n3) Insert forceps to gently pierce the embryo chorion (Sup. 1 – 1).\n\n4) Gently open forceps to tear the chorion (Sup. 1 – 2).\n\n5) Open forceps until embryo is free of the chorion (Sup. 1 – 3).\n\n\n\n1) Collect fli1-EGFP embryos and transfer to a petri dish of E3 media then incubate at 28°C to 30 hpf.\n\n2) Dechorionate embryos.\n\n3) Fix in 4% PFA overnight at 4°C – now protect samples from light as far as possible until imaging.\n\n4) Wash in room temperature PBST (Phosphate buffered saline with 0.1% Tween-20) on a rocker for three 10-min washes.\n\n5) Wash in room temperature TRIS buffer (150 mM pH 8.0) on a rocker for 5 min.\n\n6) Equilibrate embryos in TRIS buffer at 70°C for 15 min.\n\n7) Wash in room temperature PBST on a rocker for two 5-min washes.\n\n8) Rinse with distilled water on ice.\n\n9) Wash in chilled acetone (−20°C) for 15 min.\n\n10) Rinse with distilled water for three 5-min washes.\n\n11) Block with goat serum (10%), BSA (2%), and PBT (phosphate buffered saline with 0.1% Triton X-100) for 4 h at 4°C.\n\n12) Incubate with primary antibody overnight at 4°C (Dilute PCNA 1:250 in blocking solution).\n\n13) Wash in PBT for four 30-min washes.\n\n14) Incubate with secondary antibody (AF-568 anti-rabbit diluted 1:500 in blocking solution).\n\n15) Wash in room temperature PBST five times for 5 min.\n\n16) Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.\n\n17) Image using a confocal microscope as described above.\n\n\n\n1) Collect Tg fli1a:nls-mCherry embryos and incubate at 28°C until 30 hpf.\n\n2) Dechorionate embryos.\n\n3) Incubate embryos in labelling solution (500 uM EdU, 15% DMSO, E3) at 28°C for 1 h.\n\n4) Fix in 4% PFA at 4°C overnight.\n\n5) Wash with PBST for three 5-min washes, rock samples each time.\n\n6) Permeabilise in 1× saponin solution (Saponin solution is part of kit. diluted in PDT – Phosphate buffered saline with 1% DMSO, 0.1% Triton X-100) for 1 h at room temperature while rocking samples.\n\n7) Incubate with reaction cocktail (219 μL PBS, 5 μL copper protectant, 1.25 μL fluorescent azide, 25 μL buffer additive) for 1 h in the dark at room temperature whilst rocking.\n\n8) Wash five times in PBST for 5 min whilst rocking.\n\n9) Mount as described in ‘Mounting individual zebrafish embryos for confocal imaging’.\n\n10) Image using a confocal microscope as described above.\n\n\n\n1) Take treated embryos and mount on glass slides in vectashield (RRID:AB_2336789). Embryos must be laid flat on their side. This occurs naturally for young embryos (~30 hpf). If older embryos are used, the yolk may prevent embryos from lying flat, in this case the embryo yolk and head can be cut off using a scalpel.\n\n\n\n1) Collect Tg fli1a:nls-mCherry embryos and incubate at 28°C until 26 hpf.\n\n2) Dechorionate embryos using forceps.\n\n3A) For live imaging using LSM, suspend embryo(s) in melted low-melt agarose (37°C 0.8–1.2%), then use a plunger to draw the embryo(s) into a glass capillary. Wait 2 min for the agarose to set, then suspend the embryos (within the agarose column) into the LSM by gently pushing the plunger.\n\n3B) For confocal imaging, add embryo(s) to melted low melt agarose (37°C 0.8–1.2%) then draw up embryo(s) and decant into an Ibidi μ-Dish 35mm (Ibidi cat no – 81156). Use thin-tip forceps to gently push the embryo into a side-mounted position, then wait for agarose to set.\n\n4) Image using appropriate microscope ensuring 587 nm excitation is active. Adjust magnification to visualise as much of the trunk as possible whilst retaining nuclear resolution (for the LSM880 and AxioObserver SPIM, magnification should be set to 20×). Z-range should traverse the entire trunk vasculature and Z-interval should be set no lower than 8 μm.\n\n5) For euthanasia, discard embryos into bleach. For revival, return embryos to E3.\n\n\n\n1) Collect embryos and transfer to 28°C E3 media.\n\n2) Take a glass slide and place it in a petri dish so it is unable to move further to one side.\n\n3) With a Pasteur pipette, draw up embryos then, holding the glass slide petri dish slightly tilted, dispense embryos along the edge of the glass slide (Sup. 2).\n\n4) Prepare a glass needle (World Precision Instruments, cat no TW100F-4) using a Sutter P1000 glass capillary puller. Suitable settings are heat 475, pull 60, vel 80, time 135, pressure 500. Refer to the user manual for capillary pulling optimization.\n\n5) Add injection media containing 3 ng/nL of tnnt2a morpholino to a glass needle and calibrate the ejection volume using a graticule (Breckland Scientific, Precision Stage Micrometer – S1).\n\n6) Using a manipulator (World Precision Instruments M3301) and PicoPump (H. Saur PV820), inject 1 nL of morpholino into each embryo until desired number of treated embryos are obtained (some extra embryos should be treated to account for failure to develop and death).\n\n\nResults\n\nWe analysed EC proliferation in the trunk vasculature of zebrafish embryos because this vascular bed allows simultaneous imaging of the intersegmental vessels and dorsal aorta (Figure 1). We attempted to quantify EC proliferation in the zebrafish trunk using immunohistochemical, DNA labelling and transgenic approaches.\n\nDA – dorsal aorta. DLAV - dorsal longitudinal anastomotic vessel. CV - caudal vein. CVP - caudal vein plexus. PCV - posterior cardinal vein. ISVs - intersegmental vessels.\n\nImmunohistochemistry can be used to label mitotic markers found in proliferating cells. We chose PCNA as a marker of proliferation for this study as an anti-PCNA antibody had been previously validated for immunostaining zebrafish kidney sections13 and whole mount embryos.14 To determine whether whole mount immunohistochemistry could detect EC proliferation, transgenic embryos expressing endothelial GFP (Tg fli1a:nls-EGFP) were fixed at 30 hpf then treated with anti-PCNA antibody. Imaging of the embryo trunk detected many PCNA+ cells in the caudal vein plexus (CVP) and none in the ISVs (Figure 2).\n\nConfocal imaging was used to generate a z-stack of fli1a-GFP and PCNA expression in the trunk. Orthogonal views are presented alongside the image. The caudal vein plexus and intersegmental vessels were imaged to determine whether endothelial cell proliferation was detected in these vascular beds. At 30 hpf anti-PCNA was colocalised with fli1a positive cells (labelled with yellow arrows). Since flow modifies EC proliferation, we studied embryos lacking blood flow (tnnt2a morphants; shown) and controls with normal blood flow. PCNA staining did not detect proliferating endothelial cells in either group. The pattern of staining suggests a deficiency of penetration of anti-PCNA antibodies into the centre of the embryo.\n\nThe cell labelling approach works by incorporation of thymidine derivatives such as EdU into newly synthesised DNA. The modified thymidine is labelled with a fluorescent azide thereby enabling the identification of nuclei which have undergone proliferation.15 An advantage of EdU labelling is that proliferation can be measured over a period of time (i.e. the time that embryos are exposed to EdU), whereas immunostaining is restricted to detecting markers of proliferation at a single time point. To assess the effectiveness of EdU staining, transgenic embryos expressing endothelial nuclear localised mCherry (Tg fli1a:nls-mCherry) were treated with EdU from 30 to 31 hpf. Embryos were then fixed and treated with fluorescent azide. Between zero and three proliferating ECs were detected in the trunk of treated embryos, with detection occurring in the DA, but not the ISVs (Figure 3).\n\nProliferating endothelial nuclei are marked (yellow arrows).Orthagonal views are presented for the X and Y axes.\n\nAs previous approaches lacked specificity or very rarely detected EC proliferation,we sought a method for quantifying EC proliferation over a longer timeframe. EdU staining over prolonged time periods was not feasible because EdU can only reach vascular nuclei in the presence of permeabilization solvent dimethyl sulfoxide (DMSO) which has well described toxic effects above the 2% level.16 As the 1-h EdU incubation protocol called for 15% DMSO, longer incubation times were not possible without reducing the DMSO concentration. We chose to pursue an alternative approach rather than further optimize EdU labelling. To do this, we carried out time-lapse imaging of transgenic embryos expressing endothelial nuclear localised mCherry (fli1a:nls-mCherry) and cytoplasmic LifeAct-mClover (fli1a:LifeAct-mClover) using a light sheet microscope. Using this approach, EC proliferation was quantified by counting the division of EC nuclei in all visible ISVs from 26 to 30 hpf (Figure 4).\n\nThe trunk of WT zebrafish was studied from 26 to 30 hpf in 15-minute intervals using a light sheet microscope. Endothelial nuclei were labelled using the transgenic marker fli1a:nls-mCherry (red) whilst endothelial F-actin was labelled using fli1a:lifeAct-mClover (green). EC proliferation was defined as where one nucleus visibly divides into two (examples are shown and dividing cells are labelled with yellow arrows; compare upper left with upper right; compare lower left with lower right).\n\nDetection of EC proliferation in the intersegmental vessels was compared for each method. The ISVs are isolated from other tissues, so it was easiest to spot proliferation in this vascular bed. There are also relatively few nuclei in the ISVs, and ISV nuclei are less densely packed than nuclei in the DA/CVP. PCNA did not detect any proliferation events in the ISVs, EdU labelling detected fewer than one proliferation event on average, whereas time-lapse imaging detected an average of nine proliferation events (Figure 5). Two-way ANOVA of the number of proliferation events detected by each method revealed a significant difference in detection across methods and no significant difference in detection across individual zebrafish embryos (p = 0.0002).\n\nEndothelial cell proliferation was studied at 30 hpf in PCNA treated embryos, 30 to 31 hpf in EdU treated embryos, and 29–45 hpf in time-lapse imaged embryos. Time-lapse imaging detected EC proliferation more effectively than other methods. Statistics were derived from analysis by ordinary one-way ANOVA.\n\n\nDiscussion\n\nWe tested three different methods for quantifying EC proliferation in the developing zebrafish embryos. Whole mount immunostaining for PCNA was effective for detecting proliferation per se, as evidenced by detection of PCNA positive nuclei (Figure 2). A large number of PCNA positive nuclei were detected in the CVP at 30 hpf, however these cells were likely embryonic progenitors rather than ECs, which was confirmed in EdU labelling data where a large population of proliferating, non-endothelial cells were detected in the CVP. 30 hpf was chosen as at this point the embryo has a functional heart and has a functionally developed trunk vasculature. Study at later time points was avoided to minimize embryo size as others have shown that immunostaining for PCNA in zebrafish requires tissue sectioning, and adding this step would have made the protocol undesirably low throughput. Our findings are consistent with Luo et al14 which suggested that use of antibodies to label deep tissues in whole mount zebrafish embryos may be difficult. This led us to seek a small-molecule approach for labelling proliferating ECs.\n\nA DNA-labelling approach was tested in parallel. EdU is a small molecule that readily diffuses into cells in the presence of DMSO, during mitosis EdU is incorporated into newly synthesised DNA after which EdU can be detected by chemically bonding it to a fluorescent azide. As all components of the EdU labelling system were small molecules, we hypothesized that this technique would not be limited by penetration of reagents into deep tissues. Indeed, this was supported by our data as EdU labelling identified a population of proliferating cells in the caudal vein plexus that was not identified by the immunostaining approach (Figure 3). Notably, whilst some EdU+ ECs were identified, most EdU labelled cells were negative for the endothelial marker mCherry, and were likely haematopoietic progenitors which develop in the caudal vein plexus.\n\nEdU labelling revealed that EC proliferation occurred at 0.25%–1% (1–3 nuclei per field of view containing 300–400 nuclei). This observation is comparable to those observed in vitro and mammalian systems which revealed EC proliferation at 0.67–0.75% of nuclei per hour17,18; thus suggesting that EC proliferation rates are conserved between zebrafish embryos and mammalian systems.\n\nDespite this, EdU labelling is unsuitable to analyse the effects of interventions on proliferating ECs because of the limited window of detection and low number of proliferating cells detected. Therefore, we sought a method to capture greater numbers of proliferating EC from zebrafish embryos. We found that light sheet time-lapse microscopy of transgenic embryos (Tg fli1a:nls-mCherry, fli1a:lifeAct-mClover) enabled direct visualisation of EC nuclei division in proliferation (Figure 4). As this transgenic line expressed fluorescent protein exclusively in ECs it was relatively trivial to count the total number of EC proliferation events. This approach has several major advantages:\n\n• Lower material costs, which eliminates the need for antibody or cell labelling products used in immunostaining or cell labelling approaches.\n\n• Shorter experiment runtime, as experiments can be done as soon as embryos reach the desired stage of development compared to other approaches which require an additional period of time for fixation and treatment.\n\n• Non-lethal procedure, embryos do not need to be fixed to conduct the experiment.\n\n• Greater detection of EC proliferation compared to other zebrafish approaches (IHC, cell labelling) because of a larger detection window (Figures 6 and 7).\n\n• Greater breadth of data collected. Time-lapse data could also be analysed for cell migration and apoptosis as desired.\n\nThe PCNA approach uses a small (instantaneous) detection window, thus rarely detects proliferation events. EdU is more likely to detect proliferation but the window of detection remains too small to capture many events. Time-lapse imaging covers a much greater window of time and can therefore detect significantly more proliferation events.\n\nImmunostaining (for PCNA) is limited by antibody penetration. EdU labelling is limited by low detection of EC proliferation, the toxic effects of EdU treatment (which necessitates DMSO) prevent longer incubation times. Nuclei imaging by time-lapse microscopy is effective for studying EC proliferation.\n\nThe limitations of this approach are the relatively low throughput of time-lapse proliferation analysis, which was done manually in this case, however computer assisted quantification might be possible. Another limitation specifically regarding use of zebrafish to study genes involved in proliferation is that not all human genes have zebrafish orthologues, which may restrict candidates for study in this model.\n\nThis study demonstrates the utility of time-lapse imaging zebrafish embryos to measure EC proliferation. Comparable in vitro models (HUVECs, HCAECs) allow real-time imaging of EC proliferation, but lack biological relevance. Whereas established in vivo models (mouse, pig) are biologically relevant yet cannot be used for real-time in situ monitoring of EC proliferation, and must be culled to isolate vascular tissue for analysis. By using the zebrafish embryo to study EC proliferation, researchers can achieve real-time in situ monitoring of EC proliferation in a biologically relevant model whilst replacing mammals and reducing or eliminating the use of terminal procedures, thus achieving partial replacement and refinement of use of animals in CVD research.\n\nThis time-lapse approach is more suitable than other methods for quantifying EC proliferation in zebrafish larvae and has applications for studying the mechanisms of proliferation and for screening to detect compounds that modify this process, we are currently using this approach to use zebrafish larvae to screen for genes involved in EC proliferation in response to blood flow. Whilst the manual counting approach used currently is low-throughput, future work could automate detection of proliferation using image macros to enable widespread use of this system for identifying genes involved in atherosclerosis\n\n\nData availability\n\nOSF: Underlying data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’. https://www.doi.org/10.17605/OSF.IO/NV9XE.\n\nThe project contains the following underlying data:\n\nRaw images of three embryos imaged using the PCNA method, three embryos imaged using the EdU method, and three embryos imaged using the timelapse method.\n\nOSF: Extended data for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’. https://www.doi.org/10.17605/OSF.IO/Y2SCW.\n\nThe project contains the following extended data:\n\nSupplementary Figure 1: Dechorionation of zebrafish embryos. 1) Insert forceps into embryo chorion, be careful not to damage the embryo. 2) Gently open forceps to tear the chorion further. 3) Widen the forceps until the embryo has escaped the chorion.\n\nSupplementary Figure 2: Preparing zebrafish embryos for microinjection. 1) Place a glass slide in a petri dish and tilt as shown. 2) Draw up embryos in a Pasteur pipette and decant onto the edge of the glass slide as shown. 3) Use the Pasteur pipette to draw up any excess liquid from the petri dish, leaving a row of zebrafish embryos against the glass slide.\n\n\nReporting guidelines\n\nOSF: ARRIVE 2.0 checklist for ‘Quantifying endothelial cell proliferation in the zebrafish embryo’. https://www.doi.org/10.17605/OSF.IO/Y2SCW.\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 Dr Rob Wilkinson (University of Nottingham, UK) for providing fli1a:nls-mCherry, fli1a:lifeAct-mClover and fli1:EGFP lines.\n\n\nEthical statement\n\nApproval for research in zebrafish was carried out under the University of Sheffield establishment licence and Professor Tim Chico’s project licence (200624_Chico).\n\n\nReferences\n\nWeis SM, Cheresh DA: Pathophysiological consequences of VEGF-induced vascular permeability. Nature. 2005; 437: 497–504. PubMed Abstract \| Publisher Full Text\n\nKimmel CB, Ballard WW, Kimmel SR, et al.: Stages of embryonic development of the zebrafish. Dev Dyn. 1995; 203: 253–310, PubMed Abstract \| Publisher Full Text\n\nEkker SC: Morphants: a new systematic vertebrate functional genomics approach. Yeast. 2000; 17: 302–306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAblain J, Durand EM, Yang S, et al.: A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev Cell. 2015; 32: 756–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGilbert LA, Larson MH, Morsut L, et al.: CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154: 442–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerbanovic-Canic J, de Luca A, Warboys C, et al.: Zebrafish Model for Functional Screening of Flow-Responsive Genes. Arterioscler Thromb Vasc Biol. 2017; 37: 130–143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeijts B, Gutierrez E, Saikin SK, et al.: Blood flow-induced Notch activation and endothelial migration enable vascular remodeling in zebrafish embryos. Nat Commun. 2018; 9: 5314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKugler EC, van Lessen M, Daetwyler S, et al.: Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish. EMBO Rep. 2019; 20: e47047. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLister JA, Robertson CP, Lepage T, et al.: nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development . 1999; 126: 3757–3767. PubMed Abstract\n\nSavage AM, Kurusamy S, Chen Y, et al.: tmem33 is essential for VEGF-mediated endothelial calcium oscillations and angiogenesis. Nat Commun . 2019; 10: 732. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeckel E, Boselli F, Roth S, et al.: Oscillatory Flow Modulates Mechanosensitive klf2a Expression through trpv4 and trpp2 during Heart Valve Development. Curr Biol. 2015; 25: 1354–13561. PubMed Abstract \| Publisher Full Text\n\nRoman BL, Pham VN, Lawson ND, et al.: Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development. 2002; 129: 3009–3019. PubMed Abstract\n\nLeung AY, Leung JC, Chan LY, et al.: Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis. Histochem Cell Biol. 2005; 124: 105–111. PubMed Abstract \| Publisher Full Text\n\nLuo N, Li H, Xiang B, et al.: Syndecan-4 modulates the proliferation of neural cells and the formation of CaP axons during zebrafish embryonic neurogenesis. Sci Rep. 2016; 6: 25300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalic A, Mitchison TJ: A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A. 2008; 105: 2415–2420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiong X, Luo S, Wu B, et al.: Comparative Developmental Toxicity and Stress Protein Responses of Dimethyl Sulfoxide to Rare Minnow and Zebrafish Embryos/Larvae. Zebrafish. 2017; 14: 60–68. PubMed Abstract \| Publisher Full Text\n\nLi W, Li H, Sanders PN, et al.: The multifunctional Ca2+/calmodulin-dependent kinase II delta (CaMKIIdelta) controls neointima formation after carotid ligation and vascular smooth muscle cell proliferation through cell cycle regulation by p21. J Biol Chem. 2011; 286: 7990–7999. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Song H, Wu F, Mu Q, Jiang M, Wang F, Zhang W, Li L, Shao L, Li S, Yang L, Zhang M, Wu Q, Tang D: Irisin Inhibits Atherosclerosis by Promoting Endothelial Proliferation Through microRNA126-5p. J Am Heart Assoc. 2016; 5. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "118456", "date": "07 Feb 2022", "name": "Christopher George", "expertise": [ "Reviewer Expertise Network behaviour of cardiovascular cell types / cell imaging / imaging technologies / signal decoding. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact." ], "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’ clearly set out the context for their study; developing a non-invasive experimental system to accurately capture spatial and temporal information on EC proliferation in the vasculature would be important step forward. Three techniques are explored for achieving these aims. Two (PCNA immunostaining, EdU incorporation) do not appear to work for the intended purpose but it is not immediately clear from the data shown why. More ‘negative’ data would be very helpful in setting out the limitations of these techniques so that others can appreciate why these approaches ‘failed’. One technique, time-lapse imaging of nuclear division (where labelling driven by a EC-specific promoter) does appear to have some future applicability, although the technique, at present, is limited to manual counting and and very low numbers of experiments are reported in support of this approach.\nThere are a number of issues with the current version of the manuscript that require attention:\nAbstract\nThe abstract refers specifically to n=24 experimental units, where in the example given, n=4 comprise controls and then four groups of n=5 ‘treated’ groups. These numbers seem rather arbitrary and are not referred to in the paper.\nIntroduction / General\nIt would be helpful to include more detail on how the ZF embryo CV system recapitulates key features of mammalian circulatory system. Please provide clarity (and perhaps illustrative detail) on how ZFe’s “complete cardiovascular system” has a “simplicity comparable with that of two dimensional models in vitro” and “comparable relevance to mammalian in vivo models”. This might help uptake by those researchers typically using mammalian models of vascular plasticity.\nBlebbing of nuclei is not a unique marker of apoptosis. Autophagy and other activation of modes of cellular degradation are characterised by blebbing. The authors imply that apoptosis is the sole mechanism of cell death in ZF embryos. Is this correct? No data on cell death is reported in the manuscript so the references to apoptosis don’t seem to have much relevance in the context of the paper.\nMethods\nMore methodological detail / explanation should be included:\nHow are male and female zebrafish distinguished? Does the Nacre line have identical vascularization as wild-type? You state that “both transgenic lines use Nacre as background” but the study uses three TG lines. How leaky, or not, is the EC-specific promoter. Can you be sure that expression is only in EC lineages? This is a key issue since your method relies on typing ‘EC-specific’ expression. A reference to the inhibition of contractility by tnnt2a morpholino should be given. It is presumed that that TG-EGFP was used for PCNA and TG-mCherry was used for EdU to avoid the spectral overlap of fluorophores in each instance? This should be stated. More details on how the commercial EdU labelling kit results in the generation of a detectable 488nm signal should be included. What is the concentration of saponin? What is the range of EdU signal developed (i.e. time-dependent accumulation)? How homogeneous is this across cells/FACS populations? Brightfield microscopy is included as the means for discriminating dead or undeveloped embryos are identified. How? No brightfield imaging is included in the paper. Please define Light Sheet microscopy as LSM (to avoid confusion with the common use of LSM as (confocal) laser scanning microscopy. The statement that “587nm excitation is active”. Please include the excitation source and what is meant by “active”. The methods include non-terminal anaesthesia using E3 medium (“Fluorescent microscopy”) or E3 medium and tricaine (“Light-sheet microscopy”). Please clarify. What is the final concentration of tricaine used in anaesthesia? Statistics. Please check that two-way ANOVA is an appropriate statistical test for data where n=3 (i.e. is distribution normal?). Microinjection - it is appreciated that the settings are specific for the Sutter puller but it would be helpful to include units as applicable (e.g. heat, 475 degrees C?). Into which ZF embryo region is the morpholino injected?\n\nResults\nFigure 1. Perhaps this cartoon could be shown alongside actual staining of these sections in a representative embryo. Figure 2. According to the Methods section, confocal imaging fields were approximately 869 x 869 microns. Figure 2 is approx. 250 x 250 microns. It would be helpful to show the entire imaging field and then the ‘zoom’ into the region shown in Figure 2. In my copy for review, Figure 2 has thin yellow lines which are not explained and the yellow arrows do not appear to correspond to co-stained cells. Figure 2 does not support the use of PCNA as being a good marker for EC proliferation (indeed you state this, indirectly, in the Discussion) but its not immediately clear what it does show. It would be helpful too if regions, like in the cartoon in Figure 1 were included on Figure 2. Figure 3 should be presented at the same magnification/zoom as Figure 2. Figure 3 should also be better integrated with your conclusion that EdU labelling was ruled out of contention as a technique for quantifying EC proliferation. Do you have any images showing (visually) the effect of 15% DMSO on sample morphology beyond 1 hour? It is also somewhat misleading to state, as you do in Figure 7, that EdU labelling is limited by low detection of EC proliferation. The problem seems to be that the reagent is toxic which prevents longer-term imaging. It needs to made more clear that EdU is of limited use for mapping cellular proliferation occurring on a time-scale incompatible with reagent toxicity. Figure 4. Your findings suggest that to capture sufficient EC proliferation, an experimental window of up to 12 hours is required. Figure 4 only shows a plus-15min window. Some idea of how time-lapse capture of EC proliferation changes with time would be very helpful. Also, the arrowheads appear squashed and point away from the area you mean to depict. Figure 5 shows the number of proliferation events but this is not that useful. It would be much better if you showed this the number of as the proportion or percentage of mCherry cells scrutinized. ‘n’ is very low here and statistical analysis is not convincing (although the effect is obvious).\n\nDiscussion\nIt is not possible to conclude, from the data shown, that nuclei-imaging is “accurate” (Figure 7). This will need much more data and deeper modes of analysis. As you note, “computer assisted quantification” would augment your approach, but this phrase is vague. What would you want to focus on, and what would need to be taken into account / eliminated etc. More discussion on how you see this approach being potentially worked-up into a medium- to high-throughput screening platform would be helpful.\n\nOther\nZebrafish welfare and use section. The second-to-last sentence in the first paragraph needs splitting into two sentences. It would be helpful to define what constitutes an “adverse event” here. PCNA and EDU should be described in full at first mention. Has PCNA ever been used as a marker of proliferation during zebrafish CV development? (You state kidney but what about CV…….) The phrase “humanely destroyed using bleach” is inappropriate. It might be better worded as “embryos were destroyed using bleach”. Ditto “euthanised” on pages 5 and 8. The Supplemental Figures do not add to the article and should be omitted. Videos of the actual procedures would be much better.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\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? 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? Partly", "responses": [] }, { "id": "96625", "date": "01 Nov 2022", "name": "Tzung Hsiai", "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\nIn this method article, Bowley et al. determine the feasible method to quantify endothelial cell (EC) proliferation which regulates vascular formation and homeostasis in zebrafish embryos to investigate development via a comparison between three different comparative analyses: antibody, DNA labeling, and time-lapse imaging. Although rodent models are predominantly used to explore the mechanism of EC proliferation in cardiovascular disease or development, the authors pointed out there is a limitation - time-consuming, costly, and detrimental to animal welfare. The authors concluded that time-lapse imaging is accurate, precise, scalable, simple, and effective than the other two methods: antibody (PNCA) or DNA labeling (EdU) in EC proliferation study in the zebrafish embryo model. This method paper is necessary to approach EC proliferation in zebrafish and is well designed to compare three different methods, however, there are major and minor points that need to be improved.\nThe authors are focusing on the quantification of EC proliferation using three different methods, however, vascular endothelial growth factor (VEGF) is the most common marker for EC proliferation. Please address why the authors chose PCNA for detecting proliferating cell nuclear antigens and EdU for measuring DNA proliferation instead of VEGF, and it is necessary to be discussed.\n\nEdU labeling is a well-established method to quantify S-phase cells. The authors performed EdU labeling in zebrafish embryos for 1 hour and showed that it did not label as many cells as they observed via tracking nuclear division over 6 hours (Figure 5). This does not translate to EdU labelling being unable to provide accurate measurements. Instead, it simply prompts a longer period of EdU incubation. The authors argue that the high concentration of DMSO used in the labeling solution is toxic to embryos for long-term incubation, however, there is no necessity of using DMSO during EdU incubation as EdU is soluble in aqueous buffers. The authors should perform extra experiments on this.\n\nOn the other hand, there are large amounts of EdU+ cells (green) colocalization with ECs (red) in Figure 3. The authors should also test their hypothesis at different regions of the vascular network, not just ISV.\n\nIn Figure 2, to see the clear effects of tnnt2 morphant (inhibition of heart contraction) in EC proliferation, the control group (control morphant) should be presented. The authors also need to provide negative control images with only secondary antibodies to validate the staining, as virtually every cell in the image was red, meaning PCNA+. However, PCNA staining should give fewer labeled cells than EdU staining and time-lapse tracking, according to the authors. It can also be that the PCNA antibody is not working well. The authors need to address this.\n\nIn addition, to rely on the comparison, Figures 2 and 3 should be offered the same enlargement.\n\nIn Figure 5, two-way ANOVA is an advisable statistical method among the groups.\n\nThe graphical abstract (Figure 7) is arduous to comprehend because of a lack of a rationale for the limitation of immunostaining and EdU labeling. The authors should offer the reference or evidence to elaborate the experimental design.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre the 3Rs implications of the work described accurately? Yes\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? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "156027", "date": "21 Feb 2023", "name": "Stephen J. White", "expertise": [ "Reviewer Expertise Endothelial biomechanics and endothelial dysfunction" ], "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 by Bowley et al. describes a novel approach to quantify the relatively slow rates of endothelial proliferation in vivo, using zebrafish embryos, rather than in vitro models. The approach has several advantages over using other animal models to examine endothelial proliferation in vivo, including the potential use of zebrafish lines that use fluorescent reports for cell-type differentiation, the ability to examine proliferation pre-post heart beat, to determine any effects of flow and easy genetic manipulation through genome editing or knockdown with morpholinos. As such, this model provides a flexible platform to examine drug or gene regulation of (endothelial) proliferation.\nThe text and methodology is clearly described and in sufficient detail to allow other scientists to replicate the experiments described here. As such it is a useful step forward to quantify in vivo proliferation while minimising the use of protected species. The platform is sufficiently flexible to be used to advance the field on this topic.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\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": [] } ]	1	https://f1000research.com/articles/10-1032
https://f1000research.com/articles/10-1030/v1	11 Oct 21	{ "type": "Research Article", "title": "Prediction of Tensile Strength of 3D Printed Bronze PLA Part Using Response Surface Modelling", "authors": [ "Chockalingam Palanisamy", "Sugendran Nagarajan", "Sugendran Nagarajan" ], "abstract": "Background - 3D printing is a dynamic process with many process parameters influencing the product, including the type of the material; it is often difficult to understand the combined influence of these parameters.\n\nPurpose - The tensile strength of 3D printed parts is important for the functionality of components. The effects of process parameters on tensile strength must therefore be examined. The objective of this study is to develop a response surface model (RSM) to predict the final quality of a 3D printed bronze part from a different set of input parameters.\n\nMethods - The tensile test specimen was built in a Makerbot 3D printer with bronze polylactic acid (PLA) material. The three controllable input parameters were; thickness of layers, number of shells, and infill density. The three levels of layer thickness were 0.1mm, 0.2mm and 0.3mm. The number of shells was 2, 3 and 4. The infill densities were 20%, 30% and 40%. A tensile experiment tested the strength of the specimens. RSM is a statistical approach for modelling and analyzing how different variables affect the response of interest, and for optimizing it.\n\nResults - The result obtained shows that the specimen with a high layer thickness of 0.3mm and infill density of 40% is the best among all the other parameters. Finally, the regression equation produced was used for random values of layer thickness, the number of shells, and infill density, to see whether the values obtained from the tests fall into the range of experimental data.\n\nConclusion - Infill density and layer thickness are the two criteria that significantly influence the tensile property. The number of shells has the least influence on the tensile property. However, the best tensile strength is the part printed with higher infill density, a greater number of shells, and higher layer thickness.", "keywords": [ "3D Printing", "RSM", "Bronze PLA", "Tensile Strength" ], "content": "Introduction\n\nCompetitiveness in the global marketplace is increasing and manufacturers must seek ways to increase production output and quality and reduce the costs of production. 3D printing, a very recent innovation and state-of-the-art technology, is a solution that could meet their requirements.1 Bronze - polylactic acid (PLA) is the most widely used material in the 3D printing of most mechanical parts. 3D printing produces quality and defect-free objects. However, 3D printed parts do not have high tensile properties. With a combination of input parameters, parts can be printed with enhanced tensile properties. A large number of parameters influence the properties of the product; it is often difficult to understand the combination of these parameters.2 Among other considerations, printing parameters such as the thickness of each layer, number of shells and density of the infill have a major impact on the quality and performance of 3D printed components. Since tensile properties are important for the functioning of components, the effect of process parameters on mechanical properties will be studied. In this research, bronze material is used because it has not been thoroughly studied, unlike ABS or PLA. Finally, a response surface model (RSM) using Minitab 18 (Minitab, RRID:SCR_014483) software (alternatively, R Software) is developed.\n\n\nMethods\n\nThe three variable input parameters are layer thickness, number of shells, and infill density. In this study, the levels of the input parameters are: layer thickness (0.1 mm, 0.2 mm and 0.3 mm); infill percentage density (10%, 20%, and 40%); number of shells (2, 3, and 4)—all with a constant print speed of 50 mm/s. For the design of the experiment (DOE), central composite design (CCD) (Figure 1) with three input parameters (Table 1)—layer thickness, number of shells, and infill density with three levels—was used in this investigation.3 Based on the CCD Design Matrix (Table 2), 15 sets of samples were 3D printed using Makerbot Replicator with bronze PLA material.\n\nTensile testing was carried out on the samples and data was recorded.4 Using this data, a response surface model was created using Minitab 18 software (alternatively R Software). An Analysis of Variance (ANOVA) was also performed, with the independent variables, layer thickness, number of shells and infill density, to find out the factor settings that optimize the dependent variable tensile strength. Finally, a Pareto chart of the standardized effects was created to compare the relative magnitude and the statistical significance of main, square, and interaction effects of independent variables which contribute to the most variability to the dependent variable tensile strength, which also plots a reference line to indicate which effects are statistically significant.\n\n\nResults\n\nTensile testing was conducted on specimens that were printed according to the ASTM D256 specification, using the InstraonTensile tester, Instron 3360 Series dual-column table-frame equipment; the data was collected using Instron Bluehill 3 software. The results obtained from the tensile test are shown in Table 2. The results from the testing were then fed into Minitab Software to develop the response surface model of the Bronze-PLA specimen. The regression equation was obtained from the software to compute the theoretical data. Instron’s 3360 Series Dual Column Table Frames were used to conduct the tensile test. This equipment is capable of running both tensile and compression tests.\n\n\nDiscussion\n\nBased on the ANOVA analysis (Table 3), the Pareto chart (Figure 2) indicates that the factor square terms of the layer thickness, number of shells, and infill density are significant factors.5\n\nA regression equation was developed from the ANOVA analysis. The equation was developed using the coefficient of the terms of the response surface model.6\n\nTensile Test = 0.6648 + 0.0795 A + 0.0540 B + 0.0582 C − 0.0960 AA + 0.0693 BB + 0.0455 CC − 0.0818 AB + 0.0686 AC − 0.0763 BC\n\nTensile test = 0.6648 + 0.0795 * Thickness of layer + 0.0540 * Number of shells + 0.0582 * Infill density − 0.0960 * Square of layer thickness + 0.0693 * Square of number of shells + 0.0455 * Square of infill density − 0.0818 * Thickness of layer * Number of shells + 0.0686 * Layer thickness * Infill density − 0.0763 * No of shells * Infill density\n\nThe response surface model was developed successfully. Using this model, tensile test data for any different combination can be found.\n\n\nConclusions\n\nWith DOE of CCD with three input parameters—three levels each of layer thickness, number of shells, and infill density—the test specimens were printed and tensile strength experiments were conducted on specimens. Tensile test data were collected to create a response surface model to successfully predict the tensile strength of 3D printed bronze PLA. Based on the tensile strength data, we can conclude that the stronger specimen has a 0.3 mm of layer thickness, two shells and 40% infill density. The best specimen is that with higher infill density, a higher number of shells and lower layer thickness. In conclusion, infill density played a vital role in the tensile strength of the specimen.\n\n\nAuthor contributions\n\nChockalingam conceived of the idea, developed the method and performed the verification. Chockalingam encouraged Sugedran to investigate and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.\n\n\nEthical approval\n\nAll procedures used in this project have been approved by Research Ethics Committee (REC) Multimedia University (EA2892021). This work does not involve data collection from human or animal experiments or vulnerable communities.\n\n\nData availability\n\nOSF: Input parameter and their levels, experiment plan, tensile test results, Response Surface Model (RSM) model, ANOVA result and chart.\n\nOSF: Prediction of Tensile Strength of 3D Printed Bronze PLA Part Using Response Surface Modelling.\n\nhttps://doi.org/10.17605/OSF.IO/NHBDT.7\n\nThis project contains the following underlying data:\n\n• Dataset 1: Input parameters, levels, experiment plan. tensile strength results.\n\n• Dataset 2: RSM analysis using Minitab 18 (alternatively R Software), ANOVA results.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nAuthors acknowledge Mr. Wan Johannes, Senior lab technician, Faculty of Engineering and Technology, Multimedia University, Melaka, Malaysia for his help during experiments. Furthermore, ‘thank you’ to the Research Synergy Foundation for the recommendations and support.\n\n\nReferences\n\nTofail SA, Koumoulos EP, Bandyopadhyay A, et al.: Additive manufacturing: scientific and technological challenges, market uptake and opportunities. Mater. Today. 2018; 21(1): 22–37. Publisher Full Text\n\nCamargo JC, Machado ÁR, Almeida EC, et al.: Mechanical properties of PLA-graphene filament for FDM 3D printing. Inter. J. Adv. Manufact. Tech. 2019; 103(5): 2423–2443. Publisher Full Text\n\nKim H, Park E, Kim S, et al.: Experimental Study on Mechanical Properties of Single- and Dual-Material 3D Printed Products. 45th SME North American Manufact. Research Conf. Los Angeles:2017.\n\nPalanisamy C, Krishnan GK: Experimental classification and response surface modelling of compression property of 3D printed polylactic acid parts. AIP Conference Proceedings. AIP Publishing LLC.2021; Vol. 2339(No. 1): p. 020070.\n\nHafsa MN, Ibrahim M, Wahab MS: Evaluation of FDM pattern with ABS and PLA material. App. Mech. Mater. 2014; 465-466(5): 55–59. Publisher Full Text\n\nPalanisamy C, Chinnasamy N, Muthu K: Influence of Process Parameters on Rapid Prototype Part Using Response Surface Methodology. J. Eng. Tech. App. Physics. 2019; 1(1): 4–7. Publisher Full Text\n\nPalanisamy C: Prediction of Tensile Strength of 3D Printed Bronze PLA Part Using Response Surface Modelling.2021, September 6. Publisher Full Text" }	[ { "id": "97370", "date": "02 Nov 2021", "name": "Bhagat Singh", "expertise": [ "Reviewer Expertise Mechanical Engineering" ], "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\nOverview: In the present work, authors have tried to investigate the effect of tensile strength on the functionality of the 3D printed parts. They have tried to explore the effects of various process parameters on the tensile strength of these printed parts. In order to achieve this, a response surface model has been developed in terms of the input parameters viz. thickness of layers, number of shells, and infill density. They inferred that infill density and layer thickness are the two prominent parameters governing the tensile strength significantly. The number of shells has the least influence as compared to the other two parameters.\nThe manuscript is well written. It can be accepted for indexing in the current form.\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": "98499", "date": "03 Feb 2022", "name": "Raveendran Sundararajan", "expertise": [ "Reviewer Expertise Thermofluids", "pipeflow and sustainable energy" ], "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 finding could lead to the 3D printer sample production for tensile testing for the bronze part only. Layer thickness and infill density were the two main parameters chosen. Wherever possible a graph should be included e.g. Tensile load vs infill thickness and number of layers. Statistical analysis was done in Minitab and this could be more explicit in the report as to how RSM was achieved statistically.\n\nThis finding could be taken further by using different materials and comparing the data.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-1030
https://f1000research.com/articles/10-1029/v1	11 Oct 21	{ "type": "Research Article", "title": "Motion and Geometric Feature Analysis for Real-time Automatic Micro-expression Recognition Systems", "authors": [ "Adamu Muhammad Buhari", "Chee-Pun Ooi", "Vishnu Monn Baskaran", "Wooi-Haw Tan", "Chee-Pun Ooi", "Vishnu Monn Baskaran", "Wooi-Haw Tan" ], "abstract": "The trend of real-time micro-expression recognition systems has increased with recent advancements in human-computer interaction (HCI) in security and healthcare. Several studies in this field contributed towards recognition accuracy, while few studies look into addressing the computation costs. In this paper, two approaches for micro-expression feature extraction are analyzed for real-time automatic micro-expression recognition. Firstly, motion-based approach, which calculates motion of subtle changes from an image sequence and present as features. Then, secondly, a low computational geometric-based feature extraction technique, a very popular method for facial expression recognition in real-time. These approaches were integrated in a developed system together with a facial landmark detection algorithm and a classifier for real-time analysis. Moreover, the recognition performance were evaluated using SMIC, CASME, CAS(ME)2 and SAMM datasets. The results suggest that the optimized Bi-WOOF (leveraging on motion-based features) yields the highest accuracy of 68.5%, while the full-face graph (leveraging on geometric-based features) yields 75.53% on the SAMM dataset. On the other hand, the optimized Bi-WOOF processes sample at 0.36 seconds and full-face graph processes sample at 0.10 seconds with a 640x480 image size. All experiments were performed on an Intel i5-3470 machine.", "keywords": [ "micro-expression recognition", "facial feature extraction", "real-time classification", "geometric-based features", "facial graph analysis", "emotion classification" ], "content": "Introduction\n\nA micro-expression is a brief, spontaneous facial expression that occurs on a human face in response to the emotions they are experiencing. The micro-expression contains a significant amount of information, and has attracted the interest of computer vision researchers because of its potential uses in security, interrogation, and healthcare.1-3 However, due to the speed of facial muscle movement, it is difficult to extract this information, and the features must be more detailed. A typical period of micro-expression is 200 milliseconds or less.4 For real-time micro-expression analysis and emotion recognition, the process involves pre-processing, feature extraction, and recognition. This paper examines two popular feature extraction approaches: motion-based features and geometric-based features. These approaches are reported to have reliable details from uncontrolled image data, which makes it feasible for real-time analysis.\n\nA motion-based feature is constructed based on non- rigid motion changes of subtle expressions where motion changes are extracted for spotting purposes. Facial motion analysis was first presented in5 using optical flow to spot micro-expressions. Since then, several studies in this field have explored this approach for facial landmark detection and micro-expression recognition. In,6 authors proposed an optical flow features from Apex- frame network (OFF-ApexNet), which combines optical flow guided context with the convolutional neural network (CNN) to compute features. Then, authors in7 presented a novel algorithm that combines a deep multi-task convolutional network for detecting facial landmarks and a fused deep convolutional network for micro-expression features. In another study,8 authors suggested the Riesz pyramid and a multi-scale steerable Hilbert transform. While, Merghani and Yap9 proposed a new region-based method with an adaptive mask. However, the motion-based features reported recognition accuracy from the current studies peaks at 74.06% over the CASMEII using leave-one-subject-out cross validation (LOSOCV).6\n\nOn the other hand, geometric facial analysis deals with the locations and shapes of facial components. As highlighted in Liu et al.,10 performance of landmark detection algorithms is limited, where only a few studies utilized landmarks in early facial graph representations. However, with recent advancements in face analysis study, improved facial landmark detection algorithms are presented in several studies.11-14 Towards facial landmark graph features, Lei et al.,15 presented a method that only employed 28 brow and lip landmarks, which contributed significantly to micro-expressions. While, other studies16-19 presented graph-based methods using AU to define landmarks of interest. The reported recognition accuracies from these methods proved that micro-expression features can be extracted using facial graph approaches. However, the general problem with graph-based micro-expression recognition is the lack of large-scale in-the-wild datasets. To date, the recognition accuracy peaks at 87.33% over the SAMM dataset with LOSOCV as reported in Buhari et al.18\n\n\nMethods\n\nAn automatic micro-expression recognition system is implemented for real-time facial analysis by integrating face landmark detection, feature extraction, and then classification. In the developed system, a trained model is generated using publicly available spontaneous micro-expression datasets. For micro-expression feature analysis, two different methods were implemented. Firstly, a Bi-Weighted Oriented Optical Flow (Bi-WOOF) feature descriptor by Liong et al.,20 is used. This method is a motion-based approach that uses optical flow to compute features, and it requires an apex-frame spotting before the feature computation. The BiWOOF is considered in this study as the results of its performance improvement over the textural feature extraction methods such as local binary patterns on three orthogonal planes (LBP-TOP) as reported in Liong et al.20 However, the computational cost poses challenges for real-time recognition as it require apex-frame spotting. The second feature descriptor considered is a full-face graph by Buhari et al.18 This geometric-based feature computation method requires only facial landmarks to compute features. The full-face graph is considered in this study because the computational time is significantly low in comparison with motion-based. However, it is reported that the earlier geometric-based methods could not detect hidden changes in facial components due to its subtleness and briefness.\n\nFigure 1 illustrates the implemented real-time micro-expression recognition system developed using Bi-WOOF. This feature extractor requires at least two frames (i.e, neutral frame and apex-frame) to compute. Firstly, the system captures images of faces using dlib-19.4.21 Next, apex-frame spotting is applied using an automatic apex frame spotting method by Liong et al.,22 to identify the frame with the highest facial expression within the captured image sequences (i.e., processing sample). As reported by the authors in Liong et al.,22 the performance of their method improved over the annotated apex frames provided in micro-expression databases. Here, image sequences from spontaneous micro-expression datasets were utilized. Upon identifying the onset and apex frames, optical flow vectors are computed to define the face motion patterns: (i) magnitude: pixel movement intensity; (ii) orientation: flow motion direction; and (iii) optical strain: modest deformation intensity. Then, using the computed optical flow vectors (i.e., the magnitude, orientation, and the optical strain), Bi-WOOF features are formed. Step-by-step details of this method can be found in Liong et al.20 Figure 1 shows a framework of the real-time micro-expression recognition system using apex-frame spotting and BiWOOF feature extraction method.\n\nFigure 2 illustrates the implemented real-time micro-expression recognition system developed using full-face graph. At first, facial landmark detection is applied to detect coordinates of facial components using dlib-19.4. Then, segments of lines are generated using the detected coordinates of facial components by connecting each landmark point (denoted as p) with subsequent landmark points (denoted as q), for p∈{1,2,…,N} and q∈{1,2,…,p}, where N=68. This concept is described as a full-facial graph using landmark points, and segments are generated as follows:\n\nThe indexes (i.e., p,q) of every landmark with subsequent landmark points are determined and stored as segments in ℑ for feature computations. After the graph is generated, features are computed by calculating the Euclidean distance and gradient of every segment, an idea presented in Buhari et al.18 The total number of features computed using this technique is N×(N−1)→K, which translates to 4,556 features at N=68.\n\nTo further analyse the potential performance improvement of the geometric-based features, Eulerian motion magnification (EMM) is applied to the images to amplify the micro-expressions prior to the landmark detection process. Eulerian-inspired approaches23,24 do not require explicit motion vectors but emulate motion magnification by magnifying property changes, such as amplitude (denoted as A-EMM) or phase (denoted as P-EMM). According to Le et al.24 A-EMM outperformed P-EMM in terms of recognition rates over a broad range of magnification factors. Thus, this paper considered the A-EMM to the images before the feature computations. Details of the methods for A-EMM are detailed in Le et al.24 Figure 3 illustrates the principle of the integrated a magnification sub-process to the implemented single-frame sample with a geometric-based features system.\n\nThe experiments were performed using four spontaneous datasets: (i) spontaneous micro-expression dataset (SMIC) dataset,25 (ii) Chinese Academy of Sciences Micro-expression (CASMEII) dataset,26 (iii) spontaneous macro-expressions and micro-expressions (CAS (ME)2) dataset,27 and (iv) spontaneous actions and micro-movements (SAMM) dataset.28 These are spontaneous micro-expression datasets which were used in this study with full details of these datasets in Li et al.,25 Yan et al.,26 Qu et al.,27 and Davison et al.28 Details to acquire these datasets used in this study are available at www.oulu.fi/cmvs/node/41319 for SMIC,25 fu.psych.ac.cn/CASME/casme2-en.php for CASMEII,26 fu.psych.ac.cn/CASME/cas (me)2-en.php for CAS (ME)2,27 and personalpages.manchester.ac.uk/staff/adrian.davison/SAMM.html for SAMM.28 Moreover, to evaluate the performance using a larger dataset, this paper merged the four datasets to form a COMBINED dataset. The COMBINED dataset is created from the raw images of all the four datasets. The steps for generating the COMBINED datasets includes; face detection, face cropping, colour-space conversion to grayscale, and image re-scaling to 140×170. From these steps, colour-space conversion is applied to adopt the SAMM dataset samples as provided in grayscale format, where the sample re-scaling to 140×170 adopts the SMIC dataset cropped image sizes (i.e., the smallest cropped image size considered to provide reliable features description, and achieve high speed performance for real-time micro-expression recognition). The image re-scaling utilises a down-sampling technique by Buhari et al.,29 in order to re-produce a high quality down-scaled samples. In addition, the COMBINED dataset adopted the SMIC dataset sample labelling by re-grouping the seven classes of emotions (i.e., happiness, sadness, anger, surprise, fear, contempt, and disgust) to three classes (i.e., positive, negative, and surprise). Here, positive ∈{happiness}, negative ∈{sadness, anger, fear, contempt, disgust}, and surprise ∈{surprise}. Figure 4 illustrates the COMBINED dataset formation from the four spontaneous datasets. Note that the participant images utilised in Figure 4 are the publishable images with the consent of participants, as stated in the documentation from each study. While, Table 1 summarises the selected spontaneous micro-expression datasets used in this study. In this table, the COMBINED dataset is denoted as δ.\n\nTable 2 records the recognition accuracy of the baseline Bi-WOOF,20 (denoted as BBW), optimized Bi-WOOF, (denoted as OBW), full-face graph, (denoted as FFG) and full-face graph with A-EMM, (denoted as FFG+M). The baseline Bi-WOOF is referred to the original method by Liong et al.,20 which was implemented using MATLAB, then the optimized Bi-WOOF refers to the implemented C++ version that accelerates the computation performance for real-time analysis. All the four experimental setups utilized the Support Vector Machines (SVM) classifier with a Radial basis function (RBF) kernel. The SVM hyper-parameter selection is based on the recommendation in Bergstra and Bengio.30 This is described as an optimised hyper-parameter selection technique for best classification performance in comparison to the sequential tuning in a context of a model with many hyper-parameters. In addition, all measured accuracies are based on LOSOCV. Similarly, the COMBINED dataset is denoted as δ in Table 2.\n\n\nResults\n\nTable 2 presents the recognition accuracies of the baseline Bi-WOOF (denoted as BBW), the optimized Bi-WOOF (denoted as OBW), full-face graph (denoted as FFG) and the full-face graph with A-EMM (denoted as FFG+M). Here, the BBW and OBW yield the highest recognition accuracies of 66.01% and 69.15%, respectively, over the COMBINED dataset. Similarly, FFG and FFG+M yield the highest recognition accuracy of 77.05% and 77.85%, respectively, over the COMBINED dataset. From these results, it is observed that the OBW improved the performance of the BBW by up to 3.28% over the SAMM dataset. On the other hand, the implemented full-face graph with A-EMM improved the performance by up to 1.20% over the SAMM dataset. Then, in comparison with optimized Bi-WOOF and full-graph with A-EMM improved the performance by 9.72%, 11.23%, 18.32%, 7.03% and 8.7% over SMIC, CASMEII, CAS (ME)2, SAMM and COMBINED datasets, respectively. Moreover, Table 3 compares the accuracies of the optimized BiWOOF with other motion-based methods, where Table 4 compares the accuracies of the full-face graph with other geometric-based methods.\n\n\nDiscussion\n\nTable 3 lists the performance of benchmark motion-based methods,6-9,20,31,32 with the optimized Bi-WOOF. The best reported accuracy is 74.06% over the CASMEII dataset.6 Looking at Bi-WOOF+Phase,32 the highest performance reported is 68.29% over the SMIC dataset, which outperformed the baseline and the optimized Bi-WOOFs. However, the optimized Bi-WOOF outperformed the reported accuracies in other studies,7-9,31 as shown in Table 3.\n\nOn the other hand, Table 4 lists the benchmark geometric-based methods15-19 with the full-face graph and full-face graph + A-EMM, which are denoted as experiment I and experiment II, respectively. From these results, Buhari et al.,18 reported the highest accuracies of 76.67%, 75.04%, 81.41%, and 87.33% over the SMIC, CASMEII, CAS (ME)2, and SAMM datasets, respectively. In Buhari et al.,18 the full-face graph utilized 68 landmarks from the raw images (denoted as ℝ). While, the full-face graph in experiment I and experiment II yield 74.62%, 74.41%, 75.11%, 74.33% and 75.01%, 74.55%, 76.21%, 75.53% over the SMIC, CASMEII, CAS (ME)2 and SAMM datasets, respectively. From these results, the full-face graph in experiment II outperformed the full-face graph presented in18 with 8.11%, 1.1%, and 3.38% over the SMIC, CASMEII, and CAS (ME)2 datasets, respectively. While, on the other hand, Buhari et al.,18 outperformed the results in experiment II with 4.7% over the SAMM dataset. While, in comparison with reported accuracies presented in Table 3, the full-face graph in experiment II achieved the highest performance.\n\nLooking at the performance presented in Tables 3 and 4, the performance reported in Buhari et al.,18 registered the highest accuracy of 87.33% over SAMM datasets, respectively. While, the full- face with A-EMM outperformed the full-face graph performance presented in Buhari et al.18 From these results, the conclusion can be drawn that the geometric-based features are competing closely with the motion-based features. In terms of the computational time, the optimized Bi-WOOF running time is 0.36 seconds per sample (i.e., 2.7fps), while the full-face graph running time is 0.10 seconds per sample (i.e., 10fps), with a 640×480 image resolution, on an Intel i5-3470 machine. The reported running times include facial landmark detection and classification.\n\n\nConclusions\n\nThis paper analyzed the performance of motion-based features (i.e., Bi-WOOF) and geometric-based features (i.e., Full-face graph) for real-time micro-expression recognition systems. The results indicate that the optimized Bi-WOOF improved recognition accuracy of the baseline Bi-WOOF by up to 3.28% over the SAMM dataset. While, on the other hand, the full-face graph performance is improved by up to 1.20% with A-EMM over the SAMM dataset. Moreover, the full-face graph and full-face graph with A-EMM exhibit significant performance improvement over the baseline and the optimized Bi-WOOF by up to 18.32%. Though the full-face graph improved performance of recognition accuracy, the processing time could limit the readiness of the full-face graph features for real-time systems using high-speed cameras.\n\n\nData availability\n\nThe experiments were performed using four spontaneous datasets: (i) spontaneous micro-expression dataset (SMIC) dataset,25 (ii) Chinese Academy of Sciences Micro-expression (CASMEII) dataset,26 (iii) spontaneous macro-expressions and micro-expressions (CAS (ME)2) dataset,27 and (iv) spontaneous actions and micro-movements (SAMM) dataset.28 These are spontaneous micro-expression datasets which were used in this study with full details of these datasets in Li et al.,25 Yan et al.,26 Qu et al.,27 and Davison et al.28 Details to acquire these datasets used in this study are available at www.oulu.fi/cmvs/node/41319 for SMIC,25 fu.psych.ac.cn/CASME/casme2-en.php for CASMEII,26 fu.psych.ac.cn/CASME/cas (me)2-en.php for CAS (ME)2,27 and personalpages.manchester.ac.uk/staff/adrian.davison/SAMM.html for SAMM.28 Moreover, to evaluate the performance using a larger dataset, this paper merged the four datasets to form a COMBINED dataset. The COMBINED dataset is created from the raw images of all the four datasets with the source code available under extended data.\n\nZenodo: Implementation of COMBINED micro-expression dataset and Setup files for real-time micro-expression recognition using motion and geometric features. https://doi.org/10.5281/zenodo.5524141.33\n\nThe project contains the following extended data:\n\n• Real-time micro-expression recognition using biwoof features (executable setup for micro-expression recognition using motion-based features).\n\n• Real-time micro-expression recognition using full-face graph features (executable setup for micro-expression recognition using geometric-based features).\n\n• Image re-scaler for COMBINED micro-expression dataset formation (Visual Studio 2010 source code written in C++).\n\nData are available under the terms of the Creative Commons Zero (CC0 v1.0 Universal).\n\nZenodo: Performance analysis of micro-expression recognition over different sample image sizes. https://doi.org/10.5281/zenodo.5379773.34\n\nThis project contains the following extended data:\n\n• Performance improvement over 140×170 sample size.\n\n• Performance improvement over 240×340 sample size.\n\n• Performance improvement over 560×680 sample size.\n\n• Performance improvement over 1120×1360 sample size.\n\nData are available under the terms of the Creative Commons Zero (CC0 v1.0 Universal).\n\n\nAuthor contributions\n\nConceptualization, Investigation, Methodology, Formal Analysis, Software, Visualization, Writing – Original Draft Preparation, Review & Editing; AM Buhari: Conceptualization, Resources, Formal Analysis, Methodology, Supervision, Writing – Review & Editing; CP Ooi: Conceptualization, Formal Analysis, Supervision, Validation, Writing – Review & Editing; VM Baskaran. Conceptualization, Methodology, Writing – Review & Editing; WH Tan.\n\n\nCompeting interests\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.", "appendix": "Acknowledgements\n\nThis work is supported in part by Research Management Centre (RMC), Multimedia University (MMU) Cyberjaya. In addition, I would like to thank Professor RaphaÃ«l C. W. Phan and Dr. KokSheik Wong for their guidance, assistance and support in all aspects of this study.\n\n\nReferences\n\nO’Sullivan M, Frank MG, Hurley CM, et al.: Police lie detection accuracy: The effect of lie scenario. Law Hum Behav. 2009; 330(6): 530. PubMed Abstract \| Publisher Full Text\n\nFrank MG, Maccario CJ, Govindaraju V: Behavior and security. protecting airline passengers in the age of terrorism.2009.\n\nCohn JF, Kruez TS, Matthews I, et al.: Detecting depression from facial actions and vocal prosody. 2009 3rd Int Conf Affective Computing Intelligent Interaction Workshops. IEEE;2009; pages 1–7. Publisher Full Text\n\nEkman P, Friesen WV: Nonverbal leakage and clues to deception. Psychiatry. 1969; 32(1): 88–106. Publisher Full Text\n\nShreve M, Godavarthy S, Manohar V, et al.: Towards macro-and micro-expression spotting in video using strain patterns. 2009 Workshop on Applications of Computer Vision (WACV). IEEE;2009; pages 1–6. Publisher Full Text\n\nSze-Teng L, Gan YS, Yau W-C, et al.: Off-apexnet on micro-expression recognition system. arXiv preprint arXiv. 2018a: 1805.08699. Publisher Full Text\n\nLi Q, Zhan S, Xu L, et al.: Facial micro-expression recognition based on the fusion of deep learning and enhanced optical flow. Multimedia Tools and Applications 2019; 78 (20):0 29307–29322. Publisher Full Text\n\nDuque CA, Alata O, Emonet R, et al.: Mean oriented riesz features for micro expression classification. Pattern Recognition Letters. 2020; 135: 382–389. Publisher Full Text\n\nMerghani W, Yap MH: Adaptive mask for region-based facial micro-expression recognition. 2020 15th IEEE Int Conf Automatic Face and Gesture Recognition (FG 2020)(FG). IEEE Computer Society;2020; pages 428–433. Publisher Full Text\n\nYang L, Zhang X, Zhou J, et al.: Graph-based facial affect analysis: A review of methods, applications and challenges. CoRR, abs/2103. 2021a: 15599.Reference Source\n\nKazemi V, Sullivan J: One millisecond face alignment with an ensemble of regression trees. Proc IEEE conf computer vision pattern recognition. 2014: 1867–1874. Publisher Full Text\n\nMohseni S, Zarei N, Ramazani S: Facial expression recognition using anatomy based facial graph. 2014 IEEE Int Conf Systems, Man, and Cybernetics (SMC). IEEE;2014; pages 3715–3719. Publisher Full Text\n\nBaltrušaitis T, Robinson P, Morency L-P: Openface: an open source facial behavior analysis toolkit. 2016 IEEE Winter Conf Applications Computer Vision (WACV). IEEE;2016; pages 1–10. Publisher Full Text\n\nDapogny A, Bailly K, Dubuisson S: Confidence-weighted local expression predictions for occlusion handling in expression recognition and action unit detection. Int J Computer Vision. 2018; 126 (2): 255–271. Publisher Full Text\n\nLei L, Li J, Chen T, et al.: A novel graph-tcn with a graph structured representation for micro-expression recognition. Proc 28th ACM Int Conf Multimedia. 2020: 2237–2245.\n\nXie H-X, Lo L, Shuai H-H, et al.: Au-assisted graph attention convolutional network for micro-expression recognition. Proc 28th ACM Int Conf Multimedia. 2020: 2871–2880. Publisher Full Text\n\nZhou L, Mao Q, Dong M: Objective class-based micro-expression recognition through simultaneous action unit detection and feature aggregation. arXiv preprint arXiv. 2020: 2012.13148.\n\nBuhari AM, Ooi C-P, Baskaran VM, et al.: Facs-based graph features for real-time micro-expression recognition. J Imaging. 2020; 6(12): 130. Publisher Full Text\n\nLiu Y-J, Li B-J, Lai Y: Sparse mdmo: Learning a discriminative feature for micro-expression recognition. IEEE Transactions on Affective Computing. 2021b. Publisher Full Text\n\nLiong S-T, See J, Wong KS, et al.: Less is more: Micro-expression recognition from video using apex frame. Signal Processing: Image Communication. 2018b; 62: 82–92. Publisher Full Text\n\nKing DE: Dlib-ml: A machine learning toolkit. J Machine Learning Res. 2009; 100 (Jul): 1755–1758.\n\nLiong S-T, See J, Wong KS, et al.: Automatic apex frame spotting in micro-expression database. 2015 3rd IAPR Asian conf pattern recognition (ACPR). IEEE;2015; pages 665–669. Publisher Full Text\n\nWadhwa N, Rubinstein M, Durand F, et al.: Riesz pyramids for fast phase-based video magnification US Patent 9,338,331. May 10 2016.\n\nLe Ngo AC, Yee-Hui O, Phan RC-W, et al.: Eulerian emotion magnification for subtle expression recognition. 2016 IEEE Int Conf Acoustics, Speech Signal Processing (ICASSP). IEEE;2016; pages 1243–1247. Publisher Full Text\n\nLi X, Pfister T, Huang X, et al.: A spontaneous micro-expression database: Inducement, collection and baseline. 2013 10th IEEE Int Conf Workshops Automatic Face Gesture Recognition (FG). IEEE;2013: 1–6. Publisher Full Text\n\nYan W-J, Li X, Wang S-J, et al.: Casme ii: An improved spontaneous micro-expression database and the baseline evaluation. PloS one. 2014; 90(1): e86041. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFangbing Q, Wang S-J, Yan W-J, et al.: Cas (me)2: A database for spontaneous macro-expression and micro-expression spotting and recognition. IEEE Transactions on Affective Computing. 2018; 90(4): 424–436. Publisher Full Text\n\nDavison AK, Lansley C, Costen N, et al.: Samm: A spontaneous micro-facial movement dataset. IEEE Transactions on Affective Computing. 2018; 90(1): 116–129. Publisher Full Text\n\nBuhari AM, Ling H-C, Baskaran VM, et al.: Real-time high-resolution downsampling algorithm on many-core processor for spatially scalable video coding. J Electronic Imaging. 2015; 24(1): 013025. Publisher Full Text\n\nBergstra J, Bengio Y: Random search for hyper-parameter optimization. J Machine Lear Res. 2012; 130(2).\n\nLi X, Hong X, Moilanen A, et al.: Towards reading hidden emotions: A comparative study of spontaneous micro-expression spotting and recognition methods. IEEE transactions on affective computing. 2017; 90(4): 563–577. Publisher Full Text\n\nLiong S-T, Wong KS: Micro-expression recognition using apex frame with phase information. 2017 Asia-Pacific Signal Information Processing Association Annual Summit and Conference (APSIPA ASC). IEEE;2017; pages 534–537. Publisher Full Text\n\nBuhari AM, Ooi C-P, Baskaran VM, et al.: Implementation of COMBINED micro-expression dataset and Setup files for real-time micro- expression recognition using motion and geometric features.September 2021a. Publisher Full Text\n\nBuhari AM, Ooi C-P, Baskaran VM, et al.: Performance analysis of micro-expression recognition over different sample image sizes.September 2021b. Publisher Full Text" }	[ { "id": "96609", "date": "25 Oct 2021", "name": "Madhumita Takalkar", "expertise": [ "Reviewer Expertise Computer Vision", "Deep Learning", "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 paper talks about optimizing Bi-WOOF and using geometric based features to detect and recognise facial micro-expressions. Please find my comments as below.\n\nThe idea does not sound novel. There have been further developments in the detection and recognition of micro-expressions which includes deep learning models which exhibit higher accuracies than presented here in the paper. Why would the authors not compare with the latest methods? Following this comment, there are two papers the authors can refer: Madhumita et al. 20211 and Thuseethan et al. 20212.\n\nBuhari et al. (2020)3 performs better and presents higher accuracy than the proposed methods. What is the message behind the proposed method? Is the computation time reduced with the proposed method if not the accuracy improvement?\n\nSMIC database does not have the apex frame annotated. Did the author perform any methods to identify the apex frame? If so that description is missing.\n\nThe paper says Buhari et al. (2020)3 performs detection and recognition on the raw images. How are the authors comparing raw images with processed images? This may mean that processing the images is reducing the accuracy.\n\nConsidering the limited samples in the datasets, only accuracy may not be sufficient to prove the efficiency of the developed model. It would be advisable to include other performance evaluation metrics such as AUC, F1 score, and Confusion matrix.\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": "101402", "date": "06 Dec 2021", "name": "Harshadkumar B. Prajapati", "expertise": [ "Reviewer Expertise Machine learning", "image processing", "distributed computing" ], "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 manuscript performs experimental evaluation of two different methods: motion based and geometric based for facial expression classification. The manuscript contains some experiments and results, but the aims of the experiments appear to be random.\n\nThe focus is on image sequences, which is addressed by motion based feature, but why geometric based feature? Does it deal with image sequences?\n\nThe authors straightway use Bi-WOOF, a descriptor proposed by some other author. It is not justified why Bi-WOOF has been used; there are many other features. This choice should be scientifically justified.\n\nThere are many descriptors that can specify motion in image sequences. The authors should discuss such descriptors.\n\nCurrent trend is applying Deep learning for ML problems; however, the authors use SVM (traditional ML model) for classification. Why? The readers must be convinced of the choice of SVM.\n\nThe authors present results in Table 2 and Table 3, but it is not conveyed what are authors’ contributions in those.\n\nIn Table 3, at many places (-) is marked. Why? Have the authors have done these experiments? If YES, then why are many blank (-), if NO, then why are others' results included? Where are the results of the authors?\n\nIntroduction section needs to be revised. An introduction section generally includes paragraphs on the following: (1) Motivation for the work, (2) Background/Technical information or terms, (3) Problem being addressed, (4) Existing major solutions and research gaps, and (5) Proposal of this paper and its significance.\n\nAs Section (Methods) directly starts proposal using Bi-WOOF without any background concepts or theory, readers cannot follow/understand/appreciate the proposed work.\n\nThe manuscript should discuss the proposal with suitable diagram(s), showing frame/video/image processing.\n\nThe authors should show frame sequences of at least one sample from any one dataset.\n\nPresentation\nThe manuscript is very difficult to read for beginners; when any section starts, it must contain a brief paragraph discussing the content. E.g., in Section (Method), the authors directly discuss three different methods in Fig. 1, Fig. 2, and Fig. 3, but it is not clear how these are related to their proposal.\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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-1029
https://f1000research.com/articles/10-514/v1	29 Jun 21	{ "type": "Systematic Review", "title": "The effectiveness of EMR implementation regarding reducing documentation errors and waiting time for patients in outpatient clinics: a systematic review", "authors": [ "Salem Albagmi" ], "abstract": "Background: Electronic medical records (EMRs) refer to the digital copies of paper notes prepared in the physician’s office, outpatient clinics and other departments in health care institutes. EMRs are considered to be significant and preferable to paper records because they allow providers to keep accurate track of patient data and monitoring over time, thus reducing errors, and enhance overall health care quality. The aim of this systematic review was to highlight the significance of EMRs and the effectiveness of implementation regarding reducing documentation errors and waiting time for patients in outpatient clinics. Methods: PubMed, Central, Ovid, Scopus, Science Direct, Elsevier, Cochrane , WHO website and the McMaster University Health Evidence website from 2005-2020 were searched to identify studies that investigated the association between the EMR implementation and documentation error and waiting time for patients. A reviewer screened identified citations and extracted data according to the PRISMA guidelines and data was synthesized in a narrative manner. Results: After full text examination of the articles selected for this literature review, the major themes of relevance that were identified in the context of reducing documentation errors and waiting time for patients in outpatient clinic include: reduction of medical errors because of fewer documentation errors resulting from EMR implementation and reduction of waiting time for patients due to overall improvement of system workflow after use of EMRs. Conclusion: In summary of the reviewed evidence from published material, the implementation of an EMR system in any outpatient setting appears to reduce documentation errors (medication dose errors, issues of prescription errors). It was also seen that in many settings, waiting time for patients in outpatient clinics was reduced with EMR use, while in other settings it was not possible to determine if any significant improvement was seen in this aspect after EMR implementation.", "keywords": [ "EMR", "medical documentation", "documentation errors" ], "content": "Introduction\n\nThe general definition of electronic medical record (EMR) systems is an electronic record of health-associated data of a person which can be made, collected, organized and consulted by authorized doctors as well as health care staff in a health care institution. EMRs usually refer to digital copies of the paper notes made in the doctor’s office, outpatient clinics and other health care institutions (Hyman et al., 2012). EMRs consist of the doctor’s official notes and other data or information documented by and for the doctors within that office, clinic, or health care institution (Manca, 2015). EMRs are generally utilized by the providers for diagnostic purposes and treatment (George and Bernstein, 2009). EMRs are far more important than real paper records since they allow the providers to keep accurate track of patient data over time, help in identifying patients who have to be scheduled for preventive care or vital screenings, allow consistent monitoring of patients, and enhance the overall provided health care quality (DesRoches et al., 2008; Donsa et al., 2016; Cloete, 2015).\n\nThe EMR system once implemented has the acumen to give significant advantages to doctors and outpatient clinical health centers as well as the patients and the overall system of health care institutes (Agrawal and Wu, 2009). The EMR system has been shown to smooth-out the workflow pathways while enhancing the overall quality of patient care as well as patient safety (DesRoches et al., 2008; Franklin and Puaar, 2019). The benefits provided by the implementation of an EMR system in a health care facility are many and include the following:\n\n1) Doctors and other health care staff like the nursing staff have quick and instant access to patient medical data or records like previous diagnoses, known drug reactions as well as allergies, laboratory results, and currently prescribed drugs (DesRoches et al., 2008; Amato et al., 2017).\n\n2) It is possible for the outpatient clinic staff to gain access to new and past test results from other departments or even other providers in clinics that use multiple care services (Manca, 2015).\n\n3) Computerized provider order entry (CPOE) is an easier and quicker way of entering and sending medical, laboratorial, radiological and pharmacy prescriptions than paper documents, thus reducing the probability of errors.\n\n4) EMRs are linked to provision of computerized decision-support systems which are accurate in preventing adverse drug interactions and enhancing the overall use of best clinical practices in outpatient settings (Kavanagh, 2017).\n\n5) Use of EMRs ensures that there is safe, private electronic communication between the health care providers and patients (Kavanagh, 2017).\n\n6) The patient is also able to access their personal health records, disease management strategies as well as other needed health-related information sources which was not always possible with paper records (Noraziani et al., 2013).\n\n7) Implementation of an EMR system also computerizes the patient administration systems like their scheduling protocols (Noraziani et al., 2013).\n\n8) EMRs provide standardized and streamlined methods of storing electronic data and allow timely reporting that in turn improves patient safety as well as disease surveillance programs (Radley et al., 2013; Noraziani et al., 2013).\n\nMedication errors and increased waiting time for patients due to manual documentation on paper have been found to be significantly associated with compromised health care quality (Keers et al., 2013). There should be a system that can be used to minimize errors and improve the quality of life of patients. Literature is available discussing the magnificent contribution of EMRs and they have been found to improve the health care quality (Wali et al., 2020; Lin et al., 2020). However, limited studies are available discussing the variables that we have considered in our study as the outcome variable. Therefore, we conducted this systematic review to particularly focus on the reduction in documentation errors and patient waiting time in the outpatient clinic setting by the implementation of EMRs and provide an evidence-based infrastructure to healthcare institutions for better services and facilities.\n\n\nMethods\n\nThe Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines have been used in this literature review since this is an already validated methodology for choosing the final articles to be used from the results obtained within published literature relevant to this research topic (Albagmi, 2020). The PRISMA guidelines, as well as being the format for conducting and reporting systematic literature reviews, were selected due to the fact that the overall conceptual ideas and the main aims of the research topic of this project are well suited to the PRISMA guidelines. In addition, in order to carry out an accurate or valid literature review for the purposes of summarizing the various aspects of a chosen public health topic, reviews have been documented as being of more relevance when using already validated methodologies like the PRISMA guidelines (Moher et al., 2009; Page et al., 2021).\n\nThe search strategy was performed in a manner that the methodology can be repeated and employed (see Box 1). A systematic search of the literature published between 2005 and 2020 was performed on PubMed, Central, Ovid, Scopus, Science Direct, Elsevier, Cochrane using free keywords. The reference lists of relevant articles (including primary and secondary results), and grey literature (including PROSPERO and reports of relevant stakeholder organizations - WHO website and the McMaster University Health Evidence website) were screened. The search for relevant articles to be included in the literature review was done after first choosing the appropriate key words and key concepts pertaining to the main research goals of this literature review.\n\nSearch: efficiency or impact which EMR implementation, outpatient clinics\n\n\"efficiences\"[All Fields] OR \"efficiency\"[MeSH Terms] OR \"efficiency\"[All Fields] OR \"efficiencies\"[All Fields] OR \"efficient\"[All Fields] OR \"efficiently\"[All Fields] OR \"efficients\"[All Fields] OR ((\"impact\"[All Fields] OR \"impactful\"[All Fields] OR \"impacting\"[All Fields] OR \"impacts\"[All Fields] OR \"tooth, impacted\"[MeSH Terms] OR (\"tooth\"[All Fields] AND \"impacted\"[All Fields]) OR \"impacted tooth\"[All Fields] OR \"impacted\"[All Fields]) AND (\"empir musicol rev\"[Journal] OR \"emr\"[All Fields]) AND (\"implementability\"[All Fields] OR \"implementable\"[All Fields] OR \"implementation\"[All Fields] OR \"implementation s\"[All Fields] OR \"implementational\"[All Fields] OR \"implementations\"[All Fields] OR \"implementer\"[All Fields] OR \"implementers\"[All Fields] OR \"implemention\"[All Fields]) AND (\"ambulatory care facilities\"[MeSH Terms] OR (\"ambulatory\"[All Fields] AND \"care\"[All Fields] AND \"facilities\"[All Fields]) OR \"ambulatory care facilities\"[All Fields] OR (\"outpatient\"[All Fields] AND \"clinics\"[All Fields]) OR \"outpatient clinics\"[All Fields]))\n\nTranslations\n\nefficiency: \"efficiences\"[All Fields] OR \"efficiency\"[MeSH Terms] OR \"efficiency\"[All Fields] OR \"efficiencies\"[All Fields] OR \"efficient\"[All Fields] OR \"efficiently\"[All Fields] OR \"efficients\"[All Fields]\n\nimpact: \"impact\"[All Fields] OR \"impactful\"[All Fields] OR \"impacting\"[All Fields] OR \"impacts\"[All Fields] OR \"tooth, impacted\"[MeSH Terms] OR (\"tooth\"[All Fields] AND \"impacted\"[All Fields]) OR \"impacted tooth\"[All Fields] OR \"impacted\"[All Fields]\n\nEMR: \"Empir Musicol Rev\"[Journal:__jid101513485] OR \"emr\"[All Fields]\n\nimplementation,: \"implementability\"[All Fields] OR \"implementable\"[All Fields] OR \"implementation\"[All Fields] OR \"implementation's\"[All Fields] OR \"implementational\"[All Fields] OR \"implementations\"[All Fields] OR \"implementer\"[All Fields] OR \"implementers\"[All Fields] OR \"implemention\"[All Fields]\n\noutpatient clinics: \"ambulatory care facilities\"[MeSH Terms] OR (\"ambulatory\"[All Fields] AND \"care\"[All Fields] AND \"facilities\"[All Fields]) OR \"ambulatory care facilities\"[All Fields] OR (\"outpatient\"[All Fields] AND \"clinics\"[All Fields]) OR \"outpatient clinics\"[All Fields]\n\nThe formulated keywords and concepts were: “efficiency or impact which EMR implementation has had in terms of reducing documentation errors in outpatient clinics” and “efficiency or impact which EMR implementation has had in terms of reducing the waiting time for patients in outpatient clinics.” In accordance with chosen search terms, the study search made use of the keywords as well as the phrases in combination with the Boolean operators “AND” and “OR” in the following manner for search in relevant databases: “efficiency or impact which EMR implementation has had in terms of reducing documentation errors in outpatient clinics” and/or “efficiency or impact which EMR implementation has had in terms of reducing the waiting time for patients in outpatient clinics” (Cronin, Ryan and Coughlan, 2008; Timmins & McCabe, 2005; Wakefield, 2014).\n\nEach of the chosen databases and websites were used for the search keeping in mind the inclusion and exclusion criteria to find relevant published articles. The inclusion/exclusion criteria used were as follows.\n\nInclusion criteria:\n\n1. All disease and health settings.\n\n2. Articles in the English language.\n\n3. Peer-reviewed articles published 2005-2020.\n\n4. Studies that reported the impact of EMR in reducing documentation error and patient’s waiting time in outpatient clinic settings, either in segregation or as one of the reporting outcomes in a multicomponent studies, were included.\n\nExclusion criteria:\n\n1. Reminder services for appointment and other healthcare activities were not included.\n\n2. Conference proceeding and abstracts were also excluded.\n\nStudies were independently assessed by a single reviewer (SA) and were reported using the PRISMA flow diagram (Figure 1). Initial screening of studies was based on the information mentioned in the titles and abstracts. Full-paper screening was conducted by the same independent reviewer and extracted in to a standardized structure. A form was created, focused on the detail study characteristics. Primarily, the reviewer collected the data in the form and subsequently it was managed in a master Microsoft Excel sheet (2019) and then tabulated separately for each study. Study investigators were not approached to confirm the data as the included studies were well-defined and outcome was clearly mentioned.\n\nThe data collected for each study included the name of first author, year of publication, setting characteristics, study duration, participants and, outcomes.\n\nPrimary outcomes were any measure related to effectiveness of EMR in:\n\n1. Reducing documentation error,\n\n2. Reducing patient waiting time.\n\nDefinitions of variables that were sought out are described in Table 1.\n\nThis was a systematic review and hence did not require any statistical analysis. Data was synthesized in a narrative manner. Study quality and bias assessment was done by using a hybrid of the Downs and Black scale and the Newcastle-Ottawa Scale that includes 11 items (Downs et al., 1998; Wells et al., 2017) under four themes of reporting, external validity, internal validity for bias and internal validity for confounding to capture the observational and experimental study designs\n\n\nResults\n\nA search of the above-mentioned chosen databases and sources and the results obtained are shown in Figure 1 (with the use of the PRISMA flowchart for the search strategy for the research question). This literature search made use of many relevant and varied sources so as to obtain the most accurate articles.\n\nAfter the search was done, a total of 93 articles were found from the databases and other sources. After examining for and removing the duplicates, 30 articles were left. Through the application of the above-mentioned inclusion or exclusion criteria, nine articles were excluded. This resulted in a remainder of 21 articles which were further examined for relevance to the aims of this study’s topic. Following this, nine articles were further excluded for not having relevance in determining the effectiveness of EMR implementation in terms of decreasing or reducing documentation errors and waiting time for patients in outpatient clinics. The remaining 12 articles were utilized for this literature review on the basis of their being relevant to the main aims of this research study (see Figure 1 for the PRISMA flowchart) (Moher et al., 2009; Page et al., 2021).\n\nThe findings and data of the articles chosen for inclusion in this literature review are as shown in Table 2.\n\nThe results of the risk of bias assessment are presented in Figure 2. For question number 6, not all studies recruited participants; out of 12, three studies recruited participants and other studies evaluated databases.\n\nThe review of the selected articles revealed two key themes in the context of how effective the implementation of the EMR system is in reducing documentation errors and waiting time for patients in outpatient clinics, namely: there is a reduction in medical errors because of fewer documentation errors and there is some degree of reduction of waiting time for patients due to overall improvement of system workflow in some settings, after use of EMRs. The two themes tend to overlap and are interlinked. Thus, each aspect has an impact on the other factors and may ultimately improve care quality for patients attending outpatient visits.\n\n\nDiscussion\n\nThe main objectives of this study were to do a literature review to identify the effectiveness of EMR implementation in regard to reducing documentation errors and waiting time for patients in outpatient clinics. The data collected and the emerging themes indicate that there is a decrease in medical errors as a direct result of fewer documentation errors in EMR use (Priestman et al., 2018; DesRoches et al., 2008; Manca, 2015; Radley et al., 2013) and there is a decrease in waiting time for patients because of overall improvement of system workflow in some settings, after use of EMRs (Jabour, 2020; AlSarheed, 2016; Cho et al., 2017; Noraziani et al., 2013; Jin, Sivakumar and Lim, 2013), although a contrasting increase was observed in some outpatient studies (Vahdat et al., 2018).\n\nThe implementation of EMRs in outpatient or ambulatory clinics by providers in the US in one study showed a beneficial effect in reducing documentation errors, which in turn enhanced several factors of overall patient care. Improvements were seen in the quality of clinical decisions, quick communication between providers as well as with patients, timely access to all patient medical records in one place and prevention of medication errors in documentation (DesRoches et al., 2008). Many studies have shown that implementation of an EMR permits doctors to avoid documentation errors since there is improved access to comprehensive patient histories such as clinical data (Ruano et al., 2016). This in turn reduces waiting time for the patients as doctors spend less time looking for lab reports and other patient data.\n\nThe EMR implementation thus provides advantages like fast remote access to patient history, enhanced laboratory result access, medication error alerts, and reminders for scheduling preventive care (Manca, 2015; Radley et al., 2013). It was also observed that implementation of various forms of the EMR systems can be a vital tool in improving patient safety in outpatient and inpatient settings (Agrawal and Wu, 2009). As a point of comparison, it was seen that researchers studied and compared the impact of errors in paper-based and computerized EMR diabetes management with decision support in hospitalized patients with type 2 diabetes (Donsa et al., 2016; Vishwanath, Singh and Winkelstein, 2010). This could be applicable in outpatient clinics too. The results from one study suggest that software improvements and enhancement of the EMR system in a Saudi hospital’s outpatient clinics reduced the waiting time for their patients (Al Sarheed, 2016).\n\nHowever, some studies have shown contrasting results to those showing improvements in waiting times of outpatient clinic visitors. A study designed to quantify the downstream effect on patient wait times as well as the total duration of stay because of tiny increments in encounter times resulting from implementation of a new EMR system indicated that use of EMRs added extra documentation time in outpatient clinics and overall led to significant delays in the waiting times for patients (Vahdat et al., 2018).\n\nThis review has some limitations. First, due to the strict exclusion and inclusion criteria, the studies that were relevant according to the one of the outcomes but not to the main question were not included. Conference proceedings were excluded that might have contained findings of worth. Second, there was a lack of data within studies regarding setting, type of study and population. In addition, a large number of studies were found on documentation errors as compared to patient waiting time.\n\n\nConclusion\n\nIn summary, EMR implementation appears to enhance documentation, screening performance as well as prescription error prevention. The findings presented in this literature review will be of value to outpatient clinics and units that are looking to implement EMR systems. The waiting time for outpatient clinic visits can also be reduced by EMR use as there is implementation of new tools and functionality like the Web-based booking system, which can reduce the waiting time as well as duration of patients’ visits (Jabour, 2020; Noraziani et al., 2013). However, this needs to be further validated in future studies as some studies actually indicate that implementation of an EMR system led to increased waiting times and delays in care provision in outpatient clinics.\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 “The effectiveness of EMR implementation regarding reducing documentation errors and waiting time for patients in outpatient clinics: a systematic review” https://doi.org/10.6084/m9.figshare.14791356.v1 (Albagmi, 2021).\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\nAdurota O: Implementation of Electronic Health Records in an Outpatient Clinic in Maryland. Doctoral dissertation, Brandman University; 2019.\n\nAgrawal A, Wu WY: Reducing medication errors and improving systems reliability using an electronic medication reconciliation system. Jt Comm J Qual Patient Saf. 2009; 35(2): 106–114. PubMed Abstract \| Publisher Full Text\n\nAlbagmi S: May_PRISMA_2020_checklist_45039.pdf. figshare. Figure. 2021. Publisher Full Text\n\nAlSarheed AH: The Impact of Enhancing Outpatient Clinic Management Software in Reducing Waiting Time in Saudi Hospitals. Doctoral dissertation, Prince Sultan University; 2016. Retrieved on 20th September, 2020. Reference Source\n\nAmato MG, Salazar A, Hickman TTT, et al.: Computerized prescriber order entry–related patient safety reports: analysis of 2522 medication errors. J Am Med Inform Assoc. 2017; 24(2): 316–322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCho KW, Kim SM, Chae YM, et al.: Application of queuing theory to the analysis of changes in outpatients' waiting times in hospitals introducing EMR. Healthc Inform Res. 2017; 23(1): 35–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCloete L: Reducing medication errors in nursing practice. Cancer Nursing Practice. 2015; 14(1). PubMed Abstract \| Publisher Full Text\n\nCronin P, Ryan F, Coughlan M: Undertaking a literature review: a step-by-step approach. Br J Nurs. 2008; 17(1): 38–43. PubMed Abstract \| Publisher Full Text\n\nDowns SH, Black N: The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health. 1998; 52(6): 377–384. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesRoches CM, Campbell EG, Rao SR, et al.: Electronic health records in ambulatory care—a national survey of physicians. N Engl J Med. 2008; 359(1): 50–60. PubMed Abstract \| Publisher Full Text\n\nDonsa K, Beck P, Höll B, et al.: Impact of errors in paper-based and computerized diabetes management with decision support for hospitalized patients with type 2 diabetes. A post-hoc analysis of a before and after study. Int J Med Inform. 2016; 90: 58. PubMed Abstract \| Publisher Full Text\n\nFranklin BD, Puaar S: What is the impact of introducing inpatient electronic prescribing on prescribing errors? A naturalistic stepped wedge study in an English teaching hospital. Health Informatics J. 2019; 1460458219833112. PubMed Abstract \| Publisher Full Text\n\nHyman D, Laire M, Redmond D, et al.: The use of patient pictures and verification screens to reduce computerized provider order entry errors. Pediatrics. 2012; 130(1): e211–e219. PubMed Abstract \| Publisher Full Text\n\nJabour AM: The Impact of Electronic Health Records on the Duration of Patients’ Visits: Time and Motion Study. JMIR Med Inform. 2020; 8(2): e16502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin X, Sivakumar AI, Lim SY: A simulation based analysis on reducing patient waiting time for consultation in an outpatient eye clinic. 2013 Winter Simulations Conference (WSC). IEEE; 2013; (pp. 2192–2203). Publisher Full Text\n\nKavanagh C: Medication governance: preventing errors and promoting patient safety. Br J Nurs. 2017; 26(3): 159–165. PubMed Abstract \| Publisher Full Text\n\nKeers RN, Williams SD, Cooke J, et al.: Causes of medication administration errors in hospitals: a systematic review of quantitative and qualitative evidence. Drug Saf. 2013; 36(11): 1045–1067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin HL, Wu DC, Cheng SM, et al.: Association between Electronic Medical Records and Healthcare Quality. Medicine. 2020; 99(31). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManca DP: Do electronic medical records improve quality of care? Yes. Can Fam Physician. 2015; 61(10): 846. PubMed Abstract \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med. 2009; 151(4): 264–269. PubMed Abstract \| Publisher Full Text\n\nNoraziani K, Nurul’Ain A, Azhim MZ, et al.: An overview of electronic medical record implementation in healthcare system: Lesson to learn. World Applied Sciences J. 2013; 25(2): 323–332. Publisher Full Text\n\nPage MJ, Moher D, Bossuyt PM, et al.: PRISMA 2020 explanation and elaboration: updated guidance and exemplars for reporting systematic reviews. bmj. 2021; 372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPriestman W, Sridharan S, Vigne H, et al.: What to expect from electronic patient record system implementation: lessons learned from published evidence. J Innov Health Inform. 2018; 25(2): 92–104. PubMed Abstract \| Publisher Full Text\n\nRadley DC, Wasserman MR, Olsho LE, et al.: Reduction in medication errors in hospitals due to adoption of computerized provider order entry systems. J Am Med Inform Assoc. 2013; 20(3): 470–476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuano M, Villamañán E, Pérez E, et al.: New technologies as a strategy to decrease medication errors: how do they affect adults and children differently? World J Pediatr. 2016; 12(1): 28–34. PubMed Abstract \| Publisher Full Text\n\nSchwartzberg D, Ivanovic S, Patel S, et al.: We thought we would be perfect: medication errors before and after the initiation of Computerized Physician Order Entry. J Surg Res. 2015; 198(1): 108–114. PubMed Abstract \| Publisher Full Text\n\nSingh H, Graber ML, Kissam SM, et al.: System-related interventions to reduce diagnostic errors: a narrative review. BMJ Qual Saf. 2012; 21(2): 160–170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTimmins F, McCabe C: How to conduct an effective literature search. Nurs Stand. 2005; 20(11): 41–47. PubMed Abstract \| Publisher Full Text\n\nVahdat V, Griffin JA, Stahl JE, et al.: Analysis of the effects of EHR implementation on timeliness of care in a dermatology clinic: a simulation study. J Am Med Inform Assoc. 2018; 25(7): 827–832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVishwanath A, Singh SR, Winkelstein P: The impact of electronic medical record systems on outpatient workflows: a longitudinal evaluation of its workflow effects. Int J Med Inform. 2010; 79(11): 778–791. PubMed Abstract \| Publisher Full Text\n\nWakefield A: Searching and critiquing the research literature. Nurs Stand. 2014; 28(39): 49–57. PubMed Abstract \| Publisher Full Text\n\nWali RM, Alqahtani RM, Alharazi SK, et al.: Patient satisfaction with the implementation of electronic medical Records in the Western Region, Saudi Arabia, 2018. BMC Fam Pract. 2020; 21(1): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWells GA, Shea B, O’Connell DA, et al.: The Newcastle-Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses.2000." }	[ { "id": "88680", "date": "19 Jul 2021", "name": "Clair M. Sullivan", "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\nThank you for this paper.\nA few overarching comments:\nThis paper is very doctor centric. Although it is true that EMRs contain doctor's notes, they actually allow integration of notes from all members of a multidisciplinary team. Please review the paper to include this.\n\nThe two outcome variables chosen, reduced waiting time and reduced documentation errors, are both important; but I am curious as to why these two were selected out of the 100s of available impacts? Is there a logic behind this? If so, please outline as it appears a little random currently.\n\nThe terms ambulatory care and outpatients can be confusing. Are you referring to specialised ambulatory care services? Or primary care? These settings are vastly different and so it is difficult to compare a primary care provider in general practice to a specialised neurosurgery outpatients department.\n\nPlease review grammar and syntax as this appears a little clumsy at times e.g avoid calling EMRs magnificent\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? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "7160", "date": "11 Oct 2021", "name": "Salem Albagmi", "role": "Author Response", "response": "Comment 1: This paper is very doctor centric. Although it is true that EMRs contain doctor's notes, they actually allow integration of notes from all members of a multidisciplinary team. Please review the paper to include this. Page No. 3, Line No. 62-66, Actual Content Removed/ Edited/ Replaced Content: The following aspect is added in the introduction section Comment 2: The two outcome variables chosen, reduced waiting time and reduced documentation errors, are both important; but I am curious as to why these two were selected out of the 100s of available impacts? Is there a logic behind this? If so, please outline as it appears a little random currently. Page No. 5, Line No .97-107, Actual Content Removed/ Edited/ Replaced Content: Rationale for selecting both variables in the introduction section Comment 3: The terms ambulatory care and outpatients can be confusing. Are you referring to specialized ambulatory care services? Or primary care? These settings are vastly different and so it is difficult to compare a primary care provider in general practice to a specialized neurosurgery outpatients department. Removed/ Edited/ Replaced Content: Not catered - the paper is written and discussed in the context of specialized neurosurgery outpatients department" } ] }, { "id": "89947", "date": "03 Aug 2021", "name": "Shankar Srinivasan", "expertise": [ "Reviewer Expertise Health Outcomes Research", "Health Data Analytics" ], "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 describes the systematic review of published literature on the effectiveness of electronic health records in reducing documentation caused errors and also waiting time for ambulatory settings. The duration chosen is quite appropriate (2005 to 2020), given that the widespread implementation (at least in the US) was from 2009 onwards. The author has done due diligence with the selection of the manuscripts for the review by including (and specifying) the appropriate inclusion and exclusion criteria so that another researcher can reproduce the study accordingly. The analyses, results and conclusions drawn are robust and well written in responding specifically to the outcomes in question and also the results for those two outcomes.\nThe author, however, hasn't specified or explained why they chose those two outcomes and not others, such as prescription contraindication caused issues, quality of point of care, and so on, which are also included in much literature on EHR/EMR effectiveness.\nSecondly, the manuscript would have been better enhanced with some recommendations for future implementations of EHR/EMR.\nLastly, is there a difference in the types of ambulatory settings (surgical, physical therapy, urgent care)? These would have enhanced the quality and usefulness of the paper but they do not detract from the overall quality and rigor of the work reported in the existing manuscript and the way it was conducted.\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": [ { "c_id": "7161", "date": "11 Oct 2021", "name": "Salem Albagmi", "role": "Author Response", "response": "Comment 1: The author, however, hasn't specified or explained why they chose those two outcomes and not others, such as prescription contraindication caused issues, quality of point of care, and so on, which are also included in much literature on EHR/EMR effectiveness. Page No. 5, Line No. 97-107, Actual Content Removed/Edited/Replaced Content: Rationale for selecting both variables in the introduction section Comment 2: Secondly, the manuscript would have been better enhanced with some recommendations for future implementations of EHR/EMR. Page No. 19, Line No. 295-310, Actual Content Removed/Edited/Replaced Content: Future perspective of EHR is added Comment 3: Lastly, is there a difference in the types of ambulatory settings (surgical, physical therapy, urgent care)? These would have enhanced the quality and usefulness of the paper but they do not detract from the overall quality and rigor of the work reported in the existing manuscript and the way it was conducted. Page No. 17, Line No. 269-274, Actual Content Removed/Edited/Replaced Content: Discussion related to EMR/EHR system is added in the context of types" } ] } ]	1	https://f1000research.com/articles/10-514
https://f1000research.com/articles/10-191/v1	08 Mar 21	{ "type": "Study Protocol", "title": "Effect of Transcranial Direct Current Stimulation associated with aerobic exercise on the autonomic modulation of hemiparetic individuals due to stroke: a study protocol for a double-blind randomized controlled trial", "authors": [ "Solange Zilli Lo Presti Heinz", "Katia De Angelis", "Glauber Heinz", "Ariane Viana", "Fernanda Marciano Consolim-Colombo", "Agatha Calegari Bonfadine", "Ruthe Rodrigues Nemesio", "Claudia Gabriella Ribolli Bacalhau", "Rafael Alves de Oliveira", "Cristina Sayuri Miyamura", "João Carlos Ferrari Corrêa", "Fernanda Ishida Corrêa", "Solange Zilli Lo Presti Heinz", "Katia De Angelis", "Glauber Heinz", "Ariane Viana", "Fernanda Marciano Consolim-Colombo", "Agatha Calegari Bonfadine", "Ruthe Rodrigues Nemesio", "Claudia Gabriella Ribolli Bacalhau", "Rafael Alves de Oliveira", "Cristina Sayuri Miyamura", "João Carlos Ferrari Corrêa" ], "abstract": "Background: Individuals after a stroke have an imbalance in the autonomic nervous system, which increases the risk of death or recurrent episodes of stroke. Transcranial Direct Current Stimulation (tDCS) combined with aerobic exercise has shown an effect on the modulation of this system. Objective: The Heart Rate Variability (HRV) and the distance traveled on the exercise bike will be assessed to verify the additional impact of tDCS combined with aerobic exercise on individuals with chronic stroke sequelae. Methods: The 34 adult individuals with diagnoses of chronic stroke will be randomized into two aerobic exercise intervention groups: G1 (with active tDCS) and G2 (with sham tDCS), three times a week, for 12 weeks. Procedures: tDCS will be implemented during the aerobic exercise with the anodal electrode positioned over the lateral dorsal prefrontal left cortex, and the cathodal electrode over the contralateral supraorbital region, with 2mA, for 20 minutes. Assessments will be carried out pre, immediately after the intervention, and on the 12th, 24th, 36th interventions, and 30 days later. The HRV data that are pulse interval (PI), square root of the mean of the squares of the differences between adjacent normal RR intervals (rMSSD), absolute high frequency (HF), absolute low frequency (LF), high and low frequency ratio (LF / HF) will be collected using a cardio frequency meter. The analysis of the distance traveled on the exercise bike before and after interventions will be analyzed in meters. Discussion: The autonomic control via brain networks after a stroke can be altered and can promote an increase in sympathetic tone, and a higher risk of sudden death or relapse of stroke. It is crucial to demonstrate the effectiveness of available treatments to improve the autonomic function. Trial registration: The study is registered as a BRAZILIAN CLINICAL TEST RECORD (ReBEC): U1111-1222-4588 on the 2018/10/16", "keywords": [ "Stroke", "Autonomic Modulation", "Transcranial Direct Current Stimulation", "Physical Exercise", "Hemiplegia" ], "content": "Abbreviations\n\ntDCS: transcranial direct current stimulation\n\nF3: Left dorsolateral prefrontal cortex\n\nHRV: heart rate variability\n\nDLPFC: dorsolateral prefrontal cortex\n\nDT: linear time-domain\n\nFD: frequency-domain\n\nIP: Pulse interval\n\nrMSSD: square root of the mean of the squares of the differences between adjacent normal RR intervals\n\nHF: Absolute high frequency\n\nLF: Absolute low frequency\n\nLF / HF: low frequency and high-frequency ratio\n\nACE: Addenbrooke Cognitive Exam questionnaire\n\nSSQOL: stroke specific quality of life questionnaire\n\nBDI: beck depression inventory\n\nMMSE: mini mental state examination quiz\n\n\nIntroduction\n\nThe contribution of complications to the mortality of patients with stroke is variable between 12.5% to 22.7%1,2.\n\nIndividuals with stroke have depressed parasympathetic activity mainly in the acute phase, exacerbated cardiovascular responses with increased sympathetic activity, unbalanced heart rate and blood pressure, decreased Heart Rate Variability (HRV), arrhythmias, and a higher risk of sudden death3–6.\n\nDorrance & Fink (2015)7 observed that post-stroke Autonomic Nervous System (ANS) dysfunction increases the circulation of catecholamine levels in the heart, and enhances post-stroke patients' morbidity and mortality. However, it is not yet clear whether this disfunction is an effect of increased sympathetic nervous system (SNS) activity, reduced parasympathetic nervous system (PNS) activity, or a change in the balance of the two.\n\nThus, a great concern about the impact of physical fitness on stroke survivors to prevent cardiovascular risks has been observed in recent times, which shows the importance of physical exercise. It is notorious that physical exercise can prevent and mitigate heart problems. However, it requires prudence, since physical activity itself can increase cardiac output and increase blood pressure in these patients, even with the use of medications8–12.\n\nTherefore, supportive therapies that can improve the rehabilitation process are relevant, such as Transcranial Direct Current Stimulation (tDCS). Transcranial direct current stimulation associated with aerobic exercise has shown significant effects on autonomic modulation in athletes and healthy individuals, as noted by some researchers13–15. Heinz et al. (2019)16 observed that the application of tDCS in individuals with stroke sequelae tends to modulate parasympathetic action. However, the effectiveness of tDCS was not noticed by Nguyen et al. (2015)17 in this same population.\n\nRossi et al. (2016)18 showed in a systematic review that tDCS performance is a therapeutic option in autonomic modulation, allowing instantaneous ('online') and lasting ('offline') modulation in cortical excitability. However, the tDCS protocol to obtain the better performance of the autonomic balance of individuals affected by stroke remains unclear.\n\nTo investigate the effect of adding tDCS to aerobic training in the autonomic modulation of chronic stroke survivors immediately after the first therapy, after the 12th, 24th, and 36th interventions, and 30 days after the end of interventions.\n\nTo evaluate the additional effect of tDCS on aerobic training in the distance covered (meters), quality of life, and cognition after the 12th, 24th, and 36th interventions and 30 days after the end of the interventions.\n\nThis study assumes that the application of tDCS over the left dorsal prefrontal cortex (DPFC) will stimulate this area and, therefore, increase the effects of aerobic training on the autonomic modulation of individuals with stroke sequelae. This effect may occur because the left DPFC, when activated, has the function of inhibiting the sympathetic excitatory circuit of the amygdala, helping with autonomic regulation19.\n\nThe hypothesis is that after a single stimulation session, it will be possible to verify some effect on the ANS, as observed by Heinz et al. (2019)16, Okano et al. (2015)14, Petrocchi et al. (2017)20. However, these may be even more significant after a 3-month aerobic training, a time described in the literature to obtain effects on cardiovascular conditioning21. Another hypothesis is that these results will remain one month after the end of the training.\n\nIt is possible to observe effects on cognition since exercise can improve it and because the area that will be stimulated with tDCS is also responsible for behavior modulation, planning, temporal sequencing, language, and memory22–24.\n\nThrough the possible beneficial effects of exercise for the body, the improvement in the quality of life can be a consequence25,26.\n\n\nMethods\n\nThis is a protocol for a double-blind study (evaluator and participants), controlled by sham and randomized that will follow the recommendations of the Consolidated Standards of Reporting Trials (CONSORT) (Figure 1) and the recommendations of the standard protocol items for clinical trials (SPIRIT) (Reporting guidelines). The study was approved by the ethics committee of Universidade Nove de Julho, São Paulo, Brazil (CAAE: 97475718.5.0000.5511) - and registered in the Brazilian Registry of Clinical Trials (ReBEC) (U1111 -1222-4588) Participants will be informed about the research, procedures, risks, and benefits. If they agree, they will sign an informed consent form (Extended data: Appendix 127).\n\nThere will be enrolled 34 participants of both sexes at the physiotherapy clinics at Nove de Julho University, in São Paulo.\n\nThe inclusion criteria are as follows: individuals of both sexes, aged between 21 and 74 years old, minimum of six months of stroke injury, medical authorization to participate in the study, with the functional capacity of lower limbs that allow them to pedal the exercise bike, even if with the help of the therapist. The participants who usually ingest beta-blockers will not be excluded, but after the end of the research, an analysis will be carried out to compare the HRV of the individuals who use the medication with those who do not use them. Exclusion criteria include individuals with cognitive impairment (≤17) assessed by the mini-mental status exam (MMSE)28, severe heart problems, use of a pacemaker, and/or contraindications to the use of tDCS29.\n\nParticipants can freely withdraw from assessment and therapy at any time. Criteria for the termination in the study include participants who were absent more than a week away from the start date; who became ill or acquire any injury making it impossible to perform physical activity.\n\nThe sample size was calculated utilizing a pilot study with 8 individuals (4 for the active tDCS group and 4 for the tDCS sham group) and, with the sample power considering the rMSSD outcome variable by linear time-domain (DT) methods, assuming α of 0.05 and β of 0.80. Using the sample calculation tool on the website: calculoamostral.bauru.usp.br, the difference between two means with independent groups (t-test) was calculated. A total N of 15 individuals, considering possible losses, 10% was added totaling N of 17 individuals for each group and, therefore, 34 individuals were recruited, with an effect size of d = 0.000077 by Cohen (Figure 2).\n\nThe allocation of individuals to Group 1 (active tDCS combined with aerobic exercise on the stationary bike) and Group 2 (tDCS sham combined with aerobic exercise on the stationary bike) will take place using the website www.randomized.com by a researcher not involved in the evaluation and intervention.\n\nA study evaluation schedule with standard protocol items is provided in Table 1.\n\nNote: Heart Rate Variability (HRV); -T1: one week before intervention; T0: one day before intervention; T1: 20 minutes before intervention; T2: 1st-day session; T3: 12th-day session; T4: 24th-day session; T5: 36th-day session; T6: immediately after the session; T7: 30th-day follow-up after the last training session.\n\nThe assessments and interventions will be carried out in the morning, always at the same time, to minimize the effects of the circadian cycle. The recommendations will be to continue to use the medications in their regular schedule, to have a light diet on the test days, to abstain from caffeine or alcoholic beverages, and smoking, and to avoid moderate or excessive efforts on the day before the test day.\n\nTranscranial direct current stimulation. The therapy of tDCS DC-Stimulator Plus (NeuroConn) (active or sham) will be combined with aerobic exercise on the stationary bike. The anode electrode will be placed over the left dorsolateral prefrontal cortex (F3), and the return electrode (cathode) will be placed over the contralateral supra-orbital region, defined by the 10/20 electroencephalogram system. The intensity of current will be 2mA, applied for 20 minutes, 10-second linear ramp up / down.\n\nThe electrodes used will be of conductive rubber, anode 5x5 cm, and cathode 5x7 cm, wrapped in cellulose sponge moistened in 0.9% saline solution.\n\nFor sham stimulation, all electrode placement procedures will be performed equally with the active tDCS. Still, the stimulator will only be on for 30 seconds, considered a valid method for a control in tDCS studies30.\n\nThe NeuroConn DC-STIMULATOR PLUS device has settings that allow the selection of the active or sham stimulation mode by inserting codes. A researcher not involved in the procedures will allocate the participant. The external functioning of the device will not perceive the stimulus mode. Therefore, neither the researcher who will apply the intervention nor the individual will know what treatment will be used (double-blind).\n\nAfter using the tDCS is carried out, participants and researchers will be asked to complete questionnaires about blinding (Extended data: Appendix 227), adverse effects (Extended data: Appendix 327), and satisfaction of therapy (Extended data: Appendix 427).\n\nThe participant will performer the aerobic activity on a Reebok® RT 445 model N° RBEX49021 exercise bike, 30 minutes a day, with the initial 5 minutes of warm-up, 20 minutes of aerobic activity associated with active tDCS or sham, and the final 5 minutes of cooling.\n\nThe treatment will be carried out three times a week, for 12 weeks, totaling 36 sessions31. The initial intensity will be 50% of the reserve heart rate, as tolerated, and will be rising until 5% of the reserve heart rate each week32. The target aerobic intensity will be 50% to 70% of the reserve heart rate.\n\nThe following formula {% reserve HR = [(maximum HR - resting HR) x%] + resting HR} will be used to obtain the reserve heart rate (HR). If one of the individuals uses β-blockers, the maximum corrected HR should be calculated using the following formula [(the dosage taken with the drug + 95.58) / 9.74 =% that should be removed from the maximum HR]. The maximum heart rate (HRmax) will be estimated using the Karvonen formula33.\n\nThe HR and oxygen saturation (SpO₂) will be monitored as a protective measure by a portable pulse oximeter UT-100 Polar V800 frequency meter every two minutes of exercise; as well as blood pressure (BP) and the perception of dyspnea and fatigue of the lower limbs by the modified Borg questionnaire34.\n\nThe evaluations will be carried out before, after the 12th, 24th, and 36th interventions and 30 days after the end of the interventions. Personal data about the individual and the disease will be collected (Extended data: Appendix 527). The other data will be:\n\nEvaluation of heart rate variability (HRV). The CardioSeries software (http://www.danielpenteado.com/cardioseries) will be used to identify correct premature ectopic beats, and undesirable transients will be removed using linear interpolation that alters the signal stationarity. The variances of the pulse interval (IP) will be evaluated in the domain of time and frequency by the linear method.\n\nHRV will be measured using the Polar® V800 heart rate monitor device. The evaluation of cardiac modulation will be performed by recording the RR interval, processed using the Flow software (https://flow.polar.com), calculating the transducer indices of cardiac cycle fluctuation, high-frequency waves (0.15 and 0.4 Hz), low frequency (0.04 to 0.15 Hz), and the interrelation between low frequency and high frequency (0.15 and 0.4 Hz). The raw, unfiltered data will be exported, converted, and stored in an Excel file, used later for the domain of time and frequency.\n\nThe indexes obtained by analyzing the RR intervals in the time domain will be the average of the RR pulse interval utilizing absolute variance and the square root of the squared mean of the difference between the normal adjacent RR intervals (rMSSD), expressed in ms². As for the frequency domain, the data will be analyzed through the analysis of absolute high frequency (AF), low absolute frequency (BF), and the vagal sympathetic balance between low frequency and high frequency (BF / AF).\n\nTraveled distance. The distance covered will be measured at the end of the 30 minutes of aerobic exercise performed by the participant on the exercise bike.\n\nThe results of the distance covered will be compared intragroup (active and sham tDCS), for each moment pre, post 12th, 24th, 36th interventions, and 30 days after the end of the interventions to verify the evolution in both.\n\nCognitive Performance. The cognitive performance of chronic stroke patients will be assessed using the Addenbrooke Cognitive Exam (ACE)35 questionnaire. The evaluator will apply the questionnaire in three moments:\n\nThe first moment will be before randomization for the intervention groups, the second will be after the 36th intervention, and the third moment will be 30 days after the end of the training.\n\nQuality of life assessment. Quality of life will be measured by the Stroke Specific Quality of Life questionnaire (SSQOL)36.\n\nDepressive symptoms. The Beck Depression Inventory (BDI)37 will be used to assess the depressive symptoms. The results will be correlated with the performance of the physical activity38.\n\nStatistical analysis. The program SPSS Statistic version 17.0 will be used for statistical analysis.\n\nFor measures of central tendency and dispersion will be used descriptive statistical analysis. To measure the parametric variables will be used the mean and standard deviation, to measure the non-parametric variables will be used the median and the interquartile range, and to measure the categorical variables will be used the frequency and percentage.\n\nHRV data (linear methods) will be analyzed in the time domain with the variable rMSSD and in the frequency domain with absolute and normalized high frequency. The data will be submitted to the Shapiro-Wilk normality test, using the unpaired t-test for parametric data and Mann-Whitney for non-parametric data, considering the significance level p≤0.05 for all conditions.\n\n\nDiscussion\n\nThis article provides a detailed description of a prospective, randomized, controlled, double-blind clinical trial designed to demonstrate the effects of combining tDCS and aerobic training with an exercise bike on the autonomic modulation of individuals with hemiparesis due to chronic stroke.\n\nWe will publish the results, and the evidence found can contribute to the cardiovascular rehabilitation process of this population. In this sense, if positive, it allows a better prognosis in the cardiovascular rehabilitation of these individuals and reduces the likelihood of a stroke with more severe recurrence.\n\nSo far, participants have been enrolled, and the allocation is being made from the perspective of completion of collections in June 2021.\n\nThe results will be communicated to the public through publication as a data set and original research in the relevant scientific journals.\n\nWe consider some topics as possible limitations of our study:\n\nThe possible difficulty in recruiting patients due to their limited mobility;\n\nA possible analysis difficulty due to a decrease in vagal withdrawal and use of ß-blockers;\n\nAbsence of control over modifiable risk factors.\n\nDifficulty getting complementary examinations for an accurate diagnosis regarding the location and extent of the lesion.\n\n\nData availability\n\nNo underlying data are associated with this article\n\nHarvard Dataverse: Effect of Transcranial Direct Current Stimulation associated with aerobic exercise on the autonomic modulation of hemiparetic individuals due to stroke: a study protocol for a double-blind randomized controlled trial, https://doi.org/10.7910/DVN/MUNWDB27\n\nThis project contains the following extended data:\n\nAppendix 1: Clarified Free Consent Term\n\nAppendix 2: Blinding Questionnaire tDCS Researcher\n\nAppendix 3: Adverse effect tDCS\n\nAppendix 4: Evaluation of treatment satisfaction\n\nAppendix 5: Personal data about the individual\n\nRegister approved by the ethics committee\n\nRegistered in the Brazilian Registry of Clinical Trial (ReBEC)\n\nHarvard Dataverse: SPIRIT checklist and CONSORT flow diagram for ‘Effect of Transcranial Direct Current Stimulation associated with aerobic exercise on the autonomic modulation of hemiparetic individuals due to stroke: a study protocol for a double-blind randomized controlled trial’, https://doi.org/10.7910/DVN/MUNWDB27\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 thank the Brazilian fostering agency Coordination for the Improvement of Higher Education Personnel (CAPES) for granting a scholarship to the first author and the University Nove de Julho. 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Publisher Full Text\n\nGorenstein C, Andrade L: Validation of a Portuguese version of the Beck Depression Inventory and the State-Trait Anxiety Inventory in Brazilian subjects. Braz J Med Biol Res. 1996; 29(4): 453–7. PubMed Abstract\n\nGorczynski PF, Coyle M, Gibson K: Depressive symptoms in high-performance athletes and non-athletes: A comparative meta-analysis. Br J Sports Med. BMJ Publishing Group; 2017; 51(18): 1348–1354. PubMed Abstract \| Publisher Full Text" }	[ { "id": "81085", "date": "07 Apr 2021", "name": "Rita Khadka", "expertise": [ "Reviewer Expertise Assessment of cardiovascular autonomic function", "heart rate variability", "Blood pressure variability", "baroreflex sensitivity", "exercise physiology", "yoga", "electroencephalography" ], "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 has aimed to investigate the effect of transcranial direct current (tDCS) stimulation associated with aerobic exercise on the autonomic modulation of hemiparetic individuals due to stroke. It is a double-blind randomized controlled trial. The control group will receive sham tDCS. Both groups of patients will do aerobic exercise on an exercise bike for 30 min a day, 3 days a week for 12 weeks. Autonomic modulation will be assessed using heart rate variability (HRV).\nI find the study protocol interesting and noble. It has both research and clinical significance. The title of the study protocol is well written, clearly summarizing the main objective of the study. Similarly, the rationale and some portions of the methods of the study are well written/explained. However, I have some comments that need to be discussed for the further improvement of the study.\nComments Related to the Objectives of the study:\nIt would be better to modify the objectives of the study (page 3) for better clarity. It can be modified as:\nTo investigate the effect of transcranial direct current (tDCS) stimulation associated with aerobic exercise on the autonomic modulation of chronic stroke survivors immediately after the 1st, 12th, 24th, and 36th interventions, and 30 days after the end of the intervention.\n\nTo evaluate the effect of tDCS on the distance covered during aerobic training, quality of life, and cognition after the 12th, 24th, and 36th interventions and 30 days after the end of the intervention.\nRelated to the methods of the study :\nIn the inclusion and exclusion criteria of the study, nothing is mentioned about patients with chronic stroke along with other complications like diabetes mellitus, chronic kidney disease, Parkinsonism, and some other complications (page 4, last para & page 5, first para).\n\nIf patients with chronic stroke along with other complications (like diabetes mellitus, chronic kidney disease, Parkinsonism, and some other complications) are enrolled in the study, it needs to be noted down because these complications decrease HRV. If possible patients with these complications can be excluded from the study. Otherwise, a similar number of such patients enrolled in both the groups may nullify the effects.\n\nFor the intra-group comparisons of related data, it would be better to use repeated measure ANOVA for the parametric data and Friedman test for the non-parametric data (under statistical analysis page 7).\n\nIt is mentioned that the results of the depressive symptoms will be correlated with the performance of the physical activity (page 7). However, no statistical tool has been discussed under the statistical analysis. Please mention it.\n\nWhether participants will perform aerobic exercise 3 times/days a week successively or on an alternate day (page 7, para 4). It would be better to mention it.\n\nPlease mention how you will measure distance traveled (page 7).\n\nWhen will the quality of life and depressive symptoms be assessed? Not mentioned clearly in the protocol. It would be better to mention the time point for the assessment. Please check for the assessment of the other parameters also and improve them.\n\nEvaluation of heart rate variability is written well. However, it needs some minor modifications. It is written that \"rMSSD, expressed in ms2\" (page 7). It needs to be corrected as \"rMSSD is expressed in ms.\" It would be better to use the standard abbreviations for the absolute high-frequency and absolute low-frequency powers and their ratios (page 7). Absolute powers are expressed in ms2. It can be mentioned.\n\nIt would be better to add two more time-domain parameters of HRV; SDNN (standard deviation of R-R intervals) and pNN50 (percentage of the number of pairs of adjacent R-R intervals differing by more than 50 ms). Adding these parameters may strengthen the results.\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? Not applicable", "responses": [ { "c_id": "6847", "date": "11 Oct 2021", "name": "Solange Zilli Lo Presti Heinz", "role": "Author Response", "response": "Dear Reviewer We are grateful for yours comments and suggestions. We believe that we could improve our manuscript substantially based on these comments. We are submitting the revised version of our manuscript entitled \" Effect of Transcranial Direct Current Stimulation (tDCS) associated with aerobic exercise on the autonomic modulation of hemiparetic individuals due to stroke - Clinical trial protocol, Controlled, Randomized, Double-blind \". We address reviewer points in a point-by-point fashion as shown below. 1.It would be better to modify the study objectives (page 3) for clarity. It can be modified as: Reviewer's suggestion: a) To investigate the effect of transcranial direct current stimulation (tDCS) associated with aerobic exercise on the autonomic modulation of chronic stroke survivors immediately after the 1st, 12th, 24th and 36th interventions and 30 days after the end of the intervention. b) To evaluate the effect of tDCS on the distance covered during aerobic training, quality of life, and cognition after the 12th, 24th, and 36th interventions and 30 days after the end of the intervention. Answer: Thank you for your suggestion. Your considerations make our goals better described and clear. Suggested are in lines 93 to 98. 2. Related to the methods of the study: a) In the inclusion and exclusion criteria of the study, nothing is mentioned about patients with chronic stroke along with other complications like diabetes mellitus, chronic kidney disease, Parkinsonism, and some other complications (page 4, last para & page 5, first para). b) If patients with chronic stroke along with other complications (like diabetes mellitus, chronic kidney disease, Parkinsonism, and some other complications) are enrolled in the study, it needs to be noted down because these complications decrease HRV. If possible, patients with these complications can be excluded from the study. Otherwise, a similar number of such patients enrolled in both the groups may nullify the effects. Answer: Answer: Thank you for your suggestion. We agree with your comments. We insert this information in the eligibility criteria. However, some sequelae are very common among Stroke patients, like Diabetes, Hypertension, Hyperlecosterolemia, because of this these comorbidities will be not excluded, but they must be under the control of medication. However, after the end of the research, will be performed the regression analysis to verify if these variables may have interfered with the results. Lines 139 to 140. 3.For the intra-group comparisons of related data, it would be better to use repeated measure ANOVA for the parametric data and Friedman test for the non-parametric data (under statistical analysis page 7). Answer: Thank you for your suggestion. We agree with your comments, and we inserted this information. For HRV (linear methods) the data analyzed between the periods after the 1st, 12th, 24th, and 36th interventions, and 30 days after the end of the tDCS associated aerobic exercise will be analyzed in the time domain with the index of variance, rMSSD and in the frequency domain with absolute, normalized high frequency and the ratio of LF to HF power. The data will be submitted to the Shapiro-Wilk normality test, using the unpaired ANOVA with repetition parametric data and Friedman test for non-parametric data, considering the significance level p≤0.05 for all conditions. Line 267 to 276. 4. It is mentioned that the results of the depressive symptoms will be correlated with the performance of the physical activity (page 7). However, no statistical tool has been discussed under the statistical analysis. Please mention it. Answer: Thank you for your suggestion. The results of the depression symptoms (BDI) will be correlated with the distance performed on the ergometer bike. The data will be submitted to the Shapiro-Wilk normality test, using the Person´s correlation for parametric data and Sperman's Correlation for non-parametric data. Lines: 272 to 274. 5.Whether participants will perform aerobic exercise 3 times/days a week successively or on an alternate day (page 7, para 4). It would be better to mention it. Answer: Thank you for your suggestion. The treatment will be carried out three times a week, every other day, for 12 weeks, totaling 36 sessions. Lines: 44 and 195. 6.Please mention how you will measure distance traveled (page 7). Answer: Thank you for your suggestion. The distance covered (meters) performed by the participant will be measured by the device's digital marker, at the end of the 30 minutes of aerobic exercise, on the exercise bike. Line 233 to 235. 7.When will the quality of life and depressive symptoms be assessed? Not mentioned clearly in the protocol. It would be better to mention the time point for the assessment. Please check for the assessment of the other parameters also and improve them. Answer: Thank you for your suggestion. The cognitive performance of chronic stroke patients will be assessed using the Addenbrooke Cognitive Exam (ACE)34questionnaire. The evaluator will apply the questionnaire before and after the 1st, 12th, 24th, and 36th interventions, and 30 days after the end of the intervention. Line 240 to 243. Quality of life will be measured by the Stroke Specific Quality of Life questionnaire (SSQOL)35 . The evaluator will apply the questionnaire before and after the 1st, 12th, 24th, and 36th interventions, and 30 days after the end of the intervention. Line 245 to 247. The Beck Depression Inventory (BDI)36 will be used to assess the depressive symptoms. The evaluator will apply the questionnaire before and after the 1st, 12th, 24th, and 36th interventions, and 30 days after the end of the intervention. Line 250 to 252. 8.Evaluation of heart rate variability is written well. However, it needs some minor modifications. It is written that \"rMSSD, expressed in ms2\" (page 7). It needs to be corrected as \"rMSSD is expressed in ms.\" It would be better to use the standard abbreviations for the absolute high-frequency and absolute low-frequency powers and their ratios (page 7). Absolute powers are expressed in ms2. It can be mentioned. Answer: Thank you for your comments. We correct it. Line: 228 to 231. 9. It would be better to add two more time-domain HRV parameters; SDNN (standard deviation of RR intervals) and pNN50 (percentage of the number of pairs of adjacent RR intervals that differ by more than 50 ms). Adding these parameters can strengthen the results. Answer: We appreciate your suggestion. However, the software we will use is the CardioSerie, so it will not be possible to analyze the SDNN and pNN50 data as suggested by the reviewer. But we can add the time-domain index of variance, which equals the SDNN index, and the rMSSD, which equals pNN50. Line 261 to 265." } ] }, { "id": "87186", "date": "23 Jun 2021", "name": "Brenton Hordacre", "expertise": [ "Reviewer Expertise Stroke recovery", "neuroplasticity", "brain stim" ], "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 the opportunity to review this study. It is an interesting concept and I have raised a few points the authors should consider below.\nIn general, this manuscript will need language editing. There are sections which do not make sense and paragraphs that are not well formulated. For example, the first two paragraphs of the introduction are 1 sentence long. These could be combined to a single paragraph. Equally, it is highly unusual to see dot points within an academic manuscript. I’d suggest they are removed from the introduction and discussion.\n\nThe introduction lacks the relevant background information. As a reader, I am unaware why tDCS or aerobic exercise as therapies delivered over several sessions can modulate the autonomic nervous system. I suggest the authors expand the introduction, possibly utilising references 13-15, rather than just saying the evidence has been noted by some researchers.\n\nI am unclear what the primary and secondary aims of the study are. Is the first dot point the primary aim?\n\nThe hypothesises do not align with the aims. Why is there a hypothesis that you are stimulating the left DLPFC? Similarly, why is there a hypothesis that it is possible to observe effects on cognition – the hypothesis should be directional. Eg. we hypothesise that anodal TDCS to the left DLPFC combined with aerobic exercise will increase cognitive function compared to sham tDCS and aerobic exercise.\n\nI am unclear why the consort diagram is provided if no data is collected. Interestingly, it appears 34 participants have already been randomised.\n\nWhy is there an upper age limit of 74 years for inclusion criteria?\n\nThe cohens D value for the power calculation is extremely low – raising concerns. Please have a biostatistician review. In fact, I cannot replicate the power calculation. I have had a biostatistician perform the calculation and they determined 56 participants are required based on the data provided.\n\nWho is performing the outcome measures?\n\nWhat is the primary outcome measure(s) and why. What is the reliability/validity and sensitivity of these measures?\n\nThe statistical analysis section is inadequate. If there are multiple outcome measurement times and two groups, a repeated measures anova or linear mixed model is appropriate. T-tests are going to increase your error.\n\nThe discussion is inadequate. What gap in the literate is this study likely to address and what is the clinical value of running this study. What potential implications could it have for the field. How do you see this study informing clinical practise of scientific knowledge?\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Partly\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": [] } ]	1	https://f1000research.com/articles/10-191
https://f1000research.com/articles/10-1025/v1	08 Oct 21	{ "type": "Brief Report", "title": "Workplace ergonomics problems and solutions: Working from home", "authors": [ "Jian Ai Yeow", "Poh Kiat Ng", "Wei Yin Lim", "Wei Yin Lim" ], "abstract": "Background: Due to the COVID-19 pandemic, in 2020, many employees were required to work from home (WFH). During this WFH period, some employees encountered health issues related to sprains and neck or back pain owing to poor working conditions at home. As the WFH trend may continue over a prolonged period, the underlying causes and solutions to ergonomic issues must be addressed to reduce injuries. This study aims to identify the ergonomic issues encountered when working from home and suggests several solutions to minimise these issues. Methods: A qualitative ethnographic methodology was adopted. This study used focus group discussion and the panellists were among experts from the fields of higher education, healthcare, human resources (HR), and ergonomics patient in Malaysia. The most common ergonomic issues identified were based on diagnoses and observations in previous studies. Results: The panellists agreed on ergonomics issues, comprising the use of unergonomic chairs, incorrect sitting postures, irregular arrangement of key objects, improper reach distances of the laptop/keyboard/mouse, poor desk designs, footrest absence, distortion/noise, poor lighting, and poor work environment. Over time, WFH ergonomics issues may lead to burnout, carpal tunnel syndrome or other cumulative trauma disorders, high blood pressure, and stress on the cervical spine and neck. The proposed solutions include a complete WFH ergonomics and wellness checklist for employees and employers, webinar sessions on WFH ergonomics, meet-up sessions with ergonomics or HR experts, workspace rentals for co-workers, implementation of the 20-20-20 rule and job-sharing practices, and the involvement of employers or the government in procuring ergonomic equipment for WFH employees. Conclusions: This is a preliminary study and the researchers are exploring the root causes of WFH ergonomics issues and proposed solutions. While previous studies have examined workplace ergonomics, this study focuses on WFH ergonomic issues and solutions during the ongoing pandemic.", "keywords": [ "working from home", "ergonomics", "focus group", "global pandemic", "injuries", "occupational safety", "health" ], "content": "Introduction\n\nHealth issues related to sprains, strains, and back pain account for the highest total direct expenses for workers supported by the Social Security Organisation of Malaysia (SOCSO) since 2009.1 This finding exposes the impact of musculoskeletal injuries on the productivity of a company. Following the nationwide lockdowns since March 2020 in the face of the COVID-19 pandemic, employees were required to work from their homes. According to Bench,2 the work-from-home (WFH) setting has led to the development of health issues among employees, with ergonomic and clinical experts receiving numerous complaints about employees having upper back discomfort, neck issues, and eye strain. Hanel3 explained that many workers constantly gaze downwards at their laptop screen, resulting in neck issues.\n\nOther problematic WFH risks include sitting on ill-fitting and rigid chairs for long durations, impeding the adjustment of the sitting posture. Since WFH may become a new norm for employees, it is essential to find long-term solutions to enable employees to WFH with ergonomic office equipment and guidelines. This study recommends WFH guidelines and best practices for human resource (HR) departments and occupational safety and health workers to enhance safety rules and regulations for the reduction of musculoskeletal injuries and risks.\n\n\nCOVID-19 in Malaysia\n\nIn March 2020, Malaysia experienced its first lockdown, known as the movement control order (MCO).4 The MCO required many employers and employees to abruptly change their work environment and patterns by working from home. This new norm was shrouded in uncertainty, as many were not prepared for WFH due to lack of equipment and space at home. According to JobStreet’s Malaysia Survey Report,5 9 out of 10 people were impacted in some way by the pandemic and about 67% of the companies required their staff to WFH. The New Straits Times newspaper6 reported several experts raising concerns about WFH practices, including mental health issues, ergonomics issues, and emotional health issues. However, by July 2021, Malaysia has been hit by the third wave of COVID-19 and the cumulative number of confirmed COVID-19 cases had reached up to 1 million cases, including more than 3000 deaths.7 The COVID-19 situation in Malaysia is critical, and in most sectors WFH is compulsory. Due to the emergency lockdown, the guidelines for working from a home office have caused high uncertainty8 with numerous challenges and significant technical complications for both employees and employers. Employees are required to ensure that their home environment is a safe and healthy place to work.9 With ergonomic equipment or settings, such as a height-adjustable chair, adequate workspace, and appropriate desk positioning, WFH conditions could be improved. HR departments, employers, and employees need to place more focus on the standard operating procedures (SOPs) that facilitate WFH practices. While employees have a certain level of responsibility, employers should also diligently fulfil their roles and responsibilities according to the SOPs. Not adequately fulfilling their responsibility to ensure a safe working environment could lead to losses from compensation claims made by employees.10\n\nTable 1 shows some of the common WFH ergonomics issues raised by concerned experts in the news and several ergonomics articles.11-14\n\n\nMethods\n\nIn this inductive study, the researchers explored ideas from several case studies in The Star newspapers.11 In order to determine WFH problems and solutions, a focus group discussion was conducted.\n\nWFH is the new norm in Malaysia but many organisations have not prepared working solutions for their employees. To validate the problems and solutions for WFH ergonomics issues, this study adopted a qualitative approach for the preliminary data collection. The questions were validated through face validity by three academicians specialising in ergonomics and HR management.\n\nA focus group of four experts comprising a 55-year-old educator (male), a 44-year-old healthcare worker (male), a 38-year-old HR specialist (female), and a 41-year-old employee with ergonomics-related health issues (male) was conducted. Morgan17 argued the rule of thumb for the size of a panel is 6-10 homogeneous participants, but there may be reasons to have smaller groups. Due to unstable Internet connection and unfamiliarity with the video meeting platform, some technical issues were unavoidable. Purposive sampling was adopted in this research. The researchers contacted an academician with over 20 years of teaching experience in occupational safety and health via a telephone call. They also browsed through the medical staff list of a well-known private hospital in Melaka, Malaysia and selected the neurology doctors, physiotherapists, and rehabilitative physician to be the panellists. Finally, three medical workers accepted the interview but two of them were forced to pull out due to personal reasons. The participating medical worker recommended one of his patients as the next panellist for this discussion. The researchers contacted an HR specialist that works in a multinational company in Melaka, Malaysia. Due to restrictions on interstate travel panellists were selected from Melaka, Malaysia as the initial plan was to have a face-to-face focus group.\n\nThe Malaysian government imposed a full MCO on 1 June 2021 and in-person meetings were not allowed. The focus group discussion was therefore conducted through an online meeting via Google Meet on 2 June 2021. Due to time constraints and difficulties in arranging another slot for all panellists, the focus group was restricted to a total of four panellists. According to Nyumba,20 a minimum of three to four panellists for preliminary studies is acceptable. The discussion lasted approximately 2 h and 15 min and was recorded via note-taking and analysed step by step following practical guides for focus group research by Breen,14 Krueger,15 Pascall et al.16 and Morgan.17 The information was recorded using an inductive approach and Breen14 agrees that idea-generating research which aims to propose recommendations for future improvements is best facilitated by focus groups. Since this study aims to propose ideas and solutions for WFH ergonomics issues, the researchers selected this method of data collection.\n\nThe moderators comprised two researchers that had not met any of the panellists prior to the study in order to prevent favour of any one particular speaker’s point of view over another.\n\nThe focus group included 15 open-ended questions. The list of issues and solutions to WFH ergonomics (Table 1) were sent to the panellists a week before the date of the interview. Q1-Q2 were designed as an introduction to the home office and Q3-Q7 focused on the participants’ general perception of WFH ergonomics issues. Q8-Q10 solicited information about participants’ expertise and personal experience with the WFH ergonomics issues and the possibilities of long-term consequences. Q11-Q13 encouraged the participants to share their concerns and ideas about WFH ergonomics problems and solutions. Q14 and Q15 were framed to obtain feedback and conclusions from participants. Table 2 shows the questions asked during the focus group discussion.\n\nPrior to the interview, the researchers distributed a consent letter to the panellists requesting permission to disclose and publish their suggestions in this manuscript, which was signed by all participants. The researchers also sent Table 2 to panellists alongside the consent letter so they were aware of the scope of discussion before the focus group interview. The ethical approval for this study was granted by Technology Transfer Office of Multimedia University (EA135202).\n\n\nResults and discussion\n\nThe focus group discussion results were mainly exploratory and descriptive, with no statistical analysis. The focus group discussion information is valuable as it helps researchers gain insightful knowledge about WFH ergonomics issues and solutions. The focus group discussion was recorded and the most important themes such as WFH ergonomics issues, noteworthy suggestions or solutions, and unexpected findings or advice from the panellists were summarised using note-taking, and comparison was done after the interview (the accompanying dataset is openly available on Figshare21). The most common ergonomics issues identified were based on diagnoses and observations by Dr. Edward Laskowski, co-director of Mayo Clinic Sports Medicine, and Corey Kunzer, a Mayo Clinic physical therapist12 and several other researchers.11-13\n\nFor Q2, all panellists agreed that WFH might become a norm even after the pandemic, especially for IT-related jobs, administrative work, and graphic design. One panellist added that big companies may be stricter with WFH practices and more systematic in handling WFH issues. Furthermore, according to the New Straits Times newspaper,18 the executive director of The Malaysian Employers Federation proposed that the government amend the Employment Act 1955 to fit the context of WFH and cater to the new norms. The Small Medium Enterprise (SME) Association of Malaysia agreed that the amendment would help companies to set more effective WFH guidelines.\n\nFor Q4 and Q5, the panellists agreed with the issues regarding unergonomic chair use, incorrect posture, irregular arrangement of key objects, improper reach distances of the laptop/keyboard/mouse, poor desk designs, footrest absence, distortion/noise, poor lighting, and poor work environment. The panellists added that many employees did not expect WFH to be a long-term situation and did not invest in any ergonomic equipment. Based on Q6, one panellist shared that facing workload, stress, and burnout are common for professional workers, especially those who are married and have childcare responsibilities. He added that the stress of managing children’s online classes in addition to household work aggravates this problem. The HR specialist added that those with low technology competency would also be affected by these problems. Additionally, depression, conflicting information about the pandemic, pay cuts, and other negative issues affect emotional health while working. The panellists noted that these are some of the biggest problems resulting in mental workloads from the ergonomics perspective and affecting workers’ health. The panellists also highlighted that poor lighting, fatigue, key object distance, dehydration, working on the couch, and weight gain are resolvable issues for employees. However, problems such as mental workload, poor work layout, and pressure in the carpal tunnel may take time to address. For Q8, a panellist highlighted that in the long run, WFH ergonomics issues may lead to burnout, carpal tunnel syndrome or other cumulative trauma disorders, high blood pressure, and stress on the cervical spine and neck. For Q10, a panellist mentioned that all levels (employees, employers, and even the government) play a vital role in ensuring a safe home office. The proposed solutions include creating a WFH ergonomics and wellness checklist for employees, webinar sessions on WFH ergonomics, meet-up sessions with ergonomics or HR experts, workspace rentals for co-workers, implementation of the 20-20-20 rule proposed by Robin Sharma, job-sharing practices, teamwork, and flexible work schedules. Professional counselling is needed for some staff and employers should encourage more counselling and virtual informal meet-ups among employees. Work-rest balance should be taken into serious consideration. For example, the 20-20-20 rule means that for every 20 minutes spent looking at a screen, a person needs to stop and look at something 20 feet away for about 20 seconds. After 30-45 minutes of work, a break of 5-10 minutes is required. For Q12 and Q13, the HR department must impose several regulations, such as allowing claims for purchasing ergonomics items, employee mental health treatments, medical check-ups, and ergonomics training. One panellist suggested several free online platforms on social media or websites for employees to learn about ergonomics, obtain information about SOCSO rehab centres, receive tax deductions for ergonomic equipment purchases and obtain information about quality control checks on furniture manufacturers. Furniture manufacturers should also be encouraged to produce more ergonomic furniture over aesthetic designs.\n\n\nConclusion\n\nThis study is novel as it examines the WFH-related ergonomics issues in Malaysia. The focus group discussion proved to be useful in determining WFH ergonomics problems and solutions. Although there were only four participants, the focus group discussion’s objective was achieved. Among all the ergonomics problems listed, all panellists agreed that mental workload and stress levels are the major underlying factors leading to poor ergonomics issues, followed by workstation chair height. Malaysian Employers Federation’s (MEF) executive director argues that WFH does not suit everyone.18 There are many that love working interactively and working in isolation will affect their performance and increase their mental stress. Moreover, WFH lacks boundaries between work and home life especially for those with care responsibilities, who often feel completely overwhelmed in the present situation. This study can help HR departments develop a flexible working hours guideline for those affected. The employers should emphasise a more team-based structure or cross-functional organisational structure to enable everyone to work as a team. A shared workload can eventually help to reduce stress among those with care responsibilities in particular.\n\nIt is essential to ensure optimum work-rest durations especially after 30-45 minutes of work. In addition, the duration of online meetings should be restricted to 30-45 minutes to reduce the duration of screen time focus. Employers should be open to accepting new online discussion platforms besides virtual meetings. For example, employers should allow discussions freely after the meeting via emails, online suggestions, and online feedback forms, which employees can access at their own convenience. Moreover, employees should abide by the 20-20-20 rule and use an ergonomic chair when they are working from home. Employers can consider buying ergonomic chairs for employees or provide a checklist for employees to check their working conditions related to workplace ergonomics. As mentioned earlier, the government plays an important role in reducing ergonomic problems and amending the Employment Act to suit WFH conditions. The discussion highlighted the value of additional solutions and the responsibilities of every individual in solving these issues. These findings provided preliminary evidence on the need for future research on WFH ergonomics problems and solutions using other methods such as quantitative methods. The focus group discussion findings may be valuable to future researchers in identifying problems for a more specific WFH situation. Future researchers can consider using quantitative analysis to enhance this research.\n\n\nData availability\n\nFigshare: ‘Workplace ergonomics problems and solutions: Working from home’. https://doi.org/10.6084/m9.figshare.16550820.v2.21\n\nThis project contains the following underlying data:\n\n- Focus group summary. (Due to data protection issues, data was recorded via note-taking and excluded participants’ personal data. The summary only contains data on points, opinions, and information from the focus group discussion to establish the causes and solutions for ergonomics issues during WFH.)\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 are thankful to the anonymous focus group panellists for their insightful suggestions and information. Special thanks to the three expert panellists that were involved in face validation and the four focus group participants. The authors would also like to express their gratitude to Multimedia University and the organising committee of DIFCON 2021 for facilitating the review and submission of the work related to this study. Thanks to Ministry of Higher education (MOHE) for funding the FRGS grant\n\n\nReferences\n\nAbidin NZ, Rohani JM, Nordin AN, et al.: Financial impact and causes of chronic musculoskeletal disease cases in Malaysia based on social security organization of Malaysia claims record. Int J Eng Technol (UAE) . 2018; 7(3): 23–27. Publisher Full Text\n\nBench A: Alberta ergonomist says extended work-from-home setups causing increase in back pain, eye strain. Global News. 2021, March 23. Reference Source\n\nHanel S: Our work-from-home trend has developed ergonomic growing pains. Ottawa Business J, OBJ360(Ergo-Safety). 2021. Reference Source\n\nThe Movement Control Order (MCO) for COVID-19 Crisis and its Impact on Tourism and Hospitality Sector in Malaysia. Int Tourism Hospitality J. 2020. Publisher Full Text\n\nJobsreet.com: 67% of Malaysian Companies Required Staff to Work from Home.2020. Reference Source\n\nKrishnan DB: WFH: Experts speak about issues to be addressed. The New Straits Times. 2020, October 23. Reference Source\n\nHarun HN: Covid-19 cases surpass 1 million, new daily high. The New Straits Times. 2021, July 25. Reference Source\n\nGorgenyi-Hegyes E, Nathan RJ, Fekete-Farkas M: Workplace Health Promotion, Employee Wellbeing and Loyalty during Covid-19 Pandemic—Large Scale Empirical Evidence from Hungary. Economies . 2021; 9(2): 55. Publisher Full Text\n\nLP S: Ergonomics for Working from Home during COVID-19 Pandemic. Ergonomics Int J. 2020; 4(4), 1–4. Publisher Full Text\n\nYeow JA, Khan MKBJ, Ng PK: Enforcement of safety and health policy reduces human error in SMEs in the manufacturing industry. Advanced Science Letters . 2017; 23(11): 10656–10659. Publisher Full Text\n\nThe Star Online: Proper ergonomics matters when you work from home during the pandemic. The Star Newspaper. 2020, December 22. Reference Source\n\nBell J: WFH injuries on the rise: Why HR needs to take ergonomics seriously. Human Resources Director. 2020. Reference Source\n\nWiener R: Work-From-Home Risks: The Toll of Bad Ergonomics. Risk Management. 2020. Reference Source\n\nBreen RL: A practical guide to focus-group research. J Geography Higher Education . 2006; 30(3): 463–475. Publisher Full Text\n\nKrueger R: Developing Questions for Focus Groups. In Developing Questions for Focus Groups. 2014; Publisher Full Text\n\nPascall MA, Lee K, Fraser A, et al.: Using focus groups to study consumer understanding and experiences with tamper-evident packaging devices. J Food Sci Educ . 2009; 8(2): 53–59. Publisher Full Text\n\nMorgan DL: Focus Groups. Annu. Rev. Sociol. 1996; 22: 129–152. Reference Source\n\nTeoh PY: Work-from-home model can help companies cut costs. The New Straits Times. 2020, June 2020. Reference Source\n\nMurugesan M: The downside of WFH. The New Straits Times. July 14, 2020. Reference Source\n\nNyumba T, Wilson K, Derrick CJ, et al.: The use of focus group discussion methodology: Insights from two decades of application in conservation. Methods Ecology Evolution . 2018; 9(1): 20–32. Publisher Full Text\n\nYeow YA, Ng PK, Lim WY: Workplace Ergonomics Problems and Solutions: Working from Home.2021. (Accessed on 5 September2021)." }	[ { "id": "102080", "date": "24 Mar 2022", "name": "Rahul Jain", "expertise": [ "Reviewer Expertise Ergonomics and human factors", "workplace design", "work from home issues" ], "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 is important, particularly with regard to workplace ergonomics and solutions during the pandemic crisis. However, there are many aspects in which the authors could expand their aim and also analysis of the data.\nThe literature review is adequate. However, several articles published on COVID-19 and work from home scenarios in several countries need to be reviewed for a better overview. Also, it is important not only to describe and explain the issue of COVID-19 but also to link it with the situations that occur during the pandemic. The authors seem to have left out this important link of theory/model in relation to the issue being examined. Perhaps theories/models such as Karsh (2006)1, Sauter and Swanson (1996)2, and other relevant theories/models should be included in this section to highlight the importance of the theories/models in guiding the study. These two articles mentioned are very important to show the causation of work-related musculoskeletal disorders (WMSD) in model form. The current study deals with finding the causation of WMSDs among digital device users during work from home. These citations will help to show the authors various risk factors causing WMSDs because currently, it is not clear from the introduction section how they are devising such theories in their work.\nAdditionally, these studies: Jain et al., 2021a3, Jain et al., 2021b4, Jain et al., 2021c5, and Jain et al., 20226, were conducted by our group during this COVID-19 period where we have used expert opinion-based and stakeholder-based surveys for finding out the major risk factors for the causation of WMSDs. Also, we have tested an intervention for preventing the WMSDs problem in the student population. Currently, we are doing some posture-based studies for improving the working scenarios. Therefore, authors can review these studies for preparing the background of their work, because currently there is no such discussion that can be seen in their text about the causation of WMSDs as per the theories and practices defined in these citations as well as similar citations. It is the right way to discuss things in the meeting by involving the stakeholders and reporting it, but the scientific studies are more important as some information is derived after a large amount of investigation. Currently, the authors have written an essay based on a few references. The authors need to explore the literature fully.\nAs said, the focus group was conducted among four experts. Is there any approval process for the determination? Who are the approvers for the experts' opinions? Who are the ones making the consensus between experts?\nThe results are of some interest but the authors could elaborate further on the practical implications of their findings.\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? Yes", "responses": [] }, { "id": "163234", "date": "07 Mar 2023", "name": "Nancy L Black", "expertise": [ "Reviewer Expertise Ergonomics and human factors", "workstation design", "dynamic workstation use in sedentary work environments", "musculoskeletal discomfort", "posture" ], "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 interesting insights into detailed perceptions of a limited number of people forced to work from home (WFH) during the COVID-19 public health restrictions in Malaysia. The work addresses the perceptions of four participants to a series of 14 questions including one referencing a table of suggested WFH issues with corresponding proposed solutions in a virtual focus-group setting.\n\nUnfortunately, some of the WFH uses and proposed solutions are incomplete or exaggerated and no specific sources are cited within the table and those cited come from online articles that are not peer-reviewed (Wiener is “the CEO of a leading manufacturer of active office workstations and ergonomics accessories”, Bell quotes a physiotherapist and the Star online quotes the “co-director of Mayo Clinic Sports Medicine” and “a Mayo Clinic physical therapist”); better sources are available which are more specific and including ergonomists would make this richer. In Table 1, two elements are particularly problematic: 1) I am unaware of neck or back “sprains” occurring due to poor laptop / desktop positioning. Also, one part of the proposed solution is to “adjust the desk position to chest level” whereas the normal recommendation would be to adjust the desk position to relaxed elbow height, which is approximately equivalent to waist height, and to use a keyboard separated from the computer screen (raising the screen so that its top surface is slightly higher than eye height). Chest height for the keyboard would be too high. 2) The recommendations to compensate for “poor lighting, glare from screen or vision complications” are too vague: “to the brighter light source” does not consider visual problems due to light within the field of vision, and positioning “next to the window” should also mention 90 degrees relative to the window (not facing or back to the window).\nWhile some of the study design was clear, the paragraph describing the focus study group was difficult to follow. I did not understand how the listing of four experts at the start of the paragraph corresponds to the “contacted” people in the second half of that paragraph. This could be easily addressed by rewriting that paragraph.\nGenerally, the results and discussion section is good, although 20-20-20 should be fully defined the first time it is referenced. My understanding from the Abstract was that this article would include “a complete WFH ergonomics and wellness checklist” although this is not included in the article or in the interview data.\nThe interview data includes complete transcripts of the interview, which is useful and could support other research studies.\nConclusions are good, but again, references to the peer-reviewed literature would strengthen the recommendations (e.g. how shared workload \"can eventually help to reduce stress\").\nOverall, the article provides interesting insight into how the purposive sample of three professional men and one woman between the ages of 38 and 55 perceived and experienced WFH following extended public health restrictions (without revealing how long they'd been working in these conditions). Analysis by two ergonomists is good. The article is worthwhile but it lacks sufficient references to the relevant scientific literature (which were available at the time of the initial publication) to fully justify the recommendations included. References found in this cited article would assist: Black and St-Onge (2022)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? 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": "9431", "date": "20 Mar 2023", "name": "Jian Ai Yeow", "role": "Author Response", "response": "Thank you for the feedback and review. Will make the necessary amendment soon." } ] }, { "id": "163229", "date": "08 Jun 2023", "name": "Charles Ramendran", "expertise": [ "Reviewer Expertise Human resource management", "occupational safety and health", "ergonomic", "employment law", "organizational behaviour" ], "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 article has attempted to investigate and prove that ergonomics is very crucial in employment by focusing on qualitative methodology by conducting focus group discussion. However, the focus of the article was not precise with the objective as the authors need clarity on the definition of work from home concept and distinguish it from work at office. Furthermore, authors need to be updated on the latest employment act that has been amended recently in Malaysia and bring about how this research is related to ergonomics and WFH. Besides that, OSHA 1994 has also undergone the amendments which will take effect soon as OSHA 2022. The respondents were very limited and also are not representing the whole concept of ergonomics & WFH. Therefore, it has distracted from the findings of the investigation.\nIntroduction:\nSince 2009, the ergonomic problem has arisen arises and it already existed at the workplace since work from home was not implemented back then in Malaysia. Why then was the research not focused at the workplace instead of at home? Beginning of the article, the authors have a contradictory statement. Revising it will be a better introduction. The authors have mentioned that Since 2009, SOCSO has indicated the ergonomic problem has arisen and the cost to fix is at an alarmed stage. The 1st LINE: Health issues related to sprains, strains, and back pain account for the highest total direct expenses for workers supported by the Social Security Organisation of Malaysia (SOCSO) since 2009. My major concern here is what is this line 1 to do with their objective of study which focuses on WFH and in 2009 in Malaysia there weren't any developments about WFH. Therefore, authors need to reconsider this statement!\n\nWhat is the objective of recommending to HR dept. and OSH workers when all the employees are working from home not in the office? It is out of jurisdiction to monitor the ergonomic functions of employees for both departments while the employees are at home!\n\nCOVID-19 Malaysia: HR dept. and employers can monitor the working system and productivity of employees however it is not possible to focus on ergonomic part of WFH. They are vague and blurry objectives set by the authors. The idea of highlighting the ergonomic issue while WFH is not clear and precise which raises questions on the warrant to investigate in this area.\n\nMethod: Why were the questions not validated by anyone from Occupational Safety and Health since ergonomics is part of OSH? They are more prominent instead of HR management. Targeted respondents are NOT valid without OSH dept.\nFocus group defines expert members from the field of investigation. Only four members is not sufficient to generalize the outcome and practitioners from Occupational Safety and Health Department are missing!\nTable 1: Solution column: using earphones is not a solution as it causes more chronic or acute issues such as dizziness, ear pain, even hearing loss. Authors need to look into it!\nResults: Para 2: Employment Act 1955 was urged to be amended for flexibility in terms of working system, arrangements, psychological concerns. It is nothing to do with ergonomics. If the authors concerned with ergonomics, they have to look into Occupational Safety and Health Act 1994 which is also the government's concern to amend it!\nPara3: \"He added that the stress of managing children’s online classes in addition to household work aggravates this problem. The HR specialist added that those with low technology competency would also be affected by these problems. Additionally, depression, conflicting information about the pandemic, pay cuts, and other negative issues affect emotional health while working.\" This feedback/statement above does not reflect on the ergonomic issue. This response questions whether the authors selected the right respondents as the panel are expressing psychological issues than ergonomic. The statement is not significantly relevant to the ergonomic issue due to WFH!\n\"...obtain information about quality control checks on furniture manufacturers. Furniture manufacturers should also be encouraged to produce more ergonomic furniture over aesthetic designs.\" The statement above: This is a valid suggestion and manufacturers seriously need to look into it. The manufacturers need to produce ergonomic equipment (furniture) and such products must be affordable to employees to purchase and use at home for work purposes!\nConclusion: In my opinion, conclusions should be within 12 to 15 lines.\nOverall, title needs to be revamped as the article does not highlight the problems clearly, and also provide solutions especially for those who are working from home.\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? No\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? Not 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": [] }, { "id": "159747", "date": "14 Sep 2024", "name": "Lilis Surienty Abd. Talib", "expertise": [ "Reviewer Expertise Occupational Safety and Health", "Quality of WorkLife" ], "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 a good attempt by researchers to document WFH experiences during the MCOs in Malaysia which was long. However, the data needs to be collected responsibly as to promote guided research in the area by others in the future. A careful approach to data collection is basically needed for this attempt although it is exploratory research. It can be a small scope but the conclusion made must be strictly confined to what has been collected.\nAdditional information required:\nAdd more information as to why the panellists were suitable for the FGDs. Although they are experts in their fields, what are the indicators that what they think could happen really happened during the MCOs?\n\nClearly explain the FGD grouping because now it is not clear how many were really participating in the FGDs. There were changes and not clear whether the selections were Melaka state sensitive still. A bit confusing.\n\nRESULTS: Since there is no direct relationship between the selected panellists to the outcome of MSD or ergonomic health-related evidence/cases directly following the MCOs' practices of WFH (work from home), then there should not and can not be directly associated with WFH and MCOs. Unless these panellists hold data showing there is an increase in the number of symptoms and cases reported related to these. What can be concluded is that these are opinions and observations made by the panellists based on their expertise and that is all. Researchers can record the agreement and disagreements expressed by the panellists during the FGDs. But, to suggest that an ergonomic chair is the solution in general for WFH is too strong and general to be made by the authors. Because it depends on many factors such as nature of tasks, jobs, condition of work which lead to the use of chairs.\n\nThese analyses resulting in the conclusion should be illustrated somehow in the results 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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-1025
https://f1000research.com/articles/10-6/v1	06 Jan 21	{ "type": "Review", "title": "AI in healthcare: A narrative review", "authors": [ "Antti Väänänen", "Keijo Haataja", "Katri Vehviläinen-Julkunen", "Pekka Toivanen", "Keijo Haataja", "Katri Vehviläinen-Julkunen", "Pekka Toivanen" ], "abstract": "In this paper, we focus on providing a narrative review of healthcare services in which artificial intelligence (AI) based services are used as part of the operations and analyze key elements to create successful AI-based services for healthcare. The benefits of AI in healthcare are measured by how AI is improving the healthcare outcomes, help caregivers in work, and reducing healthcare costs. AI market in healthcare sector have also a high market potential with 28% global compound annual growth rate. This paper will collect outcomes from multiple perspectives of healthcare sector including financial, health improvement, and care outcome as well as provide proposals and key factors for successful implementation of AI methods in healthcare. It is shown in this paper that AI implementation in healthcare can provide cost reduction and same time provide better health outcome for all.", "keywords": [ "Artificial Intelligence", "Healthcare Analytics", "Machine Vision", "Machine Learning" ], "content": "Introduction\n\nThe healthcare industry is undergoing a revolution. The reasons for this revolution are the ever-increasing total spending on health care and the growing shortage of health care professionals. This drives to situation that healthcare industry wants to adopt new Information Technology based solutions and processes with advanced technology which could reduce costs and provide solutions for these emerging problems.\n\nBefore 2010, healthcare technology companies were focusing on the innovations provided by medical products providing historic and evidence-based care. Starting from 2010 development has focused on real-time medical platforms and outcome-based care. From 2020 technology is moving towards medical solutions delivering intelligent solutions for evidence- and outcome-based health where focusing is in collaborative and preventative care. These intelligent solutions can be achieved by using robotics, virtual and augmented reality and AI1. In a recent life science executives survey 69% of life science businesses are already piloting or adopted AI in their solutions and 22% are evaluating or planning to pilot AI solutions2. The annual savings potential by using AI in healthcare can be $150 billion by 2026 in US alone, and this should be also one of the factors to speed up the implementation of AI in healthcare sector3. Future studies will give answers if this savings will be achieved. There are multiple subdomains in healthcare domain where AI-based services are utilized.\n\nAI applications in healthcare can be classified to the following domains: surgery, nursing assistant, medical consultation, administration and workflow, treatment design, cybersecurity, machine vision, automatic and preliminary diagnosis, health monitoring, medication management, and clinical trials. All these domains have applications utilizing AI in their operations. We have collected some of the AI-utilizing services in the healthcare sector and made a comparative analysis of the effectiveness and outcome when utilizing AI versus services which are not utilizing AI. As a result, in this paper we introduce AI-based services for healthcare sector that have a high impact in care outcome and propose collection of services which could provide best outcome in preventive healthcare and in clinical work.\n\n\nServices utilizing AI in healthcare domain\n\nIn this section we review healthcare services which are utilizing AI. These services include healthcare services and supporting services which are shown in Figure 1. The reviewed services are collected from research papers and market analysis companies3. Services which are selected for analysis are providing instant care or providing direct support for care. These are robot-assisted surgery, clinical trial participation, virtual assistants for nursing and consultation, image diagnosis, dosage error reduction, medication management, preliminary diagnosis, and health monitoring. Some of the topics which have been recently studied in my research and which provide indirect services for healthcare processes but are not affecting directly to patient care, such as fraud detection, assistant for administration and workflow, cybersecurity, connected machines, and drug creation are excluded from this study. We also categorized healthcare services and applications based on degree of AI utilization and human involvement in clinical decision making4.\n\nBelow we provide a short description of AI-methods which are commonly utilized in healthcare applications and services. Common applications for AI methods are shown in Figure 2.\n\nMachine learning (ML) is a subfield of AI and presents AI solutions that are adaptive. The ML can be roughly categorized to three subareas. Supervised learning algorithms build a mathematical model of a set of data containing both the desired outputs and the inputs5. In unsupervised learning algorithms take a data set containing only inputs and find structure in the data, such as grouping or clustering of data points. In unsupervised learning the algorithms learn from the test data that has not been classified, labeled or categorized6. Reinforcement learning algorithms do not assume knowledge of an exact mathematical model of discrete time stochastic control process. Reinforcement learning is used, for example, in situations where a self-driving car is operating in an environment where feedback about good or bad choices is not available in real time.\n\nNatural language processing (NLP) is a subfield of AI that consist of tools and techniques to enable computers to read, understand, and derive meaning from human languages and enable natural interaction between computers and humans to make computer systems understand and manipulate natural languages to perform desired tasks. In healthcare, NLP is used, for example, to predict diseases based on patient’s own speech and electronic health records7.\n\nNeural network (NN) consists of digitized inputs, such as an image or speech, which proceed through several hidden layers of connected artificial neurons, each layer responding to different features progressively detecting features and provide an output. Deep NNs (DNN) require special mention because as a subcategory of NN with its variations, such as recurrent, convolutional, transfer, generative adversarial, reinforcement, representation, and transfer, are used in various AI solutions in field medicine. Typical use case for DNN is when there is need for interpret data to certain patterns from different types of clinical images such as pathology, skin lesions, retinal, and endoscopy images and to find out patterns from datasets, such as medical scans, electrocardiograms, and vital signs.\n\nDeep learning (DL) is a subfield of ML. DL is based on artificial NNs (ANNs), inspired by information processing and distributed communication nodes in biological systems, that use multiple layers to progressively extract higher level features from raw input. DL can be semi-supervised, supervised, or unsupervised. The term 'deep' refers to the number of layers (depth) through which the data is transformed. DL is a form of AI that enables computers to perform tasks based on existing data relationships.\n\nMachine vision/computer vision (MV/CV) include the methods and the technology which is used to automatically extract information from an image. The extracted information can be a i.e. good-part / bad-part signal or a set of data such as the identity, orientation, and position of objects in an image. Extracted information can be later used for applications like automatic inspection, security monitoring, industry robots and process guidance, and vehicle guidance.\n\nIn the next sections we provide detailed information about topics in healthcare AI. Summary of these techniques are collected to Table 1 where we provide studied information about AI based services which are in use in healthcare business. Studied applications / services information is limited to Healthcare domain and include the following information: service / application type, category of AI service, application / service type, AI features which have been used in application / service, outcome as a result of AI utilization, and metrics which have been used to present the outcome.\n\n\nRobotic-assisted surgical systems (RASS) and computer-assisted surgery (CAS)\n\nThe RASS have been used since year 19858 in multiple fields of surgery including cardiac surgery, thoracic surgery, gastrointestinal surgery, gynecology, orthopedic surgery, spine surgery, transplant surgery, urology, and general surgery9–16. RASS have been categorized to tree types based on the level of autonomous activity: supervisory controlled surgery, telesurgical, and shared control surgery.\n\nIn traditional surgery, surgeons can operate only on what their eyes can see and basically the only way to see inside a patient is to operate by open surgery. With RASS, surgeon can utilize cameras and tools which can be inserted through a small incision to perform procedures with exquisite precision. The minimally invasive approach is meant to provide faster patient recovery times and reduce postoperative complications. RASS can also lessen the physical burden of surgery staff. With RASS there is also possibility to collect all data and details such as video recording of the surgery, all movements and cutting and sewing actions of ongoing surgery, and use this collected data for further analysis and enhance and lean the surgery process. There are RASS manufacturers which are providing robotic surgery equipment worldwide. In this paper, we provide details about the one of the most used RASS at the time of writing this paper, DaVinci, developed by Intuitive Surgical Inc. DaVinci had 5406 installed bases in September 201917.\n\nCAS is a second approach to assisted surgical systems. Where RASS is focusing the physical surgery robot, its controlling and applications, and robot assisted surgery technology, CAS technology use computer technology for surgical planning and enhances surgical guidance to surgeon. CAS involves techniques that can directly participate in surgery or can assist in the navigation or positioning of surgical instruments18. CAS contain a set of applications used at the surgical workplace preoperatively, intraoperatively, as well as improving surgical efficiency and efficacy postoperative surgery19. Main CAS features contain creation of virtual image from the patient, diagnostic, image analysis and processing, preoperative planning, surgical simulations, and surgical navigation. CAS have been major factor in the development of RASS.\n\nWhen evaluating the clinical effectiveness of robotic surgery technology, a systematic review from 95 studies made in Canada in 2011 indicated that RASS in prostatectomy, hysterectomy, nephrectomy, and cardiac surgery compared to open or laparoscopic surgery there are many benefit in clinical outcomes which were: reduction of length of hospital stay, reduction of blood loss and transfusion rates, and reduction of complications20. There are some RASS operations in which operation times are reduced such as laparoscopic prostatectomy and, in some operations, increased such as open prostatectomy and open hysterectomy. In the same review, the economical evidence was also evaluated in prostatectomy, cardiac surgery, nephrectomy, and hysterectomy. Cost analysis showed that shortening the lengths of stay after robotic radical prostatectomy also produced reduction of hospitalization costs relative to laparoscopic surgery and open surgery. In other hand, acquiring and operational costs of surgical robots will lead to situation that approximately 75% of the surgeries in which robot assisted the surgery is more expensive. However, costs can be reduced by higher utilization rate of RASS.\n\nA systematic review was made in 2017 to evaluate patient benefits, cost, and surgeon conditions when using RASS in gynecological oncology21. Using RASS as part of treatment in cervical cancer, endometrial cancer, and ovarian cancer were studied by reviewing total of 76 references. Results were that safety in oncological surgery is similar compared with previous surgical methods, but RASS will also increase overall costs because of high RASS equipment application, acquisition, and maintenance costs.\n\nIn the research where cost and efficacy of robotic hepatectomy was studied there was indication that in RASS group operative time was 20% longer, length of stay was 35% shorter, perioperative costs were 10% higher, and postoperative costs 36% lower compared to group with normal open surgery. In the study, overall costs of RASS were 22% lower than in open surgery. Complications were similar between RASS and open surgery patient groups41.\n\n\nVirtual nurse assistants (VNAs) for healthcare\n\nIn modern digitalization, healthcare organizations and actors in healthcare processes have taken in use virtual assistants which have already been emerged to use in other business sectors. With virtual nurse assistants, hospitals can reduce sudden hospital visits and reduce the workload of healthcare professionals. These virtual assistant applications can listen, talk, and give advices/recommendations. Over the last two decades there have been studies of embodied conversational agents (ECAs) use in healthcare which have proven significant improvement in healthcare outcome when chatbots or conversational agents are in use. A majority of these ECAs have allowed user input which is used in common surveys such as multichoice or utterance. With latest advance in AI technologies, such as NLP, machine learning (ML), and neural networks (NN), have made possible to develop virtual assistants or virtual agents which are capable of utilizing conversational systems which can mimic human conversation42,43.\n\nOne widely used platform meeting European Medical Device Directives is Your.MD. This virtual health assistant using AI and machine learning utilizes United Kingdom National Health Service (NHS) data for providing personalized pre-primary care. Pre-primary will act before patient makes decision to access primary care. With Your.MD patients can perform home diagnosis by using mobile app or website. With Your.MD benchmark tests in verified test cases from Harvard University and Royal College of General Practitioners have shown medical accuracy of 85% for 20 most common conditions22.\n\nIn another study there was multiple symptom assessment apps studied, where the best results for condition coverage, accuracy of suggested conditions and urgency advice performance was measured with 200 vignettes representing real world scenarios and compared to five general practitioners (GPs). The best results were received by service ADA getting condition suggestion coverage of 99% and top-3 conditions suggestion 70.50% (GPs average 82.10%), 97% accuracy for safe urgency advice (GPs average, 97%)23.\n\nAnother VNA platform is Sensely. Its digital nurse avatar use machine learning algorithms. It utilizes patient’s medical history data, and it can monitor the condition of patient. Additionally, the VNA can keep track of appointments, fill the gap between doctor visits and predict follow-up treatments. Platform was trialed in 2019 with 72 chronic heart failure patients in clinical site. Findings indicated that platform was able to decrease readmission rate by 75% and patient monitoring costs by 66% compared to traditional care process24.\n\n\nMedication management and medication error reduction (MMMER)\n\nThe effective MMMER services can provide remarkable healthcare cost expenditure reduction and can minimize unnecessary injuries and deaths. The estimated annual costs of drug-related mortality and morbidity resulting from nonoptimized medication therapy was $528 Billion in US. This is equivalent to 16% of total US health care expenditures in 201644. Prescription drug errors cause substantial morbidity, mortality, and wasteful health care cost. In a National Audit Commission report there was evaluated that there are 7000 deaths annually in US due to medicine misuse and due to medication mistakes highlighting importance and urgency of preventive measures45. AI has multiple use cases in Medication management and dosage error reduction, such as improving medication safety, preventing drug overdoses, predict health risks and outcomes across large populations, reduce time and expenses, and monitor medication adherence.\n\nImproving medical safety. MedAware created system for detecting medication errors. In this ML-based system researchers concluded that it was clinically useful in flagging 75% of potential medication errors or issues, with 18.80% classified as having medium clinical value, and 56.20% of the valid alerts as having high clinical25.\n\nMonitoring medication nonadherence. Medication nonadherence is significant issue in healthcare costs and healthcare outcome. Medication nonadherences contribute between $100 and $300 Billion dollars in US46. One study using AI platform on mobile devices in measuring and increasing stroke patients’ medication adherence when ongoing anticoagulation therapy. Study indicated that in the intervention group medical adherence was 100% and in the control groups only 50%. The AI application was visually identifying the patient, the medication, and the confirmed ingestion. Adherence was later measured by plasma sampling and pill counts26. Another adherence monitoring AI platform utilizing neural network algorithm in machine vision was studied in clinical. Machine vision and neural network was used to identify visually patient, the drug, and confirm the ingestion of the drug. Study shows that adherence was 17.90% higher in AI group than standard-of-care modified directly observed therapy protocol27.\n\n\nClinical trial participation (CTP)\n\nAs described by United States Food and Drug Administration (FDA) and U.S. National Library of Medicine, in a clinical trial, participants are receiving specific interventions based on the research plan or protocol which is created by the investigators or researchers. These interventions may be medical products, such as drugs or devices, procedures, or changes to participants' behavior, such as diet. Clinical trials have three models. 1. Comparing new medical approach to a standard model or approach that is already available. 2. comparison new approach to a placebo containing no active ingredients. 3 comparison to new approach where no intervention is done47. It is also notable that on average it takes 10 - 15 years and 1.5 - 2.0 Billion US dollars to develop and bring a new drug to the market. In drug development, approximately 50% of the time and investment is used for the clinical trial phases48.\n\nAs a background to create more comprehensive clinical trials, for example, in one research done in Mayo Clinic participation for clinical trials of cancer patients was only 3-5% even though up to 20% were eligible28. AI can help in clinical trial design and it can be used in finding patterns of meaning automatically from large and unstructured datasets such as speech, text, or images. Natural language processing NLP understands and correlates content in spoken or written language, and in human-machine interfaces) allowing natural information exchange between humans and computers. These capabilities are used for correlating diverse and large datasets such as medical literature, electronic health records (EHRs), and databases for improving patient-trial match and recruitment or persons before starting actual trial. During actual clinical trial AI can be used monitor patients continuously and automatically. Moreover, AI utilization provide improved adherence, efficient endpoint assessment and increased control and yielding reliability. Based on this background information and need for enhancing the clinical trial participation rate there was a research conducted by IBM. In this research IBM utilized Watson artificial intelligence platform resulting 80% increase in oncology clinical trial enrollment. This increase was observed at Mayo Clinic. This new platform enabled high volume screening very efficiently28.\n\nMendel.ai research network has created Clinical trial participation pre-screening service for identifying patients which were potentially eligible for clinical trial. In one research Mendel.ai was used to retroactively provide pre-screening two oncology studies, one for breast cancer and one for lung cancer. In trials where Mendel.ai was used it resulted in a 24 - 50% increase compared to standard practices to correctly identify the number of patients as potentially eligible for clinical trials. All patients who were correctly identified by standard practices were also identified by Mendel.ai. With standard pre-screening practice an average of 263 days for lung cancer and 19 days for breast cancer patients elapsed between actual patient eligibility and identification. Respectively, detection of potential eligibility with Mendel.ai took only minutes29.\n\n\nPreliminary diagnosis and prediction (PDP)\n\nFor decades diagnosis services have been using health history data and diagnosis data to provide more accurate diagnosis for the patient and more accurate health prognosis. With current advances in AI research we have found that AI have outperformed physicians in speed and accuracy of medical diagnosis in some fields of healthcare sectors as described in following example studies from various fields of healthcare.\n\nDiabetes prediction can be performed with four different application types: retinal screening, clinical decision support, predictive population risk and patient self-management tools. In retinal screening application perform detection of diabetic retinopathy, maculopathy, exudates, and other abnormalities from retinal scan. In clinical decision support application or service can contain detection and monitoring of diabetes and comorbidities such as nephropathy, neuropathy, and wounds. In predictive or population risk stratification identification focus is on identification of diabetes subpopulations at higher risk for complications, hospitalization, and readmissions. There are also self-management tools in patient use which can consist of artificial pancreas, AI-improved glucose sensors and dietary and activity tracking devices.\n\nOne of the diabetes tracking system is Medtronic's Guardian Connect. It was the first AI-powered and FDA approved continuous glucose monitoring (CGM) system. With predictive Machine Learning (ML) algorithm Guardian Connect can predict significant changes in blood glucose levels. Changes can be predicted up to 60 minutes before the change event. System consist also sensor, which is placed on the abdomen. This sensor monitors blood glucose levels in 5 minutes interval. System was able to give alert of about 98.50% of hypoglycemic events. By these alerts, patients could act proactively to normalize their blood sugar30.\n\nHouston Methodist researchers in the US have developed a NLP-based application that can interpret mammography results by using free text radiology and pathology reports from 543 patients and keywords. Application could perform with 99% accuracy compared to manual review by clinicians. Time saving is remarkable when comparing to manual review taken from 10% of these 543 patients for application accuracy validation. This manual validation for 54 patients took approximately 50 – 70 hours31.\n\nFor diagnosing tuberculosis tests were performed with two different DCNNs, AlexNet and GoogLeNet, which are learning positive and negative X-rays for tuberculosis. The accuracy of the models was tested in 150 cases. The most functional AI model was achieved by combination of AlexNet and GoogLeNet with 96% accuracy. There were differences between the two DCNN models in 13 cases of 150. The diagnostic accuracy of the radiologist in these cases was 100%. Previously machine learning was able to get only 80% results but using deep learning the accuracy has been increased. Artificial intelligence in diagnosing tuberculosis can play very important role in the fight against the tuberculosis in the near future32.\n\nResearchers at Columbia University's New York State Psychiatric Institute and the IBM Watson Research Center have developed an AI application using automated NLP with ML capable of 100% accurate detection of the development of psychosis in susceptible individuals. Traditional diagnosis reaches 79% accuracy. Research Utilizing artificial intelligence to diagnose this disease has proven to be beneficial. Like psychologists, the app analyzes speech patterns to differentiate patients who are susceptible to psychosis. IBM researchers realized that if the mind of the interviewer (psychologist) began to wander for even a moment, they might have missed the signs that were essential to the development of psychosis, while the computer noticed them. An artificial intelligence diagnostic system eliminates human error and is therefore more accurate in diagnosis than experts33.\n\n\nMedical imaging and image diagnostics (MID)\n\nMedical imaging data is one of the best sources of information about patients and at the same time the most complex. Traditionally interpreting medical imaging scans is a highly skilled, manual job requiring many years of training. In the field of medical imaging the AI technologies, such as DNN and DL, can produce remarkable improvement in healthcare outcome and have proven to provide enhancement in speed, accuracy, and cost reduction in interpretation of medical images. MID can be utilized in healthcare for multiple cases, such as for identifying cardiovascular abnormalities, detecting musculoskeletal injuries, identifying neurological diseases, identifying thoracic complications, and common cancers screening. In the business perspective AI have high potential in MID and it is estimated to rise from $21 billion in 2018 to a value of $265 billion by 202649.\n\nIn one large meta-analysis and systematic review researchers compared diagnostic accuracy between DL methods (such as ANN, CNN and DCNN and healthcare professionals in medical imaging. From 31,587 identified studies on DL methods in medical imaging researchers included 69 studies. These studies provided enough data for calculation of accuracy with sensitivity with mean 79.10% and specificity with mean 88.30%. From these studies 14 used same sample for the out-of-sample validation between DL methods and healthcare professionals. With these 14 studies comparison results between DL methods and healthcare professionals was DL methods systematic review and meta-analysis of the complication rate and diagnostic accuracy compared to healthcare professionals 86.40% and DL models having pooled specificity of 92.50% compared to healthcare professionals 90.50%34.\n\nIn oncology, lung cancer and breast cancer are the leading causes of cancer deaths in the world and therefore there is a need for early detection solutions of these cancer types35. Most common method in breast cancer screening is digital mammography. Reading mammography images is a difficult task for radiologist and can result in both false negatives and false positives. These inaccuracies can cause delays in cancer detection and starting the treatment which lead to higher workload for radiologists and unnecessary stress for patients.\n\nMcKinney et al. created an AI system with a set of three deep learning models. This new system was capable of surpass human experts in breast cancer prediction. Researchers evaluated the developed system with two large datasets from UK (n=25,856 women) and from USA (n=3097 women). In USA new service produced 5.70% reduction of false positives and 9.40% reduction in false negatives. In UK, new service produced 1.20% false positives reduction and 9.40% reduction in false negatives. All comparison was made between AI system and clinical readers35.\n\nIn another research, a Stanford University research group taught the neural network with a database of 130,000 cancer images to automatically identify cancer and make diagnoses. The research team also tested the neural network with a database of 14,000 skin lesions. Neural network made valid diagnoses with 72% accuracy. The reference was dermatologists who were able to diagnose with a diagnosis accuracy of 66%. The test was extended to 25 doctors and there were 2000 skin lesion images, each with biopsies. The neural network was able to beat specialist doctors in all situations37.\n\nOCT is one of the most common imaging procedures and in US there was 5.4 Million OCT scans performed in 201436. DeepMind's DL was being taught with 14,884 OCT scan images from 7621 patients to recognize 50 common eye problems including three of the biggest eye diseases (diabetic retinopathy, glaucoma, and age-related macular degeneration). The AI correctly identified types of eye disease from OCT scans 94.50% of the time and 36.\n\nResearchers in China have developed a convolutional neural network-based AI platform that can identify, evaluate, and suggest treatment for congenital cataracts. Researchers tested the accuracy of CC-cruiser and the result was that the performance of the CC-Cruiser was comparable to that of an ophthalmologist: the CC-Cruiser was able to successfully diagnose all potential patients in 50 patients. Ophthalmologists were even slightly worse and made misdiagnoses in a few patients. The CC-Cruiser provided detailed care instructions to patients who needed surgical treatment and no misdiagnosis occurred50.\n\n\nPatient health monitoring (PHM)\n\nContinuous PHM can reduce patient length of hospital stay and can increase recovery time and reduce mortality rate. In home use, PHM can provide information, instructions, and reminders for preventive care. In this paper, PHM consist of services and solutions using AI methods for continuous healthcare monitoring, healthcare assessment tools, and symptom checking solutions which can be used by patients and healthcare professionals periodically or continuously. HM services are utilized in hospitals or in home. When evaluating the user acceptance of HM services in our earlier research we found that AI-based health condition monitoring solutions were evaluated as the best of 15 as most considerable technologies for healthcare and the second-best service among 15 services what healthcare professionals are willing to use51.\n\nIn Italy, researchers have developed a prototype of computer aided diagnosis, a computer-assisted diagnostic system for the diagnosis of heart disease, using artificial intelligence. The system is intended to assist general practitioners and nurses in clinical decision making that are not specialized in cardiology. The system receives anamnestic (pre-information) and instrumental (instrument-related) data and makes a diagnosis and prognosis relative to the patient's current state of health. The system also considers the patient's previous clinical history when making diagnoses. In addition, the system builds a database of patients’ data with heart failure by providing a valuable data repository for future utilization. The best results of all groups (mild, moderate, and severe) were obtained by using neural networks as the artificial intelligence method, which correctly classified the training population of 98/100 patients and the test group 31/36. The overall accuracy for the test population was 86.10%, which was the best of all technologies in the test38.\n\nIn one research, ANNs and ML algorithms was utilized to predict in-hospital mortality for patients undergoing repair of abdominal aortic aneurysm. Researchers used clinical variables such as patient history, medications, blood pressure, and length of stay as input to ANNs and ML algorithms. As a result, prediction system generated predictions of in-hospital mortality with sensitivity of 87%, specificity of 96.10%, and accuracy of 95.40%39.\n\nFinnish company Kaiku health has developed a health intervention platform for symptom monitoring and management for improving cancer patient’s quality of life and survival rate. In trial with 766 metastatic cancer patients made to evaluate effectiveness of symptom monitoring with Kaiku shows that digital symptom monitoring during chemotherapy provide patients 5.2 months longer overall lifetime, improve their quality of life within 31% of patients, and reduces hospitalization by 4% and ER visits by 7%40.\n\n\nKey elements for successful implementation of AI-based services in healthcare\n\nOur research was using only a fraction of healthcare services and applications which has been studied globally. In our review we focused on research projects or healthcare services which are commonly in use by healthcare service providers and from these we also focused to services in areas which are focusing on actual care processes. From these areas we found that there are thousands of research articles from each area of healthcare where AI methods are used to provide enhancement to healthcare. We also selected the services which have been evaluated as most considerable AI services by healthcare professionals51 and by general public52 in earlier studies. Some areas from healthcare administration systems, for example, fraud identification, connected machines, information management, and data security were left out from this review.\n\nBased on this review and our earlier studies we have noticed that there are multiple use cases where AI methods can be used to provide enhancement to healthcare processes. AI utilization can: 1) save time spent in healthcare work 2) give accurate diagnosis 3) make findings from medical images and medical reports 4) monitor health conditions and predict occurrence or progress of diseases 5) provide more quality to care 6) reduce complications in surgical operations 7) control medical adherence and medication misuse and 8) provide help in clinical decision making. Identified benefits are collected in Figure 3.\n\nWhen considering the success factors for implementation of AI methods to healthcare services we propose that successful implementation to actual healthcare use require at least:\n\na) A large clinically validated dataset to teach and validate AI methods.\n\nb) Scientific research where new or existing AI methods are validated together with healthcare professionals and AI methods developers.\n\nc) Clinically and scientifically proven enhancement in specific healthcare use case.\n\nd) Medical device certification for target market area.\n\ne) Clearly inform end users that AI does not make itself clinical decisions. It only give recommendations, provide help in clinical activities or support clinicians in decision making.\n\n\nConclusion and future work\n\nAs a conclusion of our systematic review we found out that AI can have remarkable possibilities in reducing healthcare costs, providing preventive healthcare, ease the work burden of healthcare professionals, and providing more accurate diagnosis faster and easier. The need for AI services arises in the facts that healthcare costs are continuously increasing. Additionally, age structure of population is changing, especially in developed countries, causing that there will be more chronic diseases within aged population needing expensive care. There will be also shortness of trained nurses and healthcare professionals. Moreover, access to modern and effective healthcare services are not available especially to poor and elderly people and for most of the population living in developing countries. When AI methods are used in healthcare research and IT processes in full scale, we can achieve remarkable savings in overall healthcare costs and same time improve health outcome and quality of life. The need for enhancement provided by AI methods can be seen in every studied healthcare service areas.\n\nMoreover as conclusion we have identified and proven that there is very high potential for the state-of-the-art AI solutions in almost every healthcare sector to reduce costs, ease healthcare professionals workload, improve quality of life for patients, provide preventive health and improve overall health outcome. There are also AI based solutions which can be utilized for population in developing countries. These services include preliminary diagnosis, preventive health services, patient health monitoring services and virtual nurse assistants. In addition, it must also be emphasized that artificial intelligence solutions can produce $150 billion savings in global healthcare industry by 20263. Based on these findings we can express that investing to AI in healthcare will pay for itself. We recommend all healthcare IT service development companies and research organizations to fully adopt scientifically validated AI methods in their research and development projects.\n\nWhen developing and implementing new applications and services for industrial purposes and especially for healthcare industry safety and quality of service are also key elements for successful new service implementation. To maintain and enhance quality AI method developers need to fulfill standards, regulations and solve possible emerging legal constraints. During the research we found out that United States Food and Drug Administration have proposed a regulatory framework for AI and ML-based technologies that consist of new issues in utilizing AI in healthcare53. This new framework should be utilized when developing new AI based technologies to EU markets until EU Medical Device Regulation (MDR) will create own regulatory framework for AI and ML-based technologies.\n\nMoreover, one recommended to healthcare AI service developers in AI methods implementation is to join into interest group and evaluation framework. There have been established the Focus Group on artificial intelligence for health which is a joint initiative of the World Health Organization and International Telecommunications Union that brings together academia, industry, and governmental stakeholders to drive the application of AI in health by establishing an evaluation framework54.\n\nIn our future work we design a model for novel state-of-the-art AI platform to be utilized by all healthcare service providers and healthcare IT services developers. Encouraged by the results of this research and as future work we continue the research and design of novel platform where AI methods and pre-trained AI methods can be utilized and adopted to healthcare IT services by an cloud-based, open access, easily integrated platform which use open access and even proprietary health data repositories, national health databases, hospital information systems and imaging databases as learning data and constantly evolving AI methods for providing fast, automated, and accurate diagnosis & prognosis.\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\nFrost & Sullivan: Transforming healthcare through artificial intelligence systems. 2016. Reference Source\n\nAccenture Technology Vision 2019: Full report, Accenture. Retirieved 19.12.2019. Reference Source\n\nArtificial Intelligence: Healhtcare’s new nervous system. 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PubMed Abstract \| Publisher Full Text\n\nHameed AM, Yao J, Allen RD, et al.: The Evolution of Kidney Transplantation Surgery Into the Robotic Era and Its Prospects for Obese Recipients. Transplantation. 2018; 102(10): 1650–1665. PubMed Abstract \| Publisher Full Text\n\nLee DI: Robotic prostatectomy: what we have learned and where we are going. Yonsei Med J. 2009; 50(2): 177–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInvestor Presentation Q4 2019. Intuitive Surgical, Inc. Retrieved 13.12.2019. Reference Source\n\nJenny JY: The history and development of computer assisted orthopaedic surgery. Orthopade. 2006; 35(10): 1038–1042. PubMed Abstract \| Publisher Full Text\n\nKenngott HG, Wagner M, Nickel F, et al.: Computer-assisted abdominal surgery: new technologies. Langenbecks Arch Surg. 2015; 400(3): 273–281. PubMed Abstract \| Publisher Full Text\n\nHo C, Tsakonas E, Tran K, et al.: Robot-assisted surgery compared with open surgery and laparoscopic surgery: clinical effectiveness and economic analyses [Internet]. 2011. PubMed Abstract\n\nKristensen SE, Mosgaard BJ, Rosendahl M, et al.: Robot-assisted surgery in gynecological oncology: current status and controversies on patient benefits, cost and surgeon conditions - a systematic review. Acta Obstet Gynecol Scand. 2017; 96(3): 274–285. PubMed Abstract \| Publisher Full Text\n\nCarr-Brown J, Berlucchi M: Pre-primary care: an untapped global health opportunity. Your.MD. 2016. Reference Source\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\nSensely: An integrated payer/provider wanted to intervene in a timelier manner with its Chronic Heart Failure (CHF) patients. 2019. Reference Source\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\n\nLabovitz DL, Shafner L, Reyes Gil M, et al.: Using Artificial Intelligence to Reduce the Risk of Nonadherence in Patients on Anticoagulation Therapy. Stroke. 2017; 48(5): 1416–1419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBain EE, Shafner L, Walling DP, et al.: Use of a Novel Artificial Intelligence Platform on Mobile Devices to Assess Dosing Compliance in a Phase 2 Clinical Trial in Subjects With Schizophrenia. JMIR Mhealth Uhealth. 2017; 5(2): e18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaddad T, Helgeson J, Pomerteau K, et al.: Impact of a cognitive computing clinical trial matching system in an ambulatory oncology practice. Abstract presented at American Society of Clinical Oncology (ASCO) Annual Meeting. 2018; 36(15): 6550. Publisher 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\nChristiansen MP, Garg SK, Brazg R, et al.: Accuracy of a Fourth-Generation Subcutaneous Continuous Glucose Sensor. Diabetes Technol Ther. 2017; 19(8): 446–456. PubMed Abstract \| Publisher Full Text \| Free 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\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\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. 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\nDe Fauw J, Ledsam JR, Romera-Paredes B: Clinically applicable deep learning for diagnosis and referral in retinal disease. Nat Med. 2018; 24(9): 1342–1350. 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(7639): 115–18. PubMed Abstract \| Publisher Full Text\n\nGuidi G, Iadanza E, Pettenati MC, et al.: Heart failure artificial intelligence-based computer aided diagnosis telecare system. In International Conference on Smart Homes and Health Telematics. Springer, Berlin, Heidelberg. 2012; 278–281. Publisher Full Text\n\nMonsalve-Torra A, Ruiz-Fernandez D, Marin-Alonso O, et al.: Using machine learning methods for predicting inhospital mortality in patients undergoing open repair of abdominal aortic aneurysm. J Biomed Inform. 2016; 62: 195–201. PubMed Abstract \| Publisher Full Text\n\nBasch E, Deal AM, Dueck AC, et al.: Overall Survival Results of a Trial Assessing Patient-Reported Outcomes for Symptom Monitoring During Routine Cancer Treatment. JAMA. 2017; 318(2): 197–198. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSham JG, Richards MK, Seo YD, et al.: Efficacy and cost of robotic hepatectomy: is the robot cost-prohibitive? J Robot Surg. 2016; 10(4): 307–313. 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. 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PubMed Abstract \| Publisher Full Text\n\nData Bringe Market Researcher: Global Artificial Intelligence in Medical Imaging Market - Industry Trends - Forecast to 2026. Reference Source\n\nLong E, Lin H, Liu Z, et al.: An artificial intelligence platform for the multihospital collaborative management of congenital cataracts. Nat Biomed Eng. 2017; 1(2): 0024. Publisher Full Text\n\nVäänänen A, Haataja K, Toivanen P: Survey to healthcare professionals on the practicality of AI services for healthcare [version 1; peer review: 1 approved with reservations]. F1000Res. 2020; 9(760): 760. Publisher Full Text\n\nPWC: Why AI and Robotics Will Define New Health. Reference Source\n\nFood and Drug Administration: Proposed regulatory framework for modifications to artificial intelligence/machine learning (AI/ML)-based software as a medical device (SaMD)-discussion paper. 2019. Reference Source\n\nInternational Telecommunications Union (ITU): Focus Group on artificial intelligence for health (FG-AI4H). Reference Source" }	[ { "id": "84684", "date": "13 May 2021", "name": "Dipak Kalra", "expertise": [ "Reviewer Expertise Health Informatics" ], "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 valuable area of research, and appears to have been conducted well. However, the organisational structure of the paper has left me confused, and I think it needs some further work before it can be indexed.\nThe title describes the paper as a narrative review, but I suspect it has been extracted from a larger document. I note that on page 1 there is mention of \"my research\". The conclusion starts by referring to this as a systematic review. What I am missing in this paper is anything about the methodology.\n\nThe first part of the paper provides a nice description of the drivers for AI adoption, the contribution areas of AI and some of the major kinds of AI that can be useful in healthcare. However, it's missing an introduction to the paper as a paper. In other words, something at the very beginning leads to tell the reader that they are being presented with some kind of literature review (Narrative? Systematic?), how it has has been scoped and indeed why it has been written.\nThe table presenting the literature appears suddenly without any methodological section to indicate how these references have been identified (search criteria, filter criteria etc).\n\nThen there is a section that seems like a nicely written results section which summarises the main findings from the literature presented in the table.\nIn other words, this is a rather nice literature review paper without its methodology! If this could be added then I think this would be a lovely paper to publish.\nWithout the methodology I don't feel that I have been able to carry out a proper review of the paper, although I did not find anything that I would like to be changed, just the methodology to be added. I would therefore be happy to review the paper again if the authors are in a position to add that methodology.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\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": [ { "c_id": "6984", "date": "02 Aug 2021", "name": "Antti Väänänen", "role": "Author Response", "response": "Dear Professor Kalra. Thank you for your review and suggestions. I will add your proposed changes to my article this week and kindly ask you to do 2nd review to new version after i have uploaded it to F1000. Best regards, Antti Väänänen" } ] } ]	1	https://f1000research.com/articles/10-6
https://f1000research.com/articles/10-1022/v1	08 Oct 21	{ "type": "Systematic Review", "title": "Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a systematic review", "authors": [ "Jorge Guillermo Morales Ramos", "Ambrocio Teodoro Esteves Pairazamán", "María Ema Soledad Mocarro Willis", "Samuel Collantes Santisteban", "Emma Caldas Herrera", "Ambrocio Teodoro Esteves Pairazamán", "María Ema Soledad Mocarro Willis", "Samuel Collantes Santisteban", "Emma Caldas Herrera" ], "abstract": "Background: The objective of this review was to evaluate the medicinal potential of Morus alba leaves on the control of type 2 diabetes mellitus (DM2). The research question was: what is the therapeutic potential of Morus alba leaves for the control of DM2? Methods: This systematic review was based on the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. The included studies were extracted from Scopus, Pubmed, ScienceDirect, Scielo, and Google Scholar; January 2015 to July 2021. Key search terms were MeSH and DeCS: Morus alba, mulberry, hypoglycemic agent. The inclusion criteria were: studies in rats administered Morus alba leaf extracts; studies that included the dimensions of lipidemia and glycemia; studies that included indicators such as fasting glucose, postprandial glucose, glycosylated hemoglobin, triglycerides, low-density lipoproteins, total cholesterol, and insulin resistance. Exclusion criteria: studies in which Morus alba leaves were administered with other plants; studies with other parts of the Morus alba plant; proteomic studies, cancer, duplicate studies, in vitro studies, and evaluation of included studies. All included investigations were evaluated for biases. Results: Of 253 studies found, 29 were included. The extracts of Morus alba leaves at the phytochemical level improve glucose uptake. Chlorogenic acid, isoquercitrin, and quercitrin, present in the leaves of Morus alba, have hypoglycemic properties and an ameliorating effect on diabetic nephropathy. This leaf has pharmacological effects such as glucose absorption, insulin secretion production, antioxidant and anti-inflammatory agent, antihyperglycemic and antihyperlipidemic activities, and obesity management. Conclusions: Morus alba leaves have pharmacological effects on DM2 that include glucose absorption, production of insulin secretion, antioxidant agent, antihyperglycemic and antihyperlipidemic activities, and obesity control. Beyond these results, there is a lack of studies on the potential and synergistic effects of Morus alba leaves' components, limiting the possibility of a more effective therapy using the plant's leaves.", "keywords": [ "Type 2 Diabetes Mellitus", "Morus alba", "T2DM control", "blood glucose" ], "content": "Introduction\n\nThe World Health Organization (WHO) in 2014 globally reported that 422 million adults had diabetes.1 Diabetes mellitus (DM) has become a major global problem with a significant health impact because the number of cases is constantly increasing and also because the long-term projections are less and less optimistic,2 becoming one of the diseases selected by the world health authorities that require priority attention. In 2019, DM was the leading cause of 1.5 million deaths, and in 2012 2.2 million people died due to hyperglycemia.2 By 2030, it is estimated that the global prevalence of diabetes will be 10.2% (578 million people) and 10.9% (700 million) by 2045,3 Of the three types of diabetes mellitus, the most common is type 2 (T2DM).\n\nT2DM is frequently linked to obesity. A predominant insulin resistance, accompanied by deficient hormone activity, leads to an increase in its secretion.4 T2DM is a disease associated with multiple factors and of a chronic nature, so its treatment must be seen integrally in the patient's life. Therefore, continuous adjustments are needed according to the specific requirements of each patient.5\n\nAn approach to diabetes care has been proposed by Kenneth S. Polonsky6 and is based on the formation of work teams that involve health professionals for patient care and on the development of care delivery models for diseases using this approach, the Diabetes Prevention Program (USA)6 demonstrated that physical exercise and weight loss could reduce the risk of diabetes in predisposed people by 58%. The main effects are also seen after treatment with metformin or pioglitazone.\n\nThe WHO, in its latest report on diabetes, points out that there are a variety of antidiabetic pharmacological therapies. However, most of them are expensive and cause adverse effects, impacting health systems, especially diabetic patients. The other problem is that only a minority in developing countries generally have insulin and drugs considered essential in controlling diabetes, such as antihypertensives and lipid-lowering drugs, within reach. Hence, it is necessary to establish regulatory policies and programs to improve equity in access.1\n\nThe National Demographic and Family Health Survey (ENDES) carried out in 2013 in Peru indicated a progressive increase in the number of T2DM cases, mainly due to the lifestyle of the Peruvian population. Epidemiological data suggest that there is a prevalence of 33.8% of overweight and 18.3% of obesity.7 In Peru, diabetes affects 7% of the population, representing 31.5% of acute myocardial infarctions and another 25% of cerebrovascular accidents. T2DM is equivalent to 96.8% of outpatient visits with this condition. The prevalence of glucose intolerance is 8.11%, and the variation in fasting glucose is 22.4%. The prevalence data for overweight, MS in adults, and obesity is 34.7%, 25%, and 17.5%, respectively.8\n\nThe present review focuses its study on the leaves of the Morus alba plant, which is distinguished by being used in traditional medicine and which is also a very promising species. During the last 20 years, the great majority of studies have addressed the isolation, and active principles of the polysaccharides of this plant have focused mainly on its leaves and fruits, which are both medicinal and edible.9 The results of preclinical investigations of the last decade suggest that Morus alba, as a therapeutic agent, is of utmost importance for treating DM; the polyhydroxy alkaloids, flavonoids, and polysaccharides of Morus may be; active components of great potentiality.10 In addition, this product, especially in oriental medicine, has been used for fever, liver damage, vision problems, and to lower blood sugar levels. However, the studies carried out to date on the antidiabetic compounds in this product are insufficient.11\n\nDifferent parts of Morus alba are used in the treatment of various diseases, are required for their medicinal properties such as antioxidant, antibacterial, antidiabetic, and antiviral. In addition, it has active components in the leaf, such as polysaccharides, that can lower the glucose level, blood, and blood pressure. The leaves are used for hypoglycemia and also as antioxidants, which contain the chemical components: scopolin, isoquercitrin, astragalin, rose oxide, benzyl glucopyranoside, and phenolic acids such as gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, syringic, p-coumaric, ferulic, and m-coumaric, also the polysaccharide deoxynojirimycin (DNJ).12\n\nThe present systematic review focuses on the active principles found in the leaves of the Morus alba plant and its medicinal properties, taking into account its use in traditional medicine. It is based on the fact that the extracts of Morus alba leaves contain flavonoids as main constituents, which also include antioxidant, antidiabetic, antihyperlipidemic, antiobesity, cardioprotective activities, and are rich in anthocyanins and alkaloids.13 The purpose of this systematic review was to evaluate the medicinal potential of Morus alba leaves on the control of T2DM. The research question was: what is the medicinal potential of Morus alba leaves in the control of T2DM?\n\n\nMethods\n\nThis review is based on the methodology established in the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement. (14, 41) All the scientific information included in the study was extracted from international databases such as Scopus, Pubmed, ScienceDirect, Scielo, and Google Scholar from January 2015 - July 2021; for identification, filtering, eligibility, and inclusion, the Mendeley Desktop 1.19.8 web application was used. The protocol of this systematic review was registered in the PROSPERO database, ID: 274164, on 18th August, 2021 (pending).\n\nThe primary auxiliary strategy (applied to all declared databases) to identify studies that correspond to the research question was the following:\n\n• TITLE-ABS-KEY (morus AND alba) AND PUBYEAR > 2013\n\n• (TITLE-ABS-KEY ( morus AND alba) OR TITLE-ABS-KEY (morus AND alba AND leaves)) AND PUBYEAR > 2013\n\n• (TITLE-ABS-KEY (morus AND alba) AND TITLE-ABS-KEY (mulberry)) AND PUBYEAR > 2013\n\n• (TITLE-ABS-KEY (morus AND alba) AND TITLE-ABS-KEY (mulberry) AND TITLE-ABS-KEY (hypoglycemic AND agent)) AND PUBYEAR > 2013\n\nThe auxiliary search strategy (applied to all declared databases) to identify other studies that correspond to the research question was the following:\n\n• (TITLE-ABS-KEY (Diabetes AND mellitus ) AND TITLE-ABS-KEY (type 2 diabetes AND mellitus) AND TITLE-ABS-KEY ( blood AND glucose) AND TITLE-ABS-KEY ( lipid-lowering AND agent ) AND TITLE-ABS-KEY (lipid-lowering AND agent) AND TITLE-ABS-KEY (blood AND lipids) AND TITLE-ABS-KEY (glycemic AND control) AND TITLE-ABS-KEY (postprandial AND hyperglycemia)) AND PUBYEAR > 2013\n\nThe inclusion criteria were: studies in rats administered Morus alba leaf extracts, studies that included dyslipidemia and glycemic dimensions, studies that included indicators such as fasting glucose, postprandial glucose, glycated hemoglobin, triglycerides, low-density lipoproteins, total cholesterol, and insulin resistance. Only empirical articles, systematic reviews, case studies were taken into account; Due to its importance, a descriptive analysis was included. The exclusion criteria were: studies in which Morus alba leaves were administered with other plants, studies with other parts of the Morus alba plant (fruits, roots, stems), proteomic analyses, cancer, duplicate studies, inward studies, in vitro and evaluation of included studies. Letters to the editor, narrative reviews, or scientific journal editorials were excluded from this study.\n\nTo correct biases, the PRISMA 2020 checklist14 was first used. The empirical articles were evaluated through an analytical rubric prepared according to the parameters declared in the SSAHS scale of López-López E, Tobón S, Juárez-Hernández LG) to consider scientific articles.15 Systematic reviews were assessed using the Quality Assessment of Systematic Reviews and Meta-Analyses, using an observation guide (checklist style according to the parameters of National Heart, Lung, and Blood Institute).16 The checklists can be found as extended data.41\n\nTwo experienced researchers on the team reviewed the systematic reviews—the original investigations by the three remaining members. The coordination and development of these activities were through the Zoom video chat software. To include the studies, the relationship of each one with the research question was verified, based –fundamentally– on the terms: Morus alba; mulberry; and hypoglycemic agent. Then, Quality Assessment of Systematic Reviews and Meta-Analyzes and a scale for evaluating scientific articles were used to guarantee strict compliance with the inclusion and exclusion criteria stated in previous paragraphs. The web application used throughout the identification, screening, eligibility, and inclusion process was Mendeley. To collect the relevant data from each report, PRISMA was used. In the final process of compiling/coding the reports already examined, the entire research team was involved. The variables for which relevant information was sought were diabetes mellitus, type 2 diabetes mellitus, blood glucose, lipid-lowering agent, lipoproteins, blood lipids, glycemic control, postprandial hyperglycemia.\n\n\nResults\n\nFigure 1 shows that of an initial sample of 261, 29 investigations (17 originals and 12 systematic reviews) were included for 11.11%. The articles rejected for not meeting the inclusion criteria were 232, mainly because they lacked theoretical-methodological support, detected through the quality assessment instruments.\n\nTable 1 shows the distribution of the search results by author, article title, and type of study/characteristics of the studies finally included. 58,62% (original research); 41,38% (systematic reviews). All investigations were included based on the inclusion and exclusion criteria provided in the study methodology.\n\nOR = original research; SR = systematic review.\n\nOf the set of excluded studies, the proposal by Hwang SH, Li HM, Lim SS, Wang Z, Hong JS, Huang B,17 despite meeting most of the inclusion criteria, was a clinical study that did not specify clearly if the extract was obtained from Morus alba leaves. Also, the research of Qi S, Li N, Tuo Z, Li J, Xing S, Li B, et al.,18 despite meeting most of the inclusion criteria, was excluded because the extract used did not specify if it had been made with Morus alba leaves. Finally, the research of Todd SB, Fallah S, Salemi Z, Seifi M.19 was excluded because it measured the work, measured the level of specific hormones, but not that of the constituents of the Morus alba leaf that participate in the obesity.\n\n\nDiscussion\n\nThe genus Morus includes several species, such as Morus alba, M. nigra, both native to Asia, and M. rubra of North American origin. With wide distribution in the world, this genus, both in temperate and tropical areas, reports various medicinal properties, one of the most studied being Morus alba.\n\nA study based on the network approach or network pharmacology found that the alkaloids, flavonoids, and polysaccharides of Morus alba leaves impart regulatory effects on blood sugar levels, identifying 202 components, of which 22 can have significant curative effects on DM.20 To study its main bioactive components such as anthocyanins, polysaccharides, phenols, alkaloids, and flavonoids, at the phytochemical level, various techniques have been used that include extraction and separation processes such as solid-liquid extraction, solid-phase extraction, pressurized liquid extraction, extraction by supercritical fluid, microwave-assisted extraction, ultrasonic-assisted extraction, enzyme assisted extraction, macroporous resin adsorption, silica gel chromatography, ion-exchange chromatography gel filtration chromatography, preparative liquid chromatography, and countercurrent chromatography.21\n\nTriterpenes and steroids are found in Morus alba leaf, fruit, and root extracts, as well as phenols and tannins and secondary metabolites with pharmacological properties for the treatment of many diseases associated with oxidative stress, such as Diabetes.22 The leaves of Morus alba contain flavonoids as main constituents, which, in addition, possess various biological activities, including antioxidant, antidiabetic, antihyperlipidemic, antiobesity, cardioprotective activities, and are rich in anthocyanins and alkaloids.13 Rutin or quercetin-3-rutinoside is a flavonoid glycoside obtained from the leaves of Morus alba, and its antiobesity effect has been verified by studying the lipid accumulation mechanism in 3T3-L1 adipocyte and C57BL mouse models.23 Both compounds, rutin, and quercetin-3-O-β-D-glucoside (Q3G), have been shown to enhance glucose uptake through the AKT-mediated insulin signaling pathway or AMP-activated protein kinase activation (AMPK) in 3T3L1 adipocytes and could potentially be used in therapy for T2DM management.11 In addition to rutin, the leaves of Morus alba contain apigenin, quercetin-3-triglyceride, lupeol, β-sitosterol, moracetin, isoquercitrin, coumarin, volatile oil, alkaloids, amino acids, and organic acids, proving the hypoglycemic properties and their effect enhancer on diabetic nephropathy.24 Four main chemical components were identified in the ethanol extract of Morus alba: chlorogenic acid, rutin, isoquercitrin, and quercitrin. MLE was found to improve hyperglycemia, insulin resistance, and dyslipidemia in T2 DM rats with a significant therapeutic effect.25\n\n1-deoxynojirimycin (DNJ) is another compound present in the leaves of Morus alba. It is an effective inhibitor of α-glucosidase, providing a significant hypoglycemic effect. Its primary mechanism of action occurs after 1-deoxynojirimycin enters the human body, with the inhibition of sucrose, maltase, α-glucosidase enzyme, and α-amylase decomposing starch, so it can block the absorption of carbohydrates in the body, inhibiting the increase in blood sugar to achieve the effect of the prevention and treatment of DM.26 DNJ controls postprandial hyperglycemia by acting as a natural inhibitor of α-glucosidase in patients with T2DM.27\n\nExperimental models and laboratory techniques that evaluate various pharmacological activities have been included in this review. Several investigators have used streptozotocin-induced Wistar rats for the empirical part, administering Morus alba leaf extract. One of them demonstrated antioxidant activity, and antidiabetic effect of ethanol extracts with high levels of phenolic acids, chlorogenic acid, and flavonol glycosides, finding efficacy in the correction of hyperglycemia in the increased secretion of insulin. Two other investigations corroborated the improvement of the antioxidant situation28 and that administering high doses (0.25%) and low doses (0.5%) of Morus alba leaf tea demonstrated hypolipidemic, hypoglycemic, and improvement on diabetic nephropathy.24,29 Another study mentions that administering Morus alba ethanol extracts in doses of 0.5-1g/kg and oxyresveratrol (0.6 g/kg), an essential compound in the extract, could be considered as a potential adjunct therapy for the management of Diabetes since both reduced the level of glucose from fasting blood and plasma glucose.30 Likewise, in another study, ethanolic extract of Morus alba leaves (100 mg/kg) was orally administered for eight weeks to female Wistar rats that were fed a high-cholesterol diet to evaluate the lowering effect of obesity, dyslipidemia, and insulin resistance, showing significant results since it decreased body weight gain, hypercholesterolemia, hypertriglyceridemia, atherogenic indices, based on lipid profile, and coronary artery, glucose level, and the insulin resistance index.31 The effect of Morus alba leaf extract (FME) on insulin resistance through IRS-1 / PI3K / Glut-4 signaling pathway in rats with T2DM was also evaluated; the results obtained showed that the FME showed improvement in insulin resistance, in addition, a decrease in body weight, blood glucose, triglycerides, total cholesterol, and low-density lipoprotein levels were observed, significant antihyperglycemic and antihyperlipidemic activities via the pathway signaling IRS-1/PI3K/Glut-4,33 Khyade (2018) obtained positive pharmacological effects on specimens of Rattus norvegicus induced with Diabetes, using a decoction of Morus alba leaves in doses of 20 g/L per day as treatment. The results found were decreased blood glucose levels and altered regulation of metabolic processes.32\n\nThe effects of dietary supplementation with Morus alba leaf extracts with acetone-water (AE) and ethanol-water (EE) were also evaluated on the situation of the trace element (Fe, Zn, and Cu) about the management of Diabetes and antioxidant indices in diabetic rats fed the high-fat STZ diet and biochemical analyzes (glucose, thiobarbituric acid reactive substances, ferric reducing ability of plasma) were performed in blood serum, which showed that EA decreased the hepatic and renal storage of EF, while the EE increased the levels.33 Laboratory tests were performed using mice induced to Diabetes and treatment with flavonoids extracted from the leaves of Morus alba on skeletal muscle cells, resulting in an improvement in mitochondrial function by activating AMPK in type 2 diabetes, improving resistance to the Skeletal muscle insulin significantly lowering blood glucose levels.34\n\nLikewise, it was investigated whether flavonoids, which are extracted from the leaves of Morus alba, can regulate glycolipid metabolism and to investigate whether flavonoids could regulate the signal of the IRS1 / PI3K / AKT pathway to affect the expression of FAS and GLUT4 membrane transfer capacity in 3T3. The results showed that the flavonoids from Morus alba leaf extracts alleviated the metabolic abnormalities of glycolipids in the IR model of 3T3-L1 adipocytes. The effect was associated with the activation of the IRS1/PI3K/AKT pathway.31\n\nA double-blind, randomized clinical trial was conducted in animals and in patients with glucose intolerance, who were administered a standardized extract to evaluate the antihyperglycemic effect, the inhibitory effect of α-glucosidase, the acute single oral toxicity, and the reduction of blood glucose in a test. The standardized extract of Morus alba leaves was found to inhibit α-glucosidase at a level four times higher than the positive control (acarbose), in a concentration-dependent manner, proving to be an excellent potential material to improve postprandial hyperglycemia.35\n\nWork has been carried out with the interaction of the leaves of Morus alba and other plants. One of them used fenugreek seed (Trigonella foenum-graecum), Morus alba Leaf, and American ginseng root (Panax quinquefolius) can improve glycemia in humans and animals with impaired glucose and T2DM metabolism, improving insulin sensitivity and glucose absorption in human adipocytes.36 Another study evaluated treatment options for obesity, applying a standard composition (UP603), composed of extracts of Morus alba, Ilex paraguariensis, and Rosmarinus Officinalis in diet-induced obese C57BL/6J mice. UP603 treated animals showed decreased body weight gain, 65.5% and 16.4% reductions in insulin and leptin, respectively, and a 2.1-fold increase in ghrelin level. In addition, there were reductions from 7.9% to 21.1% in total cholesterol, from 25.4% to 44.6% in triglycerides, and from 22.5% to 38.2% in cholesterol linked to lipoproteins. Low-density lipoprotein (LDL) in mice treated with 450-850 mg/kg of UP603.37\n\nOne method used to standardize the Morus alba extract is high-performance reversed-phase liquid chromatography (RP HPLC). The plant extract and the single bioactive molecule were studied for their potential for inhibition of combined CYP450, as well as forms of recombinant human CYP450, such as the isoenzymes CYP3A4, CYP2D6, CYP2C9, and CYP1A2, the findings suggested that the plant extract and its bioactive compound are safe to use in the management of Diabetes.38\n\nLikewise, experimental studies in diabetic rats and normal rats have shown that the fruit and bark of the Morus alba root can potentially be used as an effective treatment for DM239 and effectively improve hyperlipidemia and inhibit diacylglycerol acyltransferase.18\n\nA systematic review indicates that several studies have shown that the aqueous extracts of the bark of roots, leaves, and stems contain polyphenols and polysaccharides with antihyperglycemic and antihyperlipidemic properties; polysaccharides have been further studied to assess their antidiabetic bioactivity in T2DM-induced rats, clarifying the underlying mechanism of these activities.9\n\nA systematic review is presented on the chemical composition of the different parts of Morus alba and its pharmacological effects on DM, which include the influence on glucose absorption, production on insulin secretion as an antioxidant and anti-inflammatory agent. The study found that Morus alba leaf contains steroids and triterpene compounds, flavonoids, coumarin, essential oils, amino acids, alkaloids, and organic acids. The branch of Morus alba, for its part, contains tannin, fructose, stachyose, glucose, maltose, honeydew, and arabinose. The bark of the Morus alba root contains flavonoids, such as mulberry, mulberrochromene, and cyclomulberrine. The different parts of the plant contain active principles such as Polyhedral alkaloids, polysaccharides, and flavonoids are components that are found in all aspects of the Morus alba plant; the fruits also contain vitamin B1, B2, and carotene, also, fatty acids (linoleic, oleic and stearic).10\n\nAnother review to evaluate the potential of Morus alba as a natural agent in the treatment of obesity found that the inhibitory effects of Morus alba on digestive enzymes and adipocyte differentiation and its stimulating effects on energy expenditure and Lipid metabolism are mechanisms responsible for the management of obesity in obese patients.40\n\n\nConclusions\n\nIt has been shown that the aqueous extracts of the leaves of Morus alba at a phytochemical level contain 202 constituents, among them routine with proven antiobesity effect, rutin together with quercetin-3-O-β-D-glucoside (Q3G) improve the glucose uptake and demonstrated antioxidant activity and antidiabetic effect.\n\nThe compound in the Morus alba leaf, 1-deoxynojirimycin (DNJ), has been identified as an effective inhibitor of α-glucosidase and provides a significant hypoglycemic effect, which could improve postprandial hyperglycemia and also prevent and treat in DM, oxyresveratrol could be considered as a potential adjunct mechanism for the management of Diabetes.\n\nChlorogenic acid, routine, isoquercitrin, and quercitrin present in Morus alba leaves have been shown to have hypoglycemic properties and an ameliorating effect on diabetic nephropathy, insulin resistance, and dyslipidemia in rats.\n\nVarious systematic reviews have evaluated that Morus alba leaves have various pharmacological effects on T2DM, including glucose absorption, production in insulin secretion, antioxidant and anti-inflammatory agent, antihyperglycemic and antihyperlipidemic activities, and obesity management.\n\nThe results of this research have a theoretical and scientific relevance because it manages to systematize scientific research relevant to the cognitive needs that the need for the use of Morus alba leaf for the treatment of type 2 diabetes mellitus raises today, which, as explained in this research, is a public health problem worldwide.\n\n\nLimitations\n\nThe results achieved allow identifying a lack of studies on the potential and synergistic effects of the components of the Morus alba leaves; this limits the possibility of a more effective therapy using the leaves of the plant; Furthermore, there are few studies on the extraction methodology of the components of the Morus alba leaf. These limitations must be taken into account for the orientation and development of new research.\n\n\nRecommendations\n\nT2DM and the risk factors involved in the appearance of aggravating conditions of this disease, it should be considered that the extracts of the leaves of Morus alba constitute an effective alternative to keep glycemia and lipidemia under control in patients with T2DM, being an excellent complement to your usual treatment. It would also be necessary to expand pharmacological safety studies and preclinical and clinical studies of the use of Morus alba leaf extracts to generate more scientific evidence, which allows new forms of administration to be known.\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: Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a systematic review. https://doi.org/10.5281/zenodo.5227684.41\n\nThis project contains the following extended data:\n\n• Search Strategy.pdf\n\n• Flow diagram and included articles.pdf\n\n• Observation Guide (Quality Assessment of Case-Control Studies).pdf\n\n• Observation Guide (Quality Assessment of Systematic Reviews and Meta-Analyses).pdf\n\n• Scale to Evaluate Scientific Articles in Social and Human Sciences- SSAHS.pdf\n\n• Prospero Registration.pdf\n\nZenodo: PRISMA checklist for ‘Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a systematic review’. https://doi.org/10.5281/zenodo.5227684.41\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWHO: Global report on diabetes [Internet]. 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PubMed Abstract \| Publisher Full Text\n\nLim SH, Yu JS, Lee HS, et al.: Antidiabetic Flavonoids from Fruits of Morus alba Promoting Insulin-Stimulated Glucose Uptake via Akt and AMP-Activated Protein Kinase Activation in 3T3-L1 Adipocytes. Pharm 2021, Vol 13, Page 526. 2021 Apr 9 [cited 2021 Jul 9]; 13(4): 526. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nYounus I, Fatima A, Ali SM, et al.: A Review of Ethnobotany, Phytochemistry, Antiviral and Cytotoxic/Anticancer Potential of Morus alba Linn. Int J Adv Res Rev. 2016 [cited 2021 Jul 19]; 1(2): 84–96. Reference Source\n\nChan EW-C, Lye P-Y, Wong S-K: Phytochemistry, pharmacology, and clinical trials of Morus alba. Chin J Nat Med. 2016 Jan 1 [cited 2021 Jul 16]; 14(1): 17–30. 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Reference Source\n\nDíaz SM, Cazaña MY, Pérez HY, et al.: Qualitative evaluation of secondary metabolites in extracts of Morus alba L. (mulberry) varieties and hybrids. Rev Cuba Plantas Med. 2015 [cited 2021 Jul 16]; 20(3): 358–66. Reference Source\n\nGo EJ, Ryu BR, Yang SJ, et al.: Anti-obesity Effect of the Flavonoid Rich Fraction from Mulberry Leaf Extract. Korean J Med Crop Sci. 2020 [cited 2021 Jul 16]; 28(6): 395–411. Reference Source\n\nSwathi P, Gana Manjusha K, Vivekanand M, et al.: Effect of Morus alba against Hyperglycemic and Hyperlipidemic Activities in Streptozotocin Induced Diabetic Nephropathy. Biosci Biotechnol Res Asia. 2017 Dec 28 [cited 2021 Jul 16]; 14(4): 1441–1447. Reference Source\n\nJi S, Zhu C, Gao S, et al.: Morus alba leaves ethanol extract protects pancreatic islet cells against dysfunction and death by inducing autophagy in type 2 diabetes. Phytomedicine. 2021 Mar 1 [cited 2021 Jul 9]; 83. PubMed Abstract \| Publisher Full Text\n\nMahesh DS, Vidhathri BS, Vidyashree DN, et al.: Biochemical Composition and Pharmacological Properties of Mulberry (Morus spp.) - A Review. Int J Curr Microbiol Appl Sci. 2017 Jul 20 [cited 2021 Jul 9]; 6(7): 2207–17. Reference Source\n\nLiu C, Xiang W, Yu Y, et al.: Comparative analysis of 1-deoxynojirimycin contribution degree to α-glucosidase inhibitory activity and physiological distribution in Morus alba L. Ind Crops Prod. 2015 Aug 1 [cited 2021 Jul 16]; 70: 309–315. Reference Source\n\nJeszka-Skowron M, Flaczyk E, Jeszka J, et al.: Mulberry leaf extract intake reduces hyperglycaemia in streptozotocin (STZ)-induced diabetic rats fed high-fat diet. J Funct Foods. 2014 May 1 [cited 2021 Jul 16]; 8(1): 9–17. Reference Source\n\nWilson RD, Islam MS: Effects of white mulberry (Morus alba) leaf tea investigated in a type 2 diabetes model of rats. Acta Pol Pharm. 2015 [cited 2021 Jul 16]; 72(1): 153–160. PubMed Abstract\n\nAhn E, Lee J, Jeon Y-H, et al.: Anti-diabetic effects of mulberry (Morus alba L.) branches and oxyresveratrol in streptozotocin-induced diabetic mice. Food Sci Biotechnol 2017 266. 2017 Dec 12 [cited 2021 Jul 9]; 26(6): 1693–1702. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nMeng Q, Qi X, Chao Y, et al.: IRS1/PI3K/AKT pathway signal involved in the regulation of glycolipid metabolic abnormalities by Mulberry (Morus alba L.) leaf extracts in 3T3-L1 adipocytes. Chin Med. 2020 Jan 2 [cited 2021 Jul 19]; 15(1): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nKhyade VB: Influence of Leaf Decoction of Mulberry, Morus alba (L.) on Streptozotocin Induced Diabetes in Brown Rat, Rattus norvegicus (L.). Int J Res Sci Eng. 2018 [cited 2021 Jul 9]; 6(3): 1–23. Reference Source\n\nKról E, Jeszka-Skowron M, Krejpcio Z, et al.: The Effects of Supplementary Mulberry Leaf (Morus alba) Extracts on the Trace Element Status (Fe, Zn and Cu) in Relation to Diabetes Management and Antioxidant Indices in Diabetic Rats. Biol Trace Elem Res. 2016 Nov 1 [cited 2021 Jul 9]; 174(1): 158–165. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeng Q, Qi X, Fu Y, et al.: Flavonoids extracted from mulberry (Morus alba L.) leaf improve skeletal muscle mitochondrial function by activating AMPK in type 2 diabetes. J Ethnopharmacol. 2020 Feb 10 [cited 2021 Jul 19]: 248. PubMed Abstract \| Publisher Full Text\n\nHwang SH, Li HM, Lim SS, et al.: Evaluation of a Standardized Extract from Morus alba against α-Glucosidase Inhibitory Effect and Postprandial Antihyperglycemic in Patients with Impaired Glucose Tolerance: A Randomized Double-Blind Clinical Trial. Evid Based Complement Alternat Med. 2016 [cited 2021 Jul 9]; 2016. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKan J, Velliquette RA, Grann K, et al.: A novel botanical formula prevents Diabetes by improving insulin resistance. BMC Complement Altern Med. 2017 Jul 5 [cited 2021 Jul 19]; 17(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodrigues EL, Marcelino G, Silva GT, et al.: Nutraceutical and Medicinal Potential of the Morus Species in Metabolic Dysfunctions. Int J Mol Sci. 2019 Jan 2 [cited 2021 Jul 19]; 20(2). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKar A, Mukherjee PK, Sankarshan S, et al.: Possible herb-drug interaction of Morus alba L. - a potential antidiabetic plant from Indian traditional medicine. Indian J Tradit Knowl. 2015 [cited 2021 Jul 16]; 14(4): 626–631. Reference Source\n\nJiao Y, Wang X, Jiang X, et al.: Antidiabetic effects of Morus alba fruit polysaccharides on high-fat diet- and streptozotocin-induced type 2 diabetes in rats. J Ethnopharmacol. 2017 Feb 2 [cited 2021 Jul 9]; 199: 119–127. PubMed Abstract \| Publisher Full Text\n\nMahboubi M: Morus alba (mulberry), a natural potent compound in management of obesity. Pharmacol Res. 2019 Aug 1 [cited 2021 Jul 19]: 146. PubMed Abstract \| Publisher Full Text\n\nRamos JGM, Pairazamán ATE, Willis MESM, et al.: Medicinal properties of Morus alba for the control of type 2 diabetes mellitus: a systematic review [Data set]. Zenodo. 2021. Publisher Full Text" }	[ { "id": "96495", "date": "09 Nov 2021", "name": "José Enrique Alfonso Manzanet", "expertise": [ "Reviewer Expertise Scientific Comunication", "Pharmacology" ], "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 carries out a detailed analysis of the selected articles. The information search criteria are adequate and the results are correctly analyzed. However, it is recommended to be more explicit in the limitations of the study in question. It cannot be said that it effectively constitutes an effective treatment alternative when selected studies have been carried out in rats. I think the recommendations are aimed at expanding the clinical study in humans.\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": "97611", "date": "15 Nov 2021", "name": "Miryam Griselda Lora Loza", "expertise": [ "Reviewer Expertise Health & Wellness", "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 belongs to the research line of Biomedical Sciences, specifically phytochemistry and pharmacognosy in medicinal plants, addressing the evidence of the activity of a plant on Diabetes Mellitus 2. It is a relevant and current topic in the medical specialty; the study developed a careful screening based on the various sources of articles consulted and included in the study.\nSome recommendations:\nIn the abstract –methodology section–, the wording should be improved so that it is clearly understood which studies were found, and of the total, which ones were included.\n\nReview the publication “Declaración PRISMA 2020: una guía actualizada para la publicación de revisiones sistemáticas;” especially figure 1, page 795 1 compare it with figure 1 of the manuscript under review (Flow diagram of the identification, screening, eligibility and included articles), and make the appropriate adjustments.\n\nSynthesize the conclusion, focusing mainly on the study's main findings and their impact on the therapeutic potential of Morus alba leaves in the control of the disease addressed in the present study.\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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-1022
https://f1000research.com/articles/10-622/v1	21 Jul 21	{ "type": "Study Protocol", "title": "Screening for postoperative complications by continuous monitoring: protocol for the Biobeat-Postop cohort study", "authors": [ "Alexis Paternot", "Philippe Aegerter", "Aurélie Martin", "Jonathan Ouattara", "Sabrina Ma", "Sherifa Adjavon", "Bernard Trillat", "Pascal Alfonsi", "Marc Fischler", "Morgan Le Guen", "Alexis Paternot", "Philippe Aegerter", "Aurélie Martin", "Jonathan Ouattara", "Sabrina Ma", "Sherifa Adjavon", "Bernard Trillat", "Pascal Alfonsi", "Morgan Le Guen" ], "abstract": "Background: Postoperative hypotension associated with postoperative morbidity and early mortality has been studied previously. Hypertension and other hemodynamic, respiratory, and temperature abnormalities have comparatively understudied during the first postoperative days. Methods: This bi-centre observational cohort study will include 114 adult patients undergoing non-cardiac surgery hospitalized on an unmonitored general care floor and wearing a multi-signal wearable sensor, allowing remote monitoring (Biobeat Technologies Ltd, Petah Tikva, Israel). The study will cover the first 72 hours after discharge of the patient from the post-anaesthesia care unit. Several thresholds will be used for each variable (arterial pressure, heart rate, respiratory rate, oxygen saturation, and skin temperature). Data obtained using the sensor will be compared to data obtained during the routine nurse follow-up. The primary outcome is hemodynamic abnormality. The secondary outcomes are postoperative respiratory and temperature abnormalities, artefacts and blank/null outputs from the wearable device, postoperative complications, and finally, the ease of use of the device. We hypothesize that remote monitoring will detect abnormalities in vital signs more often or more quickly than the detection by nurses’ routine surveillance. Discussion: A demonstration of the ability of wireless sensors to outperform standard monitoring techniques paves the way for the creation of a loop which includes this monitoring mode, the automated creation of alerts, and the sending of these alerts to caregivers. Trial registration: ClinicalTrials.gov, NCT04585178. Registered on October 14, 2020", "keywords": [ "Surgery", "Monitoring", "Complications", "Perioperative medicine" ], "content": "Introduction\n\nPostoperative mortality remains a current controversial issue, as shown in 2016 by the International Surgical Outcomes Study.1 This prospective international cohort study reported that 16.8% of patients developed one or more postoperative complications, and 0.5% died. Several studies have focused specifically on the risks of postoperative hypotension,2 respiratory depression,3 and hypoxemia.4 This has been well demonstrated in particular with regard to unrecognized hypotension since the risk of myocardial ischemia is increased by cumulative durations of 2 to 4 h of hypotension (mean arterial pressure < 60 mmHg) or durations of more than 4 h with mean arterial pressure < 65 or 70 mmHg.5\n\nMost patients are hospitalized on an unmonitored general care floor (ward) where survival after a cardiac arrest is worse compared to ICU patients or patients hospitalized in a monitored ward setting.6 However, postoperative placement in high-level monitoring units for patients with risk factors is impossible in view of their number. To limit the risk of missing an abnormality in one of the physiological parameters, the National Health Service in England proposed (imposed in fact) a rigorous and repeated evaluation of the clinical condition at regular intervals with early warning scores (EWS).7 But this evolution in nursing practice does not prevent it from being spot check monitoring. Evolution of technology allows continuous and remote monitoring using either a bed-based mattress sensor, patient-worn monitor, and wearable patch sensors allowing continuous monitoring.\n\nThe most convenient system for patients, which makes them fully independent and therefore does not impede their mobility, is a skin patch that measures a wide range of vital signs at a frequent rate and automatically transmits this information to the nursing staff. Remote wireless vital sign monitoring on the ward has been reported in case series of medical or surgical patients8 and more recently in patients suffering from COVID-19.9\n\nTo promote the widespread use of these devices, it is necessary to confirm the comparative advantage of remote monitoring over conventional nursing monitoring. In the present study, we hypothesize that monitoring with a multi-signal wearable sensor will detect potentially dangerous vital sign abnormalities more often and more rapidly than routine surveillance in surgical patients hospitalized on an unmonitored general care floor.\n\n\nMethods\n\nEthics approval for this trial was obtained from the Ethical Committee Ile de France II (Paris, France) on September 28, 2020 (approval number: 2020-A01852-37) and registered at ClinicalTrials.gov under the trial identification number NCT04585178).\n\nWritten informed consent will be obtained by study staff from all study participants prior to their participation in study. Any modifications to the protocol will be sent to the Ethical Committee before they are implemented within the study and communication changes that impact the patients would require signing of a revised consent form. The current version is version n° 2; July 28; 2020. Complete protocol can be obtained on request. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendation for Clinical Interventional Trials (SPIRIT) guidelines.\n\nThis protocol seeks to quantify the benefit of remote monitoring using a multiparametric device in the detection of postoperative complications in comparison with the monitoring usually performed by nurses.\n\nThe Biobeat-Postop Protocol is a prospective observational bi-centre study that will be conducted in two private non-profit hospitals in which all types of surgical procedures, except cardiac procedures, are routinely performed. Patients will be consecutively enrolled and followed up for the first 72 postoperative hours after the patient leaves the post-anaesthesia care unit.\n\nRecruitment of patients began on December 15, 2020, and is ongoing.\n\nPatients will be included if they meet the following criteria: (1) are over 18 years, (2) require general anaesthesia for a major surgical procedure (gastro-intestinal surgery, gynaecological surgery, urologic surgery, and orthopaedic surgery) with an expected duration of intervention of more than 2 hours, (3) need a planned postoperative stay of more than 72 hours, and (4) have provided written informed consent. The exclusion criteria will be any abnormality of the skin or a very hairy skin at the location of the patch, tremor, allergy to the components of the patch, planned scanner or magnetic resonance imaging during the postoperative course, and pregnancy. Consecutive patients will be screened unless the physician responsible for providing the patient information and consent is not available in accordance with the regulations.\n\nPatients will be equipped with a portable Biobeat® sensor, a single-use patient device that consists of a skin patch placed 1 cm to the left of the sternum, just below the clavicle (Biobeat Technologies Ltd, Petah Tikva, Israel) (Figure 1). The sensor continuously records the photoplethysmographic waveform, which allows recording and calculation of several physiological parameters: heart rate (HR), respiratory rate (RR), peripheral oxygen saturation (SpO2), systolic arterial pressure (SAP) and SAP variation, diastolic arterial pressure (DAP) and DAP variation, and skin temperature. Other variables, stroke volume and cardiac output, are measured by this sensor but are not included in this protocol.\n\nBiobeat® is CE approved (N°688840; March 19, 2019), and its use was also approved by the FDA in 2019 (510K clearance for measurement of arterial pressure, oxygenation and HR in hospitals, clinics, and long-term care and at home).\n\nHigh agreement has been demonstrated for arterial pressure measurements obtained via the Biobeat® sensor and the gold-standard sphygmomanometer technique.10\n\nPatient selection will be performed during a preoperative anaesthesia consultation with one of the doctors on the research team. After verification of the inclusion and exclusion criteria, the physician will inform the patients about the study and obtain their written informed consent.\n\nThe patch will be put in place in the post-anaesthesia care unit just before the patient's return to the conventional hospitalization service. The monitoring data from the patch will be recorded continuously without being communicated to the nursing staff and doctors.\n\nPostoperative medical, surgical, and nursing care will be in accordance with medical indications depending on the clinical routine, without any study-specific restrictions, and all the nurses’ observations (clinical observations and measurements) will be noted on a computerized chart (Easily) as usual.\n\nData collection will end 72 hours after the patient's return to the hospital ward. Finally, when a nurse removes the patch, the status of the skin will be assessed: healthy skin (stage 0), redness limited to the contact area between the device and the skin (stage 1), redness extending beyond the contact surface of the device (stage 2), and the appearance of blisters (stage 3). The patients will be asked to evaluate their acceptance of the sensor using a 4-point Likert scale (from 0 = intolerable to 4 = no problem at all).11\n\nSafety assessments will consist of monitoring and recording of all adverse events.\n\nAny participant who wishes to terminate their participation in the study can withdraw from the trial at any time without the need for further explanation. Participants who withdraw from the study will be followed up according to routine clinical practice.\n\nOtherwise, patients will be excluded during the study if they must undergo an unplanned scan or magnetic resonance imaging during the period of monitoring. In this case, the patch will be removed before the radiologic examination.\n\nDemographic data, including age, sex, American Society of Anesthesiologists classification, body mass index, underlying diseases, reason for the surgical procedure, and type of surgical procedure, will be collected upon inclusion in the study.\n\nAll postoperative monitoring variables measured by the nurses (HR, RR, SpO2, SAP, DAP, and temperature) during the first 72 postoperative hours at a frequency determined by the medical indications will be collected using Easily software and recorded on a dedicated electronic case report form (eCRF). The time of occurrence of any complications noted by nurses and doctors will also be recorded on the eCRF.\n\nThe wearable device transmits the measurement data every 5 minutes to the Biobeat Gateway through Bluetooth. All data are uploaded to and stored on the Instamed (a French telemedicine company and Biobeat’s partner) certified health data hosting cloud (hosted by Amazon Web Services and General Data Protection Regulation compliant). The research team can then access data through the Instamed platform as well as request that data be exported as.csv files. Nursing staff and doctors will not have access to monitoring data during the protocol duration. The data will be deleted at the end of the study and can be deleted during the study as needed.\n\nThe main outcome is the occurrence of one or more hemodynamic complications during the first 72 hours following a major non-cardiac surgical procedure unless death or hospital discharge occurs sooner, with the 72-hour period starting when the patient returns to the surgical department. The secondary outcomes will concern the frequency of postoperative respiratory or temperature abnormalities, the frequency of artefacts and blank/null outputs from the wearable device and, more globally, the signal-level validity, the postoperative complications that occurred during the monitoring period, as determined by the healthcare team in accordance with the Dindo and Clavien classification12 and the comprehensive complication index.13 Finally, the ease of use of the device at the time of insertion and the patient’s tolerance to wearing the Biobeat® device will also be determined. Definitions of hemodynamic, respiratory, and temperature abnormalities are listed in Table 1.\n\nSample size\n\nOur main goal is to estimate the proportion of patients showing severe hypotension and, notably, to corroborate the prevalence found in Liem et al.5 i.e., 8%. To gain a 10% precision (± 5%) with a two-sided 5% alpha risk, 114 patients need to be included.\n\nDetection of artefacts\n\nThis will follow similar rules as previously published in a paper dedicated to the validation a new sensor.14 The rules selected to define artefacts may be updated according to experience and the literature.15 The successive rules will be recorded in a register, and all recordings will be reviewed in the light of these new rules.\n\nMissing values\n\nMissing data will not be replaced.\n\nStatistical analyses\n\nDescriptive summaries will be provided for each parameter and for each device. For continuous variables, the mean, median, and their 95% confidence limits, obtained using bootstrapping methods, will be provided. For discrete variables, counts, percentages, and confidence limits obtained using a bootstrap method will be provided.\n\nThe proportion of data gaps and artefacts for each parameter will be given as a percentage of the total number of measurement points and observations, respectively, with the corresponding 95% confidence interval (CI).\n\nWhen physiologic parameters (HR, SAP, DAP, for instance) are measured simultaneously by nurses and wearable devices, a Bland-Altman analysis for repeated measurements, accounting for multiple observations per individual, will be performed to draw mean-difference plots and derive accuracy or bias (mean difference), precision (standard deviation of difference), and limits of agreement (LoAs) that are expected to contain 95% of the paired differences between measurements by the nurses and the wireless patch, with their confidence intervals.\n\nAt the patient level and for each kind of clinical event, the proportion of patients with detection of at least one event (hypotension, for instance) by a nurse and wearable monitoring will be described in a two-by-two table. Agreement will be estimated by Cohen’s kappa coefficient with its 95% CI, while differences will be tested by a non-parametric McNemar test for paired nominal data.\n\nThe time (hours) to first occurrence of data loss or end of service of the device, or to the first occurrence of a clinical complication, will be described with Kaplan-Meier survival curves, and, if permitted by the number of such events, risk factors will be explored by a Cox model.\n\nInterim analyses are not planned in this study.\n\nA two-tailed p value < 0.05 will be considered statistically significant. All statistical analyses will be performed using R software (R Development Core Team, 2012. https://www.r-project.org/).\n\nQuality control is carried out by the Clinical Research Unit of the sponsor hospital. A qualified person will attest subject eligibility, monitor integrity of the source data and completion of the entries on the electronic case report form, verify the compliance with the clinical study protocol, the Good Clinical Practices, and the regulatory commitments). The monitor will also verify the report of adverse effects.\n\nAll subjects will be identified by a unique identification number. Each principal investigator will keep a list in a safe location which will allow the identification of the pseudonymised patients. Patients will be informed about data protection and the fact that data passed to other investigators or an authorized party for analysis will occur in a pseudonymised manner. Data analysis by the biostatistician will also be performed in a pseudonymised manner.\n\n\nDiscussion\n\nPost-operative monitoring of surgical patients as practiced in general wards is not entirely satisfactory due to its intermittent nature. This monitoring is further degraded in case of reduced nursing staff and during night hours with increased patient/staff ratios.16 This problem is all the more important as the number of patients at risk for postoperative complications will increase, particularly due to the growing population of elderly subjects.17 The possibility of compensating for the lack of monitored beds, whether it is intensive care units and high dependency care units, remote monitoring brings new perspectives. In addition, remote monitoring could be the solution in case of lack of monitored beds.\n\nLeenen et al. recently published a systematic review of the literature reporting the feasibility of the use of 13 devices and their validation for in-hospital continuous vital signs monitoring.18 Since this publication, telemonitoring has been studied outside the hospital either in patients who have undergone esophagectomy during the first 7 days at home after discharge19 or in patients with COVID-19.9,20 Some points remain to be clarified, notably the frequency of artefacts and false alarms and the clinical consequences of the use of remote monitoring.\n\nOur protocol seeks to specify the frequency of artefacts using both automatic detection with predefined bounds but also with review by clinicians as reported in a study concerning the feasibility of continuous monitoring of vital signs in surgical patients on a general ward using SensiumVitals patch.21 This study showed a high percentage of artefacts concerning the respiratory rate measurement (51% of the measurements), and a lower percentage concerning the heart rate measurement (19%) and temperature (9%). This point is crucial as artefacts will generate false alarms leading to the rejection of the technique by caregivers.\n\nWe aimed also to measure the gap between the nurse’s observation of an abnormality in a vital parameter and those detected by the remote monitoring. Such an extent will be a strong argument to encourage the generalization of this new type of monitoring, especially as its cost is not yet well known.\n\nThis cost has been estimated to be in the order of £400 to £550 per patient in a remote home monitoring model during the COV1D-19 pandemic.20 It will have to be weighed against the cost of a complication avoided or recognized and treated earlier.22,23\n\nOur protocol suffers from several weaknesses. The first is that this study will take place during the COVID-19 pandemic, which profoundly changes hospital activity and its functioning. In addition, patients included in the study are at intermediate risk since their procedure will last more than 2 hours and they are expected to have a postoperative stay of more than 72 hours but are not expected to be hospitalized in a monitored unit. This obviously reflects a practice specific to each health facility. We decided not to include in the study patients with very hairy thoracic skin since we decided not to ask patients to shave their chests. This induces a bias in the selection of the patients due to the non-inclusion of some men. We chose to have data registered by the nurses gathered at a frequency determined by medical indications and not by a priori determined intervals. Such choice could be considered as a weakness of the protocol, but we wanted to do a real-life study and consequently to have instructions given to nurses by the medical team which may vary from one patient to another. Finally, it is of prime importance to notice that only blood pressure measurement passed validation testing for accuracy10 and not the other parameters measured by the Biobeat® sensor to the best of our knowledge. Although this may be a limitation to this study, we decided to use this device because it received approval from the Food and Drug Administration and European Community regulators.\n\nIn conclusion, our protocol is specifically aimed to establish whether there is a benefit of remote monitoring using a multiparameter device in the detection of a postoperative complication resulting in an abnormality of one of the major vital signs. This demonstration would encourage the extension of this type of monitoring to hospitalized medical patients and patients at home, which includes many indications: follow-up after discharge, chronic pathology, or acute but not requiring hospitalization.\n\n\nPlans for dissemination of the study outcome\n\nResults of the present protocol will be published in peer-review medical journals.\n\n\nData availability\n\nNo underlying data are associated with this article but the data for this work will be publicly available in the Dryad repository keeping all data anonymous.", "appendix": "Acknowledgements\n\nThe authors would like to thank the Clinical Research Unit (Hôpital Foch, 92150 Suresnes, France) for its help in performing the study and for auditing trial conduct. The authors would like to thank Polly Gobin for her linguistic assistance.\n\n\nAuthor contributions\n\nAlexis Paternot: Conceptualization, Investigation, Writing – Review & Editing.\n\nPhilippe Aegerter: Formal Analysis, Methodology, Writing – Original Draft, Preparation Writing – Review & Editing.\n\nAurélie Martin: Investigation, Project Administration, Writing – Review & Editing.\n\nJonathan Ouattara: Investigation, Writing – Review & Editing.\n\nSabrina Ma: Investigation, Writing – Review & Editing.\n\nSherifa Adjavon: Investigation, Writing – Review & Editing.\n\nBernard Trillat: Formal Analysis, Data Curation, Writing – Review & Editing.\n\nPascal Alfonsi: Investigation, Writing – Review & Editing.\n\nMarc Fischler: Conceptualization, Methodology, Project Administration, Supervision, Writing – Original Draft, Preparation,Writing – Review & Editing.\n\nMorgan Le Guen: Conceptualization, Investigation, Methodology, Supervision, Writing – Original Draft, Writing – Review & Editing.\n\n\nReferences\n\nGroup, I.S.O.S.: Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries. Br J Anaesth. 2016; 117: 601–609. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuran A, Chang C, Cohen B, et al.: Incidence, Severity, and Detection of Blood Pressure Perturbations after Abdominal Surgery: A Prospective Blinded Observational Study. Anesthesiology. 2019; 130: 550–559. PubMed Abstract \| Publisher Full Text\n\nKhanna AK, Bergese SD, Jungquist CR, et al.: Prediction of Opioid-Induced Respiratory Depression on Inpatient Wards Using Continuous Capnography and Oximetry: An International Prospective, Observational Trial. Anesth Analg. 2020; 131: 1012–1024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun Z, Sessler DI, Dalton JE, et al.: Postoperative Hypoxemia Is Common and Persistent: A Prospective Blinded Observational Study. Anesth Analg. 2015; 121: 709–715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiem VGB, Hoeks SE, Mol K, et al.: Postoperative Hypotension after Noncardiac Surgery and the Association with Myocardial Injury. Anesthesiology. 2020; 133: 510–522. PubMed Abstract \| Publisher Full Text\n\nPerman SM, Stanton E, Soar J, et al.: Location of In-Hospital Cardiac Arrest in the United States-Variability in Event Rate and Outcomes. J Am Heart Assoc. 2016; 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoyal College of Physicians: National Early Warning Score (NEWS): Standardising the assessment of acute illness severity in the NHS. Report of a working party. (accessed on May 9). Reference Source\n\nPosthuma LM, Downey C, Visscher MJ, et al.: Remote wireless vital signs monitoring on the ward for early detection of deteriorating patients: A case series. Int J Nurs Stud. 2020; 104: 103515. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlankamenac K, Graf R, Barkun J, et al.: The comprehensive complication index: a novel continuous scale to measure surgical morbidity. Ann Surg. 2013; 258: 1–7. PubMed Abstract \| Publisher Full Text\n\nLe Guen M, Squara P, Ma S, et al.: Patch validation: an observational study protocol for the evaluation of a multisignal wearable sensor in patients during anaesthesia and in the postanaesthesia care unit. BMJ Open. 2020; 10: e040453. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasma W, Peelen LM, van Buuren S, et al.: Artifact Processing Methods Influence on Intraoperative Hypotension Quantification and Outcome Effect Estimates. Anesthesiology. 2020; 132: 723–737. PubMed Abstract \| Publisher Full Text\n\nRecio-Saucedo A, Maruotti A, Griffiths P, et al.: Relationships between healthcare staff characteristics and the conduct of vital signs observations at night: Results of a survey and factor analysis. Nurs Open. 2018; 5: 621–633. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin HS, Watts JN, Peel NM, et al.: Frailty and post-operative outcomes in older surgical patients: a systematic review. BMC Geriatr. 2016; 16: 157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeenen JPL, Leerentveld C, van Dijk JD, et al.: Current Evidence for Continuous Vital Signs Monitoring by Wearable Wireless Devices in Hospitalized Adults: Systematic Review. J Med Internet Res. 2020; 22: e18636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreteler MJM, Numan L, Ruurda JP, et al.: Wireless Remote Home Monitoring of Vital Signs in Patients Discharged Early After Esophagectomy: Observational Feasibility Study. JMIR Perioper Med. 2020; 3: e21705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVindrola-Padros C, Sidhu MS, Georghiou T, et al.: The implementation of remote home monitoring models during the COVID-19 pandemic in England. EClinical Medicine. 2021; 34: 100799. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeenen JPL, Dijkman EM, van Dijk JD, et al.: Feasibility of continuous monitoring of vital signs in surgical patients on a general ward: an observational cohort study. BMJ Open. 2021; 11: e042735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVonlanthen R, Slankamenac K, Breitenstein S, et al.: The impact of complications on costs of major surgical procedures: a cost analysis of 1200 patients. Ann Surg. 2011; 254: 907–913. PubMed Abstract \| Publisher Full Text\n\nde la Plaza Llamas R, Hidalgo Vega Á, Latorre Fragua RA, et al.: The Cost of Postoperative Complications and Economic Validation of the Comprehensive Complication Index: Prospective Study. Ann Surg. 2021; 273: 112–120. PubMed Abstract \| Publisher Full Text" }	[ { "id": "92786", "date": "03 Sep 2021", "name": "Philippe Cuvillon", "expertise": [ "Reviewer Expertise remote monitoring", "regional anesthesia" ], "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 the opportunity to review a topic that is my area of expertise.\nMedical monitoring dedicated to machines or computer systems in health care services will be an issue in the coming years. To date, the proposed systems are disappointing because they provide incomplete or irrelevant data. Monitoring aided by machine learning must offer predictive or immediate detection systems.\nIn the proposed study, the authors use a detection system. The main problem of this study is the definition of the primary endpoint, which is incomprehensible (see below)\nMinor remarks:\nIntroduction:\n\nFirst paragraph: postoperative mortality and morbidity are time dependent and severity dependent. The authors should specify this temporality (mortality at 30 days?). Similarly, the authors report the danger of hypotension, the studies of which are mainly intra-operative or in continuous care. This should be specified.\n\nParagraph 2: specify that to date, no study has been able to demonstrate the real interest of continuous monitoring systems on mortality in health care services, mainly because of automatic monitoring errors.\n\nLast paragraph: better specify the primary endpoint\nMethods:\nStudy population: were patients operated on under spinal anaesthesia included? Were patients under epidural or morphine included? These are major biases and independent risk populations for complications (respiratory depression)\n\nPrimary endpoint: very incomplete and not very descriptive \"main outcome is the occurrence of one or more hemodynamic complications\". Talk about cardiac arrest, drop in blood pressure but in relation to which baseline?\n\nThe NSN depends on the primary endpoint, which is incomplete, which calls into question the NSN\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "7187", "date": "07 Oct 2021", "name": "Marc Marc", "role": "Author Response", "response": "Responses to Reviewer 1 Thank you for giving me the opportunity to review a topic that is my area of expertise. Medical monitoring dedicated to machines or computer systems in health care services will be an issue in the coming years. To date, the proposed systems are disappointing because they provide incomplete or irrelevant data. Monitoring aided by machine learning must offer predictive or immediate detection systems In the proposed study, the authors use a detection system. Main remark : The main problem of this study is the definition of the primary endpoint, which is incomprehensible (see below). Minor remarks: INTRODUCTION Comment 1: Introduction, First paragraph: postoperative mortality and morbidity are time dependent and severity dependent. The authors should specify this temporality (mortality at 30 days?). Similarly, the authors report the danger of hypotension, the studies of which are mainly intra-operative or in continuous care. This should be specified. Response to Comment 1: The study concerns the early postoperative course (at most 72 first postoperative hours). This is written in the Abstract: “The study will cover the first 72 hours after discharge of the patient from the post-anaesthesia care unit.” We have changed the Introduction section adding a last sentence to be more precise: “Data obtained using the sensor will be compared to data obtained during the routine nurse follow-up during at most 72 first postoperative hours.” Thus, our study does not include the intraoperative period and the stay in the post anesthesia care unit where patients are continuously under monitoring. Similarly, our study concerns only conventional nursing monitoring performed on unmonitored general care floors (last paragraph of the Introduction). Comment 2: Paragraph 2: specify that to date, no study has been able to demonstrate the real interest of continuous monitoring systems on mortality in health care services, mainly because of automatic monitoring errors. Response to Comment 2: As written in the second sentence of the Introduction, postoperative complications remain quite frequent. We did not want to study postoperative mortality. Moreover, as we are interested in unmonitored general care floors and not ICU patients, the postoperative risk of death is very low (0.1% in our scheduled surgical population). A study using mortality as main outcome would require a huge number of patients. Otherwise, we agree with the comment about automatic monitoring errors; this is why a large part of the study concerns the screening of artefacts. Comment 3: Last paragraph: better specify the primary endpoint Response to Comment 3: We have written in the Outcomes measures section “The main outcome is the occurrence of one or more hemodynamic complications during the first 72 hours following a major non-cardiac surgical procedure unless death or hospital discharge occurs sooner, with the 72-hour period starting when the patient returns to the surgical department.” The main outcome needs effectively to be clarified: “The main outcome is the occurrence of one or more episodes with a mean arterial pressure below the threshold of 60 mmHg during the first 72 hours …..” This definition is in line with the reference used to calculate the number of patients to be included (see Response to comment 6). This threshold has been added to Table 1. METHODS Comment 4: Methods: Study population: were patients operated on under spinal anaesthesia included? Were patients under epidural or morphine included? These are major biases and independent risk populations for complications (respiratory depression) Response to Comment 4: We intend to do this study in real life and locoregional postoperative analgesia is not an exclusion criterion. Comment 5: Primary endpoint: very incomplete and not very descriptive \"main outcome is the occurrence of one or more hemodynamic complications\". Talk about cardiac arrest, drop in blood pressure but in relation to which baseline? Response to Comment 5: See response to Comment 3. As reported in Table 1 (Outcome definitions), abnormalities in mean arterial pressure are defined as absolute values and relative values. Occurrence of a major event, like a cardiac arrest is planned in the Outcome measures section: “… postoperative complications that occurred occur? during the monitoring period, as determined by the healthcare team in accordance with the Dindo and Clavien classification.12”. Comment 6: The NSN depends on the primary endpoint, which is incomplete, which calls into question the NSN Response to Comment 6: We have specified the primary endpoint (see response to comment 3). This allows for consistency with the number of subjects to be included which is based on the work of Liem et al. (Anesthesiology 2020, 133, 510-522) who report that “Postoperative hypotension was common, e.g., 2 cumulative hours below a threshold of 60 mmHg occurred in 144 (8%) patients while 4 h less than 75 mmHg occurred in 824 (48%) patients”. We used this 8% frequency knowing that there is probably some difference between our population and our nursing monitoring modalities and the corresponding elements of the study by Liem et al. We probably should have done a prior study to better specify the percentage of patients who had a postoperative hemodynamic complication requiring a specific treatment. In any case, including 114 patients will allow us to give a first answer to the interest of remote monitoring. We have modified the text to include a limitation of our study: “Finally, the number of subjects to be included in the study was based on the results of a previous publication on the occurrence of postoperative hypotension.5 There is probably some difference between our population and our nursing monitoring modalities and the corresponding elements of this study.5 We probably should have done a prior study to better specify the percentage of patients who had a postoperative hypotension in our center. In any case, including 114 patients will allow us to give a first answer to the interest of remote monitoring.”" } ] }, { "id": "91571", "date": "20 Sep 2021", "name": "Zhuo Sun", "expertise": [ "Reviewer Expertise perioperative management", "Pain management" ], "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 submitted a protocol that seeks to quantify the benefit of remote monitoring every 5 minutes using a multiparametric device in the detection of postoperative complications in comparison with the monitoring performed by nurses. In this proposed study, the authors hypothesize that monitoring with a multi-signal wearable sensor will detect potentially dangerous vital sign abnormalities more often and more rapidly than routine surveillance in surgical patients hospitalized on an unmonitored general care floor. Postoperative hemodynamic abnormalities are common and independently associated with increased postoperative morbidity and mortality. Anesthesiologists are interested in unique methods to safely monitor and manage postoperative patients.\nStrengths: This device looks very user friendly. The enrolled patients will be more likely to adhere to the study compared to other devices used in previous studies.\nMinor concerns:\nThe title is to 'screen for postoperative complications by continuous monitoring'. The primary outcome is the occurrence of one or more hemodynamic complication during the 1st 72 hours postoperatively. While in the outcome definition, the primary outcome is defined by different hemodynamic abnormalities. There is no postoperative mortality and morbidity evaluated in this study.\n\nBoth the occurrence and duration of hemodynamic abnormalities may impact postoperative outcomes. If the author can consider adding the duration of detected hemodynamic abnormalities into the outcomes, this study could provide more information for readers.\n\nIn the demographic data, the duration of procedure, post-op medications, and opioids/analgesics could be valuable information in the study.\n\nThe authors hypothesize that monitoring will detect potentially dangerous vital sign abnormalities more often and more rapidly than routine surveillance. It does make sense that continuous monitoring can collect more data, and may detect abnormalities earlier. However, the monitoring data from the patch will be recorded continuously without being communicated to the nursing staff and doctors per protocol. It is unlikely that the abnormal vital signs can be caught rapidly through this study by monitoring.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\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? Yes", "responses": [ { "c_id": "7188", "date": "07 Oct 2021", "name": "Marc Marc", "role": "Author Response", "response": "Responses to Reviewer 2 The Authors have submitted a protocol that seeks to quantify the benefit of remote monitoring every 5 minutes using a multiparametric device in the detection of postoperative complications in comparison with the monitoring performed by nurses. In this proposed study, the authors hypothesize that monitoring with a multi-signal wearable sensor will detect potentially dangerous vital sign abnormalities more often and more rapidly than routine surveillance in surgical patients hospitalized on an unmonitored general care floor. Postoperative hemodynamic abnormalities are common and independently associated with increased postoperative morbidity and mortality. Anesthesiologists are interested in unique methods to safely monitor and manage postoperative patients. Strengths: This device looks very user friendly. The enrolled patients will be more likely to adhere to the study compared to other devices used in previous studies. Minor concerns: Comment 1: The title is to 'screen for postoperative complications by continuous monitoring'. The primary outcome is the occurrence of one or more hemodynamic complications during the 1st 72 hours postoperatively. While in the outcome definition, the primary outcome is defined by different hemodynamic abnormalities. There is no postoperative mortality and morbidity evaluated in this study. Response to Comment 1: We agree with this comment and we have changed the title to: “Screening for postoperative vital signs abnormalities, and particularly hemodynamic ones, by continuous monitoring: protocol for the Biobeat-Postop cohort study” Comment 2: Both the occurrence and duration of hemodynamic abnormalities may impact postoperative outcomes. If the author can consider adding the duration of detected hemodynamic abnormalities into the outcomes, this study could provide more information for readers. Response to Comment 2: We agree with this comment, and we have changed the secondary outcomes to: “The secondary outcomes will concern the frequency of postoperative respiratory or temperature abnormalities and their duration, the frequency of artefacts ….” Comment 3: In the demographic data, the duration of procedure, post-op medications, and opioids/analgesics could be valuable information in the study. Response to Comment 3: We agree with this comment, and we have changed the first sentence of the Procedure: “Demographic data, including age, sex, American Society of Anesthesiologists classification, body mass index, underlying diseases, type and duration of the surgical procedure, will be collected upon inclusion in the study. Postoperative medications including opioids and other analgesics will also be collected.” Comment 4: The authors hypothesize that monitoring will detect potentially dangerous vital sign abnormalities more often and more rapidly than routine surveillance. It does make sense that continuous monitoring can collect more data, and may detect abnormalities earlier. However, the monitoring data from the patch will be recorded continuously without being communicated to the nursing staff and doctors per protocol. It is unlikely that the abnormal vital signs can be caught rapidly through this study by monitoring. Response to Comment 4: What we seek to measure is the difference between the two monitoring modalities: \"classic\" monitoring by nurses and \"modern\" remote monitoring. To do a blind study is mandatory. A significant difference would be an argument for investing in \"modern\" monitoring. As such, we consider this study to be a pilot study that should provide an initial answer to this question." } ] } ]	1	https://f1000research.com/articles/10-622
https://f1000research.com/articles/10-1019/v1	07 Oct 21	{ "type": "Method Article", "title": "Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy", "authors": [ "Peter Wookey", "Pragya Gupta", "Lucas Bittencourt", "Shane Cheung", "David Hare", "Sebastian Furness", "Pragya Gupta", "Lucas Bittencourt", "Shane Cheung", "David Hare", "Sebastian Furness" ], "abstract": "The expression of the calcitonin receptor (CT Receptor) is widespread throughout the life cycle of mammals and in many diseases, and in these contexts the functions of the common isoforms is largely unknown. The relatively recent development of anti-CT Receptor antibodies that bind separate epitopes on the CTa Receptor and CTb Receptor isoforms has advanced our knowledge and understanding of these events. CT Receptor at the protein level is upregulated in programmed cell death including apoptosis (as described in a previous publication) and autophagy, which is discussed in our upcoming, unpublished review. Incomplete data sets are cited in this review on the upregulation of CACLR (encoding CT Receptor) mRNA, in particular the insert-positive isoform (CTb Receptor), in response to cell stress. Cell stress is induced by growth in depleted foetal bovine serum (dFBS) or without FBS, both of which induce degrees of starvation and autophagy, or dFBS plus staurosporine, which induces apoptosis. Details of the methods deployed to generate these data are described here including measurement of the upregulation of CTb Receptor mRNA with qPCR and nanopore long range sequencing. An anti-CT Receptor antibody also known as CalRexinTM, which binds an epitope in the N-terminal domain, was conjugated to either fluorophore 568, which is accumulated into apoptotic cells as previously reported, or pHrodo Red, a pH dependent fluorescent dye, which is accumulated into autophagic and apoptotic cells. These conjugates are under development to image programmed cell death. The methods for conjugation and high content imaging on the Operetta platform are described. The high fluorescence intensity at low pH of CalRexin:pHrodo Red in both autophagic and apoptotic cells suggests localisation in autophago-lysosomes and lysosomes respectively. Overall, these observations and the methods that underpin them have contributed to our understanding of the widespread expression of CT Receptor isoforms.", "keywords": [ "cell stress", "autophagy", "apoptosis", "calcitonin receptor isoform", "antibody", "antibody conjugate", "high content screening", "quantitative PCR", "nanopore sequencing" ], "content": "Introduction\n\nThis article provides details of the methods developed to generate unpublished data, which is cited in an unpublished review article due for submission. The purpose is to provide the opportunity for researchers to review the methods, and if motivated, reproduce (and extend) the data. A panel of the unpublished data presented in the review is also include as Figure 1 below.\n\n(A) CalRexinTM:pHrodo Red compared to CalRexinTM:568 for imaging MG63 cells grown on Matrigel and undergoing autophagy (control [CON] starved by growth in depleted serum), or to induce apoptosis treated with 50 μM etoposide or 1 μM staurosporine, or treated with 50 μM chloroquine to inhibit formation of autophagolysosomes.4 Images were recorded every 30 minutes on the Operetta platform (Images displayed above were captured at 0, 4, 8, 12, 16, 20 & 24 hours). The scale bar in the 24-hour images is 50 μm. (B) Enlarged images recorded at 20 hours. Note that chloroquine known to inhibit the fusion of autophagosomes with lysosomes to form autophago-lysosomes inhibits fluorescence with CalRexin:pHrodo Red.\n\nThe methods used to generate unpublished data cited in an upcoming review, relate to the experiments with qPCR and nanopore sequencing in which the mRNA of the CTb Receptor (insert-positive isoform, exon 10 spliced in), which together demonstrate upregulation in stressed cells. These methods also include synthesis of the novel imaging reagents CalRexinTM:568 (RRID:AB_2893120) and CalRexinTM:pHrodo Red for imaging apoptosis and autophagy. All the data generated with these methods will be published in full in an experimental peer-reviewed manuscript that is currently being drafted and completed for publication.\n\n\nMethods\n\nThe mouse monoclonal antibody mAb2C4 (clone 46/08-2C4-2-2-4, CalRexinTM (RRID:AB_2893093) was developed using standard techniques summarised below for the cloning of high expression hybridomas. CalRexinTM binds in the N-terminal domain of the CT Receptor isoforms, CTa or CTb Receptor. Data supporting the validation of the antibody CalRexinTM have been presented in the supplementary materials linked to.1\n\nThe strategy to develop CalRexinTM is as follows. A peptide equivalent to the epitope in the N-terminal domain of the human CT receptor (C-PSEKVTKYCDEKGVWFK-NH2) was synthesized (Auspep, Tullamarine, Australia). This peptide was conjugated to Keyhole Limpet Hemocyanin (KLH) via a linker maleimidocaproyl-N-hydroxysuccininmide (Auspep, Australia). The rest of the procedure was performed by the dedicated laboratories at the Antibody Facility of the Walter & Eliza Hall Institute, Bundoora, Victoria, Australia. At the facilities of this third party provider, four mice were each immunized with the KLH conjugate essentially as described previously.2 The spleens from the mice were removed on the day of fusion and a single cell suspension was prepared. The cells were then washed three times in Dulbecco’s modified Eagle’s medium (DMEM). Cells of the myeloma line (SP2/O) were washed three times in DMEM and adjusted to 0.5-1.0 × 108 cells per fusion with a mouse spleen. Spleen and myeloma cells were mixed, pelleted and the fusion performed by addition of 1 mL warm polyethylene glycol 1500 to the pellet with gentle stirring (1 min). The fusion mixture was then added slowly to 25 mL DMEM. Cells were centrifuged and resuspended in hybridoma serum-free medium containing 10 % foetal bovine serum (FBS), interleukin-6 conditioned medium and HAT (hypoxanthine, aminopterin, thymidine), and plated out in 5 to 6 microtiter plates.\n\nAfter incubation of the cells for 8 to 9 days, supernatants were collected at day 14 and tested by enzyme-linked immunosorbent assay (ELISA), checked by immunohistochemistry, before positive cells were selected for limiting-dilution cloning. Usually two rounds of cloning were sufficient to ensure the hybridomas were clonal.\n\nCloned cells with the desired isotype and exhibiting a high titre by ELISA test were grown in bioreactors. The monoclonal antibody was partially purified by Protein A chromatography (in mouse tonicity PBS). The selected clone (46/08-2C4-2-2-4) was used to produce the anti-human CT Receptor monoclonal antibody [isotype IgG1, kappa] adjusted to a final concentration of 1 mg/mL protein (sterile filtered) and stored at −80°C until required. Freeze/thaw cycles were avoided.\n\nThe method of conjugation for CalRexin (mAb 2C4) and AF568:NHS esters to synthesise CalRexin:568 (RRID:AB_2893120) has been described previously.1 The molar ratio of antibody to dye was 1:15 and in the case of CalrexinTM:pHrodo Red (RRID:_2893121) 15 mg of antibody was conjugated with 1 mg of pHrodo Red NHS esters (Thermo Fisher Scientific, TFS).\n\nThe antibody was desalted by centrifugation using 5 mL Zeba column (TFS) and the buffer replaced with 100mM sodium bicarbonate, pH 8.3. The dye pHrodo Red NHS esters (MW 650, 1 mg, 1.54 μmoles) was dissolved in 150 μL of anhydrous DMSO and added dropwise to the antibody in bicarbonate buffer. After 1 hr gentle inversion at RT the reaction was quenched with 50 μL of 1.5 M triethanolamine (20% solution in bicarbonate buffer) for 3 hr.\n\nThe conjugate can be separated from unreacted and other small molecules on a Zeba column and the buffer exchanged for PBS.\n\nThe results of these data are included in Figure 1. MG63 cells were cultured in MEMα (Thermo Fisher Scientific, TFS) plus 10% foetal bovine serum (TFS), washed once in MEMα plus 10% depleted FBS (dFBS) and plated at 10,000 cells per well in a 96-well plate (CellCarrier-96 Ultra, PerkinElmer #6055302) in MEMα/10% dFBS.\n\nTo make dFBS, FBS centrifuged at 100,000 × g for 24 hours and the clear supernatant, devoid of exosomes, was collected as depleted FBS (dFBS) and the pellet discarded.\n\nCalRexinTM:AF568 (2 μg/mL, Apop Biosciences Pty Ltd) or CalRexinTM:pHrodo Red (2 μg/mL, Apop Biosciences Pty Ltd) was added to the appropriate wells of the 96-well plate. Staurosporine (1 μM final concentration, Sigma), etoposide (50 μM final concentration, Sigma) or chloroquine (50 μM) were added at time zero minutes.\n\nCells were imaged on an Operetta high-content screening system (Operetta CLS, RRID:SCR_018810) controlled by the Harmony software (Harmony version 4.1, RRID:SCR_018809). Cells were imaged over a 24 h period with images being acquired at a single focal plane every 30 min using a 40× high NA (Numerical Aperture) objective. The system was maintained at a constant temperature of 37 °C and 5% CO2 for optimal cell growth.\n\nThe following filter sets were used for imaging each dye: for CalRexin:pHrodo excitation was 520-550 nm and emission was 560-630 nm, and for CalRexin:AF568 excitation was 560-580 nm and emission was 590-640 nm. Excitation was provided by a Xenon lamp and transmission was set to 50 %. The brightfield channel was acquired with an exposure of 100 ms.\n\nTime-lapse videos were created and annotated using FIJI image analysis software, version 1.53c (Fiji, RRID:SCR_002285).\n\nRNA was extracted using RNeasy Mini Kit (Qiagen). The purity and concentration of the RNA was checked on a Nanodrop (A260/A280 ratio). To check RNA integrity 100 ng of RNA from each sample was electrophoresed on a 1.2 % Agarose gel and visualized using GelGreen (Fisher Biotec) on ChemiDoc MP Imaging system (Bio Rad ChemiDoc MP Imaging System, RRID:SCR_019037).\n\nEqual amounts of RNA from all the samples were used to synthesise cDNA using SuperScript VILO IV reverse transcriptase kit (Invitrogen). qPCR assay targeting exon 10 of human CALCR mRNA (CT Receptorb mRNA) was purchased from Thermo Fisher (Assay ID: Hs01016888_m1) along with GAPDH assay (Assay ID: Hs02786624_g1) as the housekeeping gene control. 20 μL duplex reaction was set up in a 96-well 0.1 mL qPCR microplate (Applied Biosystems) by mixing 20 ng of cDNA, 1 μL of CALCR assay, 0.5 μL of GAPDH assay, 10 μL of 2X TaqMan™ Fast Advanced Master Mix (Applied Biosystems) and nuclease-free water, keeping three replicates for each sample. qPCR run was conducted for 50 cycles of 95°C for 1 sec and 60°C for 10 secs on QuantStudio™ 3 (Applied Biosystems QuantStudio 12K Flex RealTime PCR System, RRID:SCR_021098). mRNA expression was quantified by calculating 2-∆∆Ct using Design and Analysis Software v2.5.1 (Thermo Fisher).\n\nPrimers specific to CT Receptor transcript 2 (NM_001742.3) were designed using SnapGene Viewer v5.0.4 (SnapGene, RRID:SCR_015052) covering the full coding sequence including most of 3′ UTR. The forward primer is specific to the sequence corresponding to the exon 2 and exon 5 boundary and the reverse primer is specific to a 3′ UTR region in exon 17. Additionally, both primers have a universal barcode recognizing sequence (written in lower case, Oxford Nanopore Technologies). Forward primer was 5′tttctgttggtgctgatattgcGTGACAGAATTCCAGGACAAAGAGATC3′ and reverse primer was 5′acttgcctgtcgctctatcttcGGGCAGAACTATGTGCAATTCTATAATAAGC3′. cDNA synthesis was carried out using GoScript reverse transcriptase (Promega). 100-300 ng of cDNA was used for PCR amplification conducted for 25 cycles of 94°C for 30 sec, 60°C for 1 min, 65°C for 3 min using LongAmp Taq polymerase and final extension at 65°C for 10 min in a 25 μL reaction.\n\nPCR products were purified using 0.4× Agencourt AMPure XP (Beckman Coulter) magnetic beads to remove short DNA fragments and the DNA concentration was measured by a Qubit 4 Fluorimeter (Thermo Fisher Qubit fluorimeter, RRID:SCR_018095). To confirm the product length, 1 μL of each DNA sample was analysed on 4200 Tapestation system (Agilent 4200 TapeStation System, RRID:SCR_019398) against D5000 DNA ladder (Agilent). DNA bands were visualised using Tapestation Controller Software v3.1.1 (Agilent). Samples having a band near 3.4 kb were selected for barcoding.\n\nEach DNA sample (2 fmol) was barcoded using primers from PCR Barcoding kit (EXP-PBC001) and PrimeStar GXL DNA Polymerase (Takara). The amplification was conducted for 12 cycles of 95°C for 15 sec, 62°C for 15 sec and 65°C for 3 min. Barcoded DNA was quantified using a Qubit 4 Fluorometer (Thermo Fisher) and purified using 0.4× Agencourt AMPure XP beads. Barcoded DNA, 15 fmol from each condition, were pooled together to make a DNA library using the 1D Nanopore Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies). DNA end repair and dA-tailing were performed using NEBNext Ultra II End-Repair/dA-tailing Module (NEB, UK) for 5 min at 20°C and 5 min at 65°C. The DNA library was quantified using Qubit and purified using 0.6× Agencourt AMPure XP beads. 50 ng (or approximately 24.19 fmol for an estimated length of 3.4 kb DNA) of the DNA library was loaded on the MinION R9.4.1 flow cell (Oxford Nanopore Technologies). In total 1100 pores were active at the start of the run.\n\nReads from the Nanopore sequencer, also known as 2D pass reads (basecall score ≥ 7) were analysed for quality and size distribution using NanoPlot v1.32.0 bioinformatic tool.\n\nTAQLoRe v1 (Transcript Annotation and Quantification using Long Reads) pipeline (https://github.com/twrzes/TAQLoRe) was used to create a CALCR hypothetical meta-gene by combining known and novel exons. Novel exons were defined as insertions (≥9 nucleotides) at a minimum distance of 6 nucleotides from known exons and having a minimum alignment proportion of 0.8. TAQLoRe then mapped the pass reads to the CALCR meta-gene as well as the human transcriptome. Mapped exons were annotated according to their location listed in the mapping output file. The only variant identified using this method was inclusion of exon 10. The abundance of exon 10 (%) was calculated for each staurosporine treatment vs. time sample (number of reads/total reads) to observe changes over time relative to the untreated control condition.\n\nThe limited data in Figure 1 will become part of a larger study to be published in a further experimental manuscript.\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": "Acknowledgements\n\nWe wish to thank Apop Biosciences Pty Ltd for access to the mAb2C4 antibody (CalRexinTM) and acknowledge the contribution made by the Biological Optical Microscope Platform at the University of Melbourne for the provision and maintenance of the high-resolution confocal microscopes and the Operetta platform.\n\n\nReferences\n\nFurness SGB, Hare DL, Kourakis A, et al.: A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death. Cell Death Disc. 2016; 2: 16062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShulman M, Wilde CD, Kohler G: A better cell line for making hybridomas secreting specific antibodies. Nature. 1978; 276(5685): 269–270. PubMed Abstract \| Publisher Full Text\n\nClark MB, Wrzesinski T, Garcia AB, et al.: Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain. Mol Psychiatry. 2020; 25(1): 37–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMauthe M, Orhon I, Rocchi C, et al.: Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy. 2018; 14(8): 1435–1455. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "96413", "date": "21 Oct 2021", "name": "Virender Singh", "expertise": [ "Reviewer Expertise Neuroscience and cancer 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 article “Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy” by Peter Wookey et al. describe methods to assess cell stress in terms of upregulation of CT receptor isoform(s) at mRNA level. The authors cover the generation and validation of anti-human CT Receptor antibody and CalRexin, later deployed to study cell stress. The authors have provided a clear context of why they are using CT Receptor as a readout of cell stress. The authors clearly described the technical details of the methods. For antibody production, the authors have used an epitope on the N-terminus, which is rational given that the N-terminus of a GPCR remains accessible to antibodies raised against; hence, it can be used on live cells. The authors have also compared different fluorophores, which confirms the flexibility of the antibody's use. Other researchers might want to use a fluorophore of their interest. The use of dFBS is not apparent. However, it is best to avoid exosomes containing serum while studying the uptake of compounds, from my understanding. The nanopore sequencing has been described well enough to follow. The authors have mentioned all minute details so that others can use the methods for their purpose. The authors have shown precise results of the techniques presented, except the nanopore data, which seems confidential at this stage.\nThis is a fascinating and timely method paper, and I have thoroughly enjoyed reading and reviewing this article. The topic is of high importance. I am rather enthusiastic about this paper and supportive of its publication. I only offer minor suggestions to improve readability and enhance the paper's message (adopting them is optional).\nMinor issue:\nScale bar for Figure 1B missing.\n\nAn illustration summarizing the method in the form of a flow chart with checkpoints will be helpful for readers to visualize and understand the importance of work.\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": "96415", "date": "22 Oct 2021", "name": "Eshan Ghosh", "expertise": [ "Reviewer Expertise cell biology", "biochemistry", "biophysics" ], "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 provided a streamlined workflow to assess the expression of calcitonin receptor isoforms under stressed cell conditions. Authors have used three different approaches to exhibit differential receptor expression. The partial data for calcitonin receptor localization shows good use of CalRexin antibody conjugates for interrogating the receptor trafficking dynamics under specific conditions. Owing to the unavailability of data due to specific reasons given by authors, it is hard to appreciate all three approaches. However, the detailed method provided herein is sufficient for the scientific community to replicate the results. I would also like to request the authors to detail the conditions in which the RNA has been extracted so the exact conditions can be replicated.\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? Partly\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/10-1019
https://f1000research.com/articles/10-1017/v1	06 Oct 21	{ "type": "Research Article", "title": "Modelling mode I failure at crack tip with verifications using digital image correlation", "authors": [ "Kok C.K.", "Eric Ren Wei Koh", "Gooi Mee Chen", "Eric Ren Wei Koh", "Gooi Mee Chen" ], "abstract": "Background: Linear elastic fracture mechanics (LEFM) applies to sharp cracks, although crack sites such as holes and slots are often blunt cracks with nonzero widths. The advancement in finite element method (FEM) has enabled the calculation of stress intensity factor (SIF) for unstable crack growth prediction, regardless of crack shape and size, but the calculations often differ from contour to contour. Hence, the purpose of this work is to determine if SIFs computed using commercial FEM have experimentally verifiable advantages over the traditional stress-based modelling approaches in predicting Mode I brittle failure at blunt crack tip. Methods: Experiments and simulations were conducted on brittle Poly(methyl methacrylate) (PMMA) plastic to compare the actual and predicted strain fields and SIFs at crack tip, and the critical force at which unstable crack growth initiates. A centrally straight cracked Brazilian disc made of PMMA was subjected to purely Mode I fracture. Its strain fields were measured from deformed speckle patterns using digital image correlation software. The same disc was modelled using plane stress model in FEM. By applying the critical force, SIFs were then computed using ANSYS pre-meshed crack method at different contours away from the crack tip. The effects of element type, mesh size and crack width on the simulated SIFs were investigated. Results: It was found that the experimental critical load agreed well with LEFM prediction based on PMMA fracture toughness in published literature. Disc failure happened at the first sign of tensile yield at the crack tip in the finite element model with a triangle mesh. Digital image correlation clearly shows the occurrence of unstable crack growth at critical force. It also shows comparable far field strain responses to the FEM model. Conclusions: The computed SIFs were inconsistent, and their usefulness in predicting unstable crack growth requires further investigation.", "keywords": [ "Stress Intensity Factor", "Opening Mode", "Fracture", "PMMA", "Ansys Fracture Tool" ], "content": "Introduction\n\nIt is widely accepted that crack tip shape and crack width have significant effects on the stress intensity factor (SIF). For a crack with finite width (i.e. blunt crack), researchers are working on correction factors to account for the deviations that result from linear elastic fracture mechanics (LEFM) predictions.1 Although literature is abundant on the use of finite element method to predict SIF,2 most work focused on comparing finite element predictions and those from LEFM on sharp crack, and often not complemented by experimental observations. Also, some modelling techniques require manual node adjustment (i.e. collapsed elements or quarter-node approach) and/or complex mesh morphing strategy,3 and therefore not feasible for routine use by simulation analysts. SIFs calculated in finite element commercial software, such as ANSYS, often differ from contour to contour. Experimental verifications are indeed important in cases of a crack with finite width.\n\nIn recent years, digital image correlation (DIC) has enabled researchers and engineers to measure strains at minute locales and view full-field data without requiring any physical contact, or installing relatively large and expensive strain gauges.4 It is convenient, simple, and accurate in measuring deformation and strains in large-scale structures.5 Open-source programme such as GOM correlate makes 2D DIC feasible and cost-effective. Some examples of DIC usage for monitoring structural behaviour and long term reliability of materials under different loading conditions in both static and dynamic situations5 are as follows: Abshirini et al.’s investigation on mode I failure of Brazilian disc using DIC method,6 Huang et al.’s7 study on the flexural behaviour and crack formation in cementitious composite, UK National Physical Laboratory’s (NPL) measuring of hard-to-detect micro-crack opening in concrete structures.8 Recently, digital image correlation was used to evaluate fracture mode-mixity.9\n\nIn this work, experiments and simulations were conducted on brittle Poly (methyl methacrylate) (PMMA) plastic to compare the actual and predicted strain fields around crack tip with finite width, and the critical force at which unstable crack growth initiates. The effects of mesh size and crack width on the simulated critical SIF were investigated. The aim of this work is to determine if SIFs computed using commercial finite element method have experimentally verifiable advantages over the traditional stress-based modelling approaches in predicting Mode I brittle failure at blunt crack tip.\n\n\nMethods\n\nAll procedures used in this project have been approved by the Research Ethics Committee (REC) Multimedia University (EA1682021). This work does not involve data collection from humans, human or animal experiments or vulnerable communities.\n\nCentrally straight crack Brazilian discs (CSCBD), commonly used to investigate the fracture toughness in various materials10 were prepared according to dimensions in Figure 1. The PMMA discs were machined to a diameter of 60 mm and 15 mm thick. The centre notch was of 2 × 15.5 mm in length and 1 mm in width. PMMA had been chosen in this study due to its brittle nature and the vast amount of related publications. Specimen dimensions were specified in compliance with ASTM D3967-95a11 requiring a thickness to diameter ratio t/d within the range of 0.2-0.75 and ASTM E 399-9012 requiring the specimen to be sufficiently large compared to the crack length and the plastic zone size. The benefit of using a Brazilian Disc specimen is that it is much easier to prepare than other specimens because of its simple geometry, and the uniaxial compression test is relatively easy to set up.\n\n(a) Brazilian disc and crack tip polar coordinate system15; (b) Actual disc dimensions.\n\nThe quality of DIC results depends mostly on the resolution of the speckle pattern.13 Fine speckle pattern, a good focusing lens and a high resolution (10 MP) camera are essential for DIC to track the random speckle pattern with accuracy.14 The specimen was first sprayed with a layer of plastic primer followed by white spray paint before the black speckle pattern was applied. Spray painting was used to apply random speckle patterns, as shown in Figure 2. Natural drying under sunlight took place before subsequent application of paints.\n\nFigure 3 shows the test setup. Compression was applied to CSCBD until fracture using Instron 3367 universal testing machine, at speeds of 1 mm/minute and 5 mm/minutes, respectively. During the compression test, the camera was mounted onto the tripod and calibrated using a built-in spirit level to ensure the image sensor was aligned perpendicular to the region of interest. In the setup, a high brightness LED light was used to illuminate the surface of the specimen. The camera was set to capture one image per second in black and white. Both the image acquisition and compression testing were carried out simultaneously. Image acquisition was started one to three-seconds before load application. Images and force data were synchronized during post-processing. GOM Correlate was used to perform the DIC.\n\nAccording to,10 critical SIF of Mode I fracture, KI, can be computed using equation (1).\n\nwhere P is the critical load at fracture, R is the radius of the disc, a is the semi crack length,B is the thickness of the disc and θ is the angle of crack relative to load, which is zero in this study. YIis a geometry factor and is a function of crack length ratio (a/R) and the crack angle θ.\n\nAnsys Mechanical Workbench was used to simulate the compression test. Plane stress model was used, as the finite element results do not show significant differences from those of plane strain model. This is expected as the specimen dimensions meet the requirement of small plastic zone for plain strain consideration (i.e. ASTM E 399-9012), and will not be further discussed in this paper. The material properties of PMMA are summarized in Table 1.15\n\nA force was applied through a rigid top plate in contact with the specimen, which was supported by yet another rigid plate at the bottom. Pre-meshed crack approach and linear elements were adopted. Ansys fracture tool, which calculates SIF using contour integration,16 was used to compute SIFs on 6 contours around the crack tip.\n\nAs the quality of the mesh directly affects the accuracy and speed of the solution, a finer mesh was used around the crack tip since it was the region of interest. In this study, the effects of element type (i.e. triangles vs. quadrilaterals), minimum element size at crack tip (i.e. 50 μm, vs. 2.5 μm) and crack width (i.e. 1.0 mm and 0 mm) on stress-strain contour and SIF were investigated. Figure 4 shows the different meshes on the CSCBD model. Quadratic elements reported lower SIF values, and will not be presented in this study.\n\nEquivalent strain fields in different meshes (a) Triangle mesh, crack width = 1.0 mm; (b) Quadrilateral mesh, crack width = 1.0 mm; (c) Zoom in at crack of (b); (d) Quadrilateral fine mesh, zoomed in at crack; (e) Triangle mesh, crack width = 0 mm, zoomed in at crack; The scales in (c), (d) and (e) are comparable.\n\n\nResults\n\nExperiments on CSCBD show consistent critical load ranging from 4212 N to 4502 N, with no significant differences due to the two different speeds. With the dimensions in Figure 1, using equation (1), KI turns out to be between 0.91 to 0.98 MPa×√m. Figure 5 shows the DIC of two samples at the initiation of unstable crack.\n\nIt is well-known that finer mesh tends to increase the predicted stresses at the singularity sites of crack tips, even well before the critical load. Therefore, predicting the critical load by studying the magnitude of stresses at crack tip had not been a viable option. The conventional practice in predicting unstable sharp crack growth is to obtain the SIF using LEFM at the crack front and compare it with the critical stress intensity factor (i.e. fracture toughness) of the material. However, in this FEM study simulating both a blunt crack and a sharp crack, the SIFs predicted by the model differ vastly from one contour to the next. These values are presented in Figure 6 for different meshes (i.e. Tri for triangles, Qua for quadrilaterals, Qua_Fine for fine quadrilaterals) and crack widths (CW). Results for blunt crack models of triangle and quadrilateral meshes almost overlapped in Figure 6.\n\nSIFs (in Pa × √m) of 5 contours around crack tip for different meshes.\n\nNevertheless, it was found that triangle mesh at the 1.0 mm-wide rounded crack was less sensitive towards stress concentration at crack tip. The onset of unstable crack in the experiment coincided with the onset of yielding at crack tip in the mesh, i.e. when the Mises stress based factor of safety is less than unity, as shown in Figure 7. The factor of safety in this case is the ratio of yield strength to Mises stress. And PMMA, being, a brittle material, fractures close to its yield strength.\n\nFactor of safety in the red regions is less than unity (a) Triangle Mesh; (b) Quadrilateral Mesh; (c) Quadrilateral Fine Mesh.\n\nFigure 8 shows the stress fields at crack tips for both blunt and sharp cracks in different meshes. They were mostly comparable for the blunt cracks, but only the quadrilateral fine mesh captured the stress singularity well at the sharp crack tip.\n\nMises stress (in Pa) around sharp and blunt crack tip in (a) triangle meshes (b) quadrilateral meshes (c) quadrilateral fine meshes.\n\nThe Mises strain field recorded in DIC at impending crack growth is now compared with that predicted in finite element models, namely the triangle and quadrilateral meshes, both with 1.0 mm crack width (See Figure 9). Comparison of the Mises strain values as recorded in the DIC and that predicted by the models were made along two different paths. Path A crosses the stress concentration at crack tip along the horizontal axis, whereas path B is parallel to path A and crosses the specimen centroid.\n\nComparison of the strain values as recorded in the DIC on two specimens (Ex1, Ex2) and those predicted by models using triangle mesh (FE-Tri) and quadrilateral mesh (FE-Rec) along paths A and B, as depicted in the insert.\n\n\nDiscussion\n\nThe critical SIFs obtained from the experiment, in the range of 0.91 to 0.98 MPa×√m, are consistent with 0.87-1.20 MPa×√m reported by Choi et al.,17 1.02 MPa×√m reported by Zhou et al.,18 and 1.17MPa×√m (37.1MPa×√mm) by Lerch et al.19 This implies the applicability of LEFM for the blunt “notch root-radius”20 of 0.5 mm of PMMA in this study and validates the experimental procedures in the present study.\n\nThe SIFs obtained from the finite element models were significantly lower from the actual values ranging from 0.87 to 1.17 Pa×√m reported elsewhere.17-19 The sharp crack models showed poorer predictions—their SIFs in the first contours being closest to the actual values. This correlates with the sharp drop of peak stresses away from the singularity of the sharp crack tips, in contrast with the more gradual drop in stress values at the blunt crack tip without singularity20 (see Figure 8). Although all models captured the rise of stress or strain amplitude at crack tips (see Figures 4, 7 and 8), the different attempts made using different element types and sizes did not improve the SIF predictions. This finding implies that the practice of predicting unstable crack growth using SIFs computed by commercial FEM, such as the pre-meshed crack approach of Ansys fracture tool in this case, begs a closer examination, and experimental verifications.21 There is evidence that the interaction integral approach used in the pre-meshed crack approach to determine SIFs may not be as accurate as that using the J-integral approach or the modified Virtual Crack Closure Technique.22\n\nFigure 7 shows that both the coarse and fine meshes of quadrilateral elements are able to capture noticeable yielding zones at crack tip equally well, whereas the triangle mesh displays yielding only at a single node. The insensitivity of triangle elements towards stress concentration at the blunt crack tip actually helps failure prediction in the present scenario. Yielding at a single node provides a clear-cut indicator for FEM analysts to determine the onset of failure. On the contrary, the larger yielding zones in quadrilateral element models obscure the precise moment of impending fracture, since the question of how large a yielding zone needs to be to signify failure cannot be answered easily. Further case studies to take advantage of the triangle mesh insensitivity towards stress concentration may lead to a simple yet experimentally verifiable practice in predicting crack tip failures on brittle materials such as PMMA.\n\nIt can be seen from Figure 9 that the equivalent strains captured by the DIC matched reasonably well (i.e. to the same order of magnitude) with those predicted by the finite element models. DIC managed to capture the stress concentration around the crack tip even better than the models. However, the small strains at far field regions seemed to chatter. The general agreement between DIC and the model predictions verifies the models.\n\n\nConclusions\n\nThe following are the conclusions:\n\n1) The critical SIF computed using LEFM, based on the critical loads of failed CSCBD specimens with blunt cracks, agreed well with the published critical SIF values in the literature.\n\n2) Despite the limitation of traditional stress-based approach in predicting the onset of failure induced by stress concentration or singularity at blunt and sharp crack tip, respectively, the triangle mesh in this study predicted a single node yielding precisely at the onset of unstable crack growth in the experiment. Other crack scenarios may be investigated to determine if triangle mesh insensitivity may lead to the deployment of a simple and practical stress-based approach to predict the onset of crack growth.\n\n3) Both the coarse and fine quadrilateral meshes captured the stress concentration at crack tip well but failed to produce accurate or consistent SIFs at the crack tip using the pre-meshed crack approach of Ansys fracture tool. The reliability of using SIFs computed by the FEM as crack growth predictors deserves scrutiny.\n\n4) SIFs computed using ANSYS pre-meshed crack method have not shown experimentally verifiable superiority over traditional stress-based modelling approaches in predicting Mode I brittle failure at the blunt crack tip.\n\n\nData availability\n\nOpen Science Framework: Modelling Opening Mode Fracture at Crack Tip with Verifications Using Digital Image Correlation, https://doi.org/10.17605/OSF.IO/D9HQT.\n\nThis project contains the following underlying data:\n\n\n\n- Figure 9-PhiM-Summary.xlsx\n\n- Ansys_Mechanical_New.zip\n\n- GOM.zip\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 grateful for the equipment and related technical assistance provided by Multimedia University.\n\n\nReferences\n\nWang W: On the finite width correction factor in prediction models for fatigue crack growth in built-up bonded structures. Engineering Fracture Mechanics. 2020; 235:107156. Publisher Full Text\n\nTeh S, Akbar A, Andriyana A, et al.: Numerical fracture mechanics as a practical failure investigatory tool: The outlook of cracked round bars. Engineering Failure Analysis. 2021; 128:105630. Publisher Full Text\n\nGroth C, Porziani S, Chiappa A, et al.: High fidelity numerical fracture mechanics assisted by RBF mesh morphing. Procedia Structural Integrity. 2020; 25:136–148. Publisher Full Text\n\nBlaber J, Adair B, Antoniou A: Ncorr: Open-Source 2D Digital Image Correlation Matlab Software. Exp. Mech. 2015; 55(6): 1105–1122. Publisher Full Text\n\nNonis C, Niezrecki C, Yu T-Y, et al.: Structural health monitoring of bridges using digital image correlation. Proc SPIE Int Soc Optical Eng. United States;2013. Publisher Full Text\n\nAbshirini M, Soltani N, Marashizadeh P: On the Mode I Fracture Analysis of Cracked Brazilian Disc Using a Digital Image Correlation Method. Opt. Lasers Eng. 2016; 78: 99–105. Publisher Full Text\n\nHuang BT, Li QH, Xu SL, et al.: Development of Reinforced Ultra-high Toughness Cementitious Composite Permanent Formwork: Experimental Study and Digital Image Correlation Analysis. Compos. Struct. 2017; 180: 892–903. Publisher Full Text\n\nSause MGR: Digital image correlation. Springer Ser. Mater. Sci. 2016; 242(12): 57–129.\n\nVormwald M, Hos Y, Freire JLF, et al.: Crack Tip Displacement Fields Measured by Digital Image Correlation for Evaluating Variable Mode-mixity During Fatigue Crack Growth. Int. J. Fatigue. 2018; 115: 53–66. Publisher Full Text\n\nAyatollahi MR, Aliha MRM: On the use of Brazilian disc specimen for calculating mixed mode I-II fracture toughness of rock materials. Eng. Fract. Mech. 2008; 75(16): 4631–4641. Publisher Full Text\n\nASTM D 3967-95a (Reapproved 2001): Standard Test Method for Splitting Tensile Strength of Intact Rock Core Specimen. ASTM Int;2004.\n\nASTM E399-90: Standard Test Method for Plane-Strain Fracture Toughness of Metallic Materials.ASTM;1990.\n\nCrammond G, Boyd SW, Dulieu-Barton JM: Speckle pattern quality assessment for digital image correlation. Opt. Lasers Eng. 2013; 51(12): 1368–1378. Publisher Full Text\n\nByrne E: CSI Application Note AN-525: Speckle Pattern Fundamentals. South Carolina:Correl. Solut;2010.\n\nMaterial Property Database. MIT. [Accessed 30 Aug 2018].Reference Source\n\nIsmail A, Jamian S, Kamarudin K, et al.: An Overview of Fracture Mechanics with ANSYS. Int J Integrated Engineering: Special issue. 2018; 10(5): 59–67. Publisher Full Text\n\nChoi S, Salem JA: Fracture toughness of PMMA as measured with indentation cracks. J. Mater. Res. 1993; 8(12): 3210–3217.\n\nZhou J, Wang Y, Xia Y: Mode-I fracture toughness of PMMA at high loading rates. J. Mater. Sci. 2006; 41: 8363–8366.\n\nLerch B, Thesken J, Bunnell C: Polymethylmethacrylate (PMMA) Material Test Results for the Capillary Flow Experiments (CFE). NASA STI Report Series. 2007.\n\nBerto F, Elices M, Lazzarin P, et al.: Fracture behaviour of notched round bars made of PMMA subjected to torsion at room temperature. Engineering Fracture Mechanics. 2012; 90: 143–160. Publisher Full Text\n\nMazzini D: Materiale per la didattica, Fracture Mechanics Classes, Class 7- Different FE calculation techniques 1_2.14 7 2015. [Accessed 13 8 2012].Reference Source\n\nSouto C, Tavares S, Correia J, et al.: Numerical determination of stress intensity factors: J-integral and modified virtual crack closure technique. Procedia Structural Integrity. 2020; 28: 146–154. Publisher Full Text" }	[ { "id": "96352", "date": "22 Oct 2021", "name": "Judha Purbolaksono", "expertise": [ "Reviewer Expertise Fracture mechanics", "fatigue and failure 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\nThe concept of linear elastic fracture mechanics (LEFM) has been widely used in practical engineering analyses to study the mechanical behavior of cracked structures/components. Numerical fracture mechanics has appeared to play an essential role in the development of LEFM and has become a useful tool to researchers. Current computational advancement with interactive features permits researchers to study the fracture phenomena in more details.\nThe Authors attempted to evaluate the SIFs of a centrally straight cracked Brazilian disc made of Polymethyl methacrylate (PMMA) under purely Mode I loading using a commercial finite element based software of ANSYS Workbench. Two-dimensional finite element models of using the quadrilateral and triangle elements were developed. Experimental works on the centrally straight cracked Brazilian disc (PMMA) were carried out. The strain fields were then measured from deformed speckle patterns using the digital image correlation (DIC) software. The pre-meshed crack method in ANSYS Workbench was used to generate meshing around the crack boundaries.\nMode I (opening mode) SIF is the predominant stress field parameter at the crack tip. Even though the numerical modelling for 2D cracked problems has been established, any updates on the numerical fracture mechanics are always sought.\nThe comparisons between the numerical modelling and experimental results are desired in the engineering analysis.\nTo benefit the readers, the following comments should be addressed for improvement:\nIn general, the Authors should take the opportunity to refine the explanations/texts on the findings for clarity, especially with regard to the blunt and sharp crack tips.\n\nIn this present manuscript, the SIFs for the sharp cracks by the FEM were observed to be inconsistent. The inconsistencies may be due to the sensitivity of the use of the pre-meshed method. Therefore, the Authors are encouraged to use the ANSYS APDL to model cracks (sharp crack tip) in which the crack boundaries may be defined coincidently. Next, the findings obtained by the ANSYS Workbench and APDL may be compared.\n\nThe use of DIC should be explained in more details and the discussion on the results should be further elaborated.\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?\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": [] }, { "id": "96351", "date": "01 Nov 2021", "name": "Ifeanyi Emmanuel Kalu", "expertise": [ "Reviewer Expertise Fitness-for-service assessment", "Failure Analysis", "Mechanical Behaviour of Materials." ], "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\nFor decades, Linear elastic fracture mechanics (LEFM) has been used to study the behaviour of cracks in mechanical components and structures. Commercial finite elements (such as ANSYS and ABAQUS) have been used to either substantiate experimental results or provide further insights in this field.\nIn this study, the authors carried out experimental test and numerical modelling on centrally straight cracked Brazilian discs (CSCBD) prepared from polymethyl methacrylate (PMMA) and compared their strain fields and stress intensity factors (SIF) generated at crack tip under purely Mode I loading. The strain fields from the experiment were measured using a digital image correlation (DIC) software. While pre-meshed crack method in ANSYS was used to obtain similar information. Effects of varied element types, mesh sizes and crack widths on the simulated SIFs were examined.\nThe following must be addressed to make this study and its presentation comprehensive and reliable to be indexed:\n1. In the introduction, the authors made some generalized/assumed statements without providing ample citations. These statements are as follows:\n(a) \"...researchers are working on correction factors to account for the deviations that result from linear elastic fracture mechanics (LEFM) predictions\"\n\n(b) \"Although literature is abundant on the use of finite element method to predict SIF\".\n\n(c) \"...some modelling techniques require manual node adjustment (i.e. collapsed elements or quarter-node approach) and/or complex mesh morphing strategy\".\nI suggest for each statement at least 3 citations should be provided to substantiate these statements.\n2. The experimental work was reasonably explained in detailed unlike the ANSYS Simulation. The authors need to provide more details on how the simulation was conducted such that it can be reproducible. For instance, boundary conditions used were not mentioned. They only reported, \"a force was applied through...\". What was the quantity of the force used and how was it applied? Was there any coefficient of friction applied at the contact points? What kind of supports were used? Was it a fixed or moveable support in certain direction? What mesh controls did they use for their meshing, including the finer mesh that was reported.\n\nAlso, on the ANSYS simulation, did the authors carry out a mesh convergence study on the meshes used. Using different types of element types without checking the quality of the meshes can always give a wrong result. Considering the disparity of the SIFs result from this study with that of the experiment and also those reported in previous articles, it is really a notable concern that must be properly addressed. I quote the authors in their words in the discussion: \"The SIFs obtained from the finite element models were significantly lower from the actual values ranging from 0.87 to 1.17 Pa×√mPa×√m reported elsewhere\".\nI strongly would propose that the authors properly revisit how their ANSYS simulation was done to ensure they do an accurate job that can be reliable and reproducible. The work they did is not that novel, hence, it is expected they should have similar results with what have been reported, if it was accurately done.\nEven though the authors claim that using plane stress model do not show significant difference in FE results, I differ with them. ANSYS workbench and mechanical were primarily designed for 3D simulations. Doing a 2D simulation may not give you the best of results. For instance, in this study, the experiment was done using a CSCBD with 15 mm thickness. This was totally ignored in the 2D FEM, which may have an effect in the results generated. Also in 2D, you cannot apply boundary conditions to mimic actual experiment scenario. Hence, you will always get a difference between experiment and computational results.\n3. The authors kept on using these wrong words: \"Mises stress and Mises strain\". There is nothing like Mises stress or strain. It is von Mises stress or von Mises Strain. In ANSYS, it is the Equivalent von Mises stress or Equivalent von Mises strain.\n4. The title of figure 6 is contrasting to the result displayed. The unit of the reported SIFs is different from what is on the title.\n\nOverall, I will strongly recommend that the authors go back and redo the ANSYS simulation in such a way that it will accurately simulate the experimental set-up, be reproducible and the results obtained can be reliable.\nTherefore, the conclusions they presented in this paper cannot be relied upon since there are unresolved concerns on how the simulation was performed.\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?\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": [] } ]	1	https://f1000research.com/articles/10-1017
https://f1000research.com/articles/10-1011/v1	05 Oct 21	{ "type": "Study Protocol", "title": "Efficacy of mesenchymal stromal cells in preclinical models of necrotizing enterocolitis: a systematic review protocol", "authors": [ "Camille Maltais-Bilodeau", "Ewa Henckel", "Kelly D. Cobey", "Nadera Ahmadzai", "Becky Skidmore", "Emanuela Ferretti", "Bernard Thébaud", "Ewa Henckel", "Kelly D. Cobey", "Nadera Ahmadzai", "Becky Skidmore", "Emanuela Ferretti", "Bernard Thébaud" ], "abstract": "Introduction: Necrotizing enterocolitis is an acute inflammatory disease of the intestine that can lead to necrosis and bowel perforation. It is a severe complication of preterm birth. It’s mortality rate is up to 50% and survival after necrotizing enterocolitis leads to long-term complications. The current treatment is supportive and includes bowel rest and decompression and antibiotics. Thus, new treatments are necessary to reduce mortality and morbidity. Mesenchymal stromal cells are known to have anti-inflammatory properties and might be a promising option for treatment. Here we present a protocol for a systematic review with the aim to explore the efficacy of cell therapies with mesenchymal stromal cells in animal models of necrotizing enterocolitis. The primary outcome is histological signs of necrotizing enterocolitis. Additional outcomes include survival, bowel perforation, gut permeability, gut motility, levels of inflammatory markers, cytokine levels and adverse events. Methods: We will conduct a systematic search of MEDLINE, Embase, and Web of Science databases. The retrieved records will be screened individually by two investigators. We will include all preclinical in vivo animal models of experimentally induced necrotizing enterocolitis that evaluate the efficacy of mesenchymal stromal cells or other cell therapy treatments. Outcome data will be extracted from each article and risk of bias assessment performed. Funnel plots and SYRCLE’s risk of bias tool for animal studies will be used. Data will be reported as ratios, divided in predefined subgroups where relevant. Conclusions: This systematic review aims to examine the efficacy of mesenchymal stromal cells in preclinical models of necrotizing enterocolitis and whether there is sufficient evidence to support a clinical trial of efficacy and safety of the treatment with mesenchymal stromal cells in infants with necrotizing enterocolitis.", "keywords": [ "necrotizing enterocolitis", "neonatal enterocolitis", "mesenchymal stromal cells", "stem cells", "preclinical", "animal models", "systematic review protocol" ], "content": "List of abbreviations\n\nMSC: mesenchymal stromal cell\n\nNEC: necrotizing enterocolitis\n\n\nIntroduction\n\nNecrotizing enterocolitis (NEC) is an acute inflammatory disease of the intestine.1,2 It was first described by Mizrahi et al. in 1965.3 This disease is characterized by inflammation and injury to the wall barrier of the intestine that can cause necrosis and that can lead to bowel perforation.2,4,5 It is a major complication of preterm birth.6,7 In Canada, the preterm birth rate is 7.7%8 and, on the basis of large databases, the mean prevalence of NEC in Canada and the United States is about 7% among infants with birth weight between 500 g and 1500 g1,9,10 with a mortality rate as high as 50% in neonates with a birth weight less than 1000 g.11,12 The pathogenesis of NEC is multifactorial.13 The most common known risk factors of NEC include low gestational age, low birth weight, exposure to infant formula-feeding and microbial dysbiosis.2,4,13,14 The classic NEC pathophysiological process of intestinal injury appears to be related to a basic immaturity of host defences, culminating in an excessive inflammatory response, with serum cytokines and chemokines, especially IL-8 significantly elevated15–17 and imbalance of the healthy microbiome.\n\nSurvivors of a NEC episode are at increased risk of long-term adverse outcomes including gastrointestinal failure and neurodevelopmental complications.4,12 Because the disease occurs during an important time of brain development, the long-term NEC-associated neurodevelopment outcomes include cerebral palsy and visual, cognitive and psychomotor impairment.4,18–23 NEC also significantly increases hospital length of stay and health care costs. Infants with NEC needing medical treatment are hospitalized on average 22 days longer in the neonatal intensive care unit than neonates without NEC, and those who require surgical treatment are hospitalized 60 days longer than infants without NEC.24 In the USA, the estimated average cost for medical and surgical treatment of NEC is $500 000 per patient.24,25\n\nCurrent treatment strategies depend on the severity of the disease and remain supportive only. They include bowel rest and decompression, fluid support, vasopressors and systemic broad-spectrum-antibiotics.4,13,26 Surgical treatment of NEC is recommended if bowel perforation occurs or in some cases of bowel necrosis.13 In order to reduce the morbidity and mortality associated with this inflammatory disease, evaluation of new therapies is required. Several treatments have been studied in preclinical trials, including the utilization of mesenchymal stromal cells (MSCs).\n\nMSCs were described in 1976 as fibroblast precursor cells with a role in the regulation of murine hematopoietic stem cells in bone marrow.27 Following this, multiple preclinical studies have shown a repair ability of MSCs in multiple injured organs and tissues of neonatal models such as in the brain,28–32 heart33 lungs34,35 gut36,37 and eyes.38 MSCs are known to have anti-inflammatory properties39 and considering that inflammation plays a key role in the pathogenesis of NEC, they may represent a promising therapeutic option for this disease. In 2020, a systematic review and meta-analysis of preclinical studies using animal models of NEC with stem cells products was published.40 This study suggests a benefit of stem cells in rodent models. However, the research in stem cells is a rapid-growing field in medicine and we believe that an update review with broad inclusion criteria is needed.\n\nWe aim to conduct a systematic review of the benefits and harms of MSC therapy in preclinical models of NEC. We seek to look for any clinical effect of this therapy in animal models. We will specifically address the question: In controlled preclinical studies of necrotizing enterocolitis, can MSCs reduce the histological signs of NEC in NEC-induced animal models? Our population of interest is all animal studies of NEC and we are interested in any such studies that use MSCs or any other cell therapy as an intervention. We are interested in all comparisons in the literature. Our primary outcome is histology showing NEC. The following additional outcomes will be measured: survival, bowel perforation, gut permeability, gut motility, levels of inflammatory markers, cytokine levels and adverse events.\n\n\nProtocol\n\nThis study protocol was developed through discussion between our research team which includes clinical and preclinical research experts (CMB, EH, BT, EF), an information specialist (BS), and research methodologists with expertise in knowledge synthesis (NA, KDC). The protocol was prepared using the PRISMA-P reporting guidelines41 and the protocol format for the preparation, registration and publication of systematic reviews of animal intervention studies published by de Vries et al.42 This protocol was registered using the Open Science Framework (osf.io/5rc6t). A completed PRISMA-P checklist for this protocol is provided. Post-hoc modifications to the current protocol incurred during the review process will be documented in the publication of the final report to ensure transparency.\n\nWe will include all study designs of research describing preclinical NEC models that evaluate the efficacy of MSCs or other cell therapy treatments that are published in English. Potentially relevant non-English materials will be provided in the appendix section but will not be included in the analysis. We will not limit our eligibility criteria by year of publication or publication status. Details on the study PICOS are provided below.\n\nPopulation\n\nThe population will include all preclinical in vivo animal models of experimentally induced NEC. To enhance the generalizability of our findings, NEC will be defined as any histological intestinal tissue injury. Various mechanisms of NEC have been reported in animal models in the literature.43,44 These include gavage formula or hyperosmolar formula gavage feeds, exposure to hypoxia and/or hypothermia and administration of bacteria.\n\nIntervention\n\nThe intervention of interest is all MSCs cell types, independent of the source, in animal models. MSCs will be defined by using the International Society for Cellular Therapy consensus statement. The minimal criteria to meet to define human MSC are: (1) adhesion to plastic; (2) expression of the cell surface markers CD105, CD73 and CD90 and lack of CD45, CD34, CD14 or CD11b, CD79ɑ or CD19 and HLA-DR surface molecules expression and (3) differentiation to osteoblasts, adipocytes and chondroblasts in vitro.45\n\nAll types of MSCs will be considered in the review to enhance the generalizability of our study findings. Any therapeutic MSCs regimen (prophylactic – before or during induction of NEC, or rescue after induction of NEC) will be considered.\n\nComparison\n\nIn our population study of experimental animal models of NEC, we will consider all types of reported comparator. We will also consider groups with no comparator.\n\nOutcome\n\nThe primary outcome is histological signs of NEC measured at any time after the administration of MSCs. The secondary outcomes measured are: survival, bowel perforation, gut permeability (e.g. disruption of thigh junction proteins, liver fatty acid-binding protein level and intestinal fatty acid binding protein level), gut motility, levels of inflammatory markers (e.g. white blood cells count, C-reactive protein level and fecal calprotectine) cytokine levels (e.g., interleukine-8 and toll-like receptor 4) and any describe adverse events.\n\nStudy design\n\nWe will include any study design in the search including both comparative and non-comparative studies as well as empirical research.\n\nWe have identified one parent of a preterm infant who suffered from NEC and its complications who engaged in the design of the project and provided feedback after reading the protocol manuscript. The engagement of this parent will be an important addition to our team for subsequent research leading to clinical trial preparation and conduct.\n\nSearch strategy\n\nThe search strategies will be developed and tested through an iterative process by an experienced medical information specialist in collaboration with the study team members. The MEDLINE strategy will be peer-reviewed by another senior information specialist prior to execution using the Peer Review of Electronic Search Strategies (PRESS) guidelines.46 Proposed databases include MEDLINE, Embase, and Web of Science. Searches will utilize a combination of controlled vocabulary (e.g., “Stem cells”, “Mesenchymal Stem Cells”, “Stromal Cells” and “Enterocolitis, Necrotizing”) and keywords (e.g., “MSCs”, “stromal cell”, “neonatal enterocolitis”). Vocabulary and syntax will be adjusted across the databases. To enhance search efficacy, we will use animal filters validated for Pubmed/MEDLINE and Embase,42,47,48 amended as appropriate to accommodate new developments in vocabulary. There will be no language or date restrictions on any of the searches. We will perform a grey literature search of conference websites and animal research organizations’ websites to identify literature not included in the aforementioned databases, and also perform general searches using Google Scholar.\n\nData management\n\nDistillerSR (Evidence Partners Inc, Ottawa, Canada) software will be used for records and data management throughout the review. The resulting search citations will be deduplicated and then imported into DistillerSR. Study screening and data extraction forms will be created on the platform to facilitate review of citations obtained from the search.\n\nSelection process\n\nTwo reviewers, independently and in duplicate, will carry out citation screening in two stages, reviewing records against the a priori eligibility criteria using an online systematic review software program (Distiller Systematic Review (DSR) Software; Evidence Partners Inc, Ottawa, Canada). At Stage 1, the titles and abstracts of all records obtained from the search will be screened in duplicate independently by CMB and EH while Stage 2 screening will encompass full-text review of the articles deemed potentially relevant at Stage 1. If there are disagreements between the reviewers, it will be resolved by a consensus or by a third member of the study team (EF or BT). Both stages of screening will begin with a calibration exercise to ensure consistent application of eligibility criteria between reviewers. This will include pilot screening of titles and 25 citations at Stage 1 and 25 full-text articles at Stage 2. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines,41 the exclusion reasons of potentially eligible studies will be recorded.\n\nData collection process\n\nThe two independent investigators (CMB and EH) conducting screening and extraction will use standardized forms (See drafts, Table 1). These forms will be piloted on a set of 10 documents and then discussed. If necessary, changes to the forms will be made and they will be re-piloted on 10 further documents. This process will be repeated until 80% agreement or higher is reached between the reviewers. We will contact the first author of the included studies once, if necessary, for any missing data.\n\nData items\n\nOnce all studies are identified, data extraction will be performed using a standardized data extraction form in Microsoft Excel (Microsoft Corporation, Seattle, Washington, USA). We will collect data related to key study items that include but not limited to variables in Table 1 below. If means and measures of dispersion are reported via figures in the primary studies, they will be approximated using online tools such as Webplotdigitizer (A. Rohatgi, 2020, Pacifica, California, USA).\n\nThe primary outcome is histological signs of NEC measured at any time after the administration of MSCs. The histological signs can be tissue inflammation, bacterial invasion, tissue necrosis and signs of micro-perforation. If applicable, we will use the standard histologic scoring previously described in the literature to assess the severity of histological changes in NEC.43,49 Histopathology of the lesions will be categorized in four classes: grade 1 focal, mild injury confined to villous types, grade II, partial loss of vili, grade III, necrosis extending to submucosa and grade IV, transmural necrosis.\n\nThe secondary outcomes measured will be: survival, bowel perforation, gut permeability, gut motility, levels of inflammatory markers, cytokine levels and any reported adverse events. Survival will be measured in number of days between the diagnosis of NEC and unintentional death. Bowel perforation will be defined as any free air in the abdominal cavity on imaging or visualization of a hole in the wall of the intestinal barrier by direct visualization or by histology. Any possible parameters to assess gut permeability (including but not restricted to disruption of thigh junction proteins, liver fatty acid-binding protein level and intestinal fatty acid binding protein level) and motility will be considered as a valid measure for these outcomes. Any kind of inflammatory markers (including but not restricted to white blood cells count (cells × 109/L), C-reactive protein (mg/L) and fecal calprotectine (mcg/g)) and cytokine levels (including but not restricted to interleukine-8 (pg/mL) and present of toll-like receptor 4) will be recorded. All reported adverse events will be considered.\n\nTwo independent investigators (CMB and EH) will evaluate the risk of bias for each study included in the review. We will use the SYRCLE’s risk of bias tool for animal studies.50 This tool was created to establish consistency and avoid discrepancies in the assessment of risk of bias in systematic reviews of animal intervention studies. Each item will be assigned a value of low, high or unclear. Elements of the SYRCLE’s tool include assessment of method used for the sequence generation, description of baseline animal characteristics, allocation concealment, random animal housing, blinding of outcome assessment, random outcome assessment, completeness of outcome data and statement of selective outcome reporting.\n\nDescriptive statistics and tables of evidence summary will be reported. Study characteristics will be summarized using frequencies and percentages. As a rule, heterogeneity that may be explained by clinical or methodological differences between studies will preclude any planned meta-analyses. If so, study results will be synthesized narratively. Outcomes with dichotomous endpoints (e.g., survival and presence of intestinal perforation) from each included study will be pooled and described using risk ratio and/or odds ratios and 95% confidence intervals that incorporate a random effect modelling approach. We will use forest plots to visualize the data. Statistical heterogeneity between studies will be quantified with I-squared statistics. Sparse data will not be meta-analyzed but described narratively. Outcomes with continuous endpoints (e.g., gut permeability, gut motility, levels of inflammatory markers and cytokine levels) will be pooled using mean difference or standardized mean difference. Meta-regression with multiple study level covariates will be attempted when there are at least 10 studies in the body of evidence. Otherwise, clinical and methodological diversity in studies will be explored in pre-specified subgroup and sensitivity analyses. We will analyze subgroups for the following: animal models, sex, experimental model of NEC (e.g., hypercaloric stress, exposure to hypoxia or exposure to hypothermia), administration route of MSC (e.g., intravenous or intraperitoneal), dose of MSC, timing of MSC administration after induction of NEC (less or equal to one hour, greater than one hour to less or equal to four hours, more than four hours or multiple doses), other treatment used and control group. To assess the potential meta-bias and publication bias, we will use the funnel plot as a graphical tool. The quality of reporting of individual preclinical studies will be assessed in accordance with the elements of the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines.51\n\nThis study is a systematic review of animal models and there will be no direct interaction with animals or patients. The systematic review is planned to be conducted between March and September 2021. The results of this study will be presented to the scientific community at pertinent national and international meetings. We will also publish the results of the systematic review in peer-reviewed scientific journals. The general public will be informed through various local and national associations and media.\n\nOur systematic review has been started in March 2021 with the literature search and the screening of the searched abstracts. We are currently working on the quality assessment of the included studies. At the time of writing this protocol, no data collection has been started.\n\n\nDiscussion and conclusions\n\nNEC is an important inflammatory disease that affects infants and the current treatment is supportive. In order to reduce the mortality and the morbidity associated with this disease, there is a need for new treatment targeting the inflammation process and MSCs may be beneficial. To our knowledge, no systematic review of the use of MSCs in preclinical models for NEC has been reported. The data from this review will provide information on whether there is sufficient rigorous evidence to support a clinical trial testing the feasibility and safety of MSC therapy in neonates with NEC. If not, this study will illustrate any gaps in the field which can guide the design of future research studies to address those issues.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nReporting guidelines\n\nOpen Science Framework: PRISMA-P checklist for ‘Efficacy of mesenchymal stromal cells in preclinical models of necrotizing enterocolitis: a systematic review protocol’, https://doi.org/10.17605/OSF.IO/YJNXP.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nAn earlier version of this article can be found on Research Square (doi: 10.21203/rs.3.rs-239448/v1).\n\n\nReferences\n\nNeu J, Walker WA: Necrotizing enterocolitis. N. Engl. J. Med. 2011; 364(3): 255–64. [published Online First: 2011/01/21].PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellodas Sanchez J, Kadrofske M: Necrotizing enterocolitis. Neurogastroenterol. Motil. 2019; 31(3): e13569. 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[published Online First: 2011/04/12].PubMed Abstract \| Publisher Full Text\n\nVillamor-Martinez E, Hundscheid T, Kramer BW, et al.: Stem Cells as Therapy for Necrotizing Enterocolitis: A Systematic Review and Meta-Analysis of Preclinical Studies. Front. Pediatr. 2020; 8(749). Publisher Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015; 349: g7647. [published Online First: 2015/01/04].Publisher Full Text\n\nde Vries RB , Hooijmans CR, Tillema A, et al.: A search filter for increasing the retrieval of animal studies in Embase. Lab. Anim. 2011; 45(4): 268–70. [published Online First: 2011/09/06].PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMendez YS, Khan FA, Perrier GV, et al.: Animal models of necrotizing enterocolitis. World J. Ped. Surg. 2020; 3(1): e000109. Publisher Full Text\n\nKovler ML, Sodhi CP, Hackam DJ: Precision-based modeling approaches for necrotizing enterocolitis. Dis. Model. Mech. 2020; 13(6). [published Online First: 2020/08/09].Publisher Full Text \| PubMed Abstract \| Free Full Text\n\nDominici M, Le Blanc K, Mueller I, et al.: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8(4): 315–7. [published Online First: 2006/08/23].PubMed Abstract \| Publisher Full Text\n\nMcGowan J, Sampson M, Salzwedel DM, et al.: PRESS Peer Review of Electronic Search Strategies: 2015 Guideline Statement. J. Clin. Epidemiol. 2016; 75: 40–6. [published Online First: 2016/03/24].PubMed Abstract \| Publisher Full Text\n\nHooijmans CR, Tillema A, Leenaars M, et al.: Enhancing search efficiency by means of a search filter for finding all studies on animal experimentation in PubMed. Lab. Anim. 2010; 44(3): 170–5. [published Online First: 2010/06/17].PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Vries RB , Hooijmans CR, Tillema A, et al.: Updated version of the Embase search filter for animal studies. Lab. Anim. 2014; 48(1): 88. [published Online First: 2013/07/10].PubMed Abstract \| Publisher Full Text\n\nCaplan MS, Hedlund E, Adler L, et al.: Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis. Pediatr. Pathol. 1994; 14(6): 1017–28. [published Online First: 1994/11/01].PubMed Abstract \| Publisher Full Text\n\nHooijmans CR, Rovers MM, de Vries RBM , et al.: SYRCLE’s risk of bias tool for animal studies. BMC Med. Res. Methodol. 2014; 14(1): 43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPercie du Sert N, Hurst V, Ahluwalia A, et al.: The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020; 18(7): e3000410. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "101320", "date": "03 Dec 2021", "name": "William W. Hay Jr", "expertise": [ "Reviewer Expertise Neonatal nutrition and metabolism" ], "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 Protocol manuscript, the authors describe their proposed systematic review that “aims to examine the efficacy of mesenchymal stromal cells in preclinical [animal] models of necrotizing enterocolitis and whether there is sufficient evidence to support a clinical trial of efficacy and safety of the treatment with mesenchymal stromal cells in infants with necrotizing enterocolitis.” Overall, the rationale and approaches proposed for the systematic review of MSC use in animal models of so called NEC should provide a comprehensive review of the literature that might provide evidence for justifying potential clinical trials of MSCs for reducing incidence and severity and adverse outcomes of human preterm infant NEC.\nThe rationale for, and objectives of, the Systematic Review are clearly stated.\n\nSufficient details of the methods and analysis are provided that would allow replication by others.\n\nThe statistical analysis and its interpretation are appropriate.\n\nThe conclusions drawn are supported by the results presented in the review, but there are a number of issues that should be further considered and revised, as noted below:\nThe authors justify their systematic review and potential for clinical trials of anti-inflammatory mesenchymal stromal cells to prevent or treat NEC with an average reported prevalence of 7% in North America and a mortality rate of about 50% in more preterm infants with lower birth weight. These rates may be higher than in many centers today, as preventative measures of exclusive mother’s milk feeding, including donor milk and human milk-derived fortifiers, are more commonly used, as well as in some centers the addition of prebiotics and probiotics.\n\nThe authors note that a similar systematic review was published only last year, but argue that the field is rapidly expanding, justifying the need for a new systematic review. It would help to show the authors’ evidence for how expanded the literature is in the past year, if it is, from the one published last year, to better justify such a short time between these reviews.\n\nSome evidence from the similar systematic review published last year should be included to help justify the potential efficacy of MSCs for the purposes of the proposed review and study. What did this previous review find?\n\nThe authors note that “To enhance the generalizability of our findings, NEC will be defined as any histological intestinal tissue injury.” This should be better defined and justified, as some of the approaches to inducing so called NEC in animal models might cause gut injuries that are not specifically characteristic of human preterm infant NEC.\n\nThe authors also should discuss the strengths but particularly the limitations of the animal models used to produce so called NEC, as they note that “various mechanisms of NEC have been reported in animal models in the literature, including… gavage formula or hyperosmolar formula gavage feeds, exposure to hypoxia and/or hypothermia and administration of bacteria”. Many have criticized these experimental methods as not that relevant to human preterm infant NEC, which clearly is multifactorial in its origins and pathogenesis (which the current authors acknowledge). For example, very few cases of true NEC are caused by hyperosmolar formulas, hypoxia, hypothermia, or inoculation with pathogenic bacteria, even though associated anemia might produce gut enterocyte “hypoxia”, pathogenic bacteria are commonly associated with NEC, and there appears to be a higher incidence of NEC in infants who are fed exclusive or mixed diets of bovine-derived formulas primarily with intact bovine protein.\n\nThe authors also should consider that many so called cases of NEC have been variably diagnosed, sometimes erroneously, e.g., including early postnatal spontaneous intestinal perforation which is not NEC, and signs of gut inflammation which may or may not be related to true NEC. The animal models therefore may be of less certain and potentially limited value for determining whether mesenchymal stromal cells would be appropriate for clinical trial in human preterm infants to prevent or treat true NEC.\n\nThere should be some expected number of studies to be considered from the literature for the different times related to NEC noted--prophylactic (before or during induction of NEC), during NEC, or rescue after induction of NEC, and since a previous systematic review was conducted only a year ago, that review might provide some insight into whether there are enough studies in each of these periods.\n\nEvidence for whether there might be benefit to MSCs once NEC is established vs. prophylactic use should be considered.\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? Not applicable", "responses": [] }, { "id": "127568", "date": "05 Apr 2022", "name": "Pekka Maattanen", "expertise": [ "Reviewer Expertise intestinal inflammation", "ER stress", "microbiome" ], "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\nMaltais-Bilodeau et al propose to carry out a systematic review of the efficacy of mesenchymal stromal cells in preclinical models of necrotizing enterocolitis. While a similar review was recently published in 2020, they suggest that since then further research has been done and there is a need for a more comprehensive review of these promising potential therapies for NEC. The authors state in their objectives that they will focus on studies that utilize MSCs or any other cell therapy as a preclinical intervention in animal models. It is suggested that they change their title to \"cell therapies\" in preclinical models of NEC to broadly include any cell, or stick with MSCs as they define them.\n\nOverall, the study proposal is very well planned and written, and the methods and outcomes are clear. Several points to address are noted below.\n\nIt would be helpful to estimate how many new studies could be included under the MSC category in the broader review they are proposing in comparison to the prior 2020 review on stem cell therapies for NEC, or describe more clearly how their review will be different. (See Villamor-Martinez et al 2020)1.\n\nFindings of the previous review are not commented on other than to say the prior meta-analysis suggested a benefit of stem cells. Given that the authors will address one primary outcome (histological) and several secondary outcomes (survival, bowel perforation, gut permeability, etc) in the proposed review, it is suggested that the prior study results in relation to these outcomes be briefly mentioned, or describe why the proposed study may provide further useful perspective than was reviewed in that study.\n\nGiven that histological scoring is their primary outcome, it might be helpful to include a description of how they will reconcile potentially different scoring schemes used by different authors, or will they exclude authors that do not use the standard histological scoring scheme they indicate on pg 6?\n\nGiven the many possible ways to experimentally induce NEC as described, do the authors consider one or more of these better representations of NEC, or will all findings with all models be given equal weight in terms of potential clinical benefit?\n\nIn their outcomes, I understand they mean fecal calprotectin and interleukin-8. (pg 4 and Table 1 Preclinical endpoints)\n\nTable 1 preclinical endpoints - tight junction proteins, C-reactive protein. (spelling)\n\nAdverse events are mentioned as a secondary outcome. Are there any potential adverse events from MSCs that are likely to be missed or unreported in NEC preclinical models (for example cancer risks)? Might these risks need further consideration in their literature review? (see Lee and Hong 2017)2\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? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-1011
https://f1000research.com/articles/10-450/v1	07 Jun 21	{ "type": "Software Tool Article", "title": "Designing and developing an app to perform Hofstee cut-off calculations", "authors": [ "Ken Masters", "Nadia Al-Wardy", "Nadia Al-Wardy" ], "abstract": "Determining a Hofstee cut-off point in medical education student assessment is problematic: traditional methods can be time-consuming, inaccurate, and inflexible. To counter this, we developed a simple Android app that receives raw, unsorted student assessment data in .csv format, allows for multiple judges’ inputs, mean or median inputs, calculates the Hofstee cut-off mathematically, and outputs the results with other guiding information. The app contains a detailed description of its functionality.", "keywords": [ "Hofstee", "Angoff", "Assessment", "Standard setting", "Android", "MARS" ], "content": "Introduction\n\nIn medical education assessment, determining the student pass/fail mark is a contentious issue.1 A range of methods can be used to determine this point and are covered in several other papers.2–4 In summary, however, most methods fall into three categories: norm-referenced (determined by the performance of the student group), criterion-referenced (pre-determined as an absolute cut-off point) and compromise methods (a compromise between the previous two methods is found).4\n\nThe Hofstee Method4–6 is a compromise method that follows four steps, and uses four variables or parameters (explained in more detail below) to determine the cut-off point.\n\nStep 1: Evaluation by judges\n\nIn Step 1, judges who are qualified to assess the test make an independent judgement about the values of the following four parameters:\n\n• cmin: The minimum cut-off score (i.e. the score that the judge feels would be the lowest possible score that would be considered as a pass/fail score).\n\n• cmax: The maximum cut-off score (i.e. the score that the judge feels would be the highest possible score that would be considered as a pass/fail score).\n\n• fmin: The lowest percentage of students that the judge feels should fail this test.\n\n• fmax: The highest percentage of students that the judge feels should fail this test.\n\nThe four parameters are often indicated with different abbreviations; in this paper, we use cmin, cmax, fmin and fmax as is used elsewhere.4 We should further note that judges will generally use the Angoff or similar method to determine these.4\n\nStep 2: Determining the arithmetic means\n\nBased upon the independent judgements, the arithmetic mean of each parameter is calculated. (Some researchers, e.g. Norcini2, have suggested that medians may also be used).\n\nStep 3: Plot on a graph\n\nAfter the test has been administered to the students, a graph (Figure 1) is then drawn, plotting the cumulative percentage of students against the scores obtained, and the four parameters.\n\nHofstee chart showing cumulative scores, where cmin (minimum cut-off score) = 35, cmax (maximum cut-off score) = 45, fmin (the lowest percentage of students that the judge feels should fail) = 6, and fmax (the highest percentage of students that the judge feels should fail) = 18.\n\nStep 4: Determining cut-off\n\nThe pass/fail cut-off point is then determined by drawing line AB and finding the intersect with the cumulative line. In the Figure 1 example, the cut-off is determined to be slightly less than 38%.\n\nApart from the fact that any cut-off method can be debated, there are practical problems associated with this method, and these include:\n\n1. The time taken to accurately draw the chart, and all the associated lines.\n\n2. Reading the cut-off point from an imperfect drawing, rather than determining it mathematically.\n\n3. One might wish to allow for some flexibility, and test other values for the parameters. On a hand-drawn chart, this is time-consuming and untidy, to the point of being impossible.\n\nHofstee produced a mathematical solution,7 but it requires sorting and frequency pre-calculation and data inspection, and the mathematics involved is not rudimentary (requiring seven steps). Van Der Vleuten developed a useful one for SPSS,8 but it uses expensive licensed software.\n\nAn Excel template designed by one of the authors (KM) already exists, and plotting the chart on Excel is certainly an improvement over the hand-drawn chart. However, it still requires the data to be pre-sorted and also requires the generation of the cumulative data. In addition, although the chart is drawn more accurately than by hand, it still requires a manual reading of the intersection point.\n\nA search in both the Apple and Android app stores (conducted in January 2020 and again in March 2020) confirmed that there was no such app in either of the stores. To meet this need for a simple and accurate method of determining the Hofstee cut-off, we designed and developed a simple Android app. The app automatically sorts the data, draws the chart, and calculates the cut-off point algebraically. The result is a process that is faster and more accurate than the other mentioned methods.\n\nFor usability and evaluation, the app was designed according to the relevant principles laid out in the Mobile App Rating Scale (MARS).9 The overall MARS scale is broad, and so does have a few weaknesses when applied to this type of app (e.g. it rates the entertainment value of the app), but it is still a useful guide. In addition, the app is available free of charge, and with no advertisements.\n\n\nMethods\n\nThe app, HofsteeCalc, was developed using MIT App Inventor Version 2 (builds nb182 through to nb186a). MIT App Inventor uses its own visual, block-based programming interface to develop Android and iOS apps. In addition to the internal code, the app uses three external sets of libraries and routines for browsing to and selecting the data file,10 sorting the data,11 and charting the data.12 No user or device information is collected. The app is optimised for Android 2.1 and higher, API level 28, and requires permission to read from and store data to the device.\n\nSee Figure 2 for workflow chart.\n\nThe app automatically creates a data folder and has a test file that the user can use for testing before they insert their data.\n\nThe app allows each judge’s individual parameters to be entered (up to a maximum of 10 judges), and then calculates the means, standard deviations, and medians (Figure 3a). The parameters are automatically stored if required and are available the next times the app runs. Alternately, the final means or medians can be entered directly into the main screen (Figure 3b).\n\nHofsteeCalc app: multiple judges’ parameters and output from Figure 1 data, where cmin is minimum cut-off score, cmax is maximum cut-off score, fmin is the lowest percentage of students that the judge feels should fail, and fmax is the highest percentage of students that the judge feels should fail.\n\nFor data input, the student data need to be in a single-column standard.csv file. If the.csv file contains more than one column of data, only the first column will be read. The app automatically sorts the data, so these do not have to be pre-sorted by the user.\n\nWhen the charts are to be drawn, the user can view either the chart of the whole data set (see Figure 3b: Draw Full Chart), or a detailed section (covering data which is within and close to the range of the parameters (see Figure 3b: Draw Detailed Chart). With pinching, users can zoom in and out of the charts.\n\nAs the focus of the app is a functional tool, it has a simple user interface, and includes a ‘Help’ screen that explains in detail how it is to be used. Although the app assumes a knowledge of the Hofstee method, it supplies additional references for the user. Allowing for personal preferences, it permits the user to change some user-interface colours to suit individual needs.\n\nAn ‘Options’ screen allows changing of the user-interface colour scheme of buttons, to suit the individual user’s needs.\n\nIn the Hofstee chart, we know the x1y1 and x2y2 coordinates of line AB (Figure 1). However, because the cumulative score line does not have an algebraic formula, calculating the intersection between this straight line and the cumulative line is not possible (using ‘best fit’ or ‘nearest neighbour’ might be possible but will not give 100% accuracy). It is for this reason that current users of the Hofstee method read the point manually from hand-drawn charts.\n\nThe data, however, are x1y1 and x2y2 coordinates of straight lines, and these coordinates are stored in an array (or list). So, the algebraic algorithm for determining the cut-off can be expressed in the following pseudo-code:\n\nFor each straight line in the array of lines forming the cumulative line\n\nRead the x1y1 and x2y2 coordinates of that line\n\nAlgebraically determine the intersection point (xi) of this straight line and line AB\n\nIF x1 ≤ xi ≤ x2 [there is no need to test the y coordinate]\n\nTHEN xi is the cut-off point\n\n(If the cut-off (xi) is a data point, then two lines would meet this condition, but that is no matter, as the point is identical.)\n\nReaders may recognise that, because the cut-off point is determined algebraically, there is no need to draw the chart for the calculation. The chart, however, has been included in the app because most users are used to it, and also because they may wish to make manual adjustments to the parameters based on the visual reading of the data.\n\nAfter various early test versions, Version 1.0 of the app was completed in February 2021, and uploaded into the Google Play Store at: https://play.google.com/store/apps/details?id=appinventor.ai_itmeded.HofsteeCalc. Since then, small updates have been performed, and the app is currently on Ver. 1.1.\n\nConforming to the requirements laid out in the Introduction above, the app is available free of charge, with no advertisements. It does not require access to the internet, and it does not collect, store, or transmit any personal information about the user or the device.\n\nThe app was alpha tested on various real and hypothetical, sorted and unsorted datasets (see Underlying data13), with up to 1,000 items, and consistently returned accurate results. For example, for the dataset used in Figure 1, the app calculated the cut-off at 37.62%, rather than “slightly less than 38%” (See Figure 3b).\n\nThe time to draw the chart and determine the cut-off from a dataset of unsorted, 1,000 randomly-generated numbers (MS-Excel 2019 RANDBETWEEN(1,100)), was approximately 2 seconds (Samsung S8, Model SM-G955FD, Android Ver. 9, Build PPR1.180610.011.G955FXXS6DTA1).\n\nUsing the Mobile App Rating Scale (MARS),9 both authors independently measured the app against the scale, and arrived at a score of 4.07 and 3.88, respectively. As detailed above, this less-than-ideal score was expected, as the MARS includes items not entirely appropriate to such an app.\n\n\nUse case\n\nFor use cases, anonymised data sets are available in Underlying data.13\n\nAn example of a use case utilised the data in the sheet HofsteeCalcRealDataClass01.csv.\n\nThe data set has 181 items, and the item values range from 43 to 97. The data set is unsorted.\n\nThe input parameters were determined as shown in Table 1.\n\nUse case input parameters for HofsteeCalcRealDataClass01.csv, where cmin is minimum cut-off score, cmax is maximum cut-off score, fmin is the lowest percentage of students that the judge feels should fail, and fmax is the highest percentage of students that the judge feels should fail.\n\nBased on this use case, Figure 4a shows the input parameters. Figure 4b shows the resultant ‘Detailed chart’, the Hofstee cut-off percentage (53.80), and the largest data gaps in the vicinity of the Hofstee cut-off percentage.\n\nHofsteeCalc app: multiple judges’ parameters and output from HofsteeCalcRealDataClass01.csv, where cmin is minimum cut-off score, cmax is maximum cut-off score, fmin is the lowest percentage of students that the judge feels should fail, and fmax is the highest percentage of students that the judge feels should fail.\n\n\nComments\n\nThis paper has described the successful design and development of a free, advertisement-free, Android app to calculate the Hofstee cut-off. The app meets basic design principles as established in the MARS scale, and alpha- and beta- testing has shown the app to be accurate and fast. The app is available in the Google Play app store (see Software availability14).\n\nFull usability and ease of use will be tested in the future through more rigorous, wide-spread testing among medical educators.\n\n\nConclusions\n\nWhen educating future health professionals, determining fair pass/fail cut-off points is crucial. The time taken to perform such procedures, however, adds to medical educators’ already over-burdened schedules, and competes with a range of other demands in this schedule, so it is inevitable that short-cuts and errors will occur. This research has traced the design and development of a tool that can both save time and improve accuracy when determining the Hofstee cut-off.\n\n\nData availability\n\nZenodo: HofsteeCalcDataSets. https://doi.org/10.5281/zenodo.4699233.13\n\nThis project contains the following underlying data:\n\n• RawDataForTesting.csv (data set that is built into the app’s assets).\n\n• TestingForAppData.csv (data set used to generate Figure 1 and Figure 3b).\n\n• HofsteeCalcRealDataClass01.csv (data set available for testing).\n\n• HofsteeCalcRealDataClass02.csv (data set available for testing).\n\n• HofsteeCalcRealDataClass03.csv (data set available for testing).\n\n• HofsteeCalcRealDataClass04.csv (data set available for testing).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International licenses (CC-BY 4.0).\n\n\nSoftware availability\n\nSoftware available from Google Play app store: https://play.google.com/store/apps/details?id=appinventor.ai_itmeded.HofsteeCalc\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.4633140.14\n\nLicence: Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors would like to acknowledge Prof. Cees van der Vleuten, Maastricht University, for sending us a copy of Hofstee 1997.7\n\n\nReferences\n\nSchauber SK, Hecht M: How sure can we be that a student really failed? On the measurement precision of individual pass-fail decisions from the perspective of Item Response Theory. Med Teach. 2020 Dec 1; 42(12): 1374–84. PubMed Abstract \| Publisher Full Text\n\nNorcini JJ: Standard setting on educational tests. Med Educ. 2003; 37(5): 464–9. PubMed Abstract \| Publisher Full Text\n\nDowning SM, Tekian A, Yudkowsky R: Procedures for establishing defensible absolute passing scores on performance examinations in health professions education. Teach Learn Med. 2006; 18(1): 50–7. PubMed Abstract \| Publisher Full Text\n\nBandaranayake RC: Setting and maintaining standards in multiple choice examinations: AMEE Guide No. 37. Med Teach. 2008; 30(9–10): 836–45. PubMed Abstract \| Publisher Full Text\n\nHofstee WKB: The case for compromise in educational selection and grading. In: Anderson SB, Helmick JS, editors. On Educational Testing . San Francisco: Jossey-Bass; 1983. p. 109–27.\n\nWyse AE, Babcock B: An investigation of undefined cut scores with the Hofstee Standard-Setting Method. Educ Meas Issues Pract. 2017; 36(4): 28–34. Publisher Full Text\n\nHofstee W: Cesuurprobleem opgelost [The standard setting problem resolved]. Onderz Van Onderwijs. 1977; 6: 6–7.\n\nVan Der Vleuten C: Setting and maintaining standards in multiple choice examinations (AMEE Supplement 37.1). Med Teach. 2010; 32: 174–6. Publisher Full Text\n\nStoyanov SR, Hides L, Kavanagh DJ, et al.: Mobile App Rating Scale: A New Tool for Assessing the Quality of Health Mobile Apps. JMIR MHealth UHealth. 2015; 3(1): e27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPura Vida Apps: File Extension [Internet]. App Inventor Extensions . 2019 [cited 2020 Mar 1]. Reference Source\n\nPevest.com: QuickSort routine for your App Inventor Apps! [Internet].2017 [cited 2020 Mar 1]. Reference Source\n\nManning K, Kager E: ChartMaker [Internet].2017 [cited 2020 Mar 1]. Reference Source\n\nMasters K: HofsteeCalcDataSets (Version Ver 1.1) [Data set]. Zenodo . 2021. Publisher Full Text\n\nMasters K: HofsteeCalc (Version 1.1). Zenodo . 2021, March 24. Publisher Full Text" }	[ { "id": "86927", "date": "14 Jul 2021", "name": "Adam E. Wyse", "expertise": [ "Reviewer Expertise Psychometrics", "Standard Setting", "Measurement", "Item Response Theory", "Assessment" ], "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 appreciate the opportunity to review the article “Designing and developing an app to perform Hofstee cut-off calculations” by Ken Masters and Nadia Al-Wardy. I support the idea of developing simple software tools that people can use to perform standard setting. This is an area of definite need, especially in medical education contexts. I am unaware of a widely available software application to perform the Hofstee method. In most cases that I have seen the Hofstee method used, the people leading the standard setting use Excel or another statistical software package, such as R, to perform the calculations and figure out the cut score. In this sense, the app offered by Masters and Al-Wardy may be beneficial to people who want to perform the Hofstee method and do not have already developed software to perform the Hofstee method. I also liked how the authors provided screen shots of how the app works and several figures and examples throughout the article.\nI do have several suggestions for changes to the article and app. First, the authors suggest in the section Step 1: Evaluation of Judges that “We should further note that judges will generally use the Angoff or similar method to determine these.” This statement is not completely accurate. Wyse (2020)1 discusses several different methods for performing the Hofstee method. One strategy is to figure out the minimum and maximum cut scores from the panel of judges using the Angoff (1971)2 method or another test-centered method, such as the Bookmark method (Lewis, Mitzel, & Green, 1996)3. However, this method is not the most common strategy I have seen used to collect these data. It is more common to directly ask panellists to answer four open-ended questions to solicit the data needed to estimate the Hofstee cut score. In addition, it should be noted that even if the Angoff method is used with the Hofstee method that the data on the lowest and high percentages of students that a judge feels should fail the test (which is sometimes alternatively phrased in terms of the highest and lowest students that should pass the test) need to be directly collected from individual judges.\nIt should also be clear that the description of using the Angoff method to provide data to calculate the cut scores appears to be inconsistent with how the app works in Figure 3. In Figure 3, the authors show a screen with input for each rater. It is not possible to use the Angoff method to provide the multiple limiter input data shown on this screen. The app could be improved if it allowed for data from an Angoff standard setting or other test-centered method to be input. This input could be either entering the minimum and maximum cut scores from a test-centered standard setting method or each judge’s cut score from such a standard setting. It would also be beneficial if the app allowed for an option to input the lowest and highest passing rates and a corresponding graph instead of failure rates. I have commonly seen the method used with passing rates instead of failure rates. Judges sometimes find it easier to conceptualize and use pass rates as many credentialing and licensing organizations as well as accrediting bodies use passing rates instead of failure rates. Finally, it appears that the app requires that cut scores needs to be expressed as a percentage correct. It would be useful if the app also allowed for raw scores to be input as an option as I have seen raw scores used in many different standard settings.\nAnother area for potential improvement is the example shown in Figure 1. The example shown in Figure 1 appears to be based on data from a single judge. While it is possible to determine the Hofstee cut score for each individual judge, this is rarely done as the authors note in Step 2: Determining the arithmetic mean. The figure would be more beneficial if it focused on data from a group of judges as this is how the method is typically implemented.\nThere is a fourth practical and very real problem that occurs with the Hofstee method that is not described by the authors. Wyse and Babcock (2017)4 illustrate that the Hofstee method can produce undefined cut scores where the Hofstee line segment does not intersect with the failure rate or pass rate curve. This problem is a more serious issue than the three issues described by the authors as it implies that a cut score cannot be estimated. This issue should be mentioned in this section. Wyse and Babcock (2017)4 offer a solution for how to simply solve this issue, which involves extending the Hofsee line segment so that it intersects with the pass rate or failure rate curve. The other three practical issues described by the authors are easy to solve with Excel or other statistical software for technical savvy standard setters.\n\nIt should also be pointed out that the way the authors describe calculating the Hofstee cut score is different than the way that I typically think about doing it. The authors description of their method based on arrays is not easy to understand and follow, especially for many educators who may use the app. Wyse and Babcock (2017)4 offer an easy way to determine the cut scores for the Hofstee method that guarantees a solution even if the Hofstee line segment does not intersect the pass rate or failure rate curve. The authors should consider implementing this method in their app and provide a corresponding description in the article. The strategy involves finding the equation for the line that passes through the two points represented by the means of the data collected from the standard-setting judges that was described in earlier section of the article by the authors. Then, one inputs range of possible scores on the exam to figure out the estimated pass rate or failure rate (depending on whether pass rate or failure rate data are collected from judges) for every possible score on the exam. The last step is to compare the estimated pass rates or failure rates to the observed pass rates or failure rates on the exam. The score with the smallest absolute difference between the observed and estimated pass rates or failure rates is the cut score.\nIn summary, I think having a simple software app to perform Hofstee calculations is useful. However, the current version of the app does not cover all possible ways that the Hofstee method may be implemented which may limit its utility. If the authors made several changes based on the suggestions in this review, I think the app and article would have more utility and be easier to make sense for users.\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? Partly\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": [ { "c_id": "6931", "date": "19 Jul 2021", "name": "Ken Masters", "role": "Author Response", "response": "We thank you for your detailed comments. We shall take them into account and address them in more detail when we have received responses from other reviewers." }, { "c_id": "7179", "date": "05 Oct 2021", "name": "Ken Masters", "role": "Author Response", "response": "We have now had the opportunity to respond in detail to this review. Below, we indicate the Reviewer’s comments and our response. Reviewer’s Comment Approved With Reservations Authors’ Response We thank the reviewer for their very detailed comments, and trust that our responses below and the changes to Version 1 of the paper will be to their satisfaction. Reviewer’s Comment I appreciate the opportunity to review the article “Designing and developing an app to perform Hofstee cut-off calculations” by Ken Masters and Nadia Al-Wardy. I support the idea of developing simple software tools that people can use to perform standard setting. This is an area of definite need, especially in medical education contexts. I am unaware of a widely available software application to perform the Hofstee method. In most cases that I have seen the Hofstee method used, the people leading the standard setting use Excel or another statistical software package, such as R, to perform the calculations and figure out the cut score. In this sense, the app offered by Masters and Al-Wardy may be beneficial to people who want to perform the Hofstee method and do not have already developed software to perform the Hofstee method. I also liked how the authors provided screen shots of how the app works and several figures and examples throughout the article. Authors’ Response We thank the reviewer for their overall frank comments. Reviewer’s Comment I do have several suggestions for changes to the article and app. First, the authors suggest in the section Step 1: Evaluation of Judges that “We should further note that judges will generally use the Angoff or similar method to determine these.” This statement is not completely accurate. Wyse (2020)1 discusses several different methods for performing the Hofstee method. One strategy is to figure out the minimum and maximum cut scores from the panel of judges using the Angoff (1971)2 method or another test-centered method, such as the Bookmark method (Lewis, Mitzel, & Green, 1996)3. However, this method is not the most common strategy I have seen used to collect these data. It is more common to directly ask panellists to answer four open-ended questions to solicit the data needed to estimate the Hofstee cut score. In addition, it should be noted that even if the Angoff method is used with the Hofstee method that the data on the lowest and high percentages of students that a judge feels should fail the test (which is sometimes alternatively phrased in terms of the highest and lowest students that should pass the test) need to be directly collected from individual judges. Authors’ Response Thank you for this comment. The published texts that we were using (Bandaranayake 2008; Wyse and Babcock 2017) indicated that the Hofstee was frequently used in conjunction with the Angoff method and so we mentioned that. As this is not central to the paper, however, the sentence has been deleted. Reviewer’s Comment It should also be clear that the description of using the Angoff method to provide data to calculate the cut scores appears to be inconsistent with how the app works in Figure 3. In Figure 3, the authors show a screen with input for each rater. It is not possible to use the Angoff method to provide the multiple limiter input data shown on this screen. The app could be improved if it allowed for data from an Angoff standard setting or other test-centered method to be input. This input could be either entering the minimum and maximum cut scores from a test-centered standard setting method or each judge’s cut score from such a standard setting. It would also be beneficial if the app allowed for an option to input the lowest and highest passing rates and a corresponding graph instead of failure rates. I have commonly seen the method used with passing rates instead of failure rates. Judges sometimes find it easier to conceptualize and use pass rates as many credentialing and licensing organizations as well as accrediting bodies use passing rates instead of failure rates. Authors’ Response Thank you for this comment. We apologise for the misunderstanding about what is being portrayed in Figure 3, and we acknowledge that the misunderstanding is because the caption in Figure 1 (raised by the Reviewer below), and then the paragraph that immediately precedes Figure 3, which gives the impression that a single judge’s information has been input (in Figure 3b). As a result, in addition to the corrections to the Figure 1 caption, we have altered the description in the paragraph preceding Figure 3 in order to clarify the process. Reviewer’s Comment Finally, it appears that the app requires that cut scores needs to be expressed as a percentage correct. It would be useful if the app also allowed for raw scores to be input as an option as I have seen raw scores used in many different standard settings. Authors’ Response Thank you for this comment. This may be an addition to Version 2 of the app, but the current literature (e.g. Bandaranayake 2008; Burr et al 2016) indicates percentage scores as the input, and so, it is prudent for Version 1 of the app to stick to the more common process as described in the dominant literature. Reviewer’s Comment Another area for potential improvement is the example shown in Figure 1. The example shown in Figure 1 appears to be based on data from a single judge. While it is possible to determine the Hofstee cut score for each individual judge, this is rarely done as the authors note in Step 2: Determining the arithmetic mean. The figure would be more beneficial if it focused on data from a group of judges as this is how the method is typically implemented. Authors’ Response Thank you for this point. Figure 1 is an illustrative example showing the chart after the arithmetic means of the parameters have been determined. As mentioned above, however, we acknowledge that the caption was erroneous, and it (and the sentence preceding it) have now been corrected. Reviewer’s Comment There is a fourth practical and very real problem that occurs with the Hofstee method that is not described by the authors. Wyse and Babcock (2017)4 illustrate that the Hofstee method can produce undefined cut scores where the Hofstee line segment does not intersect with the failure rate or pass rate curve. This problem is a more serious issue than the three issues described by the authors as it implies that a cut score cannot be estimated. This issue should be mentioned in this section. Wyse and Babcock (2017)4 offer a solution for how to simply solve this issue, which involves extending the Hofsee line segment so that it intersects with the pass rate or failure rate curve. The other three practical issues described by the authors are easy to solve with Excel or other statistical software for technical savvy standard setters. Authors’ Response We are reluctant to get into a discussion about the weaknesses of the Hofstee method or how to solve problems associated with it. To do so, and to do such a discussion justice (and view all arguments equally from all sides), is a paper by itself. In our discussion of the Hofstee Method, the aim is simply to explain it so that the functioning of the app is understood. Nevertheless, as readers may be expecting a discussion of the strengths and weaknesses of the Hofstee Method, we have inserted the sentence: “While there are weaknesses with the method, and they have been discussed elsewhere, this paper is focused on describing the method, and then describing an app that applies the method.” And the “elsewhere” in the sentence cites the Wyse & Babcock article mentioned by the reviewer. Reviewer’s Comment It should also be pointed out that the way the authors describe calculating the Hofstee cut score is different than the way that I typically think about doing it. The authors description of their method based on arrays is not easy to understand and follow, especially for many educators who may use the app. Wyse and Babcock (2017)4 offer an easy way to determine the cut scores for the Hofstee method that guarantees a solution even if the Hofstee line segment does not intersect the pass rate or failure rate curve. The authors should consider implementing this method in their app and provide a corresponding description in the article. The strategy involves finding the equation for the line that passes through the two points represented by the means of the data collected from the standard-setting judges that was described in earlier section of the article by the authors. Then, one inputs range of possible scores on the exam to figure out the estimated pass rate or failure rate (depending on whether pass rate or failure rate data are collected from judges) for every possible score on the exam. The last step is to compare the estimated pass rates or failure rates to the observed pass rates or failure rates on the exam. The score with the smallest absolute difference between the observed and estimated pass rates or failure rates is the cut score. Authors’ Response Thank you for your suggestion. The proposal for modification by Wyse and Babcock (2017) is, indeed, interesting. There are also others (e.g. Burr et al 2016). The app (and, therefore, the article), however, is designed to determine the Hofstee cut-off as it is commonly practiced (e.g. Bandaranayake 2008). All proposed modifications cannot be implemented, and, if the app were to favour one modification over another, we would be open to criticisms of favouritism and of promoting and endorsing one modification over others. In this instance, the criticisms would be particularly sharp, because this modification has been proposed by one of the modification authors, who is also a reviewer of this paper. If we were to implement this modification (and not others), we would lay ourselves open to the accusation that we had made the modification to the app and the paper primarily to gain favour from the reviewer in the hopes of a more favourable review. As the reviewer can appreciate, both the authors and the journal would then be under pressure to retract such a paper (and remove the app from the app store). That said, we could certainly envision a future version of the app that, based on a detailed literature review of all possible Hofstee variations and modification, would attempt to implement them all, allowing users to choose their favourite modification. Reviewer’s Comment In summary, I think having a simple software app to perform Hofstee calculations is useful. However, the current version of the app does not cover all possible ways that the Hofstee method may be implemented which may limit its utility. If the authors made several changes based on the suggestions in this review, I think the app and article would have more utility and be easier to make sense for users. Authors’ Response Thank you for your suggestion. We trust that our explanation and paper’s modifications detailed above satisfies the reviewer. Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Partly Authors’ Response We trust that our explanation and changes to the paper are to the reviewer’s satisfaction. Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly Authors’ Response We trust that our explanation and changes to the paper are to the reviewer’s satisfaction. Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly Authors’ Response We trust that our explanation and changes to the paper are to the reviewer’s satisfaction." } ] }, { "id": "90318", "date": "11 Aug 2021", "name": "Benedict Canny", "expertise": [ "Reviewer Expertise Education 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\nThis paper describes the development and use of the an app to help with the use of the Hofstee method. I suspect it will be of considerable use to those who adopt this method. Importantly, the app only addresses the analysis of data, once the appropriate data have been generated, and users should fully acquaint themselves with strengths and weakness of the Hofstee standard setting method prior to use. Indeed, this advice could be applied to all standard setting approaches, as all have their pluses and minuses.\nWhile aware of the Hofstee method, I have never used in practice, so I am unable to comment on the practicalities of this method when compared with performing this task long hand.\nI am concerned about some of the terminology used in the paper. The authors use the words to \"accurate\" and \"accuracy\" in the Alpha Testing and Conclusions sections of the manuscript, and report cut scores to two significant decimal figures. I suspect that they mean the term \"precision\", as accuracy is a measure of the proximity of the estimated value to the \"true\" value, and would be best determined by comparing this method to another (e.g. van der Vleuten's method referred to in the paper), or a \"gold\" standard (making calculations long hand). The authors have not reported on the effect of the app on the \"error rate\" of using the method, and these additional data would be useful, if available.\nFinally, the point is made in the conclusion that \"determining fair pass/fail cut-off points is crucial\". This method will only increase fairness if the error rate is reduced, and not the inherent \"fairness\" of Hofstee method, nor, indeed, any other standard setting method.\nIn conclusion, I suspect this app will be useful for those who use the Hofstee method. It would be nice to see an iOS 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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "7125", "date": "06 Sep 2021", "name": "Ken Masters", "role": "Author Response", "response": "We thank you for your comments. We shall take them into account and address them in more detail when we submit Version 2 of the paper." }, { "c_id": "7180", "date": "05 Oct 2021", "name": "Ken Masters", "role": "Author Response", "response": "We have now had the opportunity to respond in detail to this review. Below, we indicate the Reviewer’s comments and our response. Reviewer’s Comment Approved With Reservations Authors’ Response We thank the reviewer for their comments, and trust that our responses below and the changes to Version 1 of the paper will be to their satisfaction. Reviewer’s Comment This paper describes the development and use of the an app to help with the use of the Hofstee method. I suspect it will be of considerable use to those who adopt this method. Importantly, the app only addresses the analysis of data, once the appropriate data have been generated, and users should fully acquaint themselves with strengths and weakness of the Hofstee standard setting method prior to use. Indeed, this advice could be applied to all standard setting approaches, as all have their pluses and minuses. While aware of the Hofstee method, I have never used in practice, so I am unable to comment on the practicalities of this method when compared with performing this task long hand. I am concerned about some of the terminology used in the paper. The authors use the words to \"accurate\" and \"accuracy\" in the Alpha Testing and Conclusions sections of the manuscript, and report cut scores to two significant decimal figures. I suspect that they mean the term \"precision\", as accuracy is a measure of the proximity of the estimated value to the \"true\" value, and would be best determined by comparing this method to another (e.g. van der Vleuten's method referred to in the paper), or a \"gold\" standard (making calculations long hand). The authors have not reported on the effect of the app on the \"error rate\" of using the method, and these additional data would be useful, if available. Authors’ Response Thank you for this comment. Given that the final accurate answer is best determined by finding the intersect point of the two relevant lines in the chart, the final accurate result can be independently calculated with those known coordinates. (The main function of the app is to determine those two lines). So, for further verification, we have tested these coordinates using an online calculator (AMBrSoft, http://www.ambrsoft.com/MathCalc/Line/TwoLinesIntersection/TwoLinesIntersection.htm), and the app’s results are shown to be accurate to two decimal places. We also confirmed this with a enlarged hand-drawn calculation. Version 2 of the paper has been amended to reflect this, and the relevant coordinates have been placed in the paper, so that users can test these results independently. The reviewer is correct, though, that this level of increased accuracy is only against the methods that require manual reading of the charts, so, we feel we can now claim that the app is more accurate than the standard manual process, which could not have the same level of accuracy, and we have amended the paper to reflect this. Reviewer’s Comment Finally, the point is made in the conclusion that \"determining fair pass/fail cut-off points is crucial\". This method will only increase fairness if the error rate is reduced, and not the inherent \"fairness\" of Hofstee method, nor, indeed, any other standard setting method. Authors’ Response Yes, we agree. Given that the accuracy of the app has been confirmed (and can easily be verified by readers), we feel that the general statement that “determining fair pass/fail cut-off points is crucial” is valid. Reviewer’s Comment In conclusion, I suspect this app will be useful for those who use the Hofstee method. It would be nice to see an iOS version. Authors’ Response Yes, it would. Although an iOS app is planned, it would be premature to develop the app (or even allude to it in the paper) until the Android version has been accepted by the academic community. Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly Authors’ Response We trust that our explanation and changes to the paper are to the reviewer’s satisfaction." } ] } ]	1	https://f1000research.com/articles/10-450
https://f1000research.com/articles/10-1002/v1	04 Oct 21	{ "type": "Method Article", "title": "Performing post-genome-wide association study analysis: overview, challenges and recommendations", "authors": [ "Yagoub Adam", "Chaimae Samtal", "Jean-tristan Brandenburg", "Oluwadamilare Falola", "Ezekiel Adebiyi", "Yagoub Adam", "Chaimae Samtal", "Jean-tristan Brandenburg", "Oluwadamilare Falola" ], "abstract": "Genome-wide association studies (GWAS) provide huge information on statistically significant single-nucleotide polymorphisms (SNPs) associated with various human complex traits and diseases. By performing GWAS studies, scientists have successfully identified the association of hundreds of thousands to millions of SNPs to a single phenotype. Moreover, the association of some SNPs with rare diseases has been intensively tested. However, classic GWAS studies have not yet provided solid, knowledgeable insight into functional and biological mechanisms underlying phenotypes or mechanisms of diseases. Therefore, several post-GWAS (pGWAS) methods have been recommended. Currently, there is no simple scientific document to provide a quick guide for performing pGWAS analysis. pGWAS is a crucial step for a better understanding of the biological machinery beyond the SNPs. Here, we provide an overview to performing pGWAS analysis and demonstrate the challenges behind each method. Furthermore, we direct readers to key articles for each pGWAS method and present the overall issues in pGWAS analysis. Finally, we include a custom pGWAS pipeline to guide new users when performing their research.", "keywords": [ "PostGWAS", "pGWAS", "GWAS", "Meta-analysis" ], "content": "Introduction\n\nGenome-wide association studies (GWAS) have been used to identify genetic variants associated with specific traits or diseases of interest. One of the advantages of performing GWAS is that they do not require prior knowledge of the biological hypothesis underpinning the genetic machinery of the investigated trait. Many GWAS have revealed hundreds of common variants that are associated with various phenotypes, including common diseases.1 Edwards et al reported the first GWAS for age-related macular degeneration in 2005.2,3 In the last decade, many GWAS have been reported in scientific databases3 and these studies have revealed the presence of various single nucleotide polymorphisms (SNPs). For instance, GWAS Catalog reported 4865 publications and 247051 associations in 2021. These reported SNPs could be used to better understand the molecular mechanisms of common diseases and biological pathways of interesting traits.\n\nOver the last decade, most GWAS have been known to detect the genetic signals of a single gene or a single genetic marker, i.e., interpreting genes based on detected signals (positions) on genomic coordinates using a specified Genome Assembly.4 However, most complex diseases that have been targeted by GWAS are known to be caused by multiple genes that could be influenced by many other factors. It is known that GWAS typically report SNPs as statistically significant when their associated p-values are less than 5e-08. Accordingly, GWAS might not detect genetic variants with low or moderate risk.5,6 Thus, the ability of GWAS to detect a significant genomic position depends on heritability on phenotype, minor allele frequency (MAF), and sample size. Traditional GWAS could therefore be faced with the challenge of not being able to detect variants that are associated with low disease risk, implying that traditional GWAS results are prone to unreliable findings due to their false negative results.7 Furthermore, GWAS might fail to detect a significant signal if the effect of a variant in another gene is not taken into consideration.5 It is also limited in identifying changes in genotype as a response to environmental changes, i.e., detecting the impact of genotype interaction with the environment.8 These limitations of GWAS occur primarily because of the undetected effect of gene polymorphism. More specific challenges of GWAS have been reported in many scientific papers.1,5,7,9-12 Given all of the reported limitations of GWAS, it is crucial to perform post-GWAS (pGWAS) analysis. The overall goal of pGWAS analysis is to use the result of the association between the genotype and phenotype (summary statistics), with the following objectives:\n\n• Transferability of previous result,\n\n• Identification of new significant functional variants, i.e. lead SNPs,\n\n• Identification of novel disease susceptibility genes, genotype-phenotype associations, and biological pathway network, and\n\n• Building a polygenic risk score using summary statistics.\n\nSo far, the most common approaches to perform pGWAS analysis include the following three approaches: (i) Single-variant approach; (ii) Gene-scoring approach; and (iii) Pathway-sub-network-based approach. However, pGWAS approaches could be categorized into many classes based on their usage (see section below). Also, there are several methods/tools for pGWAS reported in different articles.\n\nIn this article, we provide an overview to performing pGWAS analysis and discuss the challenges behind each method. Furthermore, we direct readers to key articles for each pGWAS method and present the overall issues in pGWAS analysis. Finally, we include a custom pGWAS pipeline to guide new users when performing their research.\n\n\nGWAS analysis\n\nGWAS have become an indispensable approach in providing insight into understanding genotype-phenotype associations of complex disease. While the design for GWAS experiments are well established as bench-work, the computational methods for the analysis of GWAS data are still evolving. The typical computational pipeline to analyse any GWAS data consists of two essential tasks: a) upstream GWAS analysis (classic GWAS methods), and b) downstream GWAS analysis. The latter step is known as pGWAS analysis.\n\nThe upstream GWAS analysis is a multi-step task resulting in a list of statistically significant SNPs. This step often starts by checking the quality control of raw GWAS data which is a crucial step for the task of pGWAS analysis. It is obvious that unclean data lead to unreliable results. Therefore, many parameters such as quality control per sample, relatedness, replicate discordance, SNP quality control, sex inconsistencies, and chromosomal anomalies should be checked.13,14 After obtaining raw data-sets with high quality scores, the next step is to report the statistically significant SNPs. Many statistical tests are available for this, including Chi-square test, Fisher’s exact test, Cochran-Armitage trend test, Odds ratio, Logistic regression, ANOVA, Transmission Disequilibrium test, Bonferroni correction, and many other methods.\n\nMany free tools are available to perform GWAS upstream data analysis such as: Plink,15 PLATO,16 EIGENSOFT,17 and STRUCTURE.18 Several other tools are also available as packages within R software, the most popular open-source software for statistical computing.19\n\n\npGWAS approaches\n\nGenome wide significant threshold. This approach provides the statistics at a SNP level. This approach aims to score the association between SNPs and the target trait. Usually, the score defined by a β value or Odd Ratio (OR), and its standard error (SE) gives an idea of the genotype’s effect on the phenotype. Effect strength is computed using OR. Also, scientists use p-value as a measure of how likely this effect is to occur by chance. Statistically, many researchers use p-value to evaluate the hypothesis that there is no statistical evidence for SNP-trait associations. However, this hypothesis will be rejected when p-value is less than a predetermined threshold that is adjusted for a multi-test. Many methods are used for multiple test p-value correction. These methods include Bonferroni correction and false discovery rate (FDR).20 However, some of these methods do not consider linkage disequilibrium (LD) and they may be too stringent. In general, a threshold of 5e‐08 is acceptable for human association studies.21,22 This value has been computed using independent signals in genomes. However, this value is not absolute and it depends on many factors, including LD (and diversity), array type, whole genome sequencing or whole exome sequencing, position number in array or sequencing, imputation panels, and positions finally analyzed.23 For trans-ethnic populations, it is recommended to estimate the threshold based on population diversity and LD. For instance, analyzing 1000 Genomes data, the suggested significance thresholds were 3.24e‐08 for Africa, 9.26e‐08 for Europe, 1.83e‐07 for Mixed America, 1.61e‐07 for East Asia and 9.46e‐08 for South Asia.24 Although, a recent study using an African population proposed a threshold of 5e‐09.25\n\nSuggestive threshold. Some authors consider GWA threshold very stringent, so studies often include a “suggestive threshold”. This is superior to a genome-wide significant threshold, and values found in the literature are 1e‐05, 1e‐06 or lower. Studies estimated on “independant LD block” from 1000 Genomes used 1e‐05 for the Affymetrix 500K and Illumina 317K GWAS SNP panels, and 1e‐06 for HapMap CEPH Utah and Yoruba populations.26 The code below contains an R command to select significant SNPs using a cutoff value of 5e‐08.\n\n\n\nInflation factors. Population and cryptic relatedness can cause spurious associations in GWAS with p-values higher than random leading to false positive signals. Genomic control (GC) approach is extensively used to effectively control false positive signals. Genomic inflation factors (λ) can be computed as the median of the resulting chi-squared (χ2) test statistics divided by the expected median of the chi-squared distribution.27 Refer to equation 1 below\n\nZ (Beta/Se), χ2 and p-value can be used to compute inflation factor, using Z2 for Z, quantile of 1 - p-value at 1 degrees of freedom. The code below demonstrates how to compute inflation factors using the build-in R function qchisq that can be used to calculate value of quantile for a χ2 distribution.\n\n\n\nGlobal visualisation of results. A common way to visualize GWAS results is the Manhattan plot. The Manhattan plot demonstrates the physical location of SNPs distributed by chromosome in the x-axis with the degree to which a SNP is associated with the target trait, i.e. p-value scaled by log10 in the y-axis. A Manhattan plot can be done in R software using the qqman package, which includes functions for creating Manhattan plots and q-q plots from GWAS results.28 The code below demonstrates how to create Manhattan plots, and q-q plots the qqman R package.\n\n\n\nLocal visualisation of results. Local visualisation can be done using the method described by Pruim et al.29 In this method, information of LD, genes, and previous results of GWAS can be added. Zoom version 2 offers a virtual analysis of local GWAS results. On the other hand, LocusTrack from the UCSC genome-browser adds more annotation compared to LocusZoom.30 Furthermore, BigTop31 is capable of providing three-dimensional visualisation of data using allele frequency as a third dimension. The code below demonstrates how to use LocusZoom to visualize SNPs considering LD information.\n\n\n\nThis approach considers the association between a trait and all SNPs within a predefined window around genes rather than each marker individually. In many cases, this approach is more powerful than traditional individual-SNP-based GWAS.\n\nA gene score (GS) is a value given to a gene representing some measures related to a genetic trait. Thus, all the statistical summary values such as p-values and fold changes could be considered gene scoring values. For pGWAS analysis, GS is defined as the sum of all statistically significant alleles (i.e. the risk alleles) of the selected SNPs present in each individual under investigation.32 Various algorithms have been developed to calculate GCs based on GWAS summary statistics.33-35 It is a common approach to encode and adjust SNPs values prior to calculating GC during analysis.32 The process of SNP encoding aims to ensure that all SNPs are positively correlated with the outcome.32 One more essential aspect to consider while calculating GS is the effect size. Variation in the effect size reflects reduction of predictive power of GS.32 The GS method mostly used in pGWAS analysis is the gene-based p-value.\n\nGene level p-value. In many pGWAS pipelines, the first step in downstream GWAS analysis is to assign the SNPs to functional genomic features. The latter includes: coding genes, non-coding RNAs, 5’UTR, 3’UTR, proximal promoters, regulatory element, and enhancer elements.\n\nTwo common methods for assigning SNPs to their corresponding genes are GLOSSI36 and VEGAS.34,37 GLOSSI is available as an R package, while VEGAS is available as an online tool as well as a stand-alone tool to be run on a local machine. Besides VEGAS and GLOSSI, many pGWAS tools provide procedures to calculate genes’ p-values. For instance, ancGWAS provides four different methods to calculate the genes p-values: Simes, Smallest, Fisher, and Gwbon. Similar to ancGWAS, MAGMA provides three methods to calculate genes’ p-values. However, one of the MAGMA methods is similar to the VEGAS method. The other two methods are by considering either the smallest SNP p-value, or the highest SNP p-values.\n\nThis approach considers the fact that complex biological phenomena addressed by GWAS, including the molecular basis of the rare disease, often arise due to gene interaction rather than single gene effect. Therefore, scientists analyse GWAS based on the biological network theory to understand the disease-causing genes and mechanisms involved in traits and complex diseases, such as in rare diseases. This approach is based on the results obtained from the gene-based association test and provides a higher level of complexity by considering biological networks and/or genes ontology. Analysing GWAS based on biological networks allows us to capture biological interactions between various molecules such as proteins, functional DNA motifs, coding and non-coding RNA, as well as disease mechanisms and to consider epigenetic changes, including methylation states or other modifications (phosphorylation, acetylation, etc). Also, this approach aims to map genes that are associated with significant SNPs into known pathways/Gene Ontology terms. The result of this approach provides information about the over-represented pathways in a given set of genes/SNPs.\n\nGWAS will often identify a number of SNPs in LD with each other as being associated with the phenotype. The lead SNPs are those with the most significant p-value – they may be causal or not. The other SNPs in this region may only have an association because they are in LD with the causal SNP or they may be independently associated. This GWAS limitation may be observed when integrating information of variability in allele frequency, SNPs in LD block and imputation.38,39 For instance, when simulating GWAS results using OR value of 1.5 and allele frequency of 0.5, only 21% of the simulations demonstrated that the identified causal variants were not the most strongly associated variants.40 On the other hand, changing the previous parameters OR value of 1.1 and allele frequency of 5%, resulted in 2.4% concordance between causal variants and lead SNPs.40 Therefore, changing the previous parameters OR value of 1.1 and allele frequency of 5%, resulted in 2.4% concordance between causal variants and lead SNPs.40\n\nTherefore, fine-mapping methods are used to identify causal variants that are associated with the target trait and the number of putative causal variants from GWAS data. The fine-mapping approach integrates summary statistics from GWAS data, LD and functional annotations. There are two fine-mapping methods: (i) heuristic method that penalizes regression models, and (ii) Bayesian fine-mapping methods.38,39\n\nLead SNPs. Lead SNPs are defined as independent SNPs that have reached a minimum p-value threshold, i.e. they are independent of each other at the LD threshold. It is common to measure LD as r2 where the square of the correlation coefficient between any two indicator variables is r2.41 PLINK has implemented a function called’ld-clump’ that clumps independent SNPs.15 Moreover, FUMA tool42 identifies lead SNPs by double clumping method. The first clumping is used for clumping SNPs with p-value < 0.05 at genome-wide significant p-value, i.e. p-value < 5e‐08 and independent at r2< 0.6. This first clumping function reports significant independent SNPs. The second clumping is of significant independent SNPs at r2< 0.1 and reports lead SNPs. The code below demonstrates how to use the Plink tool to perform clumping to get lead SNPs.\n\n\n\nHeuristic fine-mapping approaches. These are used to identify potential causal SNPs.38 They are proposed to filter SNPs around lead SNPs considering the value of their pairwise correlation r2. They consider a hierarchical clustering technique to cluster all SNPs in a given region based on their pairwise r2. Another way is to use LD block by direct extraction of block and the selection of the same position on the block or by the visualisation representation of LD using LocusZoom29 or Haploview.43 Nonetheless, the described method is not a statistical approach to define putative causal variants, such as Bayesian methods or regression models. The code below demonstrates how to combine Plink tool and Haploview to perform heuristic fine-mapping.\n\n\n\nBayesian methods: framework. The Bayesian method is commonly used to identify causal variants in a predefined SNPs window containing n number of SNPs. Knowing data (D) the Bayesian method aimed to maximize statistical model (M) using the following conditional probability\n\nUsers first define an initial number of causal variant c (between 1,m SNPs). This number c is defined using genome-wide significant SNPs. To model Bayesian statistics, software will define a model M that contains c SNPs. This model often will restrain choice to the significant SNPs or suggest significant SNPs (m) in the windows. Considering the rule of combination Cm,c=m!c!n−c!, the probability model PDM is relatively easier to compute. The following Bayesian rules can be used\n\nMost of the tools are aimed to find a maximum probability to have the best combination of the causal variants. To model Bayesian statistics, many tools integrate various data such as LD and GWAS summary statistics.\n\nBayesian methods: posterior inclusion probability. This approach is used to compute post inclusion probability (PIP) at each SNP i. The PIP is computed by the sum of the posteriors over all models that include SNP i as a causal variant.38\n\nUsing the rank of PIP is an excellent way to select putative causal variants.38 The PIP approach should be used with caution to identify causal variants in the high LD regions. Therefore, it is recommended to estimate the posterior expected number of causal SNPs by summing the estimated PIPs for all SNPs in the region.\n\nBayesian methods: credible sets. This approach is used to define a set of variants that could have a good candidate. One way to estimate a credible set is to use PIP by ranking the values and doing the cumulative sum of PIP from the largest to the smallest. Then select all variants where the sum is less than the predefined α cutoff value. In general, researchers use 99% or 95% as a recommended value for α. Studies have shown that credible set and coverage probabilities are over-conservative in most fine-mapping situations as data sets are not randomly selected from among all causal variants. Therefore, an adjusted coverage is proposed to reduce such over-conservation in this approach.44 The code below demonstrates how to combine Plink tool and FINEMAP to perform Bayesian-based fine-mapping analysis.\n\n\n\nBayesian methods: trans ethnic fine-mapping. This is used to perform trans-ethnic fine-mapping studies using simulation results in similar fine-mapping resolution among the European and Asian ancestries. However, the inclusion of samples with African ancestry in meta-analysis leads to a significant improvement in fine-mapping resolution due to the lowest LD in the African ancestry population. The probability that the lead GWAS variants were also the causal variants increased using trans-ethnic GWAS data.40,45 It is a process that relies on disparate LD patterns in populations of diverse genetic ancestries to localize the causal variants. This approach has been successfully implemented to fine-map and leads to several common GWAS findings.46\n\nIntegrating annotation into fine-mapping. Functional annotations have been shown to improve the discovery power and fine-mapping accuracy. Therefore, some tools such as CausalDB,47 PAINTOR48 and BIMBAM49 have integrated expression quantitative trait locus (eQTL) information in fine-mapping approach.\n\nBayesian methods: software. Several Bayesian-based fine-mapping tools have been developed using summary statistics, LD and eQTL (Table 1). Also, some pipelines have integrated different Bayesian-based software in order to compare their results (fine-mapping of h3agwas, fine-mapping in FinnGen, and FM pipeline).\n\nMore descriptives can be found in.38\n\nOther fine-mapping approaches. Other approaches, such as regression models, are used with all SNPs in the lead SNPs region to analyze SNPs jointly. The comparison of various approaches including elastic net, ridge, Lasso, MCP, and the normal-exponential shrinkage prior, have shown that penalized methods outperform single marker analysis.53 Furthermore, a forward/stepwise regression can be used to test the independence of multi SNPs using the following algorithm:\n\n• Order the list of SNPs by their p-values p0p1…pn−1\n\n• Remove highest p-value\n\n• Apply models with p1…pn−1 with p0 as a co-variate and check if any is significant\n\n• Repeat process.\n\nThe R library SusieR has implemented a method of regression fine-mapping analyses.\n\nOther resources for fine-mapping analyses. Several review articles have been published for fine-mapping analyses. Nevertheless, we recommend the following scientific articles as a good source for beginners: “From genome-wide associations to candidate causal variants by statistical fine-mapping”,38 “A practical view of fine-mapping and gene prioritization in the post-genome-wide association era”,39 and “Fine-mapping genetic associations”.54\n\nThe aim of performing conditional association and imputation using summary statistics is to evaluate the association between SNPs and biological trait by combining various GWAS summaries from different studies. This method requires a reference population to estimate LD information. The imputation method performs meta-analysis to infer the missing genotypes among the studies before evaluating the association between the SNPs and the biological trait. This method estimates the effects of many variants that are not directly genotyped.\n\nThis method is used to predict disease risk using GWAS summaries.55 Polygenic risk score (PRS) could be used to predict an individual’s likelihood to develop a specific trait or to estimate the level of predictive power that the trait is associated with a particular set of variants.56 Although PRS methods are classified into Bayesian-based methods and non-Bayesian methods, there are more classifications of underlying PRS methods.57 PRS is calculated by aggregating effects from a large set of causal SNPs. Several tools were developed to calculate PRS. For performing PRS studies, we highly recommend this recent review paper.58 PRS is usually computed after the challenges associated with GWAS are carefully addressed. Here, we demonstrate key quality control (QC) measures and include sample bash scripts. Table 2 summarizes the seven QC measures and contains the guidelines on specific thresholds. Thresholds can differ depending on the study’s unique features.\n\nGWAS effect allele. As datasets for PRS come from different GWAS experiments, it is critical to ensure consistency. Knowing which allele is considered the effect allele, it is vital to get an accurate PRS score. However, the effect allele is not labeled clearly in many datasets.59 Different allele coding schemes exist, including Illumina’s TOP/BOTTOM coding concept, ALT/REF, effect/other, HapMap’s forward allele coding, Illumina’s A/B allele coding, Affymetrix’s A/B allele coding, REF (reference)/ALT (alternative), PLINK’s 1/2 allele coding, A1 (allele1)/A2 (allele2), A0 (allele 0)/A1 (allele1), effect allele/non-effect allele, effect allele/other allele, and many others.58,59 To avoid allele inconsistency, researchers should carefully read the documentation of GWAS datasets.\n\nSNPs level errors. The QC assessment at the SNP level is crucial to avoid misleading PRS. SNPs level errors include (i) mismatching SNPs, i.e., inconsistent SNPs due to position difference in genomic position or nucleotide type, (ii) existence of duplicate SNP, (iii) ambiguous SNPs, i.e., researchers have no idea about SNP strand (ambiguous SNPs usually are C/G or A/T SNPs), and (iv) missing alleles.\n\nChip heritability. The chip heritability (hSNP2)is also known as SNP-based heritability, which is defined as the portion of the phenotypic variation that the genotyped genetic marker can explain.60,61 Higher values of heritability indicate that the phenotype is explained best by the genotype, i.e. set of SNPs. Choi et al.58 recommend hSNP2>0.05 to perform PRS analysis. To estimate hSNP2, researchers should use LD score regression that could be used to distinguish polygenicity (SNPs effects) and confounding biases, including cryptic relatedness and population stratification.62\n\nThe code below demonstrates how to use Plink to perform quality control checks and calculating PRS.\n\n\n\nThe meta-analysis approach can be used to evaluate the association between SNPs and biological traits by combining various GWAS summaries from different studies. The following paragraphs will provide the key concepts for performing a meta-analysis. To have concrete information about the meta-analysis approach, we recommend this review article.63\n\nHeterogeneity of source. Heterogeneity in data could be derived using GWAS summary statistics. The standard variables to estimate heterogeneity in data include odds ratios, standardized effect sizes, other metrics along with their uncertainty (e.g. variance or 95% confidence interval) and the accompanying p-values. However, there might be many other variables for each dataset that are important to deal with in order to estimate heterogeneity in data.\n\nStandard meta-analysis. This is used to perform the meta-analysis approach which is to sum the Z-scores across all studies and weigh them appropriately using the sample sizes. See equation (3) below.\n\nwhere Zk is Z-score from Kth study, and wk weight of studies relative to population size.\n\nIndependence of the samples. Conventional meta-analysis has an assumption that assumes that effect sizes are independent. Simulation studies demonstrate that failure to account for overlapping samples could greatly inflate type I error.64 If accounting for the overlap is unavoidable, the overlap/covariance can be estimated using Z’s covariance between summary statistics. Some tools can account for overlapping samples, such as METAL software65 and ASSET.66\n\nCorrecting for population structure with genomics control. The presence of population structure in the GWAS study can impact an over-dispersion of the corresponding association test statistics. One approach to limit this problem is to correct statistics of each summary using genomic control. This correction factor is given as the inflation factor (λ) which is the test statistics’ median divided by its expectation under the null hypothesis.67\n\np-values versus Z scores. Meta-analysis methods based on p-values were widely used in different scientific fields until the 1980s. Then became unpopular and almost abandoned in biomedical sciences. Nowadays, the meta-analysis approach is performed using Z-score. There are two methods to estimate Z-score for GWAS data. The first method is demonstrated in equation (4).\n\nIn the second method (equation (5)), Z-score is estimated using p-value and the effect of allele.\n\nwhere signΔi is a sign of relation.\n\nRandom effects versus fixed effects. In the presence of variability of allelic effect as in trans-ethnic studies, it is common to perform a random-effect meta-analysis to correct variability of β effect between different studies. For instance, GWAMA tool68 computes a random-effects variance component using Cochran’s statistic (Q-value) to balance weight used in meta-analysis. On the other hand, Metasoft69 proposed two other different methods to take into account the heterogeneity, which are Random Effects model70 and binary effects model with m-value, i.e. weight of each summary study in summary statistics.71\n\nHeterogeneity test. Heterogeneity at a locus can be reflected in the variability in population or environment. It can be relevant to gene-environment interaction and the reason behind the variability in GWAS approach of each data-set, i.e. covariable, model and approximation of GWAS between summary statistics. The heterogeneity is computed between two sets of summary statistics rather than one locus.\n\nCochran’s statistic provides a test of heterogeneity of allelic effects at SNPs j using equation (6) below.\n\nwhere N denotes study number.\n\nAlternatively, we use Q statistic to quantify the extent of heterogeneity in allelic effects across studies.72 See equation (7) below\n\nOther meta-analysis approaches. Traditionally, meta-analyses of GWAS have focused on combining results of multiple studies for similar traits. The Bayesian framework has been tested to estimate β on different phenotypes.73 MetABF tool74 has implemented a method to perform meta-analysis across genome-wide association studies of diverse phenotypes. It is important to note that a recent review on cancer suggests that it is possible to obtain “noteworthy” Bayesian results at higher p-values that are not considered statistically significant in GWAS.75\n\nStudy alignment and error trapping. Meta-analysis aggregates various summary statistics. Therefore, any error to designate the effect allele and other allele or strand issue can cause an error in estimate β. Such errors might lead to misleading meta-analysis results as it increases Q of Cohran and heterogeneity between studies.\n\nEffect size. By conducting a meta-analysis, researchers often neglect the sample size variation among different studies “true effect sizes are the same across studies”. However, in some cases, researchers introduce the correct effect size by considering the posterior probability for each study. Some software for estimating effect size are given in Table 3.\n\nMeta-analysis output. Meta-analysis provides a new set of summary statistics. For each position that has not been discarded, new statistics will be calculated. These statistics include new values for β, σ and p-value. Users should be aware of the LD and the reference population used.\n\nMore resources for meta-analysis. We recommend h3bionet/h3agwas for meta-analysis pipeline. In addition, those interested in meta-analysis are directed to a review published by Zeggini et al.77 and another review article by Evangelou and Ioannidis.63 The code below demonstrates how to use the Metasoft tool to perform meta analysis.\n\n\n\nColocalization. Colocalization is an approach used to integrate annotations with GWAS results. The annotations resources include gene expression (eQTLs), protein expression (pQTLs), exon splicing (sQTLs), DNA methylation (mQTLs), and chromatin acetylation and chromatin accessibility (caQTLs).78\n\nStatistics for colocalization studies. Several parametric and non-parametric statistics can be done for colocalization studies.79\n\nResources for colocalization studies. We recommend the following scientific resources for those who are beginners in this field:\n\n• From GWAS to function: using functional genomics to identify the mechanisms underlying complex diseases.78\n\n• Colocalization analyses of genomic elements: approaches, recommendations and challenges.79\n\n• Bayesian test for colocalisation between pairs of genetic association studies using summary statistics.80\n\n• LocusFocus: web-based colocalization for the annotation and functional follow-up of GWAS colocalization of GWAS and eQTL signals detects target genes.81\n\n• A powerful and versatile colocalization test.82\n\nUsing summary statistics from multiple phenotypes and traits. The methods for GWAS are mostly focused on single variant analysis with a single phenotype or trait. Increasing evidence shows that pleiotropy, one gene’s effect on multiple phenotypes, plays a pivotal role in many complex traits. Therefore, associating different GWAS results for multiple phenotypes can provide an extensive power by aggregating multiple weak signals.83 Different approaches have been developed to integrate dependent p-values to assess the association between a gene and multiple correlated phenotypes.83 Several tools exist to perform multi traits analysis, including Multi-Trait Analysis of GWAS (MTAG)84 and CPASSOC package.85\n\nMendelian randomisation (MR) is a statistical approach that can be defined as “the use of genetic variants as instrumental variables to investigate the effects of modifiable risk factors for disease”.86 For instance, one trait (phenotype or disease) might be affected by confounding or reverse causation rather than a conventional observational variable. Therefore, MR aims to provide a statistical frame to verify the causality between locus and phenotype and exclude pleiotropy. Such methods will provide a reliable explanation of the results.\n\nAssumption of MR. There are three main assumptions for MR.87-90 These are (i) the genetic variant that is associated with the exposure (significant association), (ii) the genetic variant that is independent of the outcome given to the exposure and all confounders (measured and unmeasured) of the exposure-outcome association, (iii) the genetic variant that is independent of factors (measured and unmeasured) that confound the exposure-outcome relationship.\n\nStatistical methods for MR. MR general strategy is to compare beta values or p-value of the same position of two or more different GWAS using related/confounding phenotype. Several methodologies have been developed for MR analysis, an example is the ratio of coefficients estimator, which can be modeled using equation (8):\n\nwhere βe represents the change in exposure per variant allele, and βo represents the change in outcome per variant allele. Another model is the two-stage least squares. It employs a two-stage regression approach with two regression models where the first stage regression’s output is used as the input of the second stage regression. More methods for MR exist, including control function estimator, limited information maximum likelihood method, verse variance weighted method, and MR-Egger method.91\n\nMR software. Several tools have been developed to compute MR, e.g GSMR (Generalised Summary-data-based Mendelian Randomisation) from GCTA, TwoSampleMR (version 0.4.20), MR-PRESSO, and MR-LDP which is integrated LD information. We recommend the following tutorial: https://bioconductor.org/packages/release/bioc/vignettes/GMRP/inst/doc/GMRP.pdf.92\n\nThe code below demonstrates how to use gcta64 tool to perform Mendelian randomisation analysis.\n\n\n\nMR and gene expression. Recently, some methods have integrated MR into GWAS and eQTL to test if the effect of gene expression is zero on the trait.93-95 These methods provide a promising way to combine GWAS summary statistics and expression data.\n\n\nOur recommended pGWAS pipeline\n\nOur proposed pGWAS pipeline consists of three main steps: preprocessing, visualization, and the downstream pGWAS analysis (refer to Figure 1).\n\nStep 1, the preprocessing step, aims to control checks and ensure a correct input file format for the downstream pGWAS analysis. The main purpose of this step is to ensure that SNPs’ positions in the GWAS summary file accurately match the genomic coordinates in the downstream reference panel if any is available. The UCSC LiftOver tool (http://genome.ucsc.edu/cgi-bin/hgLiftOver) is widely used to correct genomic position mismatches between the GWAS summary file and the reference panel. However, other options exist, including: Bioconductor rtracklayer package,96 Assembly Converter,97 NCBI Remap,98 and the CrossMap tool.99\n\nStep 2, the visualization step, aims to visualize the raw input GWAS summary data pictorially, primarily through two scatter plots: Manhattan plot and quantile-quantile (Q-Q) plot. The Manhattan plot is widely used in genomics to visualize the results of GWAS studies. In the Manhattan plot, the X-axis represents the positions on chromosomes, while the Y-axis reflects genomic association strength with a given trait. The Q-Q plot is used to check the normality of data -mainly the normality of p-values distribution. Step 2 can be completed using the qqman R package.100\n\nStep 3, of downstream pGWAS analysis, can be divided into three approaches based on their underlining data heterogeneity. First, if there is homogenous data, researchers can perform a single variant pGWAS analysis such as χ2 to test the association between a particular variant and trait. Furthermore, researchers can perform gene set analysis or network/pathway analysis to understand the biological function underlying a list of statistically significant variants. For instance, the MAGMA tool can be used to conduct gene set-based pGWAS research,101 while pathway analysis could be done using the PASCAL (Pathway scoring algorithm) tool.102 Researchers can also undertake fine-mapping analysis and MR for homogenous GWAS summary files. Second, researchers can perform PRS analysis and genetic risk score if individual-level data is available. Third, if there is heterogeneous data from numerous independent studies, a meta-analysis can be performed.\n\n\nFinal remarks\n\nThis articles demonstrates various pGWAS methods. The advancement in these pGWAS techniques solves a significant problem in our efforts to understand the vast amount of data generated and explore fundamental biology. However, several issues should be taken into consideration when performing pGWAS analysis across trans-ethnic GWAS studies. Some of these issues include: (i) heritability of the trait, (ii) GWAS sample size, (iii) polygenicity of the traits, (iv) genetic architecture of the trait, and (v) genotype-environments interactions. Furthermore, we expect that in the future many pGWAS methods will be developed to address these limitations either for a particular ethnic group or for multi-ethnic groups.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nJia P, Zheng S, Long J, et al.: dmGWAS: dense module searching for genome-wide association studies in protein–protein interaction networks. Bioinformatics. jan 2011; 27(1): 95–102. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCano-Gamez E, Trynka G: From GWAS to Function: Using Functional Genomics to Identify the Mechanisms Underlying Complex Diseases. Front. Genet. 2020; 11. Publisher Full Text\n\nKanduri C, Bock C, Gundersen S, et al.: Colocalization analyses of genomic elements: approaches, recommendations and challenges. Bioinformatics. May 2019; 35(9): 1615–1624. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiambartolomei C, Vukcevic D, Schadt EE, et al.: Bayesian test for colocalisation between pairs of genetic association studies using summary statistics. PLoS Genet. May 2014; 10(5): e1004383. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanjwani N, Wang F, Mastromatteo S, et al.: LocusFocus: Web-based colocalization for the annotation and functional follow-up of GWAS. PLoS Comput. Biol. October 2020; 16(10): e1008336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeng Y, Pan W: A powerful and versatile colocalization test. PLoS Comput. Biol. April 2020; 16(4): e1007778. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeng Y, He T, Fang R, et al.: Genome-Wide Gene-Based Multi-Trait Analysis. Front. Genet. May 2020; 11. Publisher Full Text\n\nTurley P, Walters RK, Maghzian O, et al.: Multi-trait analysis of genome-wide association summary statistics using MTAG. Nat. Genet. February 2018; 50(2): 229–237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu X, Feng T, Tayo BO, et al.: Meta-analysis of Correlated Traits via Summary Statistics from GWASs with an Application in Hypertension. Am. J. Hum. Genet. January 2015; 96(1): 21–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavies NM, Holmes MV, Smith GD: Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ. July 2018; 362: k601. Publisher Full Text\n\nTeumer A: Common Methods for Performing Mendelian Randomization. Front. Cardio. Med. May 2018; 5. Publisher Full Text\n\nGlymour MM, Tchetgen Tchetgen EJ, Robins JM: Credible Mendelian Randomization Studies: Approaches for Evaluating the Instrumental Variable Assumptions. Am. J. Epidemiol. February 2012; 175(4): 332–339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDidelez V, Meng S, Sheehan NA: Assumptions of IV Methods for Observational Epidemiology. Stat. Sci. February 2010; 25(1): 22–40. Publisher Full Text\n\nBowden J, Smith GD, Burgess S: Mendelian randomization with invalid instruments: effect estimation and bias detection through Egger regression. Int. J. Epidemiol. April 2015; 44(2): 512–525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrover S, Fabiola Del Greco M, Stein CM, et al.: Mendelian Randomization. Elston RC, editor. Statistical Human Genetics: Methods and Protocols, Methods in Molecular Biology. New York, NY: Springer; 2017; pages 581–628. Publisher Full Text\n\nCheng Q, Yang Y, Shi X, et al.: MR-LDP: a two-sample Mendelian randomization for GWAS summary statistics accounting for linkage disequilibrium and horizontal pleiotropy. NAR Geno. Bioinform. June 2020; 2(lqaa028). Publisher Full Text\n\nPorcu E, Rüeger S, Lepik K, et al.: Mendelian randomization integrating GWAS and eQTL data reveals genetic determinants of complex and clinical traits. Nat. Commun. July 2019; 10(1): 3300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichardson TG, Hemani G, Gaunt TR, et al.: A transcriptome-wide Mendelian randomization study to uncover tissue-dependent regulatory mechanisms across the human phenome. Nat. Commun. January 2020; 11(1): 185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGleason KJ, Yang F, Chen LS: A robust two-sample Mendelian Randomization method integrating GWAS with multi-tissue eQTL summary statistics. bioRxiv. June 2020; page 2020.06.04.135541.\n\nLawrence M, Gentleman R, Carey V: rtracklayer: an r package for interfacing with genome browsers. Bioinformatics. May 2009; 25(14): 1841–1842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHowe KL, Achuthan P, Allen J, et al.: Ensembl 2021. Nucleic Acids Res. November 2020; 49(D1): D884–D891. Publisher Full Text\n\nChilders JW, Back AL, Tulsky JA, et al.: REMAP: A framework for goals of care conversations. J. Oncol. Pract. October 2017; 13(10): e844–e850. PubMed Abstract \| Publisher Full Text\n\nZhao H, Sun Z, Wang J, et al.: CrossMap: a versatile tool for coordinate conversion between genome assemblies. Bioinformatics. December 2013; 30(7): 1006–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurner SD: qqman: an r package for visualizing GWAS results using q-q and manhattan plots. J. Open Source Soft. May 2018; 3(25): 731. Publisher Full Text\n\nde Leeuw CA , Mooij JM, Heskes T, et al.: MAGMA: Generalized gene-set analysis of GWAS data. PLoS Comput. Biol. April 2015; 11(4): e1004219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarbach D, Lamparter D, Quon G, et al.: Tissue-specific regulatory circuits reveal variable modular perturbations across complex diseases. Nat. Meth. March 2016; 13(4): 366–370. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "98101", "date": "15 Nov 2021", "name": "Zhe Zhang", "expertise": [ "Reviewer Expertise statistical genomics", "animal breeding" ], "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 of the manuscript entitled “Performing post-genome-wide association study analysis: overview, challenges and recommendations” give a detailed overview to the methods and tools of post GWAS analysis. The principles, key factors, tools, resources, suggestions and codes were provided to facilitate researchers who need to conduct post GWAS analysis. Overall, the manuscript is well organized and well written. It would be helpful for all target readers. There are few of my concerns that should be addressed by the authors.\nMinor comments: “Introduction” section: the authors divided common pGWAS analysis approaches into “the following three approaches ...”. Actually, nine approaches were mentioned in the main text. Hence, rewording of this paragraph is suggested.\n\n“Bayesian methods: framework” section: the equation for Bayes’ rule is not correct.\n“Colocalization analysis” section: Providing more information is suggested, since the content in the current version of this section is a bit too simple. Such as “Several parametric and non-parametric statistics…”.\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": [] }, { "id": "122325", "date": "15 Feb 2022", "name": "Yasuhiro Sato", "expertise": [ "Reviewer Expertise plant ecology and genetics" ], "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 review paper entitled “Performing post-genome-wide association study analysis: overview, challenges and recommendations” provides a practical guide to post-GWAS analyses in human complex traits and diseases. To be honest, I am not a human geneticist, but find this paper helpful for a non-human geneticist to overview a wide variety of post-GWAS analyses. While the principles are not so deeply discussed, the example code helps us use software and interpret its result. I have a few concerns on the step 3 of downstream analyses.\nMajor comments:\nUsage of the heritability estimation The authors mention “…, the ability of GWAS to detect a significant genomic position depends on heritability on phenotype, minor allele frequency…” (Introduction) and “Higher values of heritability indicate that the phenotype is explained best by the genotype, i.e., set of SNPs” (Chip heritability section). This understanding seems correct, but I am wondering why the heritability estimation should be conducted ‘after’ GWAS. If the SNP heritability is a good proxy to know the extent to which a target trait is genetically controlled, we should perform the heritability estimation ‘before’ GWAS analysis. Some web-based GWAS pipelines provide SNP heritability before performing association mapping (e.g., GWA-Portal: Seren 2018 Methods Mol Biol).\n\nGene-set analysis Because a gene-set or gene ontology enrichment analysis is widely performed in omics analyses (e.g., RNA-Seq), further introduction to the gene-set analysis would gain a value of this manuscript. As far as I know, due to the LD among SNPs, gene-set enrichment analyses in GWAS are not so straightforward as those in RNA-Seq and the other omics analyses. For example, Gowinda is designed for an unbiased gene-set enrichment analysis for GWAS (Kofler & Schlötterer 2012 Bioinformatics).\nMinor Comments:\nPage 4 of 20 “Plink”, Page 6 of 20, and elsewhere: PLINK is described as “PLINK” in some lines but also as “Plink” in the other lines. The capital letters would be better.\n\nPage 5 of 20: The equation of the Bayesian rule is incorrect. P(M) should be a numerator.\n\nPage 13 of 20 “Statistics for colocalization studies”: This subsection is not informative as it consists of only a single sentence.\n\nPage 13 of 20 “Resources for colocalization studies”: This seems a list of papers and thus repetitive of References. Please explain the points of these papers, otherwise this list is unnecessary.\n\nPage 15 of 20: Letters in Figure 1 are too small to read.\n\nAs this review focuses on tools rather than theory, it would be great if the authors could deposit their tutorial (incl. codes and example data) on some open repository.\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": [] } ]	1	https://f1000research.com/articles/10-1002
https://f1000research.com/articles/10-998/v1	01 Oct 21	{ "type": "Software Tool Article", "title": "DarkQ: continuous genomic monitoring using message queues", "authors": [ "Adrian Viehweger", "Christian Brandt", "Martin Hölzer", "Christian Brandt", "Martin Hölzer" ], "abstract": "Newly sequenced genomes are often not noticed by potential stakeholders because submission to public databases is delayed, and search options are limited. However, the discovery of genomes can be vital: in pathogen outbreaks, fast updates are essential to coordinate containment efforts and prevent further spread. Here we introduce DarkQ, a message queue that allows for instant sharing and discovery of genomes. DarkQ is released under the BSD-2 license at github.com/phiweger/darkq.", "keywords": [ "outbreak", "molecular surveillance", "peer-to-peer", "pathogen" ], "content": "Introduction\n\nMany bioinformatic tasks complement newly sequenced genomes with existing, publicly available ones. For example, when reconstructing a local pathogen outbreak, screening similar genomes can discover related ones from other sampling sites, such as hospitals nearby, and can significantly affect public health responses.1,2 However, no tool exists to monitor newly sequenced genomes and automatically identify those of interest to the user. A long delay until they are publicly available explains why many outbreak studies are retrospective and offer limited practical value to the associated outbreak response. Several components are needed: first, a “publisher” needs to be able to send genomic “messages” to a “consumer” using a simple and secure interface. Second, the genomic “message” may require limited space to avoid upload problems or extensive storage infrastructure. Third, a mechanism is needed to route messages only to interested parties, e.g., consumers that search for genomes of a particular species in a specific geography. Lastly, on receiving a relevant message, download of the associated genome should be possible. Several projects currently develop ways to share genomes effectively (wort, stark). However, to our knowledge, ours is the first end-to-end solution available to users.\n\n\nMethods\n\nDarkQ is implemented using the Nextflow workflow manager to ensure a robust, reproducible, and portable application.3 The user interface of DarkQ is similar to the popular file system service “Dropbox”; the content of a “send” directory is tracked. When a genome is added to it, it is first compressed (“sketched”) using the MinHash algorithm4,5 (sourmash, v3.5). The reduction in file size by orders of magnitude allows for efficient transmission. Together with metadata and inferred taxonomy (using sourmash), the genome sketch constitutes a “message” (Figure 1A). The receiving message queue then uses the Advanced Message Queuing Protocol (AMQP)6 to route messages (implementation: RabbitMQ, v3.8.9) onto queues, i.e. sequential groups of messages. The original genome is uploaded (“pinned”) to a decentralized, peer-to-peer network (IPFS, v0.7).7 Its content-based address is part of the genome message.\n\nA router (circle) distributes the messages to queues via routing keys (annotated arrows). Consumers (C) can use these keys to receive only a subset of messages and then further filter them with target genomes using MinHash sketches. In parallel, the genomes from the publisher are uploaded on a decentralized peer-to-peer (P2P) network. Once messages pass through the consumer’s filters, they are automatically downloaded from the P2P network. This architecture allows the effective distribution of newly sequenced genomes and enables continuous monitoring, e.g, in outbreak scenarios. (B) Use case simulation: a hospital becomes aware of a local outbreak of an XDR Klebsiella pneumoniae (Kp) isolate of subtype (ST, right metadata column) 258 carrying a plasmid-encoded KPC-2 carbapenemase. Using DarkQ, we identified 431 genomes from several countries (leaf colors) from 26 studies (left metadata column) with an average nucleotide identity (ANI) > 99.98% and identical resistance and capsule patterns (not shown). A time-dated phylogeny revealed several non-local isolates, suggesting that the outbreak reached further than previously assumed. An interactive version of the data can be found at microreact.org/project/facEFbDrgwgp9aX97nvpHq. Scale in number of SNVs.\n\nThe consumer can subscribe to messages using an arbitrary number of filters, so-called “routing keys”. Each routing key is unique and has five properties: name of sender (e.g. “phiweger”), country code (e.g. “DE”), taxon status (“found” or “mystery”), taxon level (either one of superkingdom, phylum, class, order, family, genus, species, and strain) and taxon name at that level (e.g. “Klebsiella” for genus) – these are adapted from and must conform to the Genome Taxonomy Database (GTDB, release 89).8 For example, “phiweger.DE.*.genus.klebsiella” would select all isolate genomes of the stated genus from Germany sent by the author.\n\nBecause we can estimate genome similarity using MinHash sketches,4 the consumer can quickly filter the received genome messages using target genomes, for example those belonging to a local pathogen outbreak or current research project. If this filter is passed, then the genome is automatically downloaded from the peer-to-peer network using its content hash address, which at the same time locates and validates the downloaded file. If multiple users pin the genome, download speed can increase substantially. A downstream workflow can then be connected to refine these genomes’ analysis further, enabling a complete monitoring system.\n\nThe software can be run on any UNIX-based operating system. Operation of DarkQ requires less than one Gb RAM and a single core. Details of the workflow can be found in the README file.12\n\n\nUse cases\n\nTo test DarkQ in a monitoring system, we collected and sent onto DarkQ 9,415 genomes of Klebsiella pneumoniae, a pathogen considered an urgent global threat due to extensive antimicrobial drug resistance (CDC, AR threats report, 2019).9 We simulated a consumer subscribing to all messages from the Klebsiella genus and filtering the received messages using an isolate from a local outbreak at a large tertiary hospital in 2010.10 1,461 messages met both routing key and minimum genome similarity criteria of 0.97 at a k-mer size of 51, typically used to estimate the genomic distance at the strain level.11 After downloading the original genomes from the peer-to-peer network, they were further filtered and refined, resulting in a time-dated phylogeny (Figure 1B). The consumer thus received genomes from a total of 26 studies. Two of these studies contained genomes that belonged to the same outbreak clone the consumer used to filter the genomes. Further work is needed to investigate this relationship more thoroughly; however, an initial assessment was already possible by utilizing the mechanics implemented in DarkQ. All methods used in this use case are available elsewhere.12\n\n\nConclusion\n\nDarkQ allows a user to monitor genomic data with a simple user interface, efficient genome compression, filter-based message routing, and fast download of corresponding genomes using a decentralized peer-to-peer network. The proof-of-concept outlined here scales to thousands of genomes and could be particularly valuable in the context of pathogen outbreaks. However, our approach can also be used to disseminate research more broadly.\n\n\nData availability\n\nNCBI BioProject: Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae. Accession number PRJNA742413. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA742413.\n\n\nSoftware availability\n\nSource code available from: github.com/phiweger/darkq.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.5503447.13\n\nLicense: BSD-2 license.", "appendix": "Acknowledgements\n\nWe thank Luiz Irber and C. Titus Brown (University of California, Davis) for insightful discussions of the concepts discussed in this article. An earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/2020.11.12.379560).\n\n\nReferences\n\nGrubaugh ND, et al.: Tracking virus outbreaks in the twenty-first century. Nat. Microbiol. 2019; 4: 10–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArmstrong GL, et al.: Pathogen genomics in public health. N. Engl. J. Med. 2019; 381: 2569–2580. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Tommaso P, et al.: Nextflow enables reproducible computational workflows. Nat. Biotechnol. 2017; 35: 316–319. PubMed Abstract \| Publisher Full Text\n\nOndov BD, et al.: Mash: Fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016; 17: 132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPierce NT, Irber L, Reiter T, et al.: Large-scale sequence comparisons with sourmash. F1000Res . 2019; 8: 1006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Hara J: Toward a commodity enterprise middleware: Can AMQP enable a new era in messaging middleware? A look inside standards-based messaging with AMQP. Queueing Syst. 2007; 5: 48–55. Publisher Full Text\n\nBenet J: IPFS - content addressed, versioned, P2P file system.2014.\n\nParks DH, et al.: A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat. Biotechnol. 2018; 36: 996–1004. PubMed Abstract \| Publisher Full Text\n\nWyres KL, Lam MMC, Holt KE: Population genomics of klebsiella pneumoniae. Nat. Rev. Microbiol. 2020. Publisher Full Text\n\nLippmann N, Lübbert C, Kaiser T, et al.: Clinical epidemiology of klebsiella pneumoniae carbapenemases. Lancet Infect. Dis. 2014; 14: 271–272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoslicki D, Falush D: MetaPalette: A k-mer painting approach for metagenomic taxonomic profiling and quantification of novel strain variation. mSystems . 2016; 1: e00020–e00016. Publisher Full Text\n\nViehweger A, et al.: Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing. Klebsiella pneumoniae. Reference Source\n\nViehweger A, Hölzer M: phiweger/darkq: MVP (v0.1). Zenodo. 2021; Publisher Full Text" }	[ { "id": "96159", "date": "08 Nov 2021", "name": "Tessa Pierce-Ward", "expertise": [ "Reviewer Expertise Bioinformatics", "MinHash sketching." ], "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 “DarkQ,” a peer-to-peer network and workflow for pathogen genome monitoring. The COVID-19 pandemic has underlined the need and utility for quickly sharing pathogen genomes across both hospitals and researchers. The workflow and file sharing protocol described here could fill that need, and enable real-time genome sharing.\n\nQuestions and concerns:\nClarification on (& for) intended users:\nIntended users: hospitals, researchers, etc.?\n\nExpectations for skills needed by intended users? Installation and use currently requires familiarity with command-line, and may require familiarity with conda, Nextflow, and IPFS for troubleshooting.\n\nIPFS installation may require sudo access, and is not always straightforward. Is sudo access likely to be available for relevant hospital users? Is there a way to access the data (e.g. drop a single genome and find results) without needing this level of access?\n\nPlease address potential security concerns & comment on patient privacy:\nDoes the workflow intended to be continuously run (or run, e.g. daily in an automated fashion?) in order to enable the asynchronous genome download? What security concerns might this present for users?\n\nWhile similarity searches are conducted with the sourmash MinHash sketches, the whole genome is uploaded, stored, and downloaded based on similar queries, right? This may present security concerns.\nAre there protocols are in place to prevent accidental (or purposeful) sharing of private patient data? E.g. are only microbial and viral genomes uploaded? Is human data automatically excluded? What about metadata?\n\nTaxonomic classification method:\nI would encourage the authors to explore the sourmash tax function, introduced in sourmash v4.2. A sourmash gather -> sourmash tax workflow is now recommended over sourmash lca methods for taxonomic assignment. Note that the rs202 version of GTDB database is also now available here.\n\nWhat compression (scaling) is used for sketching (1000?)? How might scaling affect small genome (e.g. viral) pathogen similarity detection?\n\nA comparison with existing real-time pathogen tracking (e.g. Nextstrain) could be very helpful. Would sharing protocols from DarkQ be combined with nextstrain analysis and visualization workflows?\n\nIs there any integration of new published data (not uploaded by message queue)? E.g. what additional databases, if any, are searched, and would those genomes regularly become available for comparison? If not, does the utility of DarkQ depend on large-scale uptake and use?\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": "96160", "date": "29 Nov 2021", "name": "Rayan Chikhi", "expertise": [ "Reviewer Expertise bioinformatics", "data structures" ], "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 DarkQ, a proof of concept software for an architecture to perform microbial surveillance. The concept is interesting and original, and appears to be well-engineered. Monitoring microbes is of high interest scientifically. However, as I mentioned, DarkQ is a proof of concept that does not provide a service which end-users can use, it only provides the blueprint for creating such a service.\n\nSome remarks:\nPlease clarify whether the pipeline is designed for monitoring only bacteria, or also supports viruses.\n\nThe sentence “Using DarkQ, we identified [..]” throws me off, as it is unclear who are the actors in this analysis. It would be beneficial to develop the hospital use case by clearly telling, in this situation, who is the producer and who is the consumer, and whether there is any other third-party involved; i.e. which actor(s) run a DarkQ instance.\n\nThe text indicates that DarkQ works just like “Dropbox”, however Figure 1 looks nothing like Dropbox. Given the originality of the approach, it could be valuable to show the workflow from a user perspective, in addition to (or instead of) the behind-the-hood architecture.\n\nI understand the software is distributed as a proof of concept, but as it stands it cannot be used by its intended users (e.g. hospitals), given that there is no central instance. Essentially, to use DarkQ today, one currently needs to act as both the producer and the consumer. This should be noted somewhere in the article.\n\n“Operation of DarkQ requires less than one Gb RAM and a single core.“ looks unlikely, as I copied genomes from “data/test” to “data/send”, darkq crashed with the message “ .command.sh: line 2: 8734 Killed\n\nsourmash lca classify --db gtdb-release89-k31.lca.json.gz --query signature.json > taxonomy.csv” which is out-of-memory error. My config is WSL2 with 4 GB RAM allocated.\n\nIs it possible for the pipeline to _miss_ some genomes? I.e. those that are too distantly related to the target genome, or those for which the sourmash signatures are insufficiently similar to the target. Some discussion on this potential limitation would be helpful.\n\nRegarding scalability and the sentence “After downloading the original genomes from the peer-to-peer network”, is it intended that all newly sequenced genomes worldwide are to be uploaded to IPFS, permanently or for a short period? If the former, it seems that DarkQ will also act as a genome repository.\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": [] } ]	1	https://f1000research.com/articles/10-998
https://f1000research.com/articles/10-637/v1	22 Jul 21	{ "type": "Research Article", "title": "Pre-notification and personalisation of text-messages to retain participants in a smoking cessation pregnancy RCT: an embedded randomised factorial trial", "authors": [ "Elizabeth Coleman", "Rachel Whitemore", "Laura Clark", "Karen Daykin", "Miranda Clark", "Rachel Whitemore", "Laura Clark", "Karen Daykin", "Miranda Clark" ], "abstract": "Background: Low response rates in randomised controlled trials can compromise the reliability of the results, so ways to boost retention are often implemented. Although there is evidence to suggest that sending a text message to participants increases retention, there is little evidence around the timing or personalisation of these messages.\n\nMethods: A two-by-two factorial SWAT (study within a trial) was embedded within the MiQuit-3 trial, looking at smoking cessation within pregnant smokers. Participants who reached their 36-week gestational follow-up were randomised to receive a personalised or non-personalised text message, either one week or one day prior to the telephone follow-up. Primary outcomes were completion rate of questionnaire via telephone. Secondary outcomes included: completion rate via any method, time to completion, and number of reminders required.\n\nResults In total 194 participants were randomised into the SWAT; 50 to personalised early text, 47 to personalised late text, 50 to non-personalised early text, and 47 to non-personalised late text. There was no evidence that timing of the text message (early: one week before; or late: one day before) had an effect on any of the outcomes. There was evidence that a personalised text would result in fewer completions via telephone compared with a non-personalised text (adjusted OR 0.44, 95% CI 0.22–0.87, p=0.02). However, there was no evidence to show that personalisation or not was better for any of the secondary outcomes.\n\nConclusion Timing of the text message does not appear to influence the retention of participants. Personalisation of a text message may be detrimental to retention; however, more SWATs should be undertaken in this field.", "keywords": [ "Randomised Controlled Trial", "Embedded Trial", "SWAT", "Retention", "text", "notification", "personalisation", "SMS" ], "content": "Introduction\n\nRandomised controlled trials (RCTs) are the ‘gold standard’ for evaluating healthcare treatments. However, it is well documented that retaining participants can be difficult and low response rates to questionnaires can compromise the reliability and generalisability of the results1,2. A study within a trial (SWAT) can be used to test interventions to improve retention of participants3.\n\nThere is research to support the concept that text messages are effective at improving response rates in trials4–7. There is insufficient evidence to determine if the timing of text messages improves questionnaire response rates, and limited papers exploring if personalisation (inclusion of the participants name) impacts response rate8–11. This SWAT aims to evaluate the effectiveness of the timing and personalisation of text messages within an RCT to add to the evidence base for both of these interventions.\n\n\nMethods\n\nThis two-by-two factorial study was embedded within the MiQuit-3 RCT. MiQuit-3 (ClinicalTrials.gov NCT03231553) is an RCT evaluating the effectiveness of a text-message, smoking cessation self-help support programme for pregnant smokers (MiQuit), and the protocol has been published previously12. This SWAT was embedded at the 36-week gestational time point. The approval for this SWAT and the MiQuit-3 trial was granted by East Midlands–Nottingham 1 Research Ethics Committee (NRES reference 13/EM/0427 and 17/EM/0327). As the SWAT was considered low risk, informed consent was not obtained from participants, and they were unaware of the SWAT. However, as part of the MiQuit-3 trial all participants consented to their anonymised data being used for further research, and being published. The SWATs are also registered with the Northern Ireland Hub for Trial Methodology Research SWAT Repository (SWATs 35 and 44; both registered December 2015).\n\nAs with all SWATs, the sample size is limited by that of the host trial, and a formal power calculation has not been carried out. The SWAT was implemented mid-way through follow up for the host trial, and all participants that had not yet had their 36-week gestational follow-up were eligible to participate in the SWAT.\n\nParticipants in MiQuit-3 were blind to their participation in this SWAT; and were randomised 1:1:1:1 to each of the four groups (see Table 1). The randomisation was undertaken by a statistician independent of the host trial, and of the staff involved in sending the texts. Block randomisation, stratified by host trial allocation, and whether they had completed the previous follow-up; with varying block sizes of 4, 8, 12 and 16.\n\nThis SWAT explored two different interventions; personalisation and timing of text messages (early; one week before follow-up, or late; one day before follow-up). Details of the text sent to participants can be found in Table one. A £5 voucher was given to all participants who completed a follow-up, additionally those who provided a saliva sample were given another £30 (£35 total).\n\nThe primary outcome was completion rate; defined as the proportion of the questionnaires completed over the telephone within the follow-up window (14 days).\n\nThe secondary outcome measures included:\n\n- Completion rate where the questionnaire was completed by any method within the follow-up window (14 days)\n\n- Time to response, defined as the number of days between the due date of the 36-week gestation follow-up and the date the questionnaire was recorded as complete\n\n- Number of attempts to contact required before the questionnaire was complete, or the maximum number of calls is reached.\n\nThe data were analysed in Stata v.15 (RRID:SCR_012763) on an intention-to-treat (ITT) basis, using two-sided tests at the 2.5% level, as this is a factorial design the Bonferroni correction was applied to allow for multiple testing13,14. Participants were excluded from the analysis if they had withdrawn prior to the time point.\n\nThe primary outcome and completion for all methods were compared using a logistic regression model. Time to response (days between questionnaire due and complete) was analysed using a Cox Proportional Hazards regression, those who compared the questionnaire early had their time set to 0.1, those did not complete were censored at either last contact date or 120 days if not contacted, and those who withdrew in the course of the SWAT were set to their withdrawal date. The assumptions for this model were assessed using Schoenfeld residuals15. The number of attempts to contact was analysed using a negative binomial regression model, due to evidence of overdispersion. All models were adjusted for host trial allocation, whether the participant had completed the previous follow-up, age, and both SWAT intervention allocations. All models were repeated with the inclusion of an interaction term to explore any possible interactions between the two SWAT interventions; with a significance level of 5%.\n\nStata is proprietary software: a freely available alternative software that could be used to undertake this analysis is RStudio (RRID:SCR_000432 )16.\n\n\nResults\n\nIn total, 194 participants were randomised into the SWAT; 50 received the personalised text and early notification, 47 received the personalised text and late notification, 50 received the non-personalised text and early notification, and 47 received the non-personalised text and late notification17. Five participants withdrew prior to the implementation of the SWAT and are not included in the analysis. Additional participants were excluded from the analysis, where the covariates required for the model were not provided. Three participants were not contacted due to difficulties/adverse events associated with their pregnancy but are still included in the analysis under ITT principles. The flow of participants can be seen in Figure 1. Baseline characteristics by SWAT arm and overall, can be found in Table 2.\n\nThe overall completion rate by telephone was 66.1% (125/189) within 14 days of the due date. There were similar completion rates of the questionnaire via telephone within three groups; 50.0% for personalised early (24/48), 52.3% (23/44) for personalised late, and 58.0% (29/50) of non-personalised early and was slightly higher in the non-personalised late group, 66.0% (31/47).\n\nThere was no evidence for a difference in completion rate via telephone for the timing of the text message; adjusted odds ratio (OR) 0.86 (95% CI 0.44–1.67, p=0.65). There was evidence to suggest a difference in completion rate via telephone adjusted OR 0.44 (0.22–0.87, p=0.02) which implies those who received the non-personalised text were more likely to complete the questionnaire when completing via the telephone. Full details can be found in Table 3.\n\n* OR = Odds Ratio\n\nSecondary outcomes:\n\nFull details for all secondary outcomes can be found in Table 4.\n\n* OR = Odds Ratio, IRR = Incidence Rate Ratio, HR = Hazards Ratio\n\nResponse rates for all methods. There were similar completion rates of the questionnaire within each of the four groups; 64.6% for personalised early (31/48), 63.6% (28/44) for personalised late, 66.0% for early (33/50) and 70.2% (33/47) of non-personalised.\n\nThere is some, non statistically significant, evidence to suggest that there may be a difference in response rate for personalised versus non-personalised text reminders; adjusted OR 0.61 (95% CI 0.30–1.24, p=0.17), in favour of the non-personalised text messages. However, there was no evidence to suggest there was a difference in response rates in participants who received an early or late text message reminder; adjusted OR 1.06 (95% CI 0.52–2.15, p=0.87).\n\nNumber of attempts to contact required. The average number of calls required was 3.0 for all participants, with the average similar for each group (3.3 for both personalised early, 3.2 for personalised late, 3.1 for non-personalised early and 2.7 for non-personalised late). The maximum number of calls was reached for 55 of the 174 participants (31.3%) and was similar across three groups (38.6% for personalised and early, 31.7% for personalised and late, 31.1% for non-personalised early) and slightly lower in the non-personalised late group, 25%.\n\nThere was no evidence of a difference in number of contacts required between those who received an early text or a late text (p=0.45). There is also no evidence to suggest a difference between those who received a personalised or non-personalised text (p=0.23); adjusted incidence rate ratio (IRR)=1.14.\n\nTime to respond. The average time to respond was 6.2 days (ranging from 5 days early to 103 days late). This was similar between those who received a personalised text (8.2 days for early versus 7.1 days for late) and those who received the non-personalised text (4.9 days for early versus 4.7 days for late), but there is a slight difference between those who received personalised or non-personalised texts.\n\nThere was no evidence of a difference in time taken to respond between those who received the text early or late (p=0.99) or those who received a personalised or non-personalised text (p=0.12); suggesting that neither timing nor personalisation of the text message reminder affect the time taken to complete the questionnaire. The assumptions for the model held when examined using Schoenfeld residuals (p=0.66).\n\nInteraction terms. All of the models were re-run with the inclusion of any interaction term between the two SWAT allocations. There was no evidence of an interaction for the completion rate, both by phone only (p=0.57) and all methods (p=0.54). There was also no evidence of an interaction for the number of contacts required (p=0.69), or the time to respond (p=0.88).\n\nThere were 1002 participants who were randomised into the MiQuit-3 trial. Of the 777 who were not included in the SWAT, and were due a 36-week follow-up, 499 completed the questionnaire (64.2%). This is similar to the completion rate for the participants in the SWAT (overall 66.1%).\n\n\nDiscussion\n\nThis factorial SWAT showed that the timing of the text message reminder had no effect on the response rate, the time to response, or the number of attempted to contact required; these results mirror what Partha et al. reported in their work8. It also showed that personalised texts have no effect on response time, or number of attempts required. It did show that there was some evidence that sending a non-personalised text message reminder would have a larger increase in response than sending personalised text messages did. Cochrane et al. found no statistically significant difference in their study, but results favoured the non-personalised text11. As our work was conducted in a female-only population, who were between 17 and 41 years of age, the results here are only directly related to this population. Equally, as the SWAT was not powered to detect a difference, more SWATs should be undertaken in this area to allow the results to be combined in a pooled analysis to determine the true effect of the interventions, consider the effects on a wider population, and overall effectiveness.\n\n\nData availability\n\nFigshare: Underlying data for ‘Pre-notification and personalisation of text-messages to retain participants in a smoking cessation pregnancy RCT: an embedded randomised factorial trial’. https://doi.org/10.6084/m9.figshare.14224319.v117\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: CONSORT checklist for ‘Pre-notification and personalisation of text-messages to retain participants in a smoking cessation pregnancy RCT: an embedded randomised factorial trial’. https://doi.org/10.6084/m9.figshare.14229647.v118", "appendix": "Acknowledgements\n\nThe authors would like to acknowledge Professor Tim Coleman, the Chief Investigator for the MiQuit-3 trial, and members of the MiQuit-3 trial involved in implementing the SWAT into the trial.\n\n\nReferences\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials. 2014; 15(1)1: 399. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdamson J, Hewitt CE, Torgerson DJ: Producing better evidence on how to improve randomised controlled trials. BMJ. 2015; 351: h4923. PubMed Abstract \| Publisher Full Text\n\nTreweek S, Bevan S, Bower P, et al.: Trial Forge Guidance 1: what is a Study Within A Trial (SWAT)? Trials. 2018; 19(1): 139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMan MS, Tilbrook HE, Jayakody S, et al.: Electronic reminders did not improve postal questionnaire response rates or response times: a randomized controlled trial. J Clin Epidemiol. 2011; 64(9): 1001–1004. PubMed Abstract \| Publisher Full Text\n\nAshby R, Turner G, Cross B, et al.: A randomized trial of electronic reminders showed a reduction in the time to respond to postal questionnaires. J Clin Epidemiol. 2011; 64(2): 208–212. PubMed Abstract \| Publisher Full Text\n\nClark L, Ronaldson S, Dyson L, et al.: Electronic prompts significantly increase response rates to postal questionnaires: a randomized trial within a randomized trial and meta-analysis. J Clin Epidemiol. 2015; 68(12): 1446–1450. PubMed Abstract \| Publisher Full Text\n\nNaughton F, Riaz M, Sutton S: Response Parameters for SMS Text Message Assessments Among Pregnant and General Smokers Participating in SMS Cessation Trials. Nicotine Tob Res. 2016; 18(5): 1210–1214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarathy PP, Kottam L, Parker A, et al.: Timing of electronic reminders did not improve trial participant questionnaire response: a randomized trial and meta-analyses. J Clin Epidemiol. 2020; 122: 70–77. PubMed Abstract \| Publisher Full Text\n\nKeding A, Brabyn S, MacPherson H, et al.: Text message reminders to improve questionnaire response rates in RCTs: findings from three randomised sub-studies. Trials. 2015; 16(Suppl. 2): 103. PubMed Abstract \| Publisher Full Text\n\nMitchell AS, Cook L, Dean A, et al.: An embedded randomised controlled retention trial of personalised text messages compared to non-personalised text messages in an orthopaedic setting [version 1; peer review: 1 approved]. F1000Res. 2020; 9: 591. Publisher Full Text\n\nCochrane A, Welch C, Fairhurst C, et al.: An evaluation of a personalised text message reminder compared to a standard text message on postal questionnaire response rates: an embedded randomised controlled trial [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitemore R, Leonardi-Bee J, Naughton F, et al.: Effectiveness and cost-effectiveness of a tailored text-message programme (MiQuit) for smoking cessation in pregnancy: study protocol for a randomised controlled trial (RCT) and meta-analysis. Trials. 2019; 20(1): 280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStataCorp: Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC, 2017. Reference Source\n\nBland JM, Altman DG: Multiple significance tests: the Bonferroni method. BMJ. 1995; 310(6973): 170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoenfeld D: Partial residuals for the proportional hazards regression model. Biometrika. 1982; 69(1): 239–241. Publisher Full Text\n\nRStudio Team: RStudio: Integrated Development for R. RStudio, PBC, Boston, MA, 2020. Reference Source\n\nColeman E: MiQuit_SWAT_Data.csv. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14224319.v1\n\nColeman E: MiQuit SWAT CONSORT Checklist. figshare. Journal contribution. 2021. http://www.doi.org/10.6084/m9.figshare.14229647.v1" }	[ { "id": "90364", "date": "29 Jul 2021", "name": "Phil J. Edwards", "expertise": [ "Reviewer Expertise Evidence-based Data Collection" ], "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 reports the results of a 2x2 factorial trial nested within an RCT of MiQuit, a text-message, smoking cessation self-help support programme for pregnant smokers. The nested factorial trial sought to evaluate the effect on response to a questionnaire administered over the telephone of two interventions applied to a pre-notification text message: (i) personalisation (text begins “Hi [name]”, or not), and (ii) timing of text messages (early: one week before follow-up, or late: one day before follow-up).\n194 participants who had not yet had their 36-week gestational follow-up were randomised into this nested trial. Analysis of intervention effects was conducted using a logistic regression model.\nThe study found some evidence that personalised text messages reduced response (OR = 0.44; 95% CI 0.22 to 0.87; p=0.02); and no evidence that the earlier timing of text messages had an effect on response (OR = 0.86; 95% CI 0.44 to 1.67; p=0.65).\nThe manuscript is a clear and concise account of the study, citing the current literature. The study design is appropriate and the work appears to be technically sound. The authors appropriately recognise that their results are only generalisable to their study population (females aged 17 to 41 years).\nThe conclusions are adequately supported by the results, and the study makes a useful contribution to the data collection literature.\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": "7136", "date": "30 Sep 2021", "name": "Elizabeth Coleman", "role": "Author Response", "response": "Thank you for reviewing this article." } ] }, { "id": "90184", "date": "04 Aug 2021", "name": "Frances Shiely", "expertise": [ "Reviewer Expertise Epidemiology", "Trial Methodology", "SWATs" ], "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 This was a SWAT within the MiQuit-3 trial. The purpose of the SWAT was to establish if the timing or personalisation of text messages increases completion of a questionnaire. Study design was a 2 x 2 factorial SWAT. Participants who reached their 36-week gestational follow-up were randomised to receive a personalised or non-personalised text message, either one week or one day prior to the telephone follow-up. Primary outcome was completion rate of questionnaire via telephone. Secondary outcomes included: completion rate via any method, time to completion, and number of reminders required. The authors concluded that timing of the text message did not appear to influence the retention of participants. The authors concluded that personalisation of a text message may be detrimental to retention; however, more SWATs should be undertaken in this field.\nMajor Comments My first comment is on the title, the purpose of the study and the conclusions drawn. The title, correctly refers to the retention of participants in the smoking cessation pregnancy RCT. The purpose of the SWAT though is to evaluate completion of a questionnaire, not retention in the host trial, as claimed in the conclusion in the abstract. I think this is conflated throughout and the authors need to consider this carefully and amend their paper. In fact, the registered SWATs in the NI SWAT repository give the outcomes in both as questionnaire completion. So therefore the conclusions drawn in the abstract and in the discussion are not supported by the data.\n\nAbstract\n\"There was evidence that a personalised text would result in fewer completions via telephone compared with a non-personalised text (adjusted OR 0.44, 95% CI 0.22–0.87, p=0.02)\". This statement is confusing. The research question is personalised text message versus non-personalised text message. If using via telephone, then it should be also say non-personalised text via telephone.\n\nAlso, use text message, rather than text, throughout.\n\n\"Personalisation of a text message may be detrimental to retention\". I don't think your results support a statement this strong. Firstly, this is not a trial with an adequate sample size to make this claim. Secondly, when you included all methods of receiving the questionnaire (I think this is correct interpretation but it is challenging to establish in the current version of the paper as the detail on all methods is unclear) you did not find that personalised or non-personalised texts mattered. At best you can say, personalisation of a text message appears to affect questionnaire completion via telephone.\nIntroduction\nI like the succinct introduction.\nMethods\nThe methods says \"The SWATs are also registered with the Northern Ireland Hub for Trial Methodology Research SWAT Repository (SWATs 35 and 44; both registered December 2015). It's a little confusing to the reader. It looks like you're conducting two SWATs. I understand what you have taken both of these SWATS and conducted the two in a 2 x 2 factorial design. However, make this clear to the reader.\n\nReplace the phrase \"carried out\" with conducted throughout. I know the phrase carried out is used all the time in literature but it is not correct unless you are describing physically carrying an object.\n\nI would use the word women rather than participants, given that it is a trial of pregnant women.\n\nThe phrase \"all participants that had not yet had their 36-week gestational follow-up were eligible to participate\" suggests that some people were more than 36 weeks pregnant. Is this the case? Otherwise you could simplify it and say women who were 36 weeks pregnant. It's important to be clear on the timing of the intervention given that this is one of your outcomes.\n\n\"Participants in MiQuit-3 were blind to their participation in this SWAT\". Blinding is different to being unaware. Were they both blinded and unaware?\n\n\"The randomisation was undertaken by a statistician independent of the host trial, and of the staff involved in sending the texts\". Explain how the statistician did the randomisation, e.g. computer generated. Also, explain how he communicated that randomisation to the researcher assigning the women to each group.\n\n\"Block randomisation, stratified by host trial allocation, and whether they had completed the previous follow-up; with varying block sizes of 4, 8, 12 and 16\". This is not a sentence.\n\n\"A £5 voucher was given to all participants who completed a follow-up…\". Was this part of the host trial or the SWAT?\n\n\"…additionally those who provided a saliva sample were given another £30 (£35 total).\" Where does the saliva sample come into it? Is this part of the host trial? Explain in the paper.\n\nHow did you decide on how many to include for the SWAT. I accept that a sample size calculation is not required but a line in the paper on why you decided on (it appears to be 200) would be useful.\n\nIn the secondary outcome, explain what you mean by completed by any method.\n\n\"Time to response, defined as the number of days between the due date of the 36-week gestation follow-up and the date the questionnaire was recorded as complete\". Are you certain all follow-up calls were made in 24 hours?\nStatistical Analysis\n\"The primary outcome was completion rate; defined as the proportion of the questionnaires completed over the telephone within the follow-up window (14 days)\". However, you then go on to say that you used logistic regression to analyse this. Logistic regression is not suitable for four categories. I suspect what you mean is that you compared the completion rates across the two personalised/not personalised and again early/late. I can see you did this from the table. However, you need to articulate that in the text because it is currently confusing.\n\nIn statistical analysis, a full stop after level and a new sentence for \"As this is a factorial...\"\n\nFull stop required in this sentence too. Also, I suspect the word \"compared\" in this sentence should read completed. - \"Time to response (days between questionnaire due and complete) was analysed using a Cox Proportional Hazards regression, those who compared the questionnaire early had their time set to 0.1, those did not complete were censored at either last contact date or 120 days if not contacted, and those who withdrew in the course of the SWAT were set to their withdrawal date\".\n\nAgain the following is not a sentence, \"All models were repeated with the inclusion of an interaction term to explore any possible interactions between the two SWAT interventions; with a significance level of 5%.\"\nResults\n\"Additional participants were excluded from the analysis, where the covariates required for the model were not provided\". What additional participants? Quantify and explain please. Why were the covariates not 'provided'? Explain please.\n\n\"Three participants were not contacted due to difficulties/adverse events associated with their pregnancy but are still included in the analysis under ITT principles\". Commas are required to make sense of the sentence.\n\nIn your flow chart, what does primary refer to, and proximity? Add an explanation or use a term that explains a little better. Also in the flow chart, you say response rate but provide the number of participants. This is not a rate.\n\nIn the primary outcome, continue with your phraseology - the 14-day follow-up window rather than \"within 14 days of the due date\".\n\nI find the writing of the results very confusing. This sentence below suggests the outcome was completion rate via telephone versus completion rate via something else. \"There was evidence to suggest a difference in completion rate via telephone adjusted OR 0.44 (0.22–0.87, p=0.02) which implies those who received the non-personalised text were more likely to complete the questionnaire when completing via the telephone\". I'm wondering why you keep saying via telephone. It is particularly confusing when explaining the results. The last part above again says … those who received the non-personalised text were more likely to complete the questionnaire when completing via the telephone\". It looks like the method of completion is the purpose of the study.\n\nWhen you use the phrase \"were more likely to\", you must give the details of the comparison, i.e., more likely than who?\n\nIt is implied, but not adequately explained, that some women completed the questionnaire by some other means. It is not clear how this was handled in terms of the numbers analysed throughout the study and this needs to be explained.\n\nFor your tables 3 and 4, add the number of women. Why does the left column say \"response rate for all methods\" when the primary outcome is defined as the proportion of the questionnaires completed over the telephone…\"\n\nThe heading in the results section, \"Response rates for all method\", do you mean completion rates for all methods? A response rate is different.\n\nIf you hang your hat on statistically significant evidence, by quoting CIs and p-values, to establish if your SWAT was effective, or not, then the following has no place in your paper. \"There is some, non statistically significant, evidence to suggest that there may be a difference in response rate for personalised versus non-personalised text reminders; adjusted OR 0.61 (95% CI 0.30–1.24, p=0.17), in favour of the non-personalised text messages\". You cannot say there is non-statistically significant evidence and then support that statement with statistics! Remove this please.\n\nThe heading, \"Number of attempts to contact required\". Replace with, Number of attempts required to contact the women.\n\nWhat do you mean by the maximum number of calls, as stated here \"The maximum number of calls was reached for 55 of the 174 participants…\"?\n\nContacts required is a new term introduced here \" There was no evidence of a difference in number of contacts required\". What do you mean by it?\n\n\"The average time to respond was 6.2 days (ranging from 5 days early to 103 days late)\". Respond to what, the phone call or the text, or the questionnaire?\n\n\"This was similar between those who received a personalised text (8.2 days for early versus 7.1 days for late) and those who received the non-personalised text (4.9 days for early versus 4.7 days for late), but there is a slight difference between those who received personalised or non-personalised texts\". If it is similar, how can there be a slight difference? What point are you making here?\n\nInclude the number of participants in the MiQuit Trial earlier in the paper when discussing the 200 randomised for the SWAT.\nDiscussion\n\"It did show that there was some evidence that sending a non-personalised text message reminder would have a larger increase in response than sending personalised text messages did\". This is misleading because it was not the case when all methods of questionnaire were included. Please amend the statement.\n\nThe final sentence of the discussion needs to be reviewed. What do you mean by overall effectiveness?\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?\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": [ { "c_id": "7135", "date": "30 Sep 2021", "name": "Elizabeth Coleman", "role": "Author Response", "response": "The authors would like to thank you for a very thorough review of this article - please see responds to your comments and suggestions below. We have amended the article to incorporate your corrections and suggestions were we pleased it was suitable to do so. Summary This was a SWAT within the MiQuit-3 trial. The purpose of the SWAT was to establish if the timing or personalisation of text messages increases completion of a questionnaire. Study design was a 2 x 2 factorial SWAT. Participants who reached their 36-week gestational follow-up were randomised to receive a personalised or non-personalised text message, either one week or one day prior to the telephone follow-up. Primary outcome was completion rate of questionnaire via telephone. Secondary outcomes included: completion rate via any method, time to completion, and number of reminders required. The authors concluded that timing of the text message did not appear to influence the retention of participants. The authors concluded that personalisation of a text message may be detrimental to retention; however, more SWATs should be undertaken in this field. Thank you for this well written summary of the paper. Major Comments My first comment is on the title, the purpose of the study and the conclusions drawn. The title, correctly refers to the retention of participants in the smoking cessation pregnancy RCT. The purpose of the SWAT though is to evaluate completion of a questionnaire, not retention in the host trial, as claimed in the conclusion in the abstract. I think this is conflated throughout and the authors need to consider this carefully and amend their paper. In fact, the registered SWATs in the NI SWAT repository give the outcomes in both as questionnaire completion. So therefore the conclusions drawn in the abstract and in the discussion are not supported by the data. The authors have amended the title of the paper, and changed references to retention within the paper to completion of questionnaires throughout. Alongside making the other suggested changes by the reviewer, we hope to have addressed any concerns in relation to the conclusions drawn. Abstract \"There was evidence that a personalised text would result in fewer completions via telephone compared with a non-personalised text (adjusted OR 0.44, 95% CI 0.22–0.87, p=0.02)\". This statement is confusing. The research question is personalised text message versus non-personalised text message. If using via telephone, then it should be also say non-personalised text via telephone. The wording has been amended to attempt to make this statement clearer, and also reference the results for any data collection method. Also, use text message, rather than text, throughout. The term ‘text’ has been changed to ‘text message’ throughout the paper. \"Personalisation of a text message may be detrimental to retention\". I don't think your results support a statement this strong. Firstly, this is not a trial with an adequate sample size to make this claim. Secondly, when you included all methods of receiving the questionnaire (I think this is correct interpretation but it is challenging to establish in the current version of the paper as the detail on all methods is unclear) you did not find that personalised or non-personalised texts mattered. At best you can say, personalisation of a text message appears to affect questionnaire completion via telephone. This sentence has been altered to include ‘data was collected only via the telephone’, and now also references that the results do not hold for data collection via all methods. The results are correct as finding of this work, and the conclusion of the abstract references that more work is needed in this area so no other alterations have been made in regards to this comment. Introduction I like the succinct introduction. Thank you for this comment. Methods The methods says \"The SWATs are also registered with the Northern Ireland Hub for Trial Methodology Research SWAT Repository (SWATs 35 and 44; both registered December 2015). It's a little confusing to the reader. It looks like you're conducting two SWATs. I understand what you have taken both of these SWATS and conducted the two in a 2 x 2 factorial design. However, make this clear to the reader. Additional words have been added to this paragraph to make it clear that this was a factorial SWAT involving these two SWATs. Replace the phrase \"carried out\" with conducted throughout. I know the phrase carried out is used all the time in literature but it is not correct unless you are describing physically carrying an object. The phase carried out has been replaced with conducted. I would use the word women rather than participants, given that it is a trial of pregnant women. We thank you for the suggestion – however, the authors have decided not to make this change. The protocol for the MiQuit-3 trial refers to those involved both as participants, and as women. The term participants is not incorrect, and as it is made clear that this work is being done in pregnant women in both the introduction and conclusions of this paper, we feel it does not need to be re-emphasised throughout the whole paper. The phrase \"all participants that had not yet had their 36-week gestational follow-up were eligible to participate\" suggests that some people were more than 36 weeks pregnant. Is this the case? Otherwise you could simplify it and say women who were 36 weeks pregnant. It's important to be clear on the timing of the intervention given that this is one of your outcomes. This sentence references those who were eligible to be included in the SWAT. Some participants in the host trial would have been more than 36 weeks pregnant (or have had their baby/babies) when the SWAT was implement, and thus they were not eligible to be included in the SWAT. The wording of this paragraph has been altered in relation to this comment, and the reviewer comment regarding the number of participants involved in the SWAT, hopefully this has made it clearer. \"Participants in MiQuit-3 were blind to their participation in this SWAT\". Blinding is different to being unaware. Were they both blinded and unaware? A correction has been made in regards to the unawareness and blinding of participants. \"The randomisation was undertaken by a statistician independent of the host trial, and of the staff involved in sending the texts\". Explain how the statistician did the randomisation, e.g. computer generated. Also, explain how he communicated that randomisation to the researcher assigning the women to each group. Details have been added to explain how the randomisation was generated (Stata) and how the randomisation sequence was used. \"Block randomisation, stratified by host trial allocation, and whether they had completed the previous follow-up; with varying block sizes of 4, 8, 12 and 16\". This is not a sentence. Corrections have been made to this sentence. \"A £5 voucher was given to all participants who completed a follow-up…\". Was this part of the host trial or the SWAT? Detail has been added to clarify that this is part of the host trial \"…additionally those who provided a saliva sample were given another £30 (£35 total).\" Where does the saliva sample come into it? Is this part of the host trial? Explain in the paper. Detail has been added to explain why a saliva sample was given, and that it, and associated voucher were part of the host trial. How did you decide on how many to include for the SWAT. I accept that a sample size calculation is not required but a line in the paper on why you decided on (it appears to be 200) would be useful. Further details have been added to explain why only 200 were included. In the secondary outcome, explain what you mean by completed by any method. Details have been added to explain what other methods of completion could be used. \"Time to response, defined as the number of days between the due date of the 36-week gestation follow-up and the date the questionnaire was recorded as complete\". Are you certain all follow-up calls were made in 24 hours? The date of each attempt to contact was recorded. However this outcome captures completion by any follow-up method. Statistical Analysis \"The primary outcome was completion rate; defined as the proportion of the questionnaires completed over the telephone within the follow-up window (14 days)\". However, you then go on to say that you used logistic regression to analyse this. Logistic regression is not suitable for four categories. I suspect what you mean is that you compared the completion rates across the two personalised/not personalised and again early/late. I can see you did this from the table. However, you need to articulate that in the text because it is currently confusing. A logistic model can only deal with two levels of an outcome; it can handle multiple levels of a covariate. The outcome was completion (yes/no), and the swat allocations are included as covariates in the model. Each swat allocation is include as a separate covariate – as stated in the analysis section, providing two, two-level covariates. Thus the model does not include a four level variable, as you have suggested. Had swat allocation been included as a four level covariate, the model would have been able to do this – but it would not have provided an OR for each of the two SWAT interventions separately. This method is the appropriate method for analysing a binary outcome for a factorial SWAT, as such no changes have been made to the text. In statistical analysis, a full stop after level and a new sentence for \"As this is a factorial...\" Thank you for suggesting this correction – it has been made. Full stop required in this sentence too. Also, I suspect the word \"compared\" in this sentence should read completed. - \"Time to response (days between questionnaire due and complete) was analysed using a Cox Proportional Hazards regression, those who compared the questionnaire early had their time set to 0.1, those did not complete were censored at either last contact date or 120 days if not contacted, and those who withdrew in the course of the SWAT were set to their withdrawal date\". Corrections to this sentence have been made. Again the following is not a sentence, \"All models were repeated with the inclusion of an interaction term to explore any possible interactions between the two SWAT interventions; with a significance level of 5%.\" Additional words have been added. Results \"Additional participants were excluded from the analysis, where the covariates required for the model were not provided\". What additional participants? Quantify and explain please. Why were the covariates not 'provided'? Explain please. The text in this sentence has been altered to make this clearer. \"Three participants were not contacted due to difficulties/adverse events associated with their pregnancy but are still included in the analysis under ITT principles\". Commas are required to make sense of the sentence. Changes have been made to this sentence. In your flow chart, what does primary refer to, and proximity? Add an explanation or use a term that explains a little better. Also in the flow chart, you say response rate but provide the number of participants. This is not a rate. The flow diagram has been updated to include consistent terminology. The numbers in the flow diagram are referencing the numbers within each analysis. In the primary outcome, continue with your phraseology - the 14-day follow-up window rather than \"within 14 days of the due date\". This term has been made consistent throughout the paper. I find the writing of the results very confusing. This sentence below suggests the outcome was completion rate via telephone versus completion rate via something else. \"There was evidence to suggest a difference in completion rate via telephone adjusted OR 0.44 (0.22–0.87, p=0.02) which implies those who received the non-personalised text were more likely to complete the questionnaire when completing via the telephone\". I'm wondering why you keep saying via telephone. It is particularly confusing when explaining the results. The last part above again says … those who received the non-personalised text were more likely to complete the questionnaire when completing via the telephone\". It looks like the method of completion is the purpose of the study. The main data collection method for MiQuit-3 was via the telephone. As such, within the SWAT protocol for this factorial SWAT, it was decided that the primary outcome would focus specifically on data collected via this method, and as a secondary explore data collected via any method. The results for the primary and associated secondary outcome have been rewritten slightly to clarify that the results are only including those who completed it via the telephone, as opposed to comparing those who did it via the telephone with something else. When you use the phrase \"were more likely to\", you must give the details of the comparison, i.e., more likely than who? Additional words have been added for clarification. It is implied, but not adequately explained, that some women completed the questionnaire by some other means. It is not clear how this was handled in terms of the numbers analysed throughout the study and this needs to be explained. For the secondary outcomes (time to completion, and number of attempts to contact) there have been no additional adjustments or changes to the analysis due to the method of data collection. The completion method is only relevant to the primary outcome, where the trial team detailed that their main method of data collection was via the telephone, and thus the primary outcome explores this. However, to explore the effectiveness of the interventions on any data collection method (as is more typically done in RCTs where a number of methods are utilised) a secondary outcome was to explore this via any method. For your tables 3 and 4, add the number of women. Why does the left column say \"response rate for all methods\" when the primary outcome is defined as the proportion of the questionnaires completed over the telephone…\" Response rate, completion rate and retention rate are all frequently used synonymously within the SWAT world. As such the terminology within this paper has switched between the terms. In response to this comment the terminology has been made consistent. The heading in the results section, \"Response rates for all method\", do you mean completion rates for all methods? A response rate is different. Response rate has been changed to completion rate throughout. If you hang your hat on statistically significant evidence, by quoting CIs and p-values, to establish if your SWAT was effective, or not, then the following has no place in your paper. \"There is some, non statistically significant, evidence to suggest that there may be a difference in response rate for personalised versus non-personalised text reminders; adjusted OR 0.61 (95% CI 0.30–1.24, p=0.17), in favour of the non-personalised text messages\". You cannot say there is non-statistically significant evidence and then support that statement with statistics! Remove this please. This section has been reworded to reflect your suggestion. The heading, \"Number of attempts to contact required\". Replace with, Number of attempts required to contact the women. The authors have declined to make this change. In some instances the participant was not contacted, therefore it would be incorrect for the heading to referring the number required to contact them, when some were not contactable. Additionally, as explained in a previous comment we will not be changing participant to women. What do you mean by the maximum number of calls, as stated here \"The maximum number of calls was reached for 55 of the 174 participants…\"? This has been clarified both when describing the outcome, and in the results. The researcher tried to contact the participant a maximum of 6 times. Contacts required is a new term introduced here \" There was no evidence of a difference in number of contacts required\". What do you mean by it? This is referring the number of attempts to contact – a word had been omitted, and has been corrected. \"The average time to respond was 6.2 days (ranging from 5 days early to 103 days late)\". Respond to what, the phone call or the text, or the questionnaire? Terminology has been changed. \"This was similar between those who received a personalised text (8.2 days for early versus 7.1 days for late) and those who received the non-personalised text (4.9 days for early versus 4.7 days for late), but there is a slight difference between those who received personalised or non-personalised texts\". If it is similar, how can there be a slight difference? What point are you making here? The wording of the sentence has been changed to clarify this point. The sentence was meant to emphasis the difference in time between the personalised/non-personalised, whilst highlighting the similarities between early/late. Include the number of participants in the MiQuit Trial earlier in the paper when discussing the 200 randomised for the SWAT. The authors have included this figure (n=1002) earlier on in the paper. Discussion \"It did show that there was some evidence that sending a non-personalised text message reminder would have a larger increase in response than sending personalised text messages did\". This is misleading because it was not the case when all methods of questionnaire were included. Please amend the statement. Additional words have been added to explain that these results are only valid for telephone completion The final sentence of the discussion needs to be reviewed. What do you mean by overall effectiveness? These words have been removed." } ] } ]	1	https://f1000research.com/articles/10-637
https://f1000research.com/articles/10-87/v1	10 Feb 21	{ "type": "Research Article", "title": "Recommendations from long-term care reports, commissions, and inquiries in Canada", "authors": [ "Eric K. C. Wong", "Trina Thorne", "Carole Estabrooks", "Sharon E. Straus", "Eric K. C. Wong", "Trina Thorne", "Carole Estabrooks" ], "abstract": "Background: Multiple long-term care (LTC) reports have issued similar recommendations for improvement across Canadian LTC homes. Our primary objective was to identify the most common recommendations made over the past 10 years. Our secondary objective was to estimate the total cost of studying LTC issues repeatedly from 1998 to 2020. Methods: The qualitative and cost analyses were conducted in Canada from July to October 2020. Using a list of reports, inquiries and commissions from The Royal Society of Canada Working Group on Long-Term Care, we coded recurrent recommendations in LTC reports. We contacted the sponsoring organizations for a cost estimate, including direct and indirect costs. All costs were adjusted to 2020 Canadian dollar values. Results: Of the 80 Canadian LTC reports spanning the years of 1998 to 2020, 24 (30%) were based on a national level and 56 (70%) were focused on provinces or municipalities. Report length ranged from 4 to 1491 pages and the median number of contributors was 14 (interquartile range, IQR, 5–26) per report. The most common recommendation was to increase funding to LTC to improve staffing, direct care and capacity (67% of reports). A median of 8 (IQR 3.25–18) recommendations were made per report. The total cost for all 80 reports was estimated to be $23,626,442.78. Conclusions: Problems in Canadian LTC homes and their solutions have been known for decades. Despite this, governments and non-governmental agencies continue to produce more reports at a monetary and societal cost to Canadians.", "keywords": [ "long-term care", "nursing home", "older adults", "health policy", "research waste", "Canada", "commissions", "inquiries", "cost analysis", "quantitative analysis", "COVID-19" ], "content": "Introduction\n\nThe COVID-19 pandemic led to a high proportion of deaths in Canadian long-term care homes (LTCH) compared with those of other developed countries. The proportion of deaths from COVID-19 in LTCH in Canada was 81% compared with a mean of 42% in other Organisation for Economic Cooperation and Development (OECD) countries in the initial months of the pandemic1. This statistic is surprising since Canada is considered to have a relatively low number of COVID-19 deaths overall2. The difference in mortality rate was attributed to pandemic preparedness, integration of services (LTCH, public health and hospitals), funding and resources, daily care hours for residents, and comprehensiveness of inspections3.\n\nDuring the early months of COVID-19, the media reported a shortage of direct care providers and personal protective equipment (PPE) in Canadian LTCHs, which led to residents suffering from a lack of basic personal care and delays in identifying medical problems4. Reduced staffing levels and wage compression in Canada’s LTC sector compelled individuals to work at more than one facility to make a living wage. Working at multiple nursing homes contributed to the spread of COVID-19 infection5, and although restricting employment to one facility reduced the number of outbreaks, it also exacerbated pre-existing work force shortages6. Furthermore, lower staffing levels and direct care hours are associated with increased rates of infection and hospital admission among residents7, with this same trend observed in LTCHs with COVID-19 outbreaks3,5,8.\n\nIn April, the Royal Society of Canada Working Group on Long-Term Care was tasked with reviewing the current state of LTC in the face of COVID-199. Their report found 103 LTC reports, commissions, and inquiries, 80 of which were unique reports based in Canada. The review found recurrent themes of longstanding deficiencies in the LTC sector that contributed to the magnitude of the COVID-19 crisis in LTCHs. Despite the wealth of evidence, policy changes were not made. Instead, new LTC commissions and inquiries are being held in response to the pandemic, only to find the same problems already known from past reports10–12. Given the number of reports in the past, it would be helpful to know which recommendations were made recurrently. Furthermore, the cost of studying LTC using reports, commissions and inquires in Canada is also not known.\n\nThe primary objective of this study was to examine the recurring recommendations over the past 10 years. Our secondary objective was to calculate the total costs of generating all of the LTC reports, commissions, and inquiries in Canada from 1998 to 2020. We aimed to put the cost of repeatedly studying the same problems into context of the current pandemic.\n\n\nMethods\n\nQuantitative analyses were based on Canadian LTC reports and commissions identified from The Royal Society of Canada Working Group on Long-Term Care9, which was done using a environmental scan from 1998 to 2020, a hand search of the report citations and the identification of reports during communication with the affiliated organizations. Online resources including Google Scholar, government, professional regulatory organizations, unions, and association websites were searched using terms such as “long-term care”, “nursing home”, “residential care”, “report”, “commission”, “recommendation, and “inquiry”. The reports were sponsored by various governmental and nongovernmental organizations and authored by researchers in the field. Even though the reports are not peer-reviewed, the Royal Society working group reviewed the reports and found them to be an accurate reflection of the state of LTC9.\n\nOur analysis took place from July to October 2020. Recommendations were identified from reports published in the past 10 years to increase relevance to current practice. We only counted recommendations that were specifically stated, either in a heading, in a list, or in a “recommendations” section of the report. After the first five reports were reviewed by two reviewers, a discussion was held to decide on grouping of the recommendations. The same two reviewers regularly discussed the recommendation categories through the review process. Recommendation categories were chosen to reflect meaningful differences in concept. For example, even though improving staffing was a common theme, we separated out the recommendations into (i) increasing funding for more staff, (ii) improving staff education and training, (iii) optimizing the mix of staff professions, and (iv) increasing emotional and wellness supports for staff. For the cost analysis, we excluded follow-up reports and included cost estimates in the original larger report.\n\nAn earlier version of this article can be found on medRxiv (DOI: https://doi.org/10.1101/2020.11.17.20233114).\n\nWe contacted the author or sponsoring organizations of the 80 Canadian LTC reports to inquire about the estimated cost of producing each report. Using a standardized email or script for telephone calls, we requested both direct and indirect costs. Direct costs included consultancy fees, salaries, compensation for expert witnesses, graphics, layout, printing, and dissemination. Indirect costs included time donated from authors who volunteered their time to produce the report. In some cases, multiple conversations with the sponsoring organization were required to gather all of the pertinent details. When an estimated budget was not available, we searched online for global budget reports from the sponsoring organization. Total annual expenses for research, advocacy, or reports were divided by the number of reports published that year by the organization to generate an estimated cost. We also searched for media reports about costs of commissions or coroner’s inquests. If no costs were available, the estimate was based on length, depth of research, inclusion of external experts/witnesses, and the reported cost of similar LTC reports. Costs were in Canadian dollars and adjusted to 2020 values according to the Bank of Canada Inflation Calculator13.\n\nReport characteristics were extracted, including title, sponsoring organization, publication year, geographic region, scope of report, number of contributors, number of pages, and duration of the project. The sponsoring organization was defined as the group funding the report. The geographic region of the report was categorized into national, provincial, or municipal jurisdictions. We extracted the specific province or municipality. The report scope was categorized into one of the following: health system, care of older adults, continuing care (home care, assisted living, and LTC) and LTC only. The number of contributors was the total of unique authors, researchers, panel members, witnesses, and consultants, depending on the report type. The project duration was defined as the period from the date of project initiation to report completion. Data extraction and content analysis was completed by EW and TT. Accuracy of the characteristics was reviewed by both investigators and agreement on the content analysis were confirmed by discussion. Descriptive statistics incluing cost data were presented as means (standard deviation), median (interquartile range), and proportion, as appropriate. The primary outcome was recurrent recommendations. Secondary outcomes included contributors, total costs of producing the reports and median page count.\n\nNo ethics approval was required for this analysis.\n\n\nResults\n\nThe list of Canadian LTC reports from the Royal Society commission spans the years 1998 to 2020 (n=80). There was an increase in the number of reports over time, with 10 reports in the first half of 2020 (Figure 1). Most of the reports were focused at a provincial level (n=55, 68.8%), 24 reports were based on a national level (30.0%). We found one municipal report and no reports from the territories. Ontario (n=31, 55.0%) produced the majority of the provincial reports, followed by British Columbia (n=11, 19.6%) (Table 1). More reports were funded by provincial governments (n=26) compared with the federal government (n=9). Non-profit organizations (e.g. Canadian Association for Long Term Care, Canadian Institute of Health Information) and professional unions (e.g. Canadian Federation of Nurses Unions, Canadian Union of Public Employees) or associations (e.g. Registered Nurses' Association of Ontario, Canadian Medical Association) authored the remaining 45 reports. No reports were funded by the private sector. Most of the reports focused solely on LTC (68.8%), but some reports focused on continuing care, older adults, or the health care system as well. The median report length was 40 pages (interquartile range, IQR, 21–84), with 16 reports (20.0%) over 100 pages. The median number of contributors, including authors, witnesses, and consultants, was 14 (IQR 5–26).\n\nThe year of publication, geographic focus, funding organization, scope (topics covered), page count and number of contributors were extracted for each report. Costs were adjusted to 2020 Canadian dollar values. IQR, interquartile range.\n\n*Some reports had more than 1 funding organization.\n\nReviewing the reports from the last 10 years (n=48), we identified a median of 8 (IQR 3.25–18) recommendations per report. Numerous recommendations were repeated in these reports (Table 2). Overall, the most frequent recommendations were: (i) to increase or redistribute funding to improve staffing, direct care, and capacity (66.7%), (ii) to standardize, regulate and audit LTC quality of care (58.3%), and (iii) to standardize, regulate or reform education and training for LTC staff (52%). Improving staff education and training, increasing behavioural supports, and modernizing infection control measures were universally recommended in reports by governments, non-profits, professional association and unions.\n\nThe full list of reports is available in Extended data, Supplementary Tables 1 and 214.\n\nGovernment-authored reports (n=35) focused on improving data collection (n=8, 22.9%), improving education and training of staff (n=8, 22.9%) and standardizing LTC quality of care (n=8, 22.9%). Professional union reports (n=11) focused on regulating for-profit LTCHs (n=4, 36.4%) and standardizing LTC quality of care (n=4, 36.4%). The most common recommendation made by non-profits (n=19) was to improve training and education for staff (n=9, 47.4%). Professional association reports (n=20) most commonly recommended standardizing staffing mix (n=10, 50.0%) and increasing funding for direct care (n=10, 50.0%). The least common recommendation made by professional associations was for improved transparency, reporting and tracking of critical incidents (n=1, 5.0%).\n\nCritical incidents received the most attention from governments (n=6, 17.1%), followed by non-profit organizations (n=2, 10.5%) and a professional association (n=1, 5.0%). Recommendations regarding quality of care or data utilization were more likely to be made by government (n=18, 51.4%) or professional associations and unions (n=20, 64.5%) than by non-profits (n=3, 15.8%). Staff wellness support was most recommended by non-profits (n=7, 36.8%), followed by professional associations or unions (n=6, 19.4%), then government (n=3, 8.6%). The appointment of commissions or inquiries was mostly recommended by professional associations or unions (n=7, 22.6%) then by government (n=2, 5.7%) and no commissions were recommended by non-profit organizations\n\nAlthough recommendations to improve resident quality of life were mentioned in 12 reports, they mostly overlapped with other recommendations such as increasing direct care, optimizing staff mix, increasing quality of life data collection, and improving quality of care. Only one report discussed specific recommendations to improve quality of life, such as increasing decision-making capacity (e.g. choice over when to bath), improving privacy with single rooms, and prioritizing relational care over medical tasks and interventions15.\n\nThe full list of reports is available in Extended data, Supplementary Table 114\n\nNearly half of the reports (45%) had cost estimates by the sponsoring organization or by publicly available budget or media reports. The total cost for all 80 reports was estimated to be $23,626,442.78 in Canadian dollars inflated to 2020 values (Table 1). The median cost per report was $15,203.48 (IQR $10,000–$53,147.81). Details of each report’s cost estimate are shown in Extended data, Supplementary Table 214. The lowest cost was estimated at $500 for each of the Canadian Army Joint Task Force reports, which accounts for the administrative costs of writing the reports even if the services were provided on military order4. If the cost of military deployment was included, the total cost would increase by $53 million16. The highest cost was $9,046,255.51 for the Public Inquiry into the Safety and Security of Residents in the Long-Term Care Homes System (The Wettlaufer Report) in 2019, which involved 79 contributors, witnesses, and experts17.\n\n\nDiscussion\n\nThis analysis highlights the efforts of multiple organizations, both governmental and non-governmental, to address the longstanding challenges and quality issues in Canada’s LTCHs. The Royal Society Briefing Report concluded that the unresolved issues in Canada’s nursing homes have been known for decades. According to our analysis, there are numerous recommendations backed by governmental and nongovernmental groups, yet little action has followed. This inaction set the stage for increased deaths during COVID-19 pandemic and to lower quality of life in LTCHs. Our analysis further showed the substantial cost of studying the problem repeatedly over the years.\n\nDuplicate investigation of known findings reduces value and increases waste18. From the LTC reports, issues regarding understaffing, undertraining, and the negative impact of for-profit LTC homes were repeatedly mentioned9. Policy change often requires persistence19, but the cost of advocating for change should be viewed from a societal context (financial and moral). Commissions and inquiries into LTC issues, like those happening or slated to begin around the country10–12, can solidify our determination for policy change, but do not replace the need for policy implementation. Only action will help LTC residents.\n\nSeveral provinces have increased wages and provided full-time employment with more appropriate compensation and benefits to stabilize the LTC workforce20. The Ontario government went further to commit 4.00 hours per day of direct care for each LTC resident by 202421, bringing the total care hours above the national average (3.30 hours)22. However, increasing direct care hours, while essential, is only one of the many recommendations from existing reports. Identifying the right staff mix and care team composition, providing proper education and training, and supporting staff wellness are also critical to developing a long-term workforce that has sufficient resilience to confront future crises23. Furthermore, we should focus more attention on resident quality of life, which should be the ultimate goal of our efforts.\n\nAlthough the total cost of $23 million in generating LTC reports may seem insignificant compared to a government budget, it represents a substantial, lost opportunity to continually improve Canadian LTCHs. Studying the same problems repeatedly means Canadian experts are confined to revisiting critical deficiencies in LTC instead of innovating new care models. While Canada ranks among the best countries in the world for health innovation24, this pandemic highlighted a contrasting story of neglected policy in LTC25.\n\nThere are several strengths of this study. We systematically tracked down the cost of each report by contacting the sponsoring organization or consulting their global budgets. The total costs, including time donated of experts authoring these reports, were accounted for. For reports that did not have available cost data, we estimated the total cost by using reports with known costs with similar length and depth.\n\nThe main limitation of this study was the lack of true cost estimates for half of the LTC reports. Some organizations lacked transparency about costing, and others lacked detailed accounting of spending. Staff turnover and record keeping practices were barriers to accessing data, particularly for the reports produced over 10 years ago. For government agencies, we often had to call multiple departments and speak with numerous representatives and noted considerable variation in the level of disclosure. For the two military reports during COVID-19, we likely underestimated the cost since they were generated as part of military duty. However, there were still considerable costs incurred by the public by having the military deployed to those LTCHs26. We also grouped reports on health system improvement and care of older adults in general in this analysis because LTC is intricately tied to the health system at large. Leaders in the care of older adults, such as Denmark, design LTC policy as in integral part of their health system27.\n\n\nConclusion\n\nOver the last two decades, Canadian governments and non-government organizations have repeatedly investigated longstanding LTC issues and have largely drawn the same conclusions. Had the recurring recommendations been implemented, we would not only have improved working conditions, quality of care and quality of life, but would also have undoubtedly prevented unnecessary deaths due to COVID-19. Instead of continuing to investigate LTC issues, we should focus our resources on implementing the recommendations in the identified reports.\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: Supplementary tables from Recommendations from long-term care reports, commissions, and inquiries in Canada. https://doi.org/10.6084/m9.figshare.1353741214.\n\nThis project contains the following extended data:\n\nSupplementary Table 1\n\nSupplementary Table 2\n\nExtended data 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 Dr. David Moher for reviewing an early draft of the manuscript.\n\n\nReferences\n\nCanadian Institute for Health Information: Pandemic Experience in the Long-Term Care Sector: How Does Canada Compare With Other Countries? Ottawa, ON. 2020. Reference Source\n\nComas-Herrera A, Zalakaín J, Lemmon E, et al.: Mortality associated with COVID-19 outbreaks in care homes Mortality associated with COVID-19 in care homes: international evidence. 2020. Reference Source\n\nLiu M, Maxwell CJ, Armstrong P, et al.: COVID-19 in long-term care homes in Ontario and British Columbia. CMAJ. 2020; 192(47): E1540–E1546. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGovernment of Canada: Report of observations CAF personnel supporting Long Term Care Facilities (LTCFs) as part of Operation LASER. CanadaCa. 2020; (accessed September 15, 2020). Reference Source\n\nStall NM, Jones A, Brown KA, et al.: For-profit long-term care homes and the risk of COVID-19 outbreaks and resident deaths. Can Med Assoc J. 2020; 192(33): E946–55. PubMed Abstract \| Publisher Full Text\n\nDuan Y, Iaconi A, Song Y, et al.: Care Aides Working Multiple Jobs: Considerations for Staffing Policies in Long-Term Care Homes During and After the COVID-19 Pandemic. J Am Med Dir Assoc. 2020; 21(10): 1390–1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchnelle JF, Simmons SF, Harrington C, et al.: Relationship of nursing home staffing to quality of care. Health Serv Res. 2004; 39(2): 225–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Temkin-Greener H, Shan G, et al.: COVID-19 Infections and Deaths among Connecticut Nursing Home Residents: Facility Correlates. J Am Geriatr Soc. 2020; 68(9): 1899–906. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEstabrooks C, Straus SE, Flood CM, et al.: Restoring Trust: COVID-19 and The Future of Long-Term Care. R Soc Canada. 2020; (accessed September 15, 2020). Reference Source\n\nLuft A: Quebec ombudsperson to investigate government COVID-19 response in long-term care facilities. CTV News. 2020; (accessed November 8, 2020). Reference Source\n\nPatton J: Ontario long-term care commission provides government recommendations for 2nd wave in homes.Glob News. 2020; (accessed November 8, 2020). Reference Source\n\nJohnson L: Alberta to commission third-party review of response to COVID-19. Fort McMurray Today. 2020; (accessed November 8, 2020). Reference Source\n\nBank of Canada: Inflation Calculator. 2020; (accessed January 9, 2020). Reference Source\n\nWong EKC, Thorne T, Estabrooks C, et al.: Supplementary tables from Recommendations from long-term care reports, commissions, and inquiries in Canada. figshare. Journal contribution. 2021. http://www.doi.org/10.6084/m9.figshare.13537412.v1\n\nArmstrong P, Daly T: Exercising Choice in Long-Term Residential Care. 2017. Reference Source\n\nBrewster M: Military mission to COVID-hit long term care homes cost taxpayers about $53 million. CBC News. 2020. (accessed November 17, 2020). Reference Source\n\nGillese EE: Final Report - The Long-Term Care Homes Public Inquiry. Long-Term Care Homes Public Inq. 2019; 1–1491, (accessed September 15, 2020). Reference Source\n\nChalmers I, Bracken MB, Djulbegovic B, et al.: How to increase value and reduce waste when research priorities are set. Lancet. 2014; 383(9912): 156–65. PubMed Abstract \| Publisher Full Text\n\nTilley H, Shaxson L, Rea J, et al.: 10 things to know about how to influence policy with research \| Overseas Development Institute (ODI). Overseas Dev Inst. 2017; (accessed September 15, 2020). Reference Source\n\nThe Canadian Press: Increasing pay for long-term care home workers high on agenda for Trudeau, premiers. GlobalnewsCa. 2020; (accessed November 8, 2020). Reference Source\n\nJeffords S: Ontario promises new care standard in long-term care, to be implemented by 2024-2025. GlobalnewsCa. 2020; (accessed November 8, 2020). Reference Source\n\nBC Care Providers Association: Filling the Gap: Determining Appropriate Staffing and Care Levels for Quality in Long Term Care. 2019. Reference Source\n\nOECD: OECD Employment Outlook 2019: The Future of Work. Paris: OECD Publishing; 2019. Publisher Full Text\n\nWorld Intellectual Property Organization: Global Innovation Index 2019: India Makes Major Gains as Switzerland, Sweden, U.S., Netherlands, U.K. Top Ranking; Trade Protectionism Poses Risks for Future Innovation. 2019. (accessed December 31, 2020). Reference Source\n\nOECD: Workforce and safety in long-term care during the COVID-19 pandemic. OECD 2020. (accessed December 31, 2020). Reference Source\n\nThe Canadian Press: Canadian troops facing risk of COVID-19 while on duty will receive hazard pay. Glob News.2020; (accessed November 16, 2020). Reference Source\n\nSchulz E, Berlin D: European Network of Economic Policy Research Institutes Assessing Needs of Care in European Nations THE LONG-TERM CARE SYSTEM IN DENMARK. 2010. Reference Source" }	[ { "id": "79291", "date": "09 Mar 2021", "name": "Lynn Chenoweth", "expertise": [ "Reviewer Expertise Aged care", "transitions in ageing", "dementia care", "heath and aged care services", "person-centered care", "carer support." ], "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 review on the breadth, costs and recommendations arising from investigative reports on the Canadian LTC system is comprehensive, insightful and plausible.\n\nIt reflects the situation in many other developed nations and provides sufficient evidence to taking global action to redress the continuing government failure to redress LTC staffing issues.\nThis well-conducted review has clearly identified the issues associated with funding support for the aged care sector, as identified in multiple commissions of inquiry and independent reviews over a decade. The review highlights the poor-level of government sponsorship of the sector, which translates to having inadequate funds allocated to professional staffing complements. This issue has led to cuts in professional staff and an over-supply of poorly paid and inadequately-educated care workers. Specialised care and therapy needs remain unmet for the majority of older people who would benefit from receiving them. The review makes clear that the funds spent on investigating and identifying these issues have been high, and have not resulted in any tangible benefits for the sector. The authors argue that if these funds had been used to actually redress the staffing issue, the benefits would be obvious. This review has international importance, since these issues are repeated across the aged care sector in both developed and under-developed nations. Clearly, it does not need any further commissioned reviews and independent inquiries to reinforce what we already know - that care standards will only improve when aged care is funded commensurate with acute care sector services for older people.\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": "79798", "date": "25 May 2021", "name": "Adelina Comas-Herrera", "expertise": [ "Reviewer Expertise Long-term care and dementia care policy and economic aspects of care." ], "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 very timely, in the context of the need to learn lessons from the COVID pandemic’s impact on long-term care systems. The pandemic has drawn wider attention to the long-standing structural problems of long-term are systems, and particularly long-term care facilities.\nThe analysis of the recommendations is particularly interesting, confirming that many of the problems identified during the pandemic were well known before and action was not taken to address them. I would have liked, ideally to see this separated by whether the reports were from before or during the COVID-19 pandemic, it would be interesting to highlight if, for example, infection control was present in the reports prior to the pandemic. In my view, some of the detailed reports on COVID outbreaks in specific facilities are very different than the reports on the structure of the LTC system and would need to be considered separately.\nThe analysis of the costs of the reports is an important contribution, but I have not been able to check the figures properly: I have checked the supplementary table 2 using the link provided and I cannot see cost estimates for any of the reports published since 2002. It may be that the table is incomplete?\nI have a major concern on the interpretation of the costs of the reports: while I do agree that it is very important to ensure that there is no replication of reports and to avoid waste, I do not think the paper has established clearly the extent to which “the same problems have been studied repeatedly”. While coming to the same broad recommendations, some of the reports may have advanced the understanding of the system and generated new data that may had broader impacts, for example informing planning at regional level of information practice by providers.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6742", "date": "04 Jun 2021", "name": "Eric KC Wong", "role": "Author Response", "response": "Thank you for reviewing the article and for providing comments. Please see our response below: Comment: The analysis of the recommendations is particularly interesting, confirming that many of the problems identified during the pandemic were well known before and action was not taken to address them. I would have liked, ideally to see this separated by whether the reports were from before or during the COVID-19 pandemic, it would be interesting to highlight if, for example, infection control was present in the reports prior to the pandemic. In my view, some of the detailed reports on COVID outbreaks in specific facilities are very different than the reports on the structure of the LTC system and would need to be considered separately. Response We added a line highlighting that similar recommendations were found in reports dated before and after the pandemic. In Table 2, we added a line to the caption indicating that reports 1-10 were from after the pandemic and 11-48 were from before the pandemic. Each recommendation category had reports from both before and after the pandemic. “Reviewing the reports from the last 10 years (n=48), we identified a median of 8 (IQR 3.25–18) recommendations per report. Numerous recommendations were repeated in these reports, and similar recommendations were made before and after the COVID-19 pandemic (Table 2).” – Results section, second paragraph Comment: The analysis of the costs of the reports is an important contribution, but I have not been able to check the figures properly: I have checked the supplementary table 2 using the link provided and I cannot see cost estimates for any of the reports published since 2002. It may be that the table is incomplete? Response: The figshare website only preview the first 2 pages of the document for some reason. If you download the Word document from their website, the full table is shown. Please let us know if you have problems with the file. Comment: I have a major concern on the interpretation of the costs of the reports: while I do agree that it is very important to ensure that there is no replication of reports and to avoid waste, I do not think the paper has established clearly the extent to which “the same problems have been studied repeatedly”. While coming to the same broad recommendations, some of the reports may have advanced the understanding of the system and generated new data that may had broader impacts, for example informing planning at regional level of information practice by providers. Response: We agree that reports may advance our understanding of the LTC system in small increments, but based on the number of times similar recommendations have been repeated, we think there is substantial overlap in problems identified in the reports. As an example, the recently published Ontario Long Term Care COVID-19 Commission had 85 recommendations [1]. Only the first 26 recommendations were about pandemic management. The remaining 59 recommendations addressed longstanding issues with the LTC system, including resident quality of life, staff shortages, lack of staff training, poor working conditions, lack of funding, inadequate regulation, insufficient accountability, and lack of performance indicators and standards. Even the first 26 recommendations included general infection control measures previously recommended during the SARS pandemic in 2003. Similarly, the authors of the included reports in our analysis chose to highlight the same broad recommendations because those are the limiting factors to improving the LTC system. Even if there were incremental lessons and improvements, the fact that recommendations were not substantively different represented a missed opportunity to correct the most important, longstanding problems. Added line in discussion: “Although the total cost of $23 million in generating LTC reports may seem insignificant compared to a government budget, it represents a substantial, lost opportunity to continually improve Canadian LTCHs. Studying the same problems repeatedly means Canadian experts are confined to revisiting critical deficiencies in LTC instead of innovating new care models. Even if there were incremental lessons and improvements from these reports, they were inadequate in preventing one crisis after another. While Canada ranks among the best countries in the world for health innovation, this pandemic highlighted a contrasting story of neglected policy in LTC.” – Discussion section, fourth paragraph Reference 1. Marrocco FN, Coke A, Kitts J. Ontario’s Long-Term Care COVID-19 Commission: Final Report [Internet]. Toronto; 2021 Apr [cited 2021 May 29]. Available from: http://www.ltccommission-commissionsld.ca/report/pdf/Ontarios_Long-Term_Care_COVID-19_Commission_Final_Report.pdf" } ] } ]	1	https://f1000research.com/articles/10-87
https://f1000research.com/articles/8-130/v1	30 Jan 19	{ "type": "Research Article", "title": "The effect of red-fleshed pitaya (Hylocereus polyrhizus) on heat shock protein 70 and cortisol expression in strenuous exercise induced rats", "authors": [ "Novita Sari Harahap", "Aznan Lelo", "Ambrosius Purba", "Awaluddin Sibuea", "Rina Amelia", "Zulaini Zulaini", "Aznan Lelo", "Ambrosius Purba", "Awaluddin Sibuea", "Rina Amelia", "Zulaini Zulaini" ], "abstract": "Background: Oxidative stress from exercise can contribute to damaging cells, increasing heat shock protein 70 (HSP70) and suppressing the immune system in the body. This research aimed to determine the antioxidant potential of red-fleshed pitaya extract on HSP70 and cortisol expression in rats which were subjected to strenuous exercise. Methods: The subjects of this research were 32 Sprague Dawley male rats, aged 3 months, with an average weight of 200 g. Red-fleshed pitaya extract was obtained from methanol extraction process; a maceration technique was performed and the extract was concentrated using an air-drying method. Rats were randomly divided into four groups. Group 1 were subjected to strenuous exercise and treated with distilled water only; while Groups 2, 3 and 4 were subjected to strenuous exercise and treated with 100 mg/kg body weight, 200 mg/kg body weight and 300 mg/kg body weight of red-fleshed pitaya extract, respectively. Strenuous exercises in rats was performed by intense swimming of 20 min/day, 3 days a week for 3 weeks. HSP70 expression and cortisol were measured with Enzyme-Linked Immune Sorbent Assay (ELISA) method. Results: There was a significant reduction of HSP70 (p=0.000) and cortisol expression (p=0.000) between the groups. Also, there was a significant difference in the average decreasing of HSP70 expression between group 4 and either groups 1 or 2 (p=0.000). However, a significant difference between groups 4 and 3 was not observed (p=0.813). Lastly, a significant difference was found in the average decrease of cortisol expression between groups 4 and 1 (p=0.000), 2 (p=0.000), and 3 (p=0.000) respectively. Conclusion: Red-fleshed pitaya is potential to be utilized as antioxidant to decrease the HSP70 and cortisol expression.", "keywords": [ "Red-fleshed pitaya", "HSP70", "cortisol", "strenuous exercise" ], "content": "Introduction\n\nPhysical activity is an activity which has various influences and significant effects on the body. The effect of regular physical activity is a positive influence on biological functions, and will improve health and the antioxidant defense system in order to protect body from the negative effects of oxidative damage1. Strenuous exercise tends to trigger free radical compound production. Moreover, this impairs the balance of free radicals and antioxidants as a result of oxidative stress2–4. Research has discovered that oxidative stress from strenuous exercise reduces performance as it damages cells5, causing pain and muscles fatigues6, lowering antioxidant levels7,8, increasing the expression of heat shock protein 70 (HSP70)9,10 and suppressing the immune system11.\n\nDuring an intense workout, self-defense and self-adaptation depend on the body condition which can be observed from HSP70 protein expression. Increases of HSP70 in muscles indicates a response to protect muscles cells from oxidative stress. HSP70 expression is an adaptation mechanism and a sign of damaging cells caused by oxidative stress12. Previous research reported that workouts increase HSP70 expression10. Strenuous exercise is a physical stressor in the body and as a result, adrenocorticotropic hormone(ACTH) is secreted by hypothalamus hypo-physis anterior and triggers the adrenal cortex to produce cortisol13. The escalation of cortisol is influenced by the intensity and duration of training that leads to a suppression of the immune system, resulting in a decline of antibody. Cortisol can be a sign that the body encounters a decline in the immune system due to heavy training14. Antioxidants can detoxify the lipid peroxide produced during exercise, which can eliminate radicals and reduce the inflammatory response to exercise. Therefore, it can prevent a muscle damage from exercise15.\n\nThe body needs exogenous antioxidants to neutralize and prevent chain reactions from free radicals formed from heavy physical trainings16. Sources of exogenous antioxidants are Vitamin E, C and also beta-carotene. External antioxidants from food or supplements can help the body to fight an excess of free radicals. In a previous study, proanthocyanidin from grape seed was given to rats for 2 weeks. As a result, it lowered malondialdehyde level and increased the superoxide dismutase and glutathione peroxidase was activated significantly. Furthermore, it reduced fatigue after physical activitie17.\n\nRed-fleshed pitaya (Hylocereus polyrhizus) is a unique fruit with a lot of benefits. The fruit is recently popular among Indonesians and appears to be a natural antioxidant. Several in vitro studies have revealed that red-fleshed pitaya extract has the potential to be an antioxidant18. This research aimed to investigate the antioxidant potential in red-fleshed pitaya extract on HSP70 and cortisol expression in rats that were subjected to strenuous exercise.\n\n\nMethods\n\nThe subjects of this research were 32 Sprague Dawley male rats, aged 3 months, with an average weight of 200 g, were obtained from the Animal Holding Unit of the Pharmacy laboratory, University of Sumatera Utara, Indonesia.\n\nAll rats were sustained and maintained in groups (four mice per cage) in experimental animal cages of the Pharmacy laboratory, University of Sumatera Utara, Indonesia. The cage is made of plastic (30 x 20 x 10 cm) and covered with fine wire mesh. The base of the cage is covered with rice husk as thick as 0.5 - 1 cm and replaced every day during the research. The room light was controlled to be exactly at 12 hours light and 12 hours dark cycle, while the temperature 25–27°C and humidity of the room were adjusted to a normal range and fed with standard rat pellets 551, and drink (tap water) was given ad libitum.\n\nThe research applied a laboratory experiment method with random group posttest-only design. The male rats were obtained from the Pharmacy Laboratory, University of North Sumatra. The experimental animals were simple random sampling divided into four groups: Group 1 was subjected to strenuous exercise and treated with distilled water only; Group 2 was subjected to strenuous exercise and treated with dosage 100 mg/kg body weight of red-fleshed pitaya extract; Group 3 was subjected to strenuous exercise and treated with dosage 200 mg/kg body weight of red-fleshed pitaya extract; Group 4 was subjected to strenuous exercise and treated with dosage 300 mg/kg body weight of red-fleshed pitaya extract.\n\nRed-fleshed pitaya fruit, obtained from farmers, in Indonesia, was peeled, washed, cut into small pieces and then dried in a drying cabinet. After that, the fruit was blended using a blender. The fruit extract was isolated through maceration method by using ethanol 96% which has been distilled as much as 10 times the weight of red-fleshed pitaya powder. Red-fleshed pitaya fruit powder in a container had 96% ethanol added to it (ratio 1:7, fruit powder: ethanol), and then was soaked for 3 days then filtered and sealed. The macerates were collected in a container and then processed with rotary evaporator at a temperature of 45°C until the extract was thickened. After that, the same process of were repeated the remaining ethanol 96% for 3 days. The less thickened extract was then evaporated in a water bath until a thick extract was obtained.\n\n100 mg red-fleshed pitaya extract was weighed. Then, it was gently ground using a pestle and mortar. After that, carboxy methyl cellulose (CMC) Na 0.5% solution was slowly added and ground until a homogeneous phase was achieved. Finally, the suspension was added to a 10 mL measuring flask until it reached the mark line. The allocation of red-fleshed pitaya extract, dosage of 100 mg/kg body weight, for instance: weight of 200 g, volume taken: 2 ml extract suspension. Dosage of 200 mg/kg body weight, for instance: weight of 200 g, volume taken: 4 ml extract suspension. Dosage of 300 mg/kg body weight, for instance: weight of 200 g, volume taken: 6 ml extract suspension.\n\nStrenuous exercise given to all rats was a morning swim between 8–9 am for 20 minutes/day, 3 days a week over 21 days. The equipment used in this research was a 10-cm length and 25-cm diameter bath as a pool. Group 1, the rats received distilled water only; Group 2, the rats received 2 ml red-fleshed pitaya extract suspension; Group 3, the rats received 4 ml red-fleshed pitaya extract suspension; Group 4, the rats received 6 ml red-fleshed pitaya extract suspension. Administration of red-fleshed pitaya extract suspension and water was performed orally once daily for 21 days.\n\nTesting for HSP70 and cortisol was conducted two days after the rats had completed a strenuous exercise, 3 days a week over 21 days. During the test, the rats were given a maximum training session by swimming as hard as they could until the rats drowned or showed fatigue symptoms such as the entire body almost dipped into water and limb movements slowed down. After that the rats were sacrificed by placing them in a jar containing cotton which was moistened with 10 ml of chloroform. After that, 2–3 ml blood was taken from the heart.\n\nBlood samples were collected in micro tubes and centrifuged at 3000 rpm for 15 minutes. The serum was separated and stored at a temperature of 20°C until the analysis process would be carried out. Cortisol was measured with enzyme-linked immune sorbent assay (ELISA); Mouse Cortisol Elisa kit (catalog: E1483Mo, Brand Bioassay TL) and UV spectrophotometry at a wavelength of 450 nm. The HSP70 expression was recorded with ELISA; Mouse HSP70 Elisa Kit (catalog: E0302Mo, Brand Bioassay TL); the absorbance was indicated at 405 nm.\n\nData was analyzed using SPSS 22 for Windows and displayed in tables and diagrams. Normality test was conducted through Shapiro-Wilk test (P > 0.05) in order to determine the average of normal distribution of sample data which is presented as mean ± SD. The result of the normality test was used for next analysis; parametric analysis was used for normal distribution, otherwise non-parametric analysis was used. The ANOVA statistical analysis was performed to indicate the effects of treatments for each group. If the significant result is obtained, then the procedure is followed by Least Significance Difference or Bonferroni tests.\n\nThe research was performed on the animal subjects were in according with the ethical standards by the Animal Research Ethics Committees/AREC, Faculty of Mathematics and Natural Sciences University of Sumatera Utara, Indonesia (approval number 0011/KEPH-FMIPA/2018).\n\nAll efforts were made to reduce any suffering of the rats was during the experiments by following careful procedures and also by anaesthetizing the animal prior to scarifice to prevent experiencing any pain.\n\n\nResults\n\nA normality test indicated that the data are normally distributed (Table 1). HSP70 expression was decreased across all groups (69.57 vs 46.04 vs 31.47 vs 27.65 pg/mL). Group 4 had the lowest expression compared with the other groups. This research reveals a significant decrease in HSP70 expression (p=0.000) between the groups (Table 2).\n\nShapiro-wilk test, P>0.05\n\nCortisol expression was also decreased across all groups (119.02 vs 86.11 vs 62.94 vs 40.86 pg/mL). Group 4 had the lowest expression compared with the other groups. An ANOVA test revealed a significant decrease in cortisol expression (p=0.000) between the groups (Table 2).\n\nThe mean ± SD HSP70 and cortisol expression in shown\n\nFigure 1 indicates a significant difference in the average decrease of HSP70 expression between group 4 and either group 1 or 2 (p=0.000). However, a significant difference between group 4 and group 3 (p=0.813) was not found. This means that group 4, with strenuous exercise and given 300 mg/kg body weight red-fleshed pitaya extract was indicated to be more effective for reducing HSP70 expression compared to group 1 and 2, with no big variance from group 3.\n\nFigure 2 confirms that a significant difference is revealed in the average decrease of cortisol expression between groups 4 and 1 (p=0.000), 2 (p=0.000), and 3 (p=0.000). It can be concluded that group 4 with strenuous exercise and 300 mg/kg body weight red-fleshed pitaya extract was more effective in reducing cortisol expression compared to the other groups.\n\n\nDiscussion\n\nBased on the results of this research, it is found that strenuous exercise combined with daily red-fleshed pitaya extract consumption contributes to a declining expression of HSP70 and cortisol. The dosage of 300 mg/kg body weight red-fleshed pitaya extract was found to be the optimum amount in decreasing cortisol compared to 100 and 200 mg/kg body weight dosage. However, for both 300 mg/kg body weight and 200 mg/kg body weight dosage of red-fleshed pitaya extract could not provide any difference to HSP70 expression. Therefore, the red-fleshed pitaya extract can be categorized as a potential exogenous antioxidant to eliminate free radicals formed during strenuous exercise.\n\nFree radicals are an element that possesses one or more unpaired electrons in its outermost orbital. Consequently, it is very reactive to cells or cell components in its surroundings. Commonly, a reactive element finds its pair by attacking and binding with adjacent electrons. Then, if this element reacts with another radical element, a new radical element will be formed. This will consistently continue to occur leading to an unavoidable chain reaction19. During strenuous exercise, oxygen consumption rises by 20 times. The excess oxygen triggers the formation of free radicals with electrons released from the respiratory chain. Free radicals production from activity, especially superoxides, increases in the mitochondria20. When an imbalance happens due to the excess of free radicals, oxidative stress occurs and damages the DNA. Moreover, proteins will lose their structure and function like enzymes and membrane receptor. Also, there will be damage in the lipid bilayer structure21.\n\nStrenuous exercise triggers oxidative stress to occur, therefore, HSP70 expression will elevate, resulting in a decrease of endogenous antioxidant activity. HSP70 is an important protein molecule for cell healing and preventing homeostasis, and also increased expression as a cyto-protective effect22. HSP70 is induced strongly due to oxidative stress as cyto-protective to prevent oxidative damage and heal broken proteins23. The increase of HSP70 expession is aimed to balance between ischemic condition, high temperature and increased production of free radical22. In this research, it is found that HSP70 expression tends to decrease in a group with strenuous exercise combined with red-fleshed pitaya extract.\n\nThe red-fleshed pitaya extract combined with strenuous exercise is proven to be able to inhibit the increasing HSP70 expression. This occured due to the potential of red-fleshed pitaya as an antioxidant that is able to balance the increasing amount of free radicals formed and impairs HSP70 synthesis induced by strenuous exercise24. Therefore, HSP70 expession is lower compared to the groups that are not given by red-fleshed pitaya extract. The results of this research is in accordance to a research condcuted by Petiz et al. (2017) which discovered that Vitamin A combined with high intensity acivities could prevent tissue damage and reduce endogenous antioxidant defense regulation in rats, which also suppressed HSP70 expression25. Other researches have also revealed that antioxidant supplementation is proven to be effective to slow down the HSP70 synthesis caused by high intensity exercise24.\n\nPhysical training combined with low to medium dosage of red-fleshed pitaya extract were measured based on the ability and lead to immunomodulation effect that affect the body immune system and protect the body from cell damages, resulting in an effective condition to reduce the oxidative stress26. Stress resulted from physical activity like oxidative stress is responded by hypothalamus to secrete corticotrophin realizing hormone (CRH) which then delivers a message to pituitary anterior. The pituitary produced adrenocorticotropic hormone (ACTH) which is useful to activate or affect adrenal cortex where cortisol hormone is secreted. Cortisol contributes a massive influence to immune responses14 as a sign that the body is suffering from oxidative stress27. In this research, cortisol hormone level decreased in the group with strenuous exercise combined with red-fleshed pitaya extract.\n\nThis occurs since the red-fleshed pitaya extract is an effective antioxidant to reduce the risk of oxidative stress and is able to decrease the secretion of cortisol by reducing ACTH secretion in the hypothalamus and CRH in the pituitary gland28.\n\n\nConclusion\n\nBased on this research, it can be concluded that red-fleshed pitaya extract has the potential to be antioxidant with its anthocyanin content, and is able to eliminate oxidative stress due to strenuous exercise. It can be observed by the decreased pattern of HSP70 and cortisol expression in the strenuous exercise combined with red-fleshed pitaya extract group. Furthermore, the optimum result was shown in the dosage of 300 mg/kg body weight red-fleshed pitaya extract.\n\n\nData availability\n\nOpen Science Framework: The effect of red-fleshed pitaya (Hylocereus polyrhizus) on heat shock protein 70 and cortisol expression in strenuous exercise induced rats, https://doi.org/10.17605/OSF.IO/MGX4K29\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 authors would like to acknowledge Ministry of Research, Technology and Higher Education of Republic of Indonesia for funding this research via Research Grant of Universitas Negeri Medan with contract number of 027/UN33.8/LL/2018.\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\nYavari A, Javadi M, Mirmiran P, et al.: Exercise-induced oxidative stress and dietary antioxidants. Asian J Sports Med. 2015; 6(1): e24898. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEscribano BM, Tunez I, Requena F, et al.: Effects of an aerobic training program on oxidative stress biomarkers in bulls. Vet Med (Praha). 2010; 55(9): 422–428. Publisher Full Text\n\nEl Abed K, Rebai H, Bloomer RJ, et al.: Antioxidant status and oxidative stress at rest and in response to acute exercise in judokas and sedentary men. J Strength Cond Res. 2011; 25(9): 2400–9. PubMed Abstract \| Publisher Full Text\n\nSteinbacher P, Eckl P: Impact of oxidative stress on exercising skeletal muscle. Biomolecules. 2015; 5(2): 356–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValko M, Leibfritz D, Moncol J, et al.: Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007; 39(1): 44–84. PubMed Abstract \| Publisher Full Text\n\nMacedo DV, Lazarim FL, Catanho da Silva FO, et al.: Is lactate production related to muscular fatigue? A pedagogical proposition using empirical facts. Adv Physiol Educ. 2009; 33(4): 302–7. PubMed Abstract \| Publisher Full Text\n\nKürkçü R, Tekin A, Özda S, et al.: The effects of regular exercıse on oxıdatıve and antıoxıdatıve parameters ın young wrestlers. African J Pharm Pharmacol. 2010; 4(5): 244–251. Reference Source\n\nAzizbeigi K, Azarbayjani MA, Peeri M, et al.: The effect of progressive resistance training on oxidative stress and antioxidant enzyme activity in erythrocytes in untrained men. Int J Sport Nutr Exerc Metab. 2013; 23(3): 230–8. PubMed Abstract \| Publisher Full Text\n\nFolkesson M, Mackey AL, Langberg H, et al.: The expression of heat shock protein in human skeletal muscle: effects of muscle fibre phenotype and training background. Acta Physiol (Oxf). 2013; 209(1): 26–33. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Gampert L, Nething K, et al.: Response and function of skeletal muscle heat shock protein 70. Front Biosci. 2006; 11: 2802–27. PubMed Abstract \| Publisher Full Text\n\nBiller-Takahashi JD, Takahashi LS, Mingatto FE, et al.: The immune system is limited by oxidative stress: Dietary selenium promotes optimal antioxidative status and greatest immune defense in pacu Piaractus mesopotamicus. Fish Shellfish Immunol. 2015; 47(1): 360–7. PubMed Abstract \| Publisher Full Text\n\nStaib JL, Tümer N, Powers SK: Increased temperature and protein oxidation lead to HSP72 mRNA and protein accumulation in the in vivo exercised rat heart. Exp Physiol. 2009; 94(1): 71–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUsui T, Yoshikawa T, Ueda SY, et al.: Effects of acute prolonged strenuous exercise on the salivary stress markers and inflammatory cytokines. Jpn J Phys Fitness Sports Med. 2011; 60(3): 295–304. Publisher Full Text\n\nHill EE, Zacki E, Battaglini C, et al.: Exercise and circulating cortisol levels: the intensity threshold effect. J Endocrinol Invest. 2008; 31(7): 587–91. PubMed Abstract \| Publisher Full Text\n\nPeternelj TT, Coombes JS: Antioxidant supplementation during exercise training: beneficial or detrimental? Sports Med. 2011; 41(12): 1043–69. PubMed Abstract \| Publisher Full Text\n\nGomez-Cabrera MC, Viña J, Ji LL: Interplay of oxidants and antioxidants during exercise: implications for muscle health. Phys Sportsmed. 2009; 37(4): 116–23. PubMed Abstract \| Publisher Full Text\n\nBelviranli M, Gökbel H: Acute exercise induced oxidative stress and antioxidant changes. Eur J Gen Med. 2006; 3(3): 126–131. Publisher Full Text\n\nNurul SR, Asmah R: Variability in nutritional composition and phytochemical properties of red pitaya (Hylocereus polyrhizus) from Malaysia and Australia. Int Food Res J. 2014; 21(4): 1689–1697. Reference Source\n\nSahlin K, Shabalina IG, Mattsson CM, et al.: Ultraendurance exercise increases the production of reactive oxygen species in isolated mitochondria from human skeletal muscle. J Appl Physiol (1985). 2010; 108(4): 780–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomes EC, Silva AN, de Oliveira MR: Oxidants, antioxidants, and the beneficial roles of exercise-induced production of reactive species. Oxid Med Cell Longev. 2012; 2012: 756132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbruzzo PM, Esposito F, Marchionni C, et al.: Moderate exercise training induces ROS-related adaptations to skeletal muscles. Int J Sports Med. 2013; 34(8): 676–87. PubMed Abstract \| Publisher Full Text\n\nKrause M, Heck TG, Bittencourt A, et al.: The chaperone balance hypothesis: the importance of the extracellular to intracellular HSP70 ratio to inflammation-driven type 2 diabetes, the effect of exercise, and the implications for clinical management. Mediators Inflamm. 2015; 2015: 249205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanneau D, Brunet M, Frisan E, et al.: Heat shock proteins: essential proteins for apoptosis regulation. J Cell Mol Med. 2008; 12(3): 743–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhassaf M, McArdle A, Esanu C, et al.: Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle. J Physiol. 2003; 549(Pt 2): 645–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetiz LL, Girardi CS, Bortolin RC, et al.: Vitamin A Oral Supplementation Induces Oxidative Stress and Suppresses IL-10 and HSP70 in Skeletal Muscle of Trained Rats. Nutrients. 2017; 9(4): pii: E353. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaghaiee B, Karimi P, Siahkouhian M, et al.: Moderate aerobic exercise training decreases middle-aged induced pathologic cardiac hypertrophy by improving Klotho expression, MAPK signaling pathway, and oxidative stress status in Wistar rats. Iran J Basic Med Sci. 2018; 21(9): 911–919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurner-Cobb JM, Palmer J, Aronson D, et al.: Diurnal cortisol and coping responses in close relatives of persons with acquired brain injury: a longitudinal mixed methods study. Brain Inj. 2010; 24(6): 893–903. PubMed Abstract \| Publisher Full Text\n\nKraemer WJ, Ratamess NA: Hormonal responses and adaptations to resistance exercise and training. Sports Med. 2005; 35(4): 339–61. PubMed Abstract \| Publisher Full Text\n\nHarahap, Novita S: “The Effect of Red-Fleshed Pitaya (Hylocereus Polyrhizus) on Heat Shock Protein 70 and Cortisol Expression in Strenuous Exercise Induced Rats.” OSF. 2019. http://www.doi.org/10.17605/OSF.IO/MGX4K" }	[ { "id": "55888", "date": "08 Nov 2019", "name": "Yusni Yusni", "expertise": [ "Reviewer Expertise Medical Physiology: exercise physiology", "endocrine and metabolic physiology" ], "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\nIntroduction:\nPlease explain why red-fleshed pitaya is thought to act as antioxidants that will affect the heat shock protein 70 and the expression of cortisol: include theoretical and other studies that support.\n\nThe novelty of the article must be clearly explained in the introduction.\n\nMethods:\nWhy use 3 doses, whereas the purpose of the study did not assess the effective dose? In the conclusion, also mention the effective dose.\n\nExplain the results of the identification of the chemical content of red-fleshed pitaya and also the results of herbarium analysis because there is a possibility that Indonesia's geographical differences will affect its chemical content.\n\nExplain the time of blood-sampling because cortisol production is influenced by circadian.\n\nDiscussion:\nIt is important to explain the role or mechanism of action of red-fleshed pitaya on heat shock protein 70 and cortisol expression.\n\nReference:\nUse the latest references (from the last 5-10 years).\n\nNote: There are some grammatical errors, you should use the proof-read service.\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": "5050", "date": "29 Nov 2019", "name": "Novita Sari Harahap", "role": "Author Response", "response": "Introduction:1. Please explain why red-fleshed pitaya is thought to act as antioxidants that will affect the heat shock protein 70 and the expression of cortisol: include theoretical and other studies that support.Red fleshed pitaya has antioxidant activity. Red Dragon Fruit contains powerful antioxidants because it consists of various antioxidants, namely flavonoids, vitamin E, vitamin C (Lianiwati, 2011). The results of several studies stated the red dragon fruit (Hylocereus polyrhizus) has an anthocyanin content (Kanner et al., 2001) around 26,457 ppm. Dragon fruit contains flavonoids (Damayanti, 2016). Anthocyanin is a pigment that is classified as a type of water-soluble flavonoids. Anthocyanins are anthocyanidin and glucose compounds in organic acids (Shipp and Abdel Aal, 2010). Anthocyanins are believed to have a very good antioxidant effect. A study conducted at the University of Michigan in America showed that anthocyanin can destroy free radicals, more effectively than vitamin E which has been known as a powerful antioxidant. Other studies in the United States prove that anthocyanin is the most powerful antioxidant among other flavonoid classes. The content of anthocyanin is believed to inhibit various free radicals such as superoxide radicals and hydrogen peroxide. Anthocyanins and various forms of their derivatives can inhibit various oxidation reactions by various mechanisms (Astawan and Kasih, 2008). The results of previous studies ripe dragon fruit contains many organic acids (Stinzing, et al., 2004), protein (Le Bellec, et al., 2006), minerals such as potassium, magnesium, calcium and iron and vitamin C (Cal et al, 2003). Increased RONS (Reactive Oxygen and Nitrogen Species) production during exercise has potential negative effects, transient increases in RONS seem to be a trigger for many exercise-induced adaptations in skeletal muscle (Powers et al. 2010). Exercise can stimulate an increased concentration of HSP70 in muscles and plasma. Oxidative stress due to exercise can cause physiological functional changes because physical exercise is considered as a stimulus received by the hypothalamus, then the hypothalamus signals to the HPA axis, the HPA axis responds and gives positive and negative responses to the body (Guilliam & Edward, 2010). Then stimulates the hypothalamus and causes secretion of the hormone corticotrophin-releasing hormone (CRH), which stimulates the hypothalamus for ACTH secretion. Increased ACTH secretion, causes increased cortisol secretion (Usui et al, 2012).Methods:1. Why use 3 doses, whereas the purpose of the study did not assess the effective dose? In conclusion, also mention the effective dose. Giving 3 doses of 100 mg/kg BW, 200 mg/kg BW and 300 mg/kg BW to find out what is the optimal dose to neutralize oxidative stress due to strenuous physical exercise. A dose of 100 mg/kg BW can also reduce oxidative stress but not as well as a dose of 300 mg/kg BW. So it is necessary to experiment with 3 doses, the purpose of the study to find out the potential of Red-fleshed pitaya as an antioxidant and once to know the optimal dose to reduce oxidative stress.2. Explain the results of the identification of the chemical content of red-fleshed pitaya and also the results of herbarium analysis because there is a possibility that Indonesia's geographical differences will affect its chemical content.Red-fleshed pitaya is the cactus fruit of the genera Hyloreceus and Selenicereus. Dragon fruit is very popular and widely planted in Indonesia because it is known by the public as herbal medicine (Evi, et al., 2007). From the results of previous studies, ripe dragon fruit contains many organic acids (Stinzing, et al., 2004), protein (Le Bellec, et al., 2006), minerals such as potassium, magnesium, calcium and iron and vitamin C (Cal et al., 2003).3. Explain the time of blood-sampling because cortisol production is influenced by circadian.Cortisol levels are affected by circadian rhythm. Blood drawn for examination of cortisol is done in the morning because it is a basal state of the body after resting at night. Also besides, our bodies have biological variations according to time, meaning that the analytical levels examined in the morning can give different results if examined in the afternoon. On examination of Cortisol, the levels will increase in the morning and reach the lowest levels in the afternoon. Then the blood draw is done in the morning according to the normal human biological clock, the most efficient and does not result in excessive fatigue.Discussion:1. It is important to explain the role or mechanism of action of red-fleshed pitaya on heat shock protein 70 and cortisol expression.Research conducted by Rebecca et al (2010) shows that red dragon fruit contains the most polyphenols compared to other species, namely 86.13 ± 17.02 mg in 0.50 g of dried extract of red dragon fruit. Red dragon fruit (Hylocereus polyrhizus) is one of the plants that can be used as a source of antioxidants. Severe physical exercise is associated with increased HSP70 expression due to oxidative stress (Paulsen et al. 2007).Oxidative stress causes damage to cells that will cause stress responses in the form of heat shock response (HSR). HSR is regulated at the level of transcription by a mechanism involving heat shock transcription factor (HSF), especially HSF-1. Increased HSF-1 will help in increasing HSP 70 expression in certain amounts/levels in serum and blood plasma (Baird et al., 2006).Physical activity appears to occur concomitantly with the up-regulation of endogenous antioxidant systems (Powers and Jackson 2008) and heat shock proteins (HSP) in skeletal muscle (Morton et al. 2009b). While the main role of the antioxidant enzymes is to reduce oxidation and prevent oxidative damage, HSPs can prevent and reverse damage to proteins. Intriguingly, the HSPs work with the antioxidant systems, and collectively they have essential roles in cell homeostasis. Up-regulation of these proteins is, therefore, important adaptations for increased protection and recovery capacity in the face of cellular stress and damage induced by high-intensity exercise (Powers and Jackson 2008; Morton et al. 2009b).Antioxidants have been shown to abolish the acute exercise-induced increases in HSP70 protein levels (Khassaf et al. 2003; Jackson et al. 2004).Increased cortisol levels after physical activity depends on the level of fitness, exercise intensity and exercise program [Leite et al., 2011]. Exercise with heavy intensity tends to produce free radicals which can cause the production of the hormone cortisol to increase (Stachowicz, 2016).The provision of red dragon fruit that contains antioxidant polyphenols can reduce the production of free radicals due to exercise thereby reducing cortisol levels." }, { "c_id": "5684", "date": "29 Sep 2021", "name": "Novita Sari Harahap", "role": "Author Response", "response": "Thank you for this thorough review of the manuscript and your insightful comments that have improved the clarity and quality of the paper. Below there is a point-by-point response to your inquiries.The difference between this new version and version 1 is that in this new version, we can explain the antioxidant activity contained in red-fleshed pitaya that can affect heat shock protein 70 and the expression of cortisol. Red fleshed pitaya contains powerful antioxidants because it consists of various antioxidants, namely flavonoids, and polyphenol (Chemah et al. 2010; Adnan, 2011). Thus Red fleshed pitaya which has the potential as an antioxidant can reduce ROS and respond to HSP70 expression.We can also explain that giving 3 doses of 100 mg/kg body weight, 200 mg/kg body weight, and 300 mg/kg body weight to find out what is the effective dose to neutralize oxidative stress due to strenuous physical exercise. So it is necessary to experiment with 3 doses, in accordance with the purpose of the study to find out the potential of Red-fleshed pitaya as an antioxidant and once to know the effective dose to reduce oxidative stress which is influence on HSP70 and cortisol expression in rats that were subjected strenuous exercise.Red-fleshed pitaya is the cactus fruit of the genera Hyloreceus and Selenicereus. Red-fleshed pitaya is very popular and widely planted in Indonesia because it is known by the public as herbal medicine. Research by Widyaningsih et al., (2017) report that red-fleshed pitaya as an alternative treatment for anemia in pregnant women. The other studies by Irmayanti and Ardiaria (2016) report that red dragon fruit (Hylocereus Polyhizus) can reduce cholesterol levels in rats. This statement explains that red-fleshed pitaya have also affect difference." } ] }, { "id": "59870", "date": "04 Mar 2020", "name": "Mohd Adzim Khalili Rohin", "expertise": [ "Reviewer Expertise Functional Food & Nutraceuticals" ], "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 an interesting manuscript. However, there are several grammatical issues and some typos that you should check very carefully.\n\nMy main concern about your manuscript is that you have not mentioned any strengths or limitations in your study.\n\nThe small number of rats per group is indeed a limitation and should be mentioned and taken into consideration before making conclusions.\n\nThis is a prospective study on the effect of red-fleshed pitaya (Hylocereus polyrhizus) on heat shock protein 70 and cortisol expression in strenuous exercise-induced rats.\n\nThe conclusion is weak in my opinion.\n\nThe study could be considered more as a pilot study than a proper study; however the results are interesting and should be indexed.\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/8-130
https://f1000research.com/articles/10-983/v1	29 Sep 21	{ "type": "Research Article", "title": "Design and prototype of TOMO: an app for improving drug resistant TB treatment adherence", "authors": [ "Anis Fuad", "Guntur Budi Herwanto", "Ariani Arista Putri Pertiwi", "Siska Dian Wahyuningtias", "Harsini Harsini", "Ahmad Watsiq Maula", "Diyah Utami Kusumaning Putri", "Ari Probandari", "Riris Andono Ahmad", "Anis Fuad", "Guntur Budi Herwanto", "Ariani Arista Putri Pertiwi", "Siska Dian Wahyuningtias", "Harsini Harsini", "Ahmad Watsiq Maula", "Diyah Utami Kusumaning Putri", "Ari Probandari" ], "abstract": "Background: Drug resistance and multi drugs tuberculosis (DR/MDR-TB) are associated with patients' low adherence to undergoing complex treatment. Driven by the increasing use and penetration of a smartphone and the End of TB Strategy that seeks for digital health solution, Center for Tropical Medicine Universitas Gadjah Mada has developed TOMO, an Android-based app for improving medication adherence in MDR-TB. Objective: This paper aims to present the sequential steps to develop the app, its general architecture, and its functionalities. Methods: It is a design thinking process involving two MDR-TB referral centers, district health offices, primary health centers, and MDR-TB patients in Central Java and Yogyakarta, Indonesia. We adopted the Principles for Digital Development to develop and design the app. MDR-TB treatment guideline from the Indonesian Ministry of Health was used to develop functionalities of the app for improving adherence.\n\nResults: TOMO app could be used by patients, primary health centers, clinical teams, and case managers. The app prototype features include adverse event records and reports, medication-taking reminders, and communication between the patient and the TB-MDR case manager. We have successfully tested the functionalities based on four use cases: patients with high adherence, patients with low adherence, patients with adverse events, and patients following treatment in the primary health center without any visit to the MDR-TB center.\n\nConclusion: TOMO app has contributed to the limited body of literature on improving TB-MDR adherence with digital health intervention, especially using a health app. The app has been tested using four scenarios. We will follow up with usability testing before implementing the app in a real setting.", "keywords": [ "m-health", "tuberculosis", "drug resistance", "medication adherence", "smartphone", "Indonesia" ], "content": "Introduction\n\nMulti-drug resistant tuberculosis (MDR-TB) is associated with high treatment failure due to low adherence of patients in` undergoing the complex treatment process.1 The prolonged duration of treatment, the huge opportunity costs incurred by daily visits to health facilities, and the wide range of adverse drug events have been associated with high default rates of MDR-TB patients.1 Stigma, feeling ashamed, and boredom are also huge barriers to improving patient adherence to treatment.2–4\n\nIn Indonesia, the country with the fourth largest tuberculosis (TB) burden, MDR-TB cases are alarming. It is estimated that there are 32,000 cases of MDR-TB of which only 8% were detected in the TB program. The performance of MDR-TB case management urgently needs to be improved. In 2016, about 29% of cases died within the first six months after diagnosis, and 19% did not continue treatment. Therefore, a patient-centered care approach is necessary to support MDR-TB patients during long treatment to ensure medication adherence, oversee potential side effects, and offer psychosocial support. Adherence to the long course of TB-MDR treatment is a complex endeavor involving multiple factors that influence patients’ motivation and behavior in treatment-taking.1,5,6\n\nPrevious studies on other chronic conditions have shown that treatment adherence can be improved using m (mobile)-health.6–10 One of the functions of m-health is to empower patients by providing information for decision-making, consultation and advice from physicians, treatment regimen schedules, and providing better opportunities to control their healthcare.11–14 In developing countries, m-health is mainly used for providing disease information, reminders for care, and telemedicine. However, few m-Health applications exist for TB. Iribarren et al. (2016) found only two apps, and none were related to TB treatment self-management or patient-provider interaction.15\n\nDigital health technologies have been considered a potential tool to improve medication adherence in tuberculosis.9,14 Through mobile health innovations, patients will be empowered to easily request consultation and advice regarding treatment regimen, scheduling, and side effects. A recent study exploring patients and medical staffs in six countries reported a high enthusiasm for e-health innovation to support TB programmes, including apps16 and video observing treatment apps.17\n\nDigital health intervention has been recommended by WHO to strengthen health systems in at least nine use cases.18 Digital health intervention is projected to contribute to substantial cost saving and improvement of TB programs.13,19 A comprehensive review of the potential for digital health in TB programmes has been published. However, there are still only a few studies that attempt to explain the role of digital health innovation in improving medication adherence.\n\nAlong with the increasing ownership of cellular phones, especially smartphones, health intervention through applications is no longer just a reminder or reciprocal communication. Health apps can also act as an integrated application that includes various functions developed for specific purposes. In this context, we intend to report on the design and technical overview of apps specifically designed to help manage the MDR-TB program.\n\nThis paper aims to:\n\n1. Describe sequential steps to develop apps involving multiple stakeholders in the MDR-TB program\n\n2. Draw technical architecture and interoperability of the app with the existing MDR-TB information system\n\n3. Test features and functionalities of the app based on scenarios in the real setting.\n\n\nMethods\n\nThis study was a collaborative work by the Center for Tropical Medicine Universitas Gadjah Mada, Dr. Sardjito hospital in Yogyakarta province, Dr. Moewardi hospital in Central Java province, and Computer Science department of Universitas Gadjah Mada. Following subsequent steps in design thinking, all collaborators teamed up to develop the best health app solution to improve MDR-TB treatment adherence.\n\nParticipants in this study were patients with MDR-TB in Dr. Moewardi hospital, healthcare professionals (pulmonologists from Dr. Sardjito and Dr. Moewardi hospital, MDR-TB case manager at hospital and primary health care level), and district health department officers. Researchers from the Center for Tropical Medicine Universitas Gadjah Mada were the principal investigators in this study and translating user needs from clinical setting perspectives into health app workflow and design.\n\n\nStudy design\n\nFive steps of design thinking were adopted in this study; empathize, define, ideate, prototype, and test.20 Design thinking is an analytic and creative approach in solving problems or fulfilling user needs in the app development context.21,22 Perspectives from multiple stakeholders or users that will use the health app were the most important data in the app development processes. The details of each step and type of data collected are depicted in Table 1.\n\nWe conducted meetings with two MDR-TB referral hospitals; Dr. Sardjito hospital in Yogyakarta province and Dr. Moewardi hospital in Central Java province. The first meeting with Dr. Sardjito hospital icluded the empathize and define processes to collect and summarize any stories and problems that providers had in monitoring MDR-TB treatment. In addition, this study also collected information from MDR-TB patients to ensure the app covered all user needs. After problems were defined, the ideate process was started by creating a list of possible solutions and the workflow of TB application features. The prototype process based on the chosen solution was conducted iteratively. Dr. Moewardi hospital was used as the primary setting to improve the workflow of the TB app. Two workshops to introduce the app for case managers, physicians, and 13 PHC in Dr. Moewardi’s area were conducted in the test process. Prior to the workshop, a simulation mimicking use cases in a real setting was conducted by researchers.\n\nIn 2017-2018, Dr. Moewardi hospitals provided treatment to 207 patients. MDR-TB clinic in this hospital is supported by one pulmonologist, one dedicated case manager, five other supporting staff. This hospital utilized TB-Manager, a web-based application from the Ministry of Health, to register, monitor, and report the MDR-TB program. Later, this program was changed with a new electronic system called SITB (Sistem Informasi TB). For internal purposes, the hospital also implemented a hospital information system for billing and recording all patients’ transactions, including MDR-TB.\n\n\nEthical considerations\n\nThis study was started in September 2019. The study protocol and procedure were approved by the Ethical Committee of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta (No: KE/FK/0990/EC/2019 and KE/FK/1285/EC/2020).\n\n\nResults\n\nA total of four workshops and seven other activities, including observation, survey, and simulation, were conducted in this study.\n\nInsight from the empathize and define steps\n\n- The MDR-TB treatment monitoring using the current system was not able to track patient treatment adherence daily. Furthermore, the system was only focused on collecting patients baseline data and drug regimens.\n\n- Primary health care performance in supervising dai drug consumption of MDR-TB patients was not monitored. Primary health care in MDR-TB treatment is vital since patients need to visit it daily to take the drugs and report any adverse events.\n\n- Communication activities related to adverse events of MDR-TB drugs were not well organized and documented. Patients could directly consult the pulmonologist, or sometimes they could ask the case manager or primary health care first. During the consultation, the information was not recorded and could cause problems for further patient care related to the adverse events.\n\n- Adverse drug effects were issues that influenced patient adherence to MDR-TB treatment. In addition, the patients also needed social support and opportinuies to share amongst patients with MDR-TB to improve treatment adherence.\n\n- The findings from the patient’s survey were that most patients owned an Android smartphone (69.23%), and that around 80% of respondents used internet browsers and social media apps (i.e., Whatsapp).\n\nThe solution identified from the ideation step\n\n- In workshop 2, users, researchers, and the app development team decided on solutions that would become the app’s main functionalities. The first function was reporting MDR-TB drug consumption for patients and cross-checking the patients’ drug consumption by healthcare workers in the hospital or primary health care. Second, a drug adverse event reporting function could be accessed by pulmonologists, case managers, and primary healthcare workers. Third, a real-time dashboard to monitor the treatment progress of TB-MDR patients.\n\n- In order to be interoperable with the national TB information system, the research team has discussed with the TB program manager and the technical team to develop an Application Programming Interface (API), so that data from the two systems can be exchanged securely without user intervention.\n\n- A MDR-TB patient forum within the app could help patients give social support to each other during treatment. Post-treatment patients could also share their experiences to help other patients who are still undergoing the treatment process.\n\nIn the ideation step, some vital app features were chosen for initial prototype development. The cross-checking drug consumption, drug adverse event report and consultation, and monitoring dashboard were chosen for the initial phase. Other important features will be developed later after the prototype’s main features are implemented in the real setting.\n\n\nPrototype and test step\n\nThe User Interface (UI) and User Experience (UX) were developed to depict the initial app workflow and design. After several tests and revisions by researchers and users (pulmonologists and the case manager), the technical architecture and workflow of the app were established.\n\nThe first application is for TB-MDR patients (TOMO). The second application is dedicated only to providers, including Primary Health Centers, the case manager, and the treating physician (TOMO CM). The first two applications are targeted as mobile-based applications. Before publication of this paper, we will have developed the Android application to be available on PlayStore. The third application is a web-based application that will be used as a dashboard by the government to see the usage overview of the application. The web-based application is also used as an admin tool for managing user and master data. These three applications work together to achieve the primary goal. Figure 1 shows the use diagram that describes the functionality of each user within the system.\n\nThe current prototype of the TOMO application was developed in an Android environment using Kotlin programming language. Furthermore, the TOMO website was developed using PHP 7 programming language. MySQL 5.6 was used as the database management system, while Django Python (Django, RRID:SCR_012855) was used to develop the API (application programming interface) in PHP language for the application and website.\n\nThe Android applications are available on the Play store, and they can be used using Android with minimal version Kitkat (4.4). The memory needed for the TOMO app was 5.3 MB memory, and for the TOMO CM was 5.8 MB memory.\n\nThe sequential workflow of the prototype (Figure 2) shows the process from patient baseline data input, drug consumption monitoring process, adverse event consultation, and the in-person visit schedule feature. Each user type has a different feature in the app. The patient will be responsible for reporting drug consumption and adverse events and for viewing any scheduled visits. The case manager is responsible for inputting patient data and drug regimens, validating drug consumption, responding to patient adverse events, and creating a schedule for physician visits. The primary health center will be mostly responsible for drug consumption validation and reporting patient adverse events. The physician (Pulmonologist) is responsible for evaluating treatment progress and responses to adverse events. The interoperability of the prototype with the existing MDR-TB app was included in the workflow. The patient baseline data will be directly retrieved from the existing app using the API provided by the existing MDR-TB app.\n\nThe iterative processes of the test were conducted to ensure the performance and feature suitability of the prototype. The app developer conducted alpha and beta testing for bug detection. The simulation by researchers showed that the app functionalities performed well in four different use cases: patients with high adherence, patients with low adherence, patients with adverse events, and patients following treatment in the primary health center without any visit to the MDR-TB center. Two workshops for prototype introduction were conducted by inviting all users. Through the process, the prototype workflow and interface were finalized, and prototype performance was evaluated. The final prototype interface is presented in Figure 3.\n\n\nDiscussion\n\nThis work represents the preliminary design of an app to support the MDR-TB program. The design thinking method in the development process was able to fulfill all user’s needs. In regard to the nine principles of digital development,23 this app development process is inline and covered all aspects of the principles (Table 2). USAID developed the principle to avoid a failure in the technology-supported program for development.23 The elaboration of the principle and design thinking method allows the MDR-TB app to be more acceptable and sustainable to improve MDR-TB treatment adherence.\n\nSuccessful experiences from other countries in terms of tuberculosis-related apps were previously reported. In Brazil this consisted of a dedicated Tuberculosis information system within the notifiable disease surveillance system.24 In Peru, a web-based laboratory information system for tuberculosis was reported in improving communication delay in tuberculosis management.25,26 This work proposes a novel innovation of tuberculosis monitoring by adopting a cross-checking method of drug consumption. Through this method, patient adherence to the treatment and healthcare provider performance as the drug consumption observer could be monitored and evaluated in a real-time fashion. The adverse event consultation session data between patient and healthcare provider were collected in the system and could be used for patient treatment planning in the future.\n\nThe study in Brazil proposes web-semantic approach for solving interoperability issues between multiple systems.27 In Indonesia, an initiative has been conducted between TB information systems and hospital information systems. However, a systematic review of the literature revealed that there is a lack of evidence-based incentives for researchers to share data.28 In this regard, an integrated approach has been proposed by Fraser et al. to improve TB information system. This includes open data standards and interoperability, integration with mHealth applications, and ability to function in resource-poor environments.29\n\n\nChallenges of app for TB\n\nMobile phones are the fastest adopted modern technology in developing countries, as has already happened in developed countries. The abilities and features of mobile phones are promising tools in improving community health and health literacy. However, despite the high incidence of mobile phone ownership in the population, the ownership among patients with TB itself is still low. One third of patients with TB in the United States do not have smartphone.30 The situation is similar for patients with hypertension.31 This phenomenon is no different in Indonesia - among poor families one mobile phone is usually used by more than one family member. In addition, as in other developing countries, the ownership of a mobile phone is sometimes not concurrent with the ability to get internet subscription.32 It is thus important for the government to ensure affordable internet access to all communities.\n\nAnother challenge is lack of literacy among communities. Many patients with TB come from poor families with low levels of education.32 As mentioned by many studies, lack of literacy is related to poverty and low educational attainment. Furthermore this situation usually leads to inadequate health literacy, which can inhibit the patient’s desire to use novel tools like mobile applications to improve their health. It is important to understand how this particular population interacts with their mobile phone to ensure that the mobile health applications are developed according to these particualr characteristics, so that they are easy to learn and use.\n\nTOMO as a mobile application has a high potential to help patients with MDR-TB in Indonesia. It has been explained in previous studies that patients have lower adherence to completion of treatment when they have had negative treatment experiences.33 These include substantial travel time to get access to care, missed earning time because they have to spend a lot of time at the healthcare facilities, and other expenses by the patients and family who accompanied patient to the facilities. TOMO is developed to be the solution to these difficult situations by functioning as a communication platform which can connect patients directly with their case managers and attending physicians.\n\n\nFuture directions\n\nWe plan to undergo several iterations of evaluation, examining both the usability and content of the app before initiating the trial in a real setting. However, a few improvements are needed based on the real use cases, including modifications of the algorithm. In the near future, data exchange between TOMO with SITB (Sistem Informasi TB) from the Ministry of Health will be exercised. After the implementation of TOMO in Dr. Moewardi Hospital, we will conduct workshops to develop a sustainability plan and initiate scaling up.\n\nOur study has several limitations. Firstly, this evaluation of the use cases was performed in a laboratory setting by the research team members, aiming to validate the functionalities and the system workflow. Secondly, the export/import functionalities were applied based on the former electronic system, namely eTB Manager. Currently, the Ministry of Health has piloted a new electronic TB information system. This new platform offers API functionality offering electronic data exchange of certain variables into our application. We expect interoperability between our prototype with the new system before testing the application in the real setting.\n\n\nConclusion\n\nThis report depicts TOMO’s design, technical architecture, and functionalities, an app for improving medication adherence in TB-MDR treatment. This app is a mobile-based platform aimed to improve communication between TB-MDR providers and patients. It contributes to the limited body of literature on improving TB-MDR adherence with digital health interventions, especially using health apps. The prototype features include the adverse event record and report system, reminders for drug taking, and a platform for communication between patients and TB-MDR case managers. The prototype will later undergo several iterations of evaluation for usability and content. Future work will involve developing this into a fully functioning app before initiating the trial in a real setting.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nRumende CM: Risk Factors for Multidrug-resistant Tuberculosis. Acta Medica Indones. 2018 Apr 20; 50(1): 1. PubMed Abstract\n\nMphothulo N, Pengpid S, Peltzer K: Factors associated with tuberculosis reinfection and treatment failure in Taung Sub-District. South Africa. Stud Ethno-Med. 2012; 6(1): 23–30.\n\nRedwood L, Mitchell EMH, Viney K, et al.: Depression, stigma and quality of life in people with drug-susceptible TB and drug-resistant TB in Vietnam. Int J Tuberc Lung Dis. 2021; 25(6): 461–467. PubMed Abstract \| Publisher Full Text\n\nNaidu T, Pillay SR, Ramlall S, et al.: Major Depression and Stigma among Individuals with Multidrug-Resistant Tuberculosis in South Africa. Am J Trop Med Hyg. 2020; 103(3): 1067–1071. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeshmukh RD, Dhande DJ, Sachdeva KS, et al.: Social support a key factor for adherence to multidrug-resistant tuberculosis treatment. Indian J Tuberc. 2018 Jan; 65(1): 41–47. PubMed Abstract \| Publisher Full Text\n\nWang Y, Chen H, Huang Z, et al.: Drug Non-Adherence and Reasons among Multidrug-Resistant Tuberculosis Patients in Guizhou, China: A Cross-Sectional Study. Patient Prefer Adherence. 2019; 13: 1641–1653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalzon D, Migliori GB, Jaramillo E, et al.: Digital health to end tuberculosis in the Sustainable Development Goals era: achievements, evidence and future perspectives. Eur Respir J. 2017 Nov; 50(5): 1701632. PubMed Abstract \| Publisher Full Text\n\nYoeli E, Rathauser J, Bhanot SP, et al.: Digital Health Support in Treatment for Tuberculosis. N Engl J Med. 2019 Sep 5; 381(10): 986–987. PubMed Abstract \| Publisher Full Text\n\nHaberer JE, Subbaraman R: Digital Technology for Tuberculosis Medication Adherence: Promise and Peril. Ann Am Thorac Soc. 2020 Apr; 17(4): 421–423. PubMed Abstract \| Publisher Full Text\n\nLee Y, Raviglione MC, Flahault A: Use of Digital Technology to Enhance Tuberculosis Control: Scoping Review. J Med Internet Res. 2020 Feb 13; 22(2): e15727. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnwar SL, Harahap WA, Aryandono T: Perspectives on how to navigate cancer surgery in the breast, head and neck, skin, and soft tissue tumor in limited-resource countries during COVID-19 pandemic. Int J Surg Lond Engl. 2020 Jul; 79: 206–212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusiimenta A, Tumuhimbise W, Mugaba AT, et al.: Digital monitoring technologies could enhance tuberculosis medication adherence in Uganda: Mixed methods study. J Clin Tuberc Mycobact Dis. 2019 Dec; 17: 100119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNgwatu BK, Nsengiyumva NP, Oxlade O, et al.: The impact of digital health technologies on tuberculosis treatment: a systematic review. Eur Respir J. 2018 Jan; 51(1): 1701596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubbaraman R, de Mondesert L, Musiimenta A, et al.: Digital adherence technologies for the management of tuberculosis therapy: mapping the landscape and research priorities. BMJ Glob Health. 2018; 3(5): e001018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIribarren SJ, Schnall R, Stone PW, et al.: Smartphone Applications to Support Tuberculosis Prevention and Treatment: Review and Evaluation. JMIR MHealth UHealth. 2016 May 13; 4(2): e25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMargineanu I, Louka C, Vincenti-Gonzalez M, et al.: Patients and Medical Staff Attitudes Toward the Future Inclusion of eHealth in Tuberculosis Management: Perspectives From Six Countries Evaluated using a Qualitative Framework. JMIR MHealth UHealth. 2020 Nov 2; 8(11): e18156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDo D, Garfein RS, Cuevas-Mota J, et al.: Change in Patient Comfort Using Mobile Phones Following the Use of an App to Monitor Tuberculosis Treatment Adherence: Longitudinal Study. JMIR MHealth UHealth. 2019 Feb 1; 7(2): e11638. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: WHO guideline: recommendations on digital interventions for health system strengthening [Internet].2019 [cited 2021 Jun 25]. Reference Source\n\nNsengiyumva NP, Mappin-Kasirer B, Oxlade O, et al.: Evaluating the potential costs and impact of digital health technologies for tuberculosis treatment support. Eur Respir J. 2018 Nov; 52(5): 1801363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStanford HPI of D at: An introduction to design thinking: process guide.2010.\n\nBrown T, Wyatt J: Design thinking for social innovation. Dev Outreach. 2010; 12(1): 29–43.\n\nHou I-C, Lan M-F, Shen S-H, et al.: The Development of a Mobile Health App for Breast Cancer Self-Management Support in Taiwan: Design Thinking Approach. JMIR MHealth UHealth. 2020 Apr 30; 8(4): e15780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaugaman A: From principle to practice: implementing the principles for digital development. Proc Princ Digit Dev Work Group. 2016; 4.\n\nBartholomay P, Pinheiro RS, Pelissari DM, et al.: Special Tuberculosis Treatment Information System (SITE-TB) in Brazil: background, description and perspectives. Epidemiol E Serviços Saúde. 2019; 28: e2018158. PubMed Abstract \| Publisher Full Text\n\nBlaya JA, Shin SS, Yagui M, et al.: Reducing communication delays and improving quality of care with a tuberculosis laboratory information system in resource poor environments: a cluster randomized controlled trial. PloS One. 2014; 9(4): e90110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlaya JA, Shin SS, Yagui MJ, et al.: A web-based laboratory information system to improve quality of care of tuberculosis patients in Peru: functional requirements, implementation and usage statistics. BMC Med Inform Decis Mak. 2007; 7(1): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPellison FC, Rijo RPCL, Lima VC, et al.: Data Integration in the Brazilian Public Health System for Tuberculosis: Use of the Semantic Web to Establish Interoperability. JMIR Med Inform. 2020; 8(7): e17176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowhani-Farid A, Allen M, Barnett AG: What incentives increase data sharing in health and medical research? A systematic review. Res Integr Peer Rev. 2017; 2(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFraser HS, Habib A, Goodrich M, et al.: E-health systems for management of MDR-TB in resource-poor environments: a decade of experience and recommendations for future work. MedInfo. 2013; p. 627–31.\n\nBommakanti KK, Smith LL, Liu L, et al.: Requiring smartphone ownership for mHealth interventions: who could be left out? BMC Public Health. 2020; 20(1): 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLangford AT, Solid CA, Scott E, et al.: Mobile phone ownership, health apps, and tablet use in US adults with a self-reported history of hypertension: cross-sectional study. JMIR MHealth UHealth. 2019; 7(1): e12228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLatif S, Rana R, Qadir J, et al.: Mobile health in the developing world: Review of literature and lessons from a case study. IEEE Access. 2017 Jun 1; 5: 11540–11556. Publisher Full Text\n\nAisyah DN, Ahmad RA, Artama WT, et al.: Knowledge, Attitudes, and Behaviors on Utilizing Mobile Health Technology for TB in Indonesia: A Qualitative Pilot Study. Front Public Health. 2020; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "255407", "date": "15 Apr 2024", "name": "Ellen Mitchell", "expertise": [ "Reviewer Expertise TB" ], "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\nTOMO App Review Thank you for the opportunity to review this manuscript about the sequential development and piloting of a mobile app designed to facilitate communication among parties in the DR-TB treatment process. There are strengths, areas for further elaboration, and questions about the manuscript that are summarized as follows: Strengths\n\nThe authors are to be commended for the thoughtful, step-wise process described in this manuscript. This manuscript will be useful for those seeking to replicate the process in other contexts. The key features of interoperability, trialability, automatic aggregation and dashboard, seem very good for lowering the work load of busy health care workers and program staff. Documenting patient reported outcomes(PRO) is so important and the DR-TB field is so far behind in this area, that it is great to see pharmacovigilance incorporated in a digital health tool. While some might argue that this is shifting the recording and reporting AE burden from the clinician to the patient, others may see it as providing a forum for patients to voice their discomfort and suffering in real time. The question then becomes: what, if any remedial actions are taken in response to the call for help. We must all stay tuned for the next paper to find out. It is only empowering if the call for help is answered with support and solutions.\nAreas for further refinement\nThe authors suggest at the beginning that adverse drug events are the main driver of non-persistence on treatment. Certainly, side effects, symptoms of the disease, stigma, catastrophic costs, low quality care, and other barriers also play a role in early withdrawal from a treatment course. Later on in the manuscript is this statement “ [drivers] include substantial travel time to get access to care, missed earning time because they have to spend a lot of time at the healthcare facilities, and other expenses by the patients and family who accompanied patient to the facilities. TOMO is developed to be the solution to these difficult situations by functioning as a communication platform which can connect patients directly with their case managers and attending physicians.” This sentences seems to imply that if they use the app they don’t need to go to the health facility daily, that the self-reported self administration of treatment (SAT) relieves them of this obligation. Is that correct? Is this a replacement for DOT? That is inferred but not explicitly stated. If it is not a form of SAT, then how exactly does TOMO solve the substantial travel time and missed earning time issue? It would be good to have a figure or a mapping of what the main reasons for forsaking treatment are (from the patient survey) and how the app’s features specifically corresponds to these.\n\nThere seems to be a disconnect at times between what is expressed as a need and what is provided as a service. From the survey, MDR patients report needing social support. The notion that an unmoderated chat group of MDR-patients would provide support and increase adherence is interesting, but I am unaware of the evidence to support this claim. Consider to offer some evidence to motivate the decision to have an unmoderated chat at the support intervention . Unmoderated chat groups can be used in many ways by their users. It would be good to know if the group is anonymous or not. Anonymous chat groups of vulnerable groups have not had a great track record. For example, peer-support apps for opioid disorder often contain misinformation, trolling, and harassment. How will you ensure that people with TB are not harmed in the group chat? The authors point out that there is a lot of enthusiasm for digital health for TB adherence. However, it would be good to point out that the results of digital health trials for modifying behavior of people with TB have been mixed. Qualitative work suggests that patients value the human element in their care and are not necessarily keen to interact with robots, electronic pillboxes, SMS messages.\nComment regarding the App development (not the manuscript)\nThe TOMO app seems to have been co-designed with pulmonologists and primary care workers. The MDR patients are surveyed at the beginning, and they are involved in testing the prototype, but the middle part of the actual design of the app features does not include them. Digital health for end users with TB that is not co-designed by people with TB can lead to them feeling even further alienated from society. It appears that health care workers are the main beneficiaries of this system as currently designed.\nAreas that are not yet clear to this reader:\nAccess to smart phones is lower among women and poorer persons. The provision of support and other benefits via smart phones and the requirement of having a bank account disadvantages women, those under 18, and the poor. Lack of electricity, internet connectivity; and low technology literacy are also more common among the poor. How can the app be modified to contribute to equity? How do the 31% without a smart phone differ from those with a smart phone? While reporting adverse events is important for the program, it is less clear how this is useful for the patient, unless there is a change in regimen or offer of symptom management. Does the app measure whether and how long it takes for there to be a clinical response? Are the reminders to respond also pinging the HCWs? Or just the patients?\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "255412", "date": "23 Apr 2024", "name": "Wilson Tumuhimbise", "expertise": [ "Reviewer Expertise digital 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\nThank you for the opportunity to review this manuscript. I have noted a few areas that require clarification. Methods How many collaborators were involved. Its wonderful to be specific about the exact number of collaborators that were part of this phase. This should be revised in the entire manuscript so that specificity and clarity and ensured.\nThere is a need to ensure consistency in wording, in some sections, you mention meetings while in others you mention workshops, these should be revised. Ideally it would be great to mention the composition of these meetings, how many members made up each of these meetings, how were they selected, what was the eligibility criteria, this needs to be clear and how was data from these meetings obtained, was it through recordings and later transcribed verbatim.\nHealth apps, deal with a huge amount of sensitive health data, how is the security of this app ensured given that unintended disclosure of health status has been cited in some literature.\nIs this app available free of charge or it requires participants to pay a certain fee additionally, does it rely on the internet to function, or it has an offline version?\nProvide the section of strengths and limitations of this study.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "255409", "date": "03 Sep 2024", "name": "Malaisamy Muniyandi", "expertise": [ "Reviewer Expertise Health Economics", "HTA", "TB" ], "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\nManuscript number F1KR00CDE F1R-VER70812-R Title: Design and prototype of TOMO: an app for improving drug resistant TB treatment adherence\nOverall comments: This manuscript focused on an important topic an app for improving drug resistant TB treatment adherence and presented the sequential steps to develop the app, its general architecture, and its functionalities. Overall this manuscript address important issue and well written paper I recommended for indexing with minor revisions and worth emphasising the following points.\nSpecific comments\n\nThe authors mentioned that already few m-Health applications exits for TB. It will be useful for the reader, provided the details on what is novel in this app or any new features added in this app. What is the treatment success rate if we use this app to increase treatment adherence for MDR-TB What way it is different than the 99DOTS, MERM boxes, What is the recommendation for us of this app to other countries to increase MDR-TB treatment adherence. Is it only for MDR-TB; What about other TB patients for this app\n\nConclusion to be based on this current study findings. The conclusion provided in this paper is not from this study findings\n(TOMO app could be used by patients, primary health centers, clinical teams, and case managers……)\nThis is the good work, needs further for implementation to see the feasibility and acceptability of this app for MDR-TB patients Minor edits required\nBackground: ‘multi drugs tuberculosis’ to be changed as ‘multi drug resistance tuberculosis’ TB-MDR to be changed as MDR-TB The terms used are m-Health, mobile health, e-TB manger, mHealth is these are same, or what is context authors describing these terms. If these are same then the same terminology to be used throughout the text. Need to provide references for: Page-3 Para 5: line 2\n\nA comprehensive review of the potential for digital health in TB programmes has been published.\n\nPage-9 Para 1: line 1\n\nSuccessful experiences from other countries in terms of tuberculosis-related apps were previously reported.\n\nPage-9 last Para: line 2\n\nAs mentioned by many studies, lack of literacy is related to poverty and low educational attainment\n\nAbbreviation TB to followed throughout text Conclusion to changed; it seems to be developed the app, it needs to checked under the field condition.\n\nWhat about cost-effeteness of this intervention\n\nOverall it is a good work needs revision considering the above 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? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-983
https://f1000research.com/articles/10-982/v1	29 Sep 21	{ "type": "Study Protocol", "title": "Study protocol for the SeMaCo study: A longitudinal regional cohort study to assess COVID-19 seroprevalence in blood donors", "authors": [ "Robert Pohl", "Stephan-Werner Krämer", "Christoph Stallmann", "Enno Swart", "Pauline Marquardt", "Achim-Jens Kaasch", "Christian-Joachim Apfelbacher", "Hans-Gert Heuft", "Stephan-Werner Krämer", "Christoph Stallmann", "Enno Swart", "Pauline Marquardt", "Achim-Jens Kaasch", "Christian-Joachim Apfelbacher", "Hans-Gert Heuft" ], "abstract": "Introduction: Serologic studies are crucial for clarifying the regional dynamics of the SARS-CoV-2 coronavirus pandemic as well as the success of a vaccination campaign against COVID-19. We describe a cohort study investigating the seroprevalence of antibodies against SARS-CoV-2 in Magdeburg (Saxony-Anhalt, Germany). Protocol and study design: The SeMaCo study (Serologische Untersuchungen bei Blutspendern des Großraums Magdeburg auf Antikörper gegen SARS-CoV-2) is a longitudinal, regional cohort study to assess the seroprevalence of COVID-19 in blood donors from Magdeburg (Capital of Saxony-Anhalt) and surrounding areas. We consider blood donors as a surrogate for the healthy, working-age population of Saxony-Anhalt. The study primarily aims to measure the prevalence and kinetics of IgG antibodies against SARS-CoV-2 in first time and repeat blood donors over a period of 21 months. The study explores four survey periods of three to four months each (January–April 21, July–October 21, February–April 22, July–October 22). At each visit, we will assess the attitude towards vaccination, the antibody response following vaccination, as well as undesired vaccination effects. Furthermore, we will collect data on occupational activities, housing conditions and the frequency of family and other social contacts.\nDiscussion: The SeMaCo study extends the spectrum of seroepidemiological investigations in Germany. A longitudinal observation with repeated testing and serial interviews can provide a more accurate view on the dynamics of COVID-19 prevalence and spread than repeated cross-sectional studies. Based on interim results from similar studies, we expect a seroprevalence of SARS-CoV-2 antibodies below 5% in the first survey period. SeMaCo will influence policy decisions and preventative measures.", "keywords": [ "SeMaCo study protocol", "COVID-19", "SARS-CoV-2 antibodies", "seroprevalence", "repeat blood donors", "COVID-vaccination", "epidemiology" ], "content": "Introduction\n\nThe SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2) has been circulating since December 2019. It is the causative agent for COVID-19 (coronavirus disease 2019). Transmission is facilitated through infectivity in pre- and asymptomatic disease (Robert Koch-Institut – SARS-CoV-2 virus – profile). In Germany, more than 3.5 million people have contracted COVID-19 and more than 84,000 people have died as of 8th May 2021 (Robert Koch-Institut – SARS-CoV-2 virus – report).\n\nAt the beginning of the pandemic, the federal state of Saxony-Anhalt (FSA) was considered a COVID-19 low prevalence area within Germany (Robert Koch-Institut SARS-CoV-2 virus – report). Meanwhile, the FSA has a seven day incidence of COVID-19 in 103 per 100,000 inhabitants, which is in line with the nationwide average (as of 14th May 2021) (Robert Koch-Institut – SARS-CoV-2 virus – case numbers). Data from Japan and Italy show that approximately 18–23 % of all individuals infected with SARS-CoV-2 are asymptomatic.1,2 Therefore, the number of COVID-19 infections in the FSA may still be substantially underestimated.\n\nPatients suffering from COVID-19 develop a cellular and humoral immune response. Detailed data on the long-term kinetics of the immune response are currently not available. Neutralizing antibodies3 and memory T-cells4 have been shown to persist for at least several months. By vaccination, high neutralizing antibody titers can be induced with vaccine efficacies of up to 95% in clinical studies.5 However, reports show that reinfections occur6 and vaccines do not offer 100% protection.7 During the course of the pandemic, new variants of the virus may lead to immune escape.\n\nEpidemics become self-limiting when herd immunity is reached through natural infection or mass vaccination. The threshold when herd immunity can be expected is influenced by various factors such as the basic reproduction number, occurrence of immune escape variants, and waning immunity. Monitoring seroprevalence and circulating variants can help to understand these mechanisms.\n\nDue to the temporary successful containment of SARS-CoV-2 in summer 2020 and despite the current exponential viral spread only a small proportion of the German population has experienced a SARS-CoV-2 infection. In March 2021, the seroprevalence of specific SARS-CoV-2 antibodies in a German population sample (93,000 blood donors) was reported to be a small proportion 7–8% (Robert Koch-Institut – Project RKI/SeBluCo Report) The use of safe and effective vaccines should protect individuals and the population from SARS-CoV-2 infection and/or COVID-19 disease. The vaccination start date in Germany was 27th December 2020. Several effective vaccines are currently available. However, a third wave with exponential growth, with variants of SARS-CoV-2, is currently being observed in Germany.\n\nThe detection of SARS-CoV-2-specific IgG antibodies in serum is a relatively reliable indicator of infection. About 90 % of patients with mild symptoms develop SARS-CoV-2 –specific IgG antibodies in serum whereas IgG is formed by all patients with severe symptoms.8 IgM and IgG antibodies are formed approximately simultaneously, 7–14 days after infection.9\n\nMost commercially available serological tests detect SARS-CoV-2 antibodies against either the spike protein or the nucleocapsid protein. The spike protein is essential for binding to and fusion of the virus with the target cell. It can elicit a protective immune response and is also used as an antigen in vaccines.\n\nThe detection of specific antibodies to SARS-CoV-2 in a representative group will provide a better estimate of the true extent of the COVID-19 epidemic in the FSA. The SeMaCo study (Serologische Untersuchungen bei Blutspendern des Großraums Magdeburg auf Antikörper gegen SARS-CoV-2) will observe blood donors in the city of Magdeburg (capital of the FSA with 240,000 inhabitants) and surrounding areas.\n\n\nObjectives\n\nThe primary objective of the study is to determine the dynamics of the antibody response to the spike protein of SARS-CoV-2 on a population level. Important secondary objectives are:\n\n1. To investigate the influence of age, gender, occupational activities, domestic conditions, and the frequency of family and other social contacts, on the incidence of SARS-CoV2 antibodies.\n\n2. To analyse differences between participants with symptomatic and asymptomatic COVID19 disease courses.\n\n3. To assess vaccination preparedness and the effect of a vaccination campaign on vaccination preparedness.\n\n4. If a new vaccine becomes available, to analyse the influence of vaccination on the antibody detection rate.\n\n5. To determine test characteristics of other serological tests for antibodies against SARS-CoV-2 spike and nucleocapsid antigens, other coronaviruses like SARS, MERS, 229E NL63, and HKU1, as well as influenza A and B antigen subtypes.\n\n\nProtocol\n\nThis prospective, longitudinal cohort study will recruit donors at the blood donation service of the University Hospital Magdeburg in the FSA. Donors of blood, plasma, and thrombocytes will be asked to participate during four survey periods. Existing blood samples from 2019 will also be tested (negative controls):\n\nBS-0: ca. 2,000 existing samples from 2019 (Q3/Q4, 2019)\n\nBS-1: ca. 2,000 donors, January–April 2021\n\nBS-2: ca. 2,000 donors, July–October 2021\n\nBS-3: ca. 2,000 donors, January–April 2022\n\nBS-4: ca. 2,000 donors, July–October 2022\n\nWe aim to test the same participants during all four survey periods. Donors who do not attend subsequent survey periods will be replaced by new donors until the sample size of 2,000 donors for each planned collection period has been reached. The first survey period (BS-1) is three and a half months (20th January 2021 – 30th April 2021).\n\nThe study will be conducted for 21 months so that vaccination effects can be reliably assessed (e.g., first approval for vaccine in the EU was on 27th December 2020).\n\nPrior to the start of the SeMaCo study, we assessed demographic factors of the blood donors at the University Hospital Magdeburg. In addition to blood sampling, the study participants will be asked to fill out two questionnaires (Q1–2) and, from the start of the second survey period, a follow-up questionnaire (Q3): Q1 for possible contacts and contact frequencies in their educational, professional and private contexts, Q2 for their attitude towards vaccination (in general and towards COVID-19) as well as recording the timing, vaccine and undesired effects of vaccination. Different questionnaires will be used at the initial and the follow-up examinations. At follow-up, the answers regarding current educational, professional, and private contact frequencies (Q3) and the current vaccination status will be updated. To ensure compatibility with other studies in Germany, questions were adapted from other COVID studies and vaccination surveys with permission of the authors10–11 (https://hzi-c19-antikoerperstudie.de/ and https://methodcov.de/). One aim was to be able to compare the results of our study with other COVID studies in Germany. The questions we used were therefore taken from other COVID and vaccination surveys conducted in Germany. Questions on socio-demographics are based on the demographic standards of the Federal Statistical Office. In addition, some questions from questionnaire 2 (vaccination attitudes) were developed by ourselves.12 All questionnaires were pretested and slightly revised for final use.\n\nThe study population are blood donors at the University Hospital Magdeburg. In 2019, 4,724 donors attended, 2,317 (49.0%) of whom were women. A total of 3,931 (83.2%) were multiple donors. The multiple donors all age groups, from 18 years upward, were rather equally represented with an increase of donors in the higher age groups older than 45 years. This resembles the general age distribution of the FSA where the average age was 47.9 years and the majority of inhabitants were older than 40 years in 2019 (https://de.statista.com/). Residents of Magdeburg represent 68% of the Magdeburg University Hospital blood donors and 32% are residents of the surrounding area. Overall, the blood donors essentially represent a cross-section of the healthy working population. It is expected that a high proportion of the blood donors will participate in the study due to their intrinsic motivation.\n\nParticipants must be a minimum of 18 years old and must provide written informed consent. Consent can be given stepwise for each study module: testing of blood samples, sociodemographic/contact survey, and the attitude towards vaccination survey.\n\nThe exclusion criteria are based on the donor exclusion criteria of the German Medical Association. There are numerous reasons that can lead to temporary or even permanent exclusion from blood donation. Typical reasons for temporary deferral are medical interventions, taking medication or travelling abroad, especially to areas in which mosquitoes can transmit malaria or viruses (e.g., Chikungunya virus, Zika virus). Reasons for temporal deferral do not result in exclusion from this study. Serious diseases are the most common reasons for a permanent ban and do result in an exclusion from this study.\n\nSample size was calculated using the GPower program (version 3.1.9.7, Heinrich- Heine-University Düsseldorf, Düsseldorf, Germany) (GPower, RRID:SCR_013726).13 Based on interim results of a similar study (Robert Koch-Institut – Project RKI/SeBluCo Report), there was an assumption that 1% of our study population was undetected infected. The sample calculation resulted in a sample size of n = 1,719 (α = 0.05, beta = 0.80, with a power of 80%). Therefore, our goal was to recruit at least 1,719 subjects.\n\nBased on the donor numbers from the blood bank, we assumed 2,000 participants during the planning. We aim to test the same participants during survey periods. Donors who do not attend subsequent survey periods will be replaced by new donors until the sample size of 2,000 donors for each planned collection period has been reached.\n\n\nLaboratory processing\n\nBlood samples (serum tubes, 8.5 ml) are collected during blood donation. Samples are pseudonymized in the Institute for Transfusion Medicine and Immunohematology with blood bank (ITIB). A label with the ITIB blood donation identification number (INR) is printed and attached to the serum tube. The samples are then registered using the laboratory information and management system (LIMS) of the Institute of Medical Microbiology and Hospital Hygiene (IMMB) and assigned an order number for every sample. The samples are transported from the ITIB to the IMMB with a pneumatic tube system.\n\nSpecifically, IgG antibodies are serially measured in first time and repeat blood donors over a period of 21 months. Upon arrival at the laboratory (IMMB), the serum tube is centrifuged and stored at 4°C for a maximum of 72 hours before being analysed. The test system ensures comparability of IgG values over all four survey periods by internal quality control procedures and assay calibration as prescribed in the analysers instruction manual and the IMMB Standard Operating Procedure. IgG antibody testing is performed according to the IMMB Standard Operating Procedure using an automated, CE-marked, accredited procedure. A uniform test system will be used throughout the study (LIAISON® SARS-CoV-2 TrimericS IgG, Diasorin).\n\nSeveral retention samples are frozen at −80°C. These samples serve for retesting, neutralization tests, and testing in case new test methods become available.\n\nThe test results are stored in the IMMB’s laboratory information and management system (LIMS) and are accessible through the order number. The test results can be accessed by the ITIB. The pseudonymized results will be stored for 30 years in the LIMS. If a test result is positive for antibodies, the participant will be informed by the ITIB.\n\nNeutralizing antibodies will be determined for a random portion of the samples at the IMMB. Additionally, samples from individuals who reported a COVID-19 infection or vaccination but do not exhibit IgG-antibodies in the serological test will be tested for neutralizing antibodies. A sample from the time prior to the infection is included in the analysis for comparison.\n\nThe samples will be stored for at least two years at −80°C at the IMMB.\n\nThe study participant’s survey will be pseudonymized during the participant’s blood donation using five-digit participant pseudonyms (ID-P) by the ITIB. Ideally, the questionnaires will be completed online using the Otto-von-Guericke University’s survey system, LimeSurvey (version 3.23.1+200825, LimeSurvey GmbH/LimeSurvey: An open-source survey tool. LimeSurvey GmbH, Hamburg, Germany). A paper version of the questionnaire will be available to participants who do not wish to complete it online. The subsequent electronic recording of the paper questionnaires will be done by the Institute of Social Medicine and Health Systems Research (ISMHSR). The ISMHSR and the IMMB will not have access to the study participants’ personal data at any time.\n\nThe data processing (collection, storage, use) in the SeMaCo study is based on the University Hospital Magdeburg’s and the ISMHSR’s data protection concepts. The data collection in the context of the (online) survey is carried out using the LimeSurvey server at the OvGU University Computing Center (UCC). The data protection concepts are in accordance with the EU General Data Protection Regulation (EU-GDPR) and the Federal Data Protection Act.\n\n\n\n1. The participant management at the ITIB uses five-digit participant pseudonyms (ID-P) for each of the study participants, which remain unchanged for each participant for the duration of the study. In addition, an eight-digit identification number (INR) is generated for each blood donation.\n\n2. The online questionnaires are generated by the ISMHSR on OvGU's LimeSurvey server and made available to the study participants for completion.\n\n3. The study participants fill in the online questionnaires, indicating their ID-P. Alternatively, a paper version of the questionnaire will be provided to participants who do not wish to complete the questionnaire online, or in the event that the survey system has a malfunction. The ID-P is also recorded on the paper version.\n\n4. The survey data is stored pseudonymously in the LimeSurvey survey system in the OvGU Computing Centre for the duration of the respective collection period (BS-1 to BS-4).\n\n5. At the end of the respective sampling period (BS-1 to BS-4), the survey data will be exported from the survey system to the ISMHSR. Intermediate data backups will also be made.\n\n6. The survey data will be stored, long-term, pseudonymously in the ISMHSR.\n\n7. Blood samples are taken from the study participants. The sample tubes are marked with the specific INR of the ITIB, assigned an order number for every sample in the IMMB’s LIMS by ITIB and sent to the IMMB.\n\n8. The IMMB performs the blood analysis and stores the results, together with the corresponding INR.\n\n9. The antibody test-result is sent to the ITIB, together with the INR in the IMMB’s LIMS.\n\n10. The serological test results will be exported into a Microsoft Excel (version 2016) (RRID:SCR_016137) spreadsheet (An open-access alternative that can provide an equivalent function is Google sheets (RRID:SCR_017679)) in the ITIB computer system (Excel spreadsheet) and stored there for the long-term. The Excel file will contain the INR, the ID-P and the antibody result.\n\n11. The antibody findings are assigned to the corresponding ID-P in the ITIB.\n\n12. A positive antibody status (test result) is extracted from the antibody findings and stored separately, together with the INR.\n\n13. The antibody status is transmitted to the ISMHSR together with the corresponding ID-P.\n\n14. The antibody status and ID-P are stored, long-term, in the ISMHSR.\n\n15. The survey data is linked to the antibody status data via the ID-P at the ISMHSR. The dataset is stored as an analysis dataset.\n\n16. This is followed by the analysis of the data by the SeMaCo researchers.\n\n\n\n• Person identifying data: ITIB Donor Information System, Excel Data Sheet ITIB\n\n• Pseudonymized blood analysis results: LIMS of the University Hospital Magdeburg\n\n• Pseudonymized survey results: Temporary storage on the LimeSurvey server of the UCC; permanent storage in the ISMHSR\n\n• Pseudonymized antibody status: permanent storage in the ITIB and ISMHSR\n\nThe main analyses will be descriptive using e.g., counts and percentages. Missing values will be excluded. We will compare groups (for example: vaccinated and non-vaccinated subjects) with the Wilcoxon test (for continuous parameters). Analyses of covariance (ANCOVA) are also included. P-values of 0.05 or less are deemed significant. All statistical analyses will be performed using Statistical Package for the Social Sciences (SPSS, version 26.0, Armonk, NY: IBM Corp (IBM SPSS Statistics, RRID:SCR_019096)) An open-access alternative that can provide an equivalent function is the R stats package (R Project for Statistical Computing, RRID:SCR_001905).\n\nParticipants can revoke consent for each module of the study in writing. If the participant revokes consent to participate in the study, the questionnaire survey data will be deleted, and the results of the antibody tests will be anonymized. The study participants will be informed of the deletion and anonymization.\n\nThe written informed consent includes information about the study objectives and content, the measuring instruments, the type and content of data collection and storage, the voluntary nature of their participation, and the option to revoke consent at any time. The blood donors also have the option not to participate in individual study modules (partial consent) and are not disadvantaged by non-participation. The blood donors will have sufficient time to consider their decision before giving their consent/refusal. The blood donation staff will be always available to answer questions. The study has been approved by the Otto-von-Guericke University Magdeburg ethics committee (No. 163/20). The study is registered in the DRKS (German Clinical Trials Register): DRKS00023263.\n\nThe results of the study will be published in peer-reviewed journals and presented to the public at a press event. In addition, social media (e.g., Instagram, Facebook, Twitter, LinkedIn) will be used to inform the public about news from the study.\n\nThe study started on 20th January 2021. At the end of the initial survey period (30th April 2021), 2237 subjects were recruited. The next survey period started on 1st July 2021.\n\n\nConclusions\n\nSeroepidemiological studies can be an important component for better estimation and classification of the prevalence of infection during the COVID-19 pandemic.10 The SeMaCo study expands the spectrum of German sero-epidemiological studies by investigating the prevalence of antibodies to SARS-CoV-2 in blood donors from Magdeburg and the surrounding areas of the capital of Saxony-Anhalt in a prospective manner. Both genders equally contribute to blood donation and the donors’ age distribution reflects the age distribution in the general population of the FSA. Moreover, older donors (60–72 years of age) are also included. For donors over 60 years of age, this is possible in accordance with criteria of the German Medical Association after an individual medical decision. Therefore, we consider blood donors as a suitable surrogate from the healthy working age until pension age population of the FSA.\n\nLongitudinal sampling in repeat donors will allow for an estimate of how the virus spreads in the general population and/or the success of a vaccination campaign during the study period of 21 months (January 2021 to October 2022). In addition to the serological examination, information about daily individual face-to-face contacts in the context of the donors’ normal professional and social activities are collected and correlated with a future COVID infection.\n\nFurthermore, the SeMaCo study assesses blood donors’ attitude towards vaccination and their COVID-19 vaccination status. A special feature of the study is that both the antibody tests and the questionnaires are conducted among repeat donors at four different time periods. This allows a more accurate view on the dynamics and course of SARS-CoV-2 antibody prevalence, contact intensities and patterns and attitudes towards vaccination over time in northern Saxony-Anhalt. Depending on the further course of the epidemic, the efficacy of containment measures and the vaccination frequency within the study population, COVID-19 antibody levels are expected to increase significantly in the three subsequent survey periods. Moreover, SeMaCo will deliver data on the persistence of the SARS-CoV-2 antibody response in a large group of healthy individuals over a study period of 21 months. We expect that the results of the SeMaCo study can influence policy decisions and prevention efforts.\n\n\nData availability\n\nFigshare: Extended data for ‘Study protocol for the SeMaCo study: A longitudinal regional cohort study to assess COVID-19 seroprevalence in blood donors’.\n\nhttps://doi.org/10.6084/m9.figshare.16546215.v2.12\n\nThis project contains the following extended data:\n\n- SeMaCo – Self-formulated questions.docx\n\n(Questionnaire 2 (Attitude towards vaccination))\n\n(Questionnaire 1 (Contact Information) and Questionnaire 3 (Follow-up):\n\nQuestions about sociodemographic data (Information about the person, school degree, occupational activities); Questions about contacts with COVID-19 infected persons and (occupational and private) contacts; questions about nursing activities; questions about participation in events (during pandemic); questions about commuting to work, housing conditions, household, family and travel used with permission from Robert Koch-Institute.10\n\nQuestions about testing for COVID-19 and physician diagnosis of COVID-19 and symptoms if test/diagnosis is positive used with permission from Robert Koch-Institute10 and Helmholz Center for Infection Research (https://hzi-c19-antikoerperstudie.de/). Questions about marital status based on MethodCOV project (https://methodcov.de/).\n\nQuestionnaire 2 (Attitude towards vaccination): Questions about attitudes toward vaccination for COVID-19 used with permission from KUNO Kids Health Study. University of Regensburg.11)\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 the technical personnel and students of the ITIB and IMMB for support in conducting the study.\n\n\nReferences\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). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuitrago-Garcia D, Egli-Gany D, Counotte MJ, et al.: Occurrence and transmission potential of asymptomatic and presymptomatic SARS-CoV-2 infections: A living systematic review and meta-analysis. PLoS Med. 2020; 17(9): e1003346. Publisher Full Text\n\nRipperger TJ, Uhrlaub JL, Watanabe M, et al.: Detection, prevalence, and duration of humoral responses to SARS-CoV-2 under conditions of limited population exposure. medRxiv 2020. Publisher Full Text\n\nKang CK, Kim M, Lee S, et al.: Longitudinal Analysis of Human Memory T-Cell Response according to the Severity of Illness up to 8 Months after SARS-CoV-2 Infection. J. Infect. Dis. 2021; 224: 39–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJackson LA, Anderson EJ, Rouphael NG, et al.: An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N. Engl. J. Med. 2020; 383(20): 1920–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeehner J, Horton LE, Pfeffer MA, et al.: SARS-CoV-2 Infection after Vaccination in Health Care Workers in California. N. Engl. J. Med. 2021; 384(18): 1774–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaden LR, El Sahly HM, Essink B, et al.: Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 2021; 384(5): 403–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarklund E, Leach S, Axelsson H, et al.: Serum-IgG responses to SARS-CoV-2 after mild and severe COVID-19 infection and analysis of IgG non-responders. PLoS One. 2020; 15(10): e0241104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo L, Ren L, Yang S, et al.: Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19). Clin. Infect. Dis. 2020; 71(15): 778–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantos-Hövener C, Busch MA, Koschollek C, et al.: Seroepidemiologische Studie zur Verbreitung von SARS-CoV-2 in der Bevölkerung an besonders betroffenen Orten in Deutschland – Studienprotokoll von CORONA-MONITORING lokal.2020. Publisher Full Text\n\nBrandstetter S, Böhmer MM, Pawellek M, et al.: Parents' intention to get vaccinated and to have their child vaccinated against COVID-19: cross-sectional analyses using data from the KUNO-Kids health study. Eur. J. Pediatr. 2021 May 17; 1–6. Publisher Full Text\n\nPohl R, Krämer S-W, Stallmann C, et al.: Study protocol of the SeMaCo study: A longitudinal regional cohort study to assess COVID-19 seroprevalence in blood donors. Figshare. 2021. Publisher Full Text\n\nFaul F, Erdfelder E, Buchner A, et al.: Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behav. Res. Methods. 2009; 41: 1149–60. PubMed Abstract \| Publisher Full Text" }	[ { "id": "140446", "date": "29 Jun 2022", "name": "Thibaud Spinetti", "expertise": [ "Reviewer Expertise 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\nIn the present study protocol, the authors aim to address the seroprevalence of SARS-CoV2 or/and SARS-CoV2 vaccination in the Saxony-Anhalt area population in Germany. In addition, the authors will also assess social behaviors and the subject's attitude towards vaccination via questionnaires. The sample population representing people from the Saxony-Anhalt area are blood donors at the University Hospital Magdeburg.\nThe rationale and objectives of the study are clearly described. The authors established an appropriate study design to address the study's objectives, including four follow-up visits and existing samples for 2019 as control (for antibody response/titration).\nInformation is missing to clearly understand the planned statistics and be able to repeat the study, i.e. :\nThe authors indicate that they « aim to test the same participant during all four survey periods .» How missing data will be handled, e.g., data of a subject are present for BS-1, BS-4 but not for BS-2 and BS-3 (missing visits due to medical condition (COVID-19 or not) or not related to medical condition).\n\nAre BS-0 subjects' specific samples needed? If yes, how are data from a new donor (no BS-0) analyzed?\n\nAre only subjects analyzed if all visits are completed (if yes, please explain how generated bias is handled)?\n\nHow are handled data of patients who die during the study period (COVID-19 related or not)?\n\nAre alternatives to obtain information if patients are not presenting to a study visit (proactive contact, at least for questionnaires)?\n\nExclusion criteria that could affect the sample population should be listed e.g. if any, according to German regulations: restriction on BMI, sexual behavior, age… (not all readers are aware of German blood donation regulations).\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "140445", "date": "01 Jul 2022", "name": "Gannon C.K. Mak", "expertise": [ "Reviewer Expertise Virology" ], "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 protocol written by Pohl et al. described the plan to investigate SARS-CoV-2 seroprevalence in the city of Magdeburg and surrounding areas. Overall, the study protocol was clear and well written. It covered different aspects, (1) objectives, (2) study population including inclusion and exclusion criteria, and (3) time frames of sampling and targeted sample size. The technical parts were described in detail from pre-examination, examination, and post-examination procedures. The study protocol correlated well with the primary objective (i.e. determination of the dynamics of the antibody response). The secondary objectives 1 to 3 (page 3) should be able to be covered by Questionnaire 1 and Questionnaire 2 (page 8). However, I do have several comments for the remaining two secondary objectives and the corresponding examination procedures.\nIt is unlikely that the SARS-CoV-2 will vanish as evidenced by the SARS-CoV-1 between 2003 and 2004. On the contrary, SARS-CoV-2 will circulate among us just like other respiratory viruses. Due to the antigenic drift/shift, the vaccines for influenza viruses have to update annually. The currently available SARS-CoV-2 vaccines are based on the prototype strain detected in late December 2019, either Wuhan-Hu-1 or WIV04. With the emergence of Omicron in late November 2021, it is reasonable to suspect that the effectiveness of COVID-19 vaccines is affected due to the unusually high number of spike protein substitutions/changes. Although Omicron was only detected after the authors completed this protocol (i.e. September 2021), the new vaccine that you mentioned in the secondary objective 4 should be referring to the new COVID-19 vaccines based on the BA.1 Omicron available later this year1.\nOmicron evolves continuously, several sub-lineages/descendent lineages, BA.2 to BA.5, BA.2.12.1 were noted. The new variants usually replace the old variants with competitive advantages (e.g. higher transmissibility, mismatched vaccines). The latest findings showed that among participants who had been infected with the BA.1 or BA.2, the neutralizing antibody titer against BA.2.12.1, BA.4, and BA.5 was lower than the old variants. It means that neutralization escape was found for the emerging variants 2,3. Undoubtedly, the incidences of COVID-19 are increased due to the relaxation of COVID-19 control policies worldwide and emerging variants. As the disease progresses, it is not an easy task to interpret the SARS-CoV-2 seroprevalence, different variables should be taken into account. To ensure a valid study, it is therefore important to rule out all sources of bias and confounding factors.\nThe test results of SARS-CoV-2 antibodies vary between assays 4 and also the targets used 5. Only one assay was used to assess the SARS-CoV-2 IgG antibody in this study protocol. Unless there are strong justifications, the authors should consider using another assay for assessing the SARS-CoV-2 antibodies and also the seroconversion. Although the authors will assess the neutralizing antibodies in a partial portion of the samples, the assay used was not known. The use of nucleocapsid (N) as the vaccine antigen target is in a dilemma. The downside of this target is that the results may be overestimated since N protein is conserved among other coronaviruses. Solving this issue should not be a concern since samples were obtained prior to SARS-CoV-2 being detected in late December 2019. However, it can help to differentiate if the results are due to vaccination or past infection 4. In addition, it can help to confirm the responses from the questionnaires, and the outcomes of the study will be more convincing. Careful and comprehensive planning is important so that secondary objective 5 could be addressed.\nThe strength of the study is the overlapping between pre- and post-omicron circulation. I am not sure if the study has generated results yet. I hope the authors may find the comments useful and design the way forward for this ongoing study. Early identification of potential deficiencies should enable the authors to overcome these flaws so that positive results are generated and concordant with the authors’ objectives.\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? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-982
https://f1000research.com/articles/10-976/v1	27 Sep 21	{ "type": "Research Article", "title": "Failure Risk Model of Sustainable Solid Waste Management at The University in Indonesia using Dynamic System Approach", "authors": [ "Dino Rimantho", "Nur Yulianti Hidayah", "Ayu Herzanita", "Anggina Sandi", "Nur Yulianti Hidayah", "Ayu Herzanita", "Anggina Sandi" ], "abstract": "Background - One of the problems encountered by all universities in realizing a sustainable Green Campus is solid waste management due to its complex and dynamic nature, which is known to have the potential risks of failure. Aim – Application of a dynamic modeling approach with the FMEA and AHP methods to eliminate the risk associated with the inability of universities to process solid waste.\n\nMethods– Data on risk factors and alternative evaluation methods were collected from five experts from various fields. Results – The results of the risk assessment by experts show some of the highest risk values are associated with Technological Infrastructure, with a risk value of around 900, and the lowest risk is associated with Regulation collisions with a risk value of around 378. The results provided adequate information on the increased risk of each factor from 2019 to 2030 with verified moderate and optimistic conditions models comprising significant values of approximately 0.009 and 0.0273, respectively. This study assists higher education management in decreasing risk failure in the region by creating simulation models that will likely be utilized for scenario analysis in the future. The System Dynamic model's entire approach proved helpful in accomplishing long-term solid waste management in universities. Other dynamic insights that assisted universities improve their knowledge of solid waste management systems. Research limitations– Further study needs to be conducted in the dynamic model to obtain a more comprehensive and detailed result. This is because the developed model provided an overview that needs further improvement, especially concerning several quantitative data, towards obtaining the adequate method for sustainable solid waste management. Originality/value – This research used the hybrid method and AHP techniques to analyze solid waste in universities which were developed into a dynamic system model. However, further research is needed to enhance this hybrid method.", "keywords": [ "Solid waste", "risk", "systems", "dynamics", "modeling", "sustainability" ], "content": "1. Introduction\n\nA green campus is described as the coexistence of environmentally conscious practice and education. It is also a location where initiatives are endorsing ecological tenets. This idea was introduced in Europe in the 1990s, with the need to intervene in “any region where human impacts had a negative effect on the environment” acknowledged during the 1992 Rio Earth Summit. Furthermore, it involves the removal of waste food inefficiencies, use of traditional energy sources for regular power needs, as well as proper disposal and efficient recycling programs. Moreover, universities need to establish a timetable for the implementation of these initiatives through solid waste management. This approach has to be integrated into institutional planning to build a clean and green campus.\n\nPresently, there is no clear legislation regulating the waste management activities of tertiary institutions in Indonesia, which poses highly complex threats. Moreover, solid waste management is aimed at minimizing or eliminating threats to human health and the environment. This method is known to work effectively when the process does not threaten human wellness or damage society. Based on this, a sustainable risk management model is needed at universities, thereby adopting a Dynamic Systems approach.\n\nSystem Dynamics (SD) is a software-based solution used to tackle complex problems in accordance with the predicted time. Moreover, the key to applying this method depends on input, stock, and flow. These factors support the assumptions related to a highly complex process. Additionally, the study carried out by Chaerul and Tanaka (2008) offers a thorough overview of dynamic system applications.\n\nThese approaches are considered effective when dealing with certain solid waste management types (Ghisolfi et al., 2017; Rimantho et al., 2020), either in hospitals or developing nations (Chaerul et al., 2008; Kum et al., 2005; Sufian & Bala, 2007). Furthermore, the study carried out by Karavezyris et al. (2002) on an integrated waste management model in Berlin City complements system dynamics through fuzzy logic to address qualitative variables. Based on a small data sample to predict solid waste generation in a fast-growing area, Dyson and Chang (2005) utilized dynamic system modeling. Also, Inghels and Dullaert (2011) developed a similar model to assess the impact of Flemish waste management prevention initiatives. Generally, previous studies only used the dynamic system method to predict potential solid waste generation to serve as a reference for future analysis. However, no research has been carried out in terms of predicting solid waste management process failure.\n\nBased on the recycling activities of solid waste, pollution has been discovered to be complex, uncertain, and rapidly changing, making it impossible for toxic control to be studied and prevented using specific methods. Therefore, a system approach and modeling, which involves combining several methods, such as FMEA, AHP, and System Dynamics, is needed. Also, pollution risk factors are observed as an input in the preparation of solid waste management scenarios. These are also expected to provide future preventive measures. Additionally, risk factor management scenarios are simulated to observe the possibilities of future occurrences through a dynamic system. Besides, specific interventions that aid in realizing an optimal scenario in system objectives are recommended for policy directives.\n\nThis study aims to create a complex system model by offering multiple choices, which helps identify the best policies for enhancing sustainable waste management. Moreover, risk management is one of the subjects discussed in this study, as it is a significant element that impacts waste performances. Based on handling changes over time, the dynamic system model was adopted.\n\n\n2. Methods\n\nThis study incorporated qualitative and quantitative analysis in order to evaluate and establish an alternative model. Another, technique known as the dominant-less design was adopted. Furthermore, a specific research methodology and analytical technique, known as the systems thinking approach (Senge, 1994) and dynamic method (Coyle, 1996), were employed.\n\nThe primary data were collected by five experienced experts familiar with the potential risks emerging from inadequate solid waste management. Also, the data was obtained from publications containing various related details.\n\nRisk assessment was also analyzed using the Failure Mode Effect Analysis (FMEA) method. The solid waste management risk model was categorized into several groups, namely the technology, social, recycling, regulatory or legal, and waste generation rate sub-models. Meanwhile, the determination of intervention as an alternative solution to optimally reduce risk was adopted. This was carried out by applying the AHP method, using the pairwise comparisons made by the five experts. The application of FMEA and AHP methods also employed the use of questionnaires distributed to seven experienced experts (FMEA (4) and (3) AHP) from indigenous universities. Additionally, the selection of three experts was approved by the Ethic committee from Pancasila University.\n\nBased on the results, the next step was to create a model to identify potential and prospective risk behaviours. This model was constructed through the application of a dynamic system method using the Powersim Studio 2005 Software. A free open-access alternative software that can perform these same functions would be Simantics System Dynamics Version 1.35.0 (http://sysdyn.simantics.org/).\n\nThis study focused on the development of possible ideas related to solid waste management through system dynamic analysis, which aims to facilitate (1) the establishment of Causal Loop and Stock Flow Diagrams (CLD & SFD), (2) Leverage analysis, and (3) Scenario specification.\n\nSystem dynamics were utilized qualitatively and quantitatively. This method is also used as a method of reasoning and understanding to explore its performance qualitatively. However, the system dynamics analysis was transformed into a quantitative simulation and optimization model to help policy development. Moreover, certain assumptions of system discourse led to the adoption of the analytical method. Jay Forrester (the father of system dynamics) described it as “the investigation of information input characteristics (managed), as well as the use of models for the design of improved organizational form and guidance policy” in 1961 (Coyle, 1996).\n\nThe qualitative and quantitative dynamic analysis of the Coyle method was used in the following stages,\n\na. Introduction to the issues- this study aims to explain the problematic failure risk of the waste management process through the use of an input-output diagram.\n\nb. Understanding the problem and descriptive system to systematically better understand the issue by presenting a summary of the solid waste management’s failure risk using Causal Loop Diagram (CLD) and Stock Flow Diagrams (SFD).\n\nc. Qualitative analysis is an effective method for obtaining a deeper understanding of difficult problems.\n\nd. Simulation modeling is the most significant step. Moreover, the conversion of CLD and SFD into this model led to rapid processing and enhancement, and development.\n\ne. Policy Testing and Design were the final steps. This involves the designation and evaluation of policies or scenarios by simulating and adjusting future systems to obtain accurate results.\n\nVerification was carried out by ensuring that the simulation model performed as expected. Based on this reason, a statistical test was carried out to determine whether the components of this model performed as desired. Moreover, the variable that needed to be tested was the time required for the components to exit the processes within the model. This was also carried out to determine if the results were significantly statistically different from the real system’s recorded time. Additionally, verification was carried out by using a 2-sample t-test.\n\nThis t-test was used to analyze the hypothesis and calculate the confidence interval of the difference between two population means and the unknown standard deviation (σ). The hypotheses are stated as follows,\n\nH0: μ1 ≤ μ2 (The actual and modeled risk values are either similar or less)\n\nH1: μ1> μ2 (The actual risk value is greater than the modeled one)\n\nWhere H0 and H1 = the null and alternative hypotheses, μ1 and μ2 = the mean values of the first and second populations, and δ0 = the difference in mean values between the 2 populations tested (Walpole et al., 2007). Statistical testing was carried out using Minitab® 16.1.1 software (Minitab, RRID:SCR_014483). A free open-access alternative software that can perform these same functions would be R (R Project for Statistical Computing, RRID:SCR_001905).\n\n\n3. Results and discussion\n\nAs earlier mentioned, the risk assessment was carried out by five experts with different backgrounds. The results are shown in Table 1.\n\nTable 1 shows information related to certain failure risks of solid waste management at the University. Based on these results, several sub-factors such as infrastructure, health, safety, and leadership support, and compliance with regulations had high-risk values of 900, 720, and 729, respectively. Additionally, Table 1 shows that the high-risk values need to be prevented.\n\nBased on this, an alternative was determined through the AHP method. The results of the pairwise comparison assessment are shown in Table 2.\n\nTable 2 provides information on the alternatives, which were observed as the risk reduction intervention program. Based on these results, the improvement of recycling technology, at a weight of 0.429, was regarded as the best. This was accompanied by the development of regulations on solid waste management, participation of the private sector, and campus residents' socialization, with a weight of 0.230, 0.223, and 0.122, respectively.\n\nBased on the immense scope of waste management, the unresolved issue of its failure in universities is a source of concern. Furthermore, it is complicated by several issues, such as lack of environmentally-friendly technologies, inadequate resources, poor leadership support, possible disputes, regional vulnerability, as well as unavailability of recycling facilities and locations. A system dynamic analysis that was focused on model testing and scenario development was adopted to potentially control the problem. According to this system dynamic analysis, the failure risk was referred to as a system. Conversely, the multiple evaluated and risk subsystems were referred to as flow rate and stock level, respectively.\n\nThe model testing analysis was also used to determine the system’s leverage by evaluating the compatibility of all subsystems. Using the Powersim Studio 2005 software, a dynamic model was developed and tested. A free open-access alternative software that can perform these same functions would be Simantics System Dynamics Version 1.35.0 (http://sysdyn.simantics.org/). Moreover, the determined subsystems shown in Table 1 were significantly evaluated with the analytical method. These were the variables that impacted the risk mechanism for waste management failure, and therefore, need to be addressed when developing scenarios.\n\nThe subsystem was developed due to several discussions and evaluations reported in studies carried out based on strong educational history and familiarity with solid waste management and its risks.\n\nAfterward, the scenario analysis was carried out through the Causal Loop Diagram and Stock Flow Diagrams (CLD & SFD), based on the discussion and risk evaluation outcome. These diagrams were crucial steps adopted in evaluating the system’s leverage, thereby creating several potential scenarios for reducing the likely occurrence of solid waste management failure. The adopted CLD and SFD processes are shown as follows in Figure 1.\n\nBased on Figure 1, the relationship between subsystems and variables related to the CLD demonstrated negativity and positivity (balancing and reinforcing loops). This derivative facilitated the assessment of factors that had a significant impact on the failure risk of the waste management process.\n\nThe CLD is used to illustrate the four threats that need to be controlled to protect the environment and human health. These include technical, social, recycling, and policy risks. Moreover, the social, policy, and technological risks, represent contextual relationships. Also, understanding this connectivity in terms of technological risk is based on the fact that “the presence of a technological hazard was highly dependent on compliance with regulations or rules, such as monitoring equipment protection and safety, as well as the creation of technical and non-technical infrastructures.” Additionally, technological use in universities was observed to affect regulatory risk, such as solid waste recycling activities. These were found still to adopt a lot of environmentally unfriendly and straightforward technology while also necessitating regulations on the recycling operation.\n\nThe practices that involved several risk factors, such as inadequate working methods and recycling sites, including public tensions and regional vulnerability, had significant social implications. Moreover, the technical factors often affected the risks associated with recycling. Additionally, numerous studies reported that the equipment used for the recycling process was primitive and environmentally unfriendly.\n\nBased on the CLD analysis, a quantitative measure with a sequential description of the risks influencing the scope of waste management failure was observed. The technical factor was also closely linked to the waste management failure risk, which resulted in poor environmental quality in universities. Therefore, the possibility of enforcement was discovered to be another support point.\n\nThis situation refers to the lack of policies, which promote the use of more environmentally-friendly technologies while still considering workers' safety (Amoyaw-Osei et al., 2011; Sas et al., 2015). Moreover, one of the shortcomings in developed countries is waste management. This is due to the fact that the construction of service facilities lags behind the availability of disposal infrastructure, which necessitates physical resources, such as land, water, energy, and location, among others (Porter 1998). The effective recycling of solid waste by processing, smelting, and refining saves costs and helps minimize greenhouse emissions.\n\nThe emergence of social risks also impacted the solid waste management process, which is one of the barriers to achieving sustainability. Assefa and Frostell (2007) carried out a study that illustrated the significance of social indicators on solid waste management. Also, Owusu (2010) published a report on the social effects of inadequate waste management. The results of these studies showed that ineffective environmental management had both direct and indirect social implications. Moreover, poor expectations and involvements, public unrest, and regional vulnerability are factors that impacted social aspects (Birhanu & Berisa 2015; Schindler et al., 2012; Oberlin & Szántó 2011).\n\nLaw enforcement or compliance is also one of the risk factors that leads to failure in the solid waste management process. Vilas (2015) reported that environmental legislation was ineffectively executed due to a lack of group enforcement. Another reason that led to this failure was policy disputes. According to research carried out by Egwurube (1983), it was reported that one of Nigeria’s environmental conservation agencies had been abolished. This was due to the enactment of incoherent policies within the solid waste management sector. Based on tertiary institutions, data processing also posed a risk. Also, the MSW characterization data availability was the initial crucial step regarding implementing an integrated solid waste management strategy aimed to protect human health and the environment (Oberlin 2013).\n\nRecycling is one of the most efficient strategies for eliminating solid waste because it reduces emissions and also increases economic activity. It has been shown that one of the main reasons for the failure of this process is the lack of expertise and competent staff. Therefore, workers' willingness to engage in the recycling process is important. This is because they need to be familiar with the practices and proper usage of recycling equipment (Sinha-Khetriwal et al., 2009). Furthermore, inadequate work facilities are another factor that triggers the occurrence of possible failure. This factor has been observed to cause bad operational practices due to a reliance on improvised equipment which is hazardous to the environment. For example, some recycling businesses in Africa, Asia, and Latin America are yet to use appropriate facilities (Sthiannopkao & Wong 2013). Moreover, inefficient recycling processes have resulted in major material and resource losses (Rao 2014). Saphores et al. (2006) revealed that the waste management process also has the potential to trigger the risk of failure.\n\nThe relationship between factors that generate risk is greatly triggered by sustainable waste management. However, universities' active role in implementation needs to be explored further by carrying out SFD research. Moreover, the development of scenario formulations reduces the risk of failure in a waste management system. Additionally, SFD settings were used to assess the system’s leverage.\n\nBased on the preliminary analysis in this research, the risk assessment will start in 2021, and is expected to continue until 2030, as shown in Table 3.\n\nAccording to Table 3, all variables experienced a growing trend in the probability of solid waste management failure in universities. Furthermore, the social factor had the highest risk growth rate, with a value of 1255 in 2030. This is similar to several previous studies which revealed social factors as one of the contributors to the failure of the waste management process (Assefa & Frostell 2007; Owusu 2010).\n\nIrrespective of the fact that there was an upward trend yearly, the regulatory risk factor did not pose a major risk in 2030, as shown by the model simulation, with a value of 19. Moreover, this demonstrated that the cost of regulatory risk is reasonable. Based on our simulation model performance, the recycling factor with the highest risk growth rate per year is important. Therefore, solid waste management decision-makers need to focus on this aspect based on present trends.\n\nAfterward, a structural suitability test was carried out to determine whether or not the modeled structure contradicted the established awareness of the real system. It was also carried out to ascertain whether or not the main structure of the real system had been modeled. Moreover, the interactions between the components in each subsystem were defined by the structure of the failure risk management model. This was further divided into several sub-models, namely technology, regulation, and social.\n\nAccording to the simulation results realized using Powersim, the infrastructural, regulatory, and social risk sub-models were displayed an escalation scenario. This fundamental trend shows that they were involved in increasing the reciprocal dominance of sub-factors. Moreover, an escalation scenario tends to occur when two or more parties are involved in a citation, with each subsystem responding to the others. However, assuming the subsystems displaying increased risk were not regulated or eliminated, the competition escalation results in a “lose-lose” situation. Additionally, an in-depth understanding of the processes that potentially pose a threat to other factors or subsystems is one of the best methods to handle this type of situational pattern. Furthermore, a consideration of the existence of delay (delay time) in the implementation phase is critical, as essential activities were likely to be easily avoided or minimized.\n\nSeveral scenarios were further generated based on the structure suitability test results. These had different policy designs that were implemented in the field under realistic conditions. Moreover, the analytical results of risk and formulated waste management techniques were used to create control scenarios. This research was carried out to plan future strategic measures by enhancing significant risk factors. The best policy was implemented based on the long-term sustainability of solid waste management risk in tertiary institutions. This was carried out after considering the actions of the most profitable model.\n\nBesides, these management scenarios were also focused on the assessments of potential risk factor conditions. Based on the respondents' predictions, these scenarios were unconditionally created in the study area. Afterward, these factors were combined through the respondents' prediction of future variable states. This led to the generation of three scenarios, namely (1) positive, (2) moderate, and (3) current (actual). Additionally, Table 4 shows several scenarios for reducing risk factors.\n\nThe model simulation, including all existing intervention results, is shown in Figure 4. The differences in the model-built scenarios in terms of pessimistic, moderate, and optimistic models are also shown in Figure 4. Moreover, the pessimistic scenario was based on the conditions of solid waste management factors, observed to experience failure. This was due to the assumption that the policy was unaltered in the initial scenario (did nothing). However, this was also compared with other alternatives, and it was discovered that several regulations were not better than the current ones. Based on this point of view, observations showed that without any intervention or modification, simulating risk models for electronic waste management factors was impossible, and all risk factors were predicted to increase, within 10 years considerably.\n\nThe moderate scenario also suggested that possible future conditions were fully considered under certain circumstances and the capacity of presently owned resources. This setup is dependent on risk factors under the following circumstances, (1) inadequate facilities, (2) recycling locations, (3) data management, (4) leadership support, as well as (5) campus residents' expectations and involvements. Based on this moderate scenario, it was stated that relatively 30% to 50% of the total activity or program was not fully implemented, as shown in Table 4. According to Table 3, the simulation model showed a significant decrease in the risk value.\n\nThe model was also enhanced by an optimistic scenario, observed to include other interventions without eliminating the regulatory types. This proves that a legal framework was provided during the implementation of solid waste management. Based on the inclusion of other interventions, better models are likely to be obtained. Moreover, more significant risk reduction values were likely to be achieved, probably in technological innovation and collaboration between actors associated with solid waste management.\n\nFigure 4 shows the simulation results of the waste management risk model, which includes various interventions, such as technological innovation, stakeholder cooperation, and regulatory or legal action. However, technical innovation interventions were included in the technology factor sub-model, responsible for reducing safety and equipment risks. Also, the system adopted by informal workers remained traditional, as it failed to prioritize the safety and security of the staff. Based on the simulation results, this model proved that interventions in several scenarios lowered the risk value of the legal factors. Therefore, this triggered the previously included risk category. Model integration was used to achieve a better reduction result.\n\nVerification was carried out by ensuring the expected performances of the simulation model were realized. Based on this reason, a statistical test was needed to determine whether the components in the model were executed according to the desired concept. Moreover, the tested variable was the time required for the component to exit the processes within the model. This was also carried out to determine whether the results were significantly similar to that of the real system. In addition, this verification test was carried out using a 2 sample t-test. Statistical testing was carried out using Minitab® 16.1.1 software (Minitab, RRID:SCR_014483). A free open-access alternative software that can perform these same functions would be R (R Project for Statistical Computing, RRID:SCR_001905).\n\nThe result showed an average of 1146.83, under the current condition, which was then changed to 898.39, after it was moderately intervened. However, the average was observed at 677.89, after being optimistically intervened. Additionally, a descriptive Pearson correlation resulted in 0.999 in the two intervention models (moderate and optimistic). This further proves that the relationship was extremely close. Based on these results, the values of t-stat for both moderate and optimistic conditions were 3.144 and 2.54306, respectively. These were similar to the paired t-test. Furthermore, the result of the t-table was 2,200, with a p-value of 0.027. Based on the fact that the p-value is less than alpha 5% (t-count> t-table), the decision was rejected H0, which indicates that there was a significant difference between the intervention results of the modeled system.\n\nThis showed that the previous studies partially solved the problem of waste management. However, this research had the potential to create new problems. According to certain implications, the potential to reduce the failure risk of the waste management process was indicated due to the implementation of various operating alternatives under moderate and optimistic conditions. This showed that the best alternative solution in waste management was the combination of several approaches into a dynamic system method. Therefore, the results need to be implemented to avoid the risk of failure in waste management. Additionally, the application of this method needs to provide future perspectives related to risk and solid waste management.\n\n\n4. Conclusions\n\nA System Dynamic model for reducing risk failure in solid waste management in universities was developed and modeled in this research. We contest that the main problem involved in achieving a green campus is solid waste management. Generally, several preliminary studies have been carried out on the partial and incomprehensive risk analysis of solid waste management. Meanwhile, no research has been carried out on the risk factors that threaten this process, such as technology, social, financial, legal, and recycling methods. Furthermore, the failure of the management process was assessed from a variety of perspectives, including technical, regulatory, social, and recycling factors. Each of these aspects contained several subsystems, which were found to be intricately linked. Based on designing simulation models likely used for scenario analysis in the future, this study aids higher education management in reducing risk failure in the area. The full perspective of the System Dynamic model was valuable in achieving sustainable solid waste management in universities. However, other dynamic insights that helped boost knowledge of solid waste management systems in universities were further reported in this study. Based on the results, a moderate and positive scenario, which included multiple risk mitigation program interventions in each aspect, was proposed. Generally, the System Dynamic model functions as an effective decision support system (DSS), for stakeholders involved in solid waste management in universities.", "appendix": "Acknowledgment\n\nThe authors are grateful to the University of Pancasila’s Faculty of Engineering, for providing research funding on Green Campus, under the contract number: 0121/FTUP/X/2018.\n\n\nData availability\n\nFigshare: FMEA Result.csv.\n\nhttps://doi.org/10.6084/m9.figshare.14669907\n\nFigshare: AHP Result.csv.\n\nhttps://doi.org/10.6084/m9.figshare.14669952\n\nThis project contains the following underlying data.\n\n• FMEA Result.csv (The results of the FMEA Questionnaire, assessing the risk of failure of the solid waste management process).\n\n• AHP Result.csv: (AHP results from the expert assessment related to solid waste management strategy in universities).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAmoyaw-Osei Y, Opoku Agyekum O, Pwamang JA, et al.: Ghana e-waste country assessment. United Nations Environment Programme (UNEP). 2011. Reference Source\n\nAssefa G, Frostell B: Social Sustainability and Social Acceptance in technology Assessment: A Case Study of Energy Technologies. Technol. Soc. 2007; (29): 63–78. Publisher Full Text\n\nBirhanu Y, Berisa G: Assessment of Solid Waste Management Practices and the Role of Public Participation in Jigjiga Town, Somali Regional State, Ethiopia. Int J Environ Pollut . 2015; 3(5): 153–168. Publisher Full Text\n\nChaerul M, Tanaka M, Shekdar AV: A system dynamics approach for hospital waste Management. Waste Manag . 2008; 28(2): 442–449. PubMed Abstract \| Publisher Full Text\n\nCoyle RG: System Dynamics Modelling: A Practical Approach . London: Chapman and Hall; 1996.\n\nDyson B, Chang N: Forecasting municipal solid waste generation in a fast growing urban region with system dynamics modelling. J. Waste Manag. 2005; 25(7): 669–679. Publisher Full Text\n\nEgwurube J: Local Government and Environmental Management. Paper presented at the National Conference on Development and the Environment held at NISER University of Ibadan, Jan 17 & 19.1983.\n\nGhisolfi V, de Lorena G, Chaves D, et al.: System dynamics applied to closed-loop supply chains of desktops and laptops in Brazil: A perspective for social inclusion of waste pickers. J. Waste Manag. 2017; 60: 14–31. Publisher Full Text\n\nInghels D, Dullaert W: An analysis of household waste management policy using systems dynamics modeling. Waste Manage. Res. 2011; 29(4). PubMed Abstract \| Publisher Full Text\n\nKaravezyris V, Timpe KV, Marzi R: Application of system dynamics and fuzzy logic to forecasting of municipal solid waste. Math. Comput. Simul . 2002; 60(3–5): 149–158. Publisher Full Text\n\nKum V, Sharp A, Harnpornchai N: Improving the solid waste management in Phnom Penh city: a strategic approach. J. Waste Manag. 2005; 25(1): 101–109. PubMed Abstract \| Publisher Full Text\n\nOberlin AS, Szántó GL: Community-level composting in a developing country: case study of KIWODET, Tanzania. Waste Manage. Res. 2011; 29(10): 1071–1077. PubMed Abstract \| Publisher Full Text\n\nOberlin AS: Characterisation of household waste in kinondoni municipality, dar es salaam. Acad. J. Interdiscip. Stud. 2013; 2(13): 35–46. Publisher Full Text\n\nOwusu G: Social effects of poor sanitation and waste management on poor urban communities: a neighborhood-specific study of Sabon Zongo, Accra. J Urbanism . 2010; 3(2): 145–160. Publisher Full Text\n\nPorter ME: The Competitive Advantage of Nations . 5th ed. Palgrave Macmillan; 1998.\n\nRao LN: Environmental Impact of Uncontrolled Disposal of E-Wastes. Int. J. ChemTech Res. 2014; 6(2): 1343–1353.\n\nRimantho D, Noor E, Eriyatno EH: Dynamic Model of Risk Management of Electronic Waste in DKI Jakarta–Indonesia. Int. J. Adv. Sci. Technol. 2020; 29(01): 899–910.\n\nSas I, Thoney KA, Joiness JA, et al.: Sustainable Fashion Supply Chain Management. [e-book]. 2015; [Accessed: 26 January 2021].Reference Source\n\nSaphores J-D, Nixon H, Ogunseitan OA, et al.: Household willingness to recycle electronic waste an application to California University of California post-prints year 2006 paper 1165.2006. Publisher Full Text\n\nSenge PM, Kleiner A, Roberts C: The Fifth Discipline Fieldbook . New York: Doubleday/Currency; 1994.\n\nSinha-Khetriwal D, Kraeuchi P, Widmer R: Producer responsibility for e-waste management: key issues for consideration—learning from the Swiss experience. J. Environ. Manage. 2009; 90: 153–165.\n\nSthiannopkao S, Wong MH: Handling e-waste in developed and developing countries: initiatives, practices, and consequences. Sci Total Environ . 2013; 463–464: 1147–1153. Publisher Full Text\n\nSufian MA, Bala BK: Modeling of urban solid waste management system: the case of Dhaka city. J. Waste Manag. 2007b; 27(7): 858–868. Publisher Full Text\n\nSufian MA, Bala BK: Modeling of urban solid waste management system: the case of Dhaka city. J. Waste Manag. 2007a; 27(7): 858–868. PubMed Abstract \| Publisher Full Text\n\nVilas MA: A Critical Overview of Legal Profile on Solid Waste Management in India. Int. J. Res. Chem. Environ. 2015; 5(1): 1–16." }	[ { "id": "95778", "date": "28 Oct 2021", "name": "John Pastor Ansah", "expertise": [ "Reviewer Expertise Health systems", "heath policy and systems 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 aim of this project is not clearly stated. The study aim “to create a complex system model by offering multiple choices, which helps identify the best policies for enhancing sustainable waste management” is poorly formulated and should be revised significantly. Models are developed to address a specific question which is missing in this manuscript.\n\nIn the methods section, the first statement “this study incorporates qualitative and quantitative analysis in order to evaluate and establish an alternative model” should be explained. It is very difficult to understand what the authors mean with that statement.\n\nWhat is dominant-less design? The authors should provide some explanation.\n\nThe methods section is poorly written. The authors were unable to clearly describe what was done in the study. Most of the materials in the results and discussion section should actually be in the methods section.\n\nIn the “results and discussion” section the authors indicated that “infrastructure, health, safety, and leadership support, and compliance with regulations had high-risk values of 900, 720 and 729, respectively, without providing unit of measurement. It is very difficult to understand what these numbers mean.\n\nSo far, I am reading the results and discussion section, and I have no idea the relevance of Table 1. This is because the authors were unable to explain what this research is about in the methods section. The presentation of the manuscript requires significant improvement.\n\nThe paper is not well organised and presented. The authors were unable to explain in the manuscript the causal loop diagram in Figure 1 and the stock and flow diagram in Figure 2. These figures should be moved to the methods section and properly explained with model equations provided in the appendix for review. Also, the causal loop diagram should help to understand the model structure. After that, the authors should conduct model validation and sensitivity analysis. All these were not done.\n\nThe results section does not read as results. The authors should refer to other papers to understand how to report results from a modelling and simulation study.\n\nThis paper is not ready for indexing and needs a complete review to improve quality and readability.\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? 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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "151696", "date": "04 Nov 2022", "name": "Muhammet Gul", "expertise": [ "Reviewer Expertise occupational health and safety risk assessment" ], "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 study presents a risk assessment model for sustainable solid waste management at the university in Indonesia. The model covers AHP, FMEA and dynamic system approach as methodology of the manuscript. Five field experts provided the data for the study. The model has merits for literature. It has the ability of injecting system dynamics modeling along with MCDM and failure mode and effect analysis. This point forms the unique contribution of the work.\nIt is hard to see the computational details of AHP and FMEA. However, SD is the main argument and on the focal point of the article, application steps of these two methods may be clearer and broader.\n\nIn the introduction, there is no clarification on the reasons of integrated usage of FMEA, AHP and SD. This must be clarified.\n\nThere are many errors in the text regarding writing style, flow of the text, and grammar. A comprehensive check and revision is needed.\n\nNumber of experts are just mentioned without giving more details and their judgments.\n\nAlso we do not see any details regarding SD simulation. No parameter values and equations are available.\n\nAbstract and conclusion sections should be rewritten.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-976
https://f1000research.com/articles/10-970/v1	27 Sep 21	{ "type": "Research Article", "title": "Stakeholder perceptions of strategies to reduce fast food consumption in Cambodian adults", "authors": [ "Samphors Sim", "Pall Chamroen", "Rebecca S. Dewey", "Vong Pisey", "Pall Chamroen", "Rebecca S. Dewey", "Vong Pisey" ], "abstract": "Background: Fast food consumption is one of the major contributing factors effecting overweightness and obesity, leading to many non-communicable diseases. Therefore, the purpose of this study was to determine strategies for reducing the fast-food consumption of Cambodian adults. Methods: This qualitative study was conducted among adults in Phnom Penh city, Cambodia, in 2018. 10 stakeholders were included from different institutions in Cambodia, mostly health institutions. The tools used in this study were a multidisciplinary meeting with stakeholders and the completion of observation forms. Using a semi-structured questionnaire, data were collected, and a thematic analysis was used. Results: Stakeholders’ viewpoints followed three identifiable themes with regard to approaches to reduce fast-food consumption among Cambodian adults. These comprised: (1) health education and health promotion (focusing on educational institutions), (2) reducing the availability and marketing impact of fast-food, and (3) implementing government policy. Conclusions: Knowing the important contributors to reduce the consumption of the fast food among Cambodia adults was the first priorities for all policy makers and other stakeholders to take action. This study provided essential findings for improving the decision-making abilities of those preparing strategy and policy for reducing fast-food consumption.", "keywords": [ "Stakeholders", "fast food", "strategies", "qualitative study", "Cambodia" ], "content": "Introduction\n\nGlobally, food-related behaviors and nutrition habits of human have been rapidly changing with globalization.1 Many people in Asia have maintained existing behaviors, traditional nutrition habits and dietary patterns, comprising consuming mostly rice and vegetables.2 Specifically, much of the food consumed is low in animal products.3 Conversely, more modern lifestyles and eating habits in Asia are often influenced by westernized diets containing high energy-density foods, large amounts of sugar, and saturated fats.4 Specifically, the increased consumption of fast-food is considered the main contributing factor to the over-nutrition of the Asian population as opposed to under-nutrition.5 This has consequently been seen to increase rates of overweightness and obesity.6 However, the trend of continuing emaciation and increasing obesity is still a big challenge for developing countries.7 According to previous studies and literature reviews, the prevalence of overweightness is significantly higher in urban, compared to rural, areas.8\n\nCambodia enjoys continued vigorous economic growth. In 2015, the actual level of growth was estimated at 7%, compared to a level of 7.1% in 2014. The garment sector, together with the services and construction sectors, are the major drivers of the Cambodian economic development. Growth is estimated to have remained stable in 2016, due to recouping from the local need and high garment exports, stagnation in the agriculture industry, and less extreme development in tourism. Poverty is steadily falling in Cambodia, reaching a rate of 7.7% in 2012, signifying a population of approximately three million people below the poverty line, and a further 8.1 million being near the poverty line. Around 90% of these people live in countryside areas.9\n\nPhnom Penh, the capital city of Cambodia, was selected as the location for the present study as it is a fast-food hotspot.10 With an area of 678.46 square kilometers (261.95 square miles), Phnom Penh is a municipality with status equal to that of Cambodian provinces. Phnom Penh comprises 12 districts, or Khans. Eight of these are considered to comprise the outskirts of the city, namely Dangkao, Meanchey, Porsenchey, Sen Sok, Russei Keo, Porsen Chey, Chroy Chong Var, and Prek Pnov. Phnom Penh is the administrative center of all 12 districts. These 12 districts are further comprised of 76 communes and 637 groups. Since 1979, the population of Phnom Penh has grown fourfold with population growth rate of 3.92%. Because of the city's growing population and economy, the metro system has been expanded to serve a larger area. Phnom Penh's population is estimated to contain 284,721 families, with a working-age population (18-59 years) of 864,310, representing 59.72% of the total population. An estimated 84.7% of these workers are in the service sector; 14% in the craft sector, and 10% in the agriculture sector.\n\nAnalyses have shown population increase and economic growth, along with significant expansion of the fast-food sector to be major underlying causes of death in Cambodia, with non-communicable diseases (NCDs) accounting for 52% of the total deaths. These comprise 24% cardiovascular diseases, 13% cancers, 4% chronic respiratory diseases, 2% diabetes, and 9% other NCDs. Reported incidence of risk factors such as raised blood pressure were 19.4% among adult males and 14.9% among adult females in 2008.11 In urban populations, around 5.4% of people were reported as living with diabetes in 2010.12 Little research has been conducted into the strategies and methodologies for reducing the consumption of fast-food, or into characterizing the perceptions of stakeholders. Therefore, the purpose of the study was to explore possible strategies for reducing fast-food consumption.\n\n\nMethods\n\nA qualitative methodology was used in this study. Previously collected survey results were presented13 to relevant stakeholders including members of the working age population (18-59 years), both male and female; doctors, nurses, public health practitioners, community leaders, school leaders and social marketing experts. These roles were selected as these are the individuals who have sufficient knowledge and experience in the crucial issues. Individuals who were willing to participate were invited to attend a multidisciplinary meeting, and a participatory observation with a member of the research team. The aim of this phase of the study was to clearly define the problem and provide recommendations for measures aimed at reducing fast-food consumption among the working age population in Phnom Penh, Cambodia.\n\nA total of 10 potential key informants were selected, representing all the above key stakeholder groups. Potential key informants were contacted in advance and informed of the goal and the protocol of the research. Purposive sampling was used to select key informants, thereby enabling the researcher to select potential key informants who had an understanding of the researchers’ expectations and could provide the necessary data. Following the initial purposive sampling, a snowball sampling method was used to expand the number of participants in the research. The 10 informants were selected thus: four individuals were recruited from the working age population, covering both men and women, and representing those who consume fast-food (a) regularly, (b) seldom, and (c) never. The rest were one doctor, one nurse, one public health practitioner, one community leader, one school leader, and one social marketing expert (see Table 1). In cases where potential key informants were difficult to identify, a snowball sampling technique was used. Most of them are the experts in the field, therefore, all information was provided specifically.\n\nValidated qualitative data collection methods were used to collect data from the sources outlined above based on the triangulation principle. Methods included a multidisciplinary meeting and participatory observations at different levels to identify factors associated with fast-food consumption among the working age population, as well as the underlying causes of these factors. A multidisciplinary meeting was conducted with stakeholders, coordinated by an interviewer or facilitator, and observed by two note takers along with sound recorders. The multidisciplinary meeting took place in a room of a university in Phnom Penh in April, 2018. The duration of the meeting was around three hours. The meeting followed a semi-structured format, consisting of open-ended questions that would elicit personal opinions, perceptions, and experiences from key informants. The format for the meeting did not change during the course of the meeting and was based on three questions:\n\n1. In your opinion, what government policies might reduce the fast-food consumption among working age adults in Phnom Penh, in the present day?\n\n2. How could the diets be improved among working age group in Phnom Penh, in the present day?\n\n3. Do you have any other ideas that you would like to express more?\n\nKey informants were contacted in advance by telephone or email to be invited to participate in the meeting. A member of the research team played the role of interviewer/facilitator. The two note-takers were trained in the process of taking clear notes throughout the meeting two days beforehand.\n\nThematic analysis was used to interrogate the data obtained from the multidisciplinary meeting and participant observations to identify factors associated with fast-food consumption and potential solutions among the working age population. The underlying causes of the problem were fully characterized so that policy recommendations for the reduction of fast-food consumption could be developed. The thematic analysis followed a six-step procedure developed by Braun and Clarke (2006).14 The six steps were implemented after all data (i.e. transcriptions from the multidisciplinary meeting and participant observations) had been translated from Khmer to English. Following this, translated data were checked by a member of the research team for completeness and consistency. Following the data check, the six-phase thematic analysis was conducted. In the first step, the researcher became familiar with the data by reading the translated transcriptions word by word and line by line. In the second step, the researcher started to assign codes which were derived from the raw data alone, and not from their opinions or ideas. Coding was conducted manually by writing notes on the transcribed texts with colored pens. This produced an initial list of ideas about the concepts present in the data. The researcher identified the codes and matched them with the appropriate data extracts. In the third stage, themes were identified. The long lists of different codes that had been identified in the previous phase was analyzed, and the researcher considered how different codes could be combined so as to create an overarching theme, and if necessary, a subtheme structure within each theme. The relationships between codes, themes, and subthemes were shown in an initial thematic map. Following this, in the next phase, the researcher reviewed the themes with the attached set of coded extracts. The initial thematic maps were then refined to reflect this phase. Further reviewing and refining of the coding was conducted by the researcher again and again until the researcher was satisfied with the coding process. The researcher then reviewed each theme individually and assigned it a name and description. Finally, the results of the thematic analysis were interpreted for report.\n\nValidity and reliability of the qualitative data collection and analysis followed the approach suggested by Mays and Pope (2000),15 which comprised triangulation of the data across multiple sources, comparing results from multiple stakeholder interviews to the findings available. Their responses were considered. Addressing differences between individual informants’ accounts formed a part of the error reduction process, in turn helping to generate further original data. A clear and definitive data collection procedure, including participant selection and data analysis procedure also contributed to the quality of the study.\n\nThe study design was received and approved by Khon Kaen research ethics committee, the human research ethics committee at the authors’ institution (Reference No. HE582071). All stakeholders/participants gave written informed consent prior to the commencement of the research process.\n\n\nResults\n\nStakeholders’ points of view regarding approaches to reduce fast-food consumption among adults in Cambodia were divided into three themes namely (1) health education and health promotion (with the primary focus on educational institutions), (2) reducing the availability and marketing impact of fast-food, and (3) implementing government policy (see Figure 1).\n\nHealth education and health promotion were considered the most critical concepts in stakeholder feedback for reducing fast-food consumption in Cambodia. Stakeholders from several sectors highlighted the need to not only provide health education and health promotion, but also for health educators and health promoters to receive comprehensive training in the identification of target audiences or prospective customers. Some stakeholders expressed the opinion that the youth or teenagers should be targeted with educational and promotional material, owing to the popularity of fast-food among the youth and teenage population. Further, some stakeholders suggested promoting traditional Khmer food because it contains significantly less fat, salt, and sweet ingredients in comparison to western food.\n\n“I think that mass media of fast food is a major challenge. Therefore, health educator and health promoters should study considerably for the contents of the campaigner education/promotion of fast-food company, fining out the tactics or tricks which attract the customers in their advertising or promotion and whether their advertising or promotion have adverse impact to the health of the consumers.” (Social marketer).\n\n“… motivating and promoting to the youth or teenagers to change their behavior from consuming fast food to local Khmer food which has less fatty, salty and sweet ingredients or deducing the tax on local Khmer food, in turn, increasing tax on fast food if possible.” (Community leader).\n\n“Currently, the mass media and social media play important roles in information distribution. Therefore, the use of platforms such as Facebook, Line messages, and television was proposed to reach the target audience quickly; however, the contents of the message must be creative to achieve penetration.”\n\n“If the campaign or promotion is attractive with a creative idea fitted with the needs of the target audiences, it will be able to catch the audience attention quickly.” (Public health practitioner).\n\n“A short video clip which can be broadcasted on social media or television about nutrition should be produced where the messages of the short video clips informing the target audiences why they should not eat this food.” (Social marketer).\n\nAll stakeholders stated that educational institutions should engage in health promotion and health education related to nutrition, especially the impact of fast-food on health.\n\n“… It will be effective if the target of education and promotion focusing on educational institution … the schools are able to prepare the projects concerning to applying nutrition lessons in the classroom as well as practice the lessons which students have learnt to practice in their real life.” (School leader).\n\n“… it is only the discipline or rules which force students to practice nutrition lessons in their real life; however, building an understanding about healthy diet along with teaching them how to be responsible in their own life.” (School leader).\n\nAll stakeholders emphasized the necessity for the government to take a role in reducing the impact of fast-food. Stakeholders advocated controlling and limiting the advertisement of fast-food in the media, especially the youth and teenagers. It was also suggested to impose health standards on food manufacture and sale that must be met, in terms of maximum salt or sugar levels in foods, and limiting the number of fast-food outlets adjacent to educational institutions.\n\n“The government should pay more attention to controlling the media advertising which revealed the meaning of unhealthy food to the people and also set or push the policy such as food labeling and food standard criteria.” (Social marketer).\n\n“If possible, the government should set or push the policy on the fast-food restaurants or shops which are located next to the academic institution especially from primary school to high school.” (Community leader).\n\nOnce government policy has been devised, the government must follow through to ensure that it is implemented and take measures to monitor and evaluate its impact. Only then will it be possible to determine whether it has achieved its goals, or whether it requires improvement.\n\n“The government should have the willingness to take actions with the policy which has been set and implement it equally for all institutions and occasions.” (Medical doctor/Nurse).\n\n“Monitoring during the implementing the policy is absolutely crucial in order for the aims of policy could be achieved.” (Public health practitioner).\n\n\nDiscussion\n\nThe findings of this study are in support of approaches proposed in internationally published literature.16 Health education and the use of promotional techniques were proposed as measures to reduce unhealthy diets.17 Children should be educated through school, and a number of suggestions were made for making educational access available to the adult population.18 Specifically, the food industry marketed the message “eat less” as part of recent policies addressing dietary issues. However, the “eat less” message appears to directly conflict with the goals of the food industry, which are to have people eat more of their products.4\n\nAn approach of increased intersectional collaboration,15 the method of enlisting the civic society and forming a collaboration between government and other stakeholders assured that all stakeholders were working together (including sellers and food companies) to provide encouragement for people to seek out support for maintaining a healthy lifestyle and healthy diet. The Cambodian government needs to take action to improve the nation’s diet, specifically with a focus on addressing the energy-dense and micronutrient-poor diets of many Cambodian children. However, the government should consider the effects of its policies on people’s right to choose, and on the food industry, as it is difficult to find a balance between health, freedom of choice, and the economic viability of the food industry.2 Moreover, the implementation of the chosen approach should avoid ambiguity and redundancy in the allocation of responsibility. The responsibilities for the implementation and monitoring of policy must specifically allocated to the relevant government agency or agencies to maximize intersectional collaboration between government agencies while ensuring the endeavors are given high priority. Stakeholders recommended the control of advertising on the television and internet to limit the marketing of fast-food to teenagers. This is a similar approach to the one recommended by some international organizations.3\n\nSimilarly, reducing the areas where fast food outlets are permitted, specifically in close proximity to schools, was a method mentioned in a forum in English related to health, whereby they advocated limiting permission for vendors of takeaway food to be placed within a given distance from schools in order to help influence the food choices students make on their way home from school.19 To mitigate the argument from the company of the fast food, new regulation must be evidence-based and provide scientific information on the importance of the relationship between public health and environmental factors such as food availability, diet, and nutrition. A similar argument was made during the introduction of a new food and nutrition policy in Norway, where the dairy and meat industry moved to support the production of low-fat milk due to the strong evidence base used in policy formation.20 School-based policies have been demonstrated to be highly effective in improving students’ dietary intake.21 However, the finding of widespread support for restrictive food policies in schools, including from the food industry, was surprising. As such, openly supporting school-based policies may gain the food industry public trust or deflect attention from the marketing of other food products to children. The food industry has lobbied for policy agenda to become more “industry-friendly”.18 The government is required to show transparency amid a strong impact of challenging when seeking and gaining industry support for policy. Government departments and officials must adhere to strict ethical guidelines in dealing with the food industry.18 This area of the investigation would benefit from an understanding of the factors influencing members of several different age groups from their own perspective.\n\n\nLimitations\n\nThere are only 10 key informants invited to join the multidisciplinary meeting. Therefore, there were some information missing. Also, the researcher had done the meeting only one time to get the information from the key informants.\n\n\nData availability\n\nThe raw data used in this study are comprised of sound, picture and video recordings of stakeholder participation. Due to confidentiality requirements stipulated in the ethical approval and consent processes, the authors are obliged to keep the identities of all stakeholders/participants anonymous, and the raw data associated with this article cannot be made publicly available. Access to the data will be considered on a case-by-case basis. Individuals who wish to access the data can contact the corresponding author Sim Samphors via mobile phone: Cambodia: (+855) 12 39 59 05/Thailand: (+66) 82 30 93 165, E-mail: simsamphors@gmail.com. Also, if you have further questions please feel free to contact Khon Kaen University Ethical Committee, Thailand. Tel: 043347057.", "appendix": "References\n\nPopkin BM: The nutrition transition in low-income countries: an emerging crisis. Nutr. Rev. 1994; 52(9): 285–98. PubMed Abstract \| Publisher Full Text\n\nKosulwat V: The nutrition and health transition in Thailand. Public Health Nutr. 2006; 5(1a): 183–9. PubMed Abstract \| Publisher Full Text\n\nAdair LS, Popkin BM: Are child eating patterns being transformed globally? Obes. Res. 2005; 13(7): 1281–99. PubMed Abstract \| Publisher Full Text\n\nPopkin BM: The nutrition transition and obesity in the developing world. J. Nutr. 2001; 131(3): 871s–3s. PubMed Abstract \| Publisher Full Text\n\nFrench SA, Story M, Neumark-Sztainer D, et al.: Fast food restaurant use among adolescents: associations with nutrient intake, food choices and behavioral and psychosocial variables. Int J Obes Relat Metab Disord: J Int Assoc Study Obesity. 2001; 25(12): 1823–33. PubMed Abstract \| Publisher Full Text\n\nPaeratakul S, Ferdinand DP, Champagne CM, et al.: Fast-food consumption among US adults and children: dietary and nutrient intake profile. J. Am. Diet. Assoc. 2003; 103(10): 1332–8. PubMed Abstract \| Publisher Full Text\n\nKim SH, Hwang J-Y, Kim MK, et al.: Dietary factors related to body weight in adult Vietnamese in the rural area of Haiphong, Vietnam: the Korean Genome and Epidemiology Study (KoGES). Nutr. Res. Pract. 2010; 4(3): 235–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMendez MA, Monteiro CA, Popkin BM: Overweight exceeds underweight among women in most developing countries. Am. J. Clin. Nutr. 2005; 81(3): 714–21. PubMed Abstract \| Publisher Full Text\n\nTHE WORLD BANK: Working for a World Free of Poverty 2016.[cited 2016 August 24]. Reference Source\n\nCanvassco: Market Analysis For Fast Food Chain Market in Cambodia, Laos, Myanmar and Vietnam 2014.[cited 2016 August 25]. Reference Source\n\nAn Y, Yi S, Fitzpatrick A, et al.Appropriate body mass index and waist circumference cutoff for overweight and central obesity among adults in Cambodia. PLoS One. 2013; 8(10): e77897. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCambodia NCDs [Internet]: 2014. Reference Source\n\nSim S, LaoHaSiriWong W: Fast Food Consumption, Overweight and Obesity among Working Age Persons in Cambodia. J. Clin. Diagn. Res. 2019; 13(7). Publisher Full Text\n\nBraun V, Clarke V: Using thematic analysis in psychology. Qual. Res. Psychol. 2006; 3(2): 77–101. Publisher Full Text\n\nMays N, Pope C: Qualitative research in health care. Assessing quality in qualitative research. BMJ. 2000; 320(7226): 50–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhulkerd S, Knai C, Lock K, editors: Stakeholdersû Perceptions of Factors Influencing Fast Food Consumption in Thai Adolescents.2012.\n\nWaxman AWHO global strategy on diet, physical activity and health. Food Nutr. Bull. 2004; 25(3): 292–302. PubMed Abstract \| Publisher Full Text\n\nLobstein T, Millstone E; University of S et al.: Policy options for responding to obesity: evaluating the options: summary report of the EC-funded project to map the views of stakeholders involved in tackling obesity - the PorGrow project.Brighton: SPRU. 2006.\n\nChildren, adolescents, and advertising: Committee on Communications. American Academy of Pediatrics. Pediatrics. 1995; 95(2): 295–297.\n\nJaime PC, Lock K: Do school based food and nutrition policies improve diet and reduce obesity? Prev. Med. 2009; 48(1): 45–53. PubMed Abstract \| Publisher Full Text\n\nNestle M, Jacobson MF: Halting the obesity epidemic: a public health policy approach. Public Health Rep. 2000; 115(1): 12–24. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "313408", "date": "16 Sep 2024", "name": "Sheetal Bhoola", "expertise": [ "Reviewer Expertise Food", "Nutrition and culinary tourism" ], "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 study is timeous and very informative. The culture of fast-food consumption globally has a direct impact on the nutritional well-being of people of all nations and ages in the world. The study can serve as a catalyst for other similar impactful studies in various areas, especially the developing world. The study is pertinent and can contribute significantly to decision making process.\nIt is important that the authors show knowledge expertise of the research area and incorporate references of recent/similar studies conducted on the topic. The authors should also extend themselves by indicating the lacunae of studies in this focal area.\nThe article encapsulates the presented research and data well, however the authors fail to mention when the last ( year) a similar study was conducted in Cambodia which will validate the study even further.\nTheoretically, there are some conceptual gaps that are prominent for the reader. For instance, this statement, “ Phnom Penh, the capital city of Cambodia, was selected as the location for the present study as it is a fast-food hotspot”\nThe authors should substantiate and support this statement with information and references. There are other similar statements which lack appropriate validation in research and theory. There are numerous references available. In addition, the authors could introduce the study and speak about the global mass media trends in relation to fast food MNC’s and the recent studies that have been conducted in the Northern Countries or First world countries. The authors do not make mention of The UN SDG’s and their goals of nutrition and healthy eating.\nThe above factors will enhance the article's validity and relevance. Overall the article is well written and the data collated and the discussion reads well. The authors should also reflect on focus areas of similar articles in neighbouring areas and indicate how this study adds to the lacunae of research if there is, or how this empirical research add value and differentiates from previous similar studies. The paper should also include information about how different this study is to other similar studies within Cambodia.\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/10-970
https://f1000research.com/articles/9-1244/v1	15 Oct 20	{ "type": "Research Article", "title": "Diagnostic accuracy of a urine dipstick for detecting albuminuria in hypertensive patients", "authors": [ "Phornwipa Panta", "Win Techakehakij", "Phornwipa Panta" ], "abstract": "Background: Screening for albuminuria is generally recommended among patients with hypertension. While the urine dipstick is commonly used for screening urine albumin, there is little evidence about its diagnostic accuracy among these patients. This study aimed to assess the diagnostic accuracy of a dipstick in Thai hypertensive patients for detecting albuminuria. Methods: This study collected the data of 3,067 hypertensive patients, with the results of urine dipstick and urine albumin-to-creatinine ratio (ACR) from random single spot urine being examined in the same day at least once, at Lampang Hospital, Thailand, during 2018. For ACR, a reference standard of ≥ 30 mg/g was applied to indicate the presence of albuminuria. Results: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of the trace result from dipsticks were 53.6%, 94.5%, 86.5%, and 75.5%, respectively. The area under the receiver operating characteristic curve of the dipstick was 0.748. Conclusion: Using the dipstick for screening albuminuria among hypertensive patients should not be recommended due to its low sensitivity. In response to high PPV, a trace threshold of the dipstick may be used to indicate presence of albuminuria.", "keywords": [ "hypertension", "albuminuria", "urine dipstick", "diagnostic test" ], "content": "Introduction\n\nStrong evidence has indicated that the presence of albuminuria in hypertensive patients is associated with the development of chronic kidney disease (CKD), which increases the risk of cardiovascular-related morbidity and mortality1,2. Early detection of CKD is important as either angiotensin-converting-enzyme inhibitor drugs or angiotensin II receptor blocker drugs can be added to a patient’s treatment regimen to slow down the progress of the disease and thus reduce all-cause mortality.\n\nDetection of albumin in urine plays an important role in diagnosing CKD in the early stages. Regarding the detection of albumin in urine, urine albumin-to-creatinine ratio (ACR) has widely been recommended to be used in diagnosing albuminuria, which is defined as the amount of urine albumin divided by urine creatinine ≥ 30 mg/g [≥ 3 mg/mmol]3–5.\n\nDespite the recommendations, performing ACR in all patients with hypertension is not always applicable, particularly in a primary care unit in rural or outreach areas where the necessitated resources may be unavailable. Practically, the urine dipstick is a test that has widely been used to identify the presence of albumin in the urine due to its low cost and high accessibility.\n\nAlthough using the urine dipstick is pragmatic, existing literature has not affirmed the accuracy of the test. Previous research has revealed a variety of diagnostic accuracy of the urine dipstick, compared with ACR. While some studies suggest that the dipstick is inappropriate for screening albuminuria6–9, others conclude that trace albuminuria from a dipstick can be used to indicate the presence of urine albumin10,11.\n\nOwing to result inconsistencies, it is still arbitrary as to whether or not positive findings of albumin from a urine dipstick could be used to confirm presence of albuminuria. Additionally, there is as yet no evidence to demonstrate if diagnostic results would be consistent across populations. Therefore, this study aimed to assess the diagnostic accuracy of a dipstick in Thai hypertensive patients for detecting albuminuria.\n\n\nMethods\n\nThis analysis is based on retrospective data from patients who visited Lampang Hospital from January to December 2018. The study included patients aged 18 years and over who were diagnosed with hypertension, ICD10 code “I10-14”, with the results of urine dipstick and ACR from random single spot urine being examined in the same day at least once. Laboratory results from the last visit were used if multiple results of a urine dipstick and ACR on the same day were presented within the same patient. Patients with urinary tract infections were excluded from the study.\n\nThis study protocol was approved by the Ethics Committee at Lampang Hospital (No.79/62). Consent of the patients to use their data in the study was waived by the ethical committee due to the retrospective nature of the study.\n\nACR was a reference standard to indicate the level of urine albumin. Evaluation of ACR was performed at Lampang Hospital using the immunoturbidimetric essay by AU5800/DxC700AU. The result of ACR ≥30 mg/g indicates the presence of albuminuria12,13.\n\nThis study employed the urine dipstick, “URiSCAN 9 SG” and the analyzer “URiSCAN SUPER+ and EH2080”, as an index test. Interpretation of the results were based on the color changes on the indicator tetrabromophenol blue in the presence of urine albumin. A positive reaction is indicated by a color change to yellow or green, reflecting the albumin results of negative, trace, 1+, 2+,3+, and 4+.\n\nDemographic characteristics including age and sex were collected for use in the analysis. Body mass index was calculated by weight in kilograms divided by squared height in centimeters14. Glomerular filtration rate (GFR) was estimated using the formula eGFR = 141 × min(SCr/κ, 1)α × max(SCr /κ, 1)-1.209 × 0.993Age × 1.018 [if female] × 1.159 [if Black]15. Information about patients’ underlying disease of diabetes was obtained from the diagnosis in the hospital’s electronic medical record with ICD10 code “E10-14”16.\n\nChi-squared test and t-test were applied to explore the association between the presence of albuminuria from ACR and covariates, with a significance level of 0.05. Sensitivity, specificity, positive predictive value, and negative predictive value of the dipstick were calculated, with 95% confidence intervals. The area under the receiver operating characteristic curve was approximated to demonstrate the test performance17. Subgroup analyses using the trace threshold of dipstick were performed to elucidate the diagnostic accuracy of the test among subgroups. Statistical analyses were performed using STATA version 1318.\n\n\nResults\n\nA total of 3,067 hypertensive patients matched the study criteria and were included in the analysis (Table 1). The mean age of the patients was 63.7 year, with ~40% being men. Diabetes appeared among 73.7% of the patients; 17.7% of them had eGFR <60 ml/min/1.73m2. Albuminuria was present in 24.5% of those with negative result from the dipsticks. Distribution of albumin-creatinine ratios with respect to results of urine dipsticks were exhibited in Figure 1.\n\nTable 2 demonstrated the sensitivity, specificity, positive and negative predictive values of urine dipstick in detecting albuminuria. It is seen that sensitivity of 53.6% was observed when the trace threshold was applied, whereas cutoff of ≥2+ and higher yields 100% test specificity. The area under the receiver operating characteristic curve was 0.7482 (Figure 2).\n\nCI, confidence interval.\n\nComparing diagnostic accuracy of the dipstick, it appears that sensitivity, specificity, along with positive and negative predictive values were approximately the same in all subgroups (Table 3).\n\nPPV, positive predictive value; NPV, negative predictive value\n\n\nDiscussion\n\nExisting studies have manifested a wide range of positive predictive values (PPVs) of urine dipsticks among patients with hypertension, ranging from 27 to 827,19. However, none have been conducted in a Thai population. Results of this study, exploring the diagnostic accuracy of the dipstick in a Thai population, not only illustrates the outcomes in this specific population, but can also be used in comparison with results from other populations for a better understanding of test accuracy.\n\nPrevious research has documented the differences in sensitivity and specificity of the dipstick across populations. A Japanese study showed sensitivity, specificity, and PPV of 37.1%, 97.3%, and 71.4%, respectively11. Another study conducted in Australian adults showed sensitivity, specificity, and PPV of 69.4%, 86.8%, and 27.1%, respectively7. One possible explanation for the difference in diagnostic accuracy of the dipstick was owing to differences in the characteristics of the populations7. The other study points out variation in the calibration of the dipstick as another explanation for differences between populations9. Compared with previous reports, diagnostic parameters shown in this study affirms variation in diagnostic performance of the dipstick across populations. This implies that the assessment of dipstick performance should be recommended for different populations.\n\nIt should be noted that false positive results of the dipstick could come from highly alkaline urine and contamination of antiseptics. Moreover, urine specimens used in this study came from random spot urine collection, which may be subjected to false positive results. Likewise, false negative results may have occurred due to excessive hydration before collecting the urine specimen, which leads to a decrease in concentration of urine albumin and subsequently a smaller chance of detecting albuminuria.\n\nSuch low sensitivity of 53% from the urine dipstick indicates that almost half of the patients with albuminuria cannot be identified using just the urine dipstick. It is also seen that among patients with a negative albumin result from the dipstick, albuminuria was found in nearly a quarter of them. This outcome well aligns with previous studies asserting low sensitivity of the dipstick in detecting albuminuria6,9,11. Given strong evidence indicating the high probability of cases being undetected, using the dipstick alone should not be recommended for use in screening of albuminuria among hypertensive patients.\n\nResults from the study revealed a rather high predictability of the dipstick in detecting urine albumin. Concerning the dipstick cutoffs, applying the trace threshold yields a PPV of 86.5%, compared with 98.2% and 100% using the 1+ and 2+ thresholds, respectively. Though a rather high chance of predicting albuminuria once hypertensive patients have these results of trace or higher from the dipstick, it should be borne in mind that albuminuria may be overly diagnosed with the application of the trace threshold, compared with using the higher cutoffs.\n\nAlthough excellent PPV can be achieved when employing higher thresholds of the dipstick, drawbacks remain when the recommendation for using the high threshold is applied due to fewer patients being applicable. Considering the trade-off between PPV and applicability of the dipstick results, the trace threshold may be recommended for indicating the presence of albuminuria in hypertensive patients.\n\nEven though the KDIGO guidelines3 have recommended the use of ACR to indicate the presence of albuminuria, this is proven to be rather costly and not readily available in some regions. Limitations, regarding the availability and costs of ACR, may arise when considering the application of ACR for routine screening of hypertensive patients. Nonetheless, evidence has demonstrated a low sensitivity of urine dipsticks, which should not be recommended for screening albuminuria. Hence, ACR is deemed the option for screening albuminuria in the setting where resources are available.\n\n\nConclusion\n\nWhile existing evidence is controversial to whether the urine dipstick should be recommended for screening albuminuria in hypertensive patients, results from this study demonstrated that the dipstick has such low sensitivity in detecting albumin in urine in the Thai population. These results suggest that the urine dipstick not be recommended for screening urine albumin in patients with hypertension. In contrast, results of trace or higher yields high PPV, indicates a very high possibility of the presence of microalbuminuria.\n\n\nData availability\n\nFigshare: Diagnostic Accuracy of a Urine Dipstick for Detecting Albuminuria in Hypertensive Patients, http://www.doi.org/10.6084/m9.figshare.1265171620.\n\nFigshare: STARD checklist for \"Diagnostic Accuracy of a Urine Dipstick for Detecting Albuminuria in Hypertensive Patients\", http://doi.org/10.6084/m9.figshare.1267315421.\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 Dr.Thanawat Vongchaiudomchoke, nephrologist, and Dr.Napat Phetkub for their valuable comments.\n\n\nReferences\n\nIseki K, Ikemiya Y, Iseki C, et al.: Proteinuria and the risk of developing end-stage renal disease. Kidney Int. 2003; 63(4): 1468–74. PubMed Abstract \| Publisher Full Text\n\nHemmelgarn BR, Manns BJ, Lloyd A, et al.: Relation between kidney function, proteinuria, and adverse outcomes. JAMA. 2010; 303(5): 423–29. PubMed Abstract \| Publisher Full Text\n\nKerry Willis P, Cheung M, Slifer S: Kidney international supplements. 2013; 3(1). Reference Source\n\nPathania M, Rathaur VK, Yadav N, et al.: Quantitative Micro-albuminuria Assessment from 'Random Voided Urinary Albumin: Creatinine Ratio' Versus '24 hours Urinary Albumin Concentration' for Screening of Diabetic Nephropathy. J Clin Diagn Res. 2013; 7(12): 2828–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Renal Association: CKD stages. 2019.\n\nZeller A, Sigle JP, Battegay E, et al.: Value of a standard urinary dipstick test for detecting microalbuminuria in patients with newly diagnosed hypertension. Swiss Med Wkly. 2005; 135(3–4): 57–61. PubMed Abstract\n\nWhite SL, Yu R, Craig JC, et al.: Diagnostic accuracy of urine dipsticks for detection of albuminuria in the general community. Am J Kidney Dis. 2011; 58(1): 19–28. PubMed Abstract \| Publisher Full Text\n\nKoeda Y, Tanaka F, Segawa T, et al.: Comparison between urine albumin-to-creatinine ratio and urine protein dipstick testing for prevalence and ability to predict the risk for chronic kidney disease in the general population (Iwate-KENCO study): a prospective community-based cohort study. BMC Nephrol. 2016; 17(1): 46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark JI, Baek H, Kim BR, et al.: Comparison of urine dipstick and albumin: creatinine ratio for chronic kidney disease screening: A population-based study. PLoS One. 2017; 12(2): e0171106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSam R, Shaykh M, Pegoraro A, et al.: The significance of trace proteinuria. Am J Nephrol. 2003; 23(6): 438–41. PubMed Abstract \| Publisher Full Text\n\nKonta T, Hao Z, Takasaki S, et al.: Clinical utility of trace proteinuria for microalbuminuria screening in the general population. Clin Exp Nephrol. 2007; 11(1): 51–55. PubMed Abstract \| Publisher Full Text\n\nPalaniappan L, Carnethon M, Fortmann SP: Association between microalbuminuria and the metabolic syndrome: NHANES III. Am J Hypertens. 2003; 16(11 Pt 1): 952–58. PubMed Abstract \| Publisher Full Text\n\nToto RD: Microalbuminuria: definition, detection, and clinical significance. J Clin Hypertens (Greenwich). 2004; 6(11 Suppl 3): 2–7. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Body mass index - BMI. 2019. Reference Source\n\nNational Kidney Foundation: CKD-EPI CREATININE EQUATION. 2009. Reference Source\n\nWorld Health Organization: International classification of diseases (ICD). World Health Organization; 2006.\n\nŠimundić AM: Measures of Diagnostic Accuracy: Basic Definitions. EJIFCC. 2009; 19(4): 203–11. PubMed Abstract \| Free Full Text\n\nStataCorp L: Stata Statistical Software: Release 13. 2013. Reference Source\n\nLim D, Lee DY, Cho SH, et al.: Diagnostic accuracy of urine dipstick for proteinuria in older outpatients. Kidney Res Clin Pract. 2014; 33(4): 199–203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanta P, Techakehakij W: Diagnostic Accuracy of a Urine Dipstick for Detecting Albuminuria in Hypertensive Patients. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12651716.v1\n\nTechakehakij W: STARD checklist for \"Diagnostic Accuracy of a Urine Dipstick for Detecting Albuminuria in Hypert ensive Patients\". figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12673154.v1" }	[ { "id": "85559", "date": "14 Jun 2021", "name": "Surendran Deepanjali", "expertise": [ "Reviewer Expertise urinary tract infections", "medical philosophy" ], "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\nPanta P et al conducted a retrospective study to evaluate the performance of urine dipstick testing for albumin compared to urine albumin-creatinine ratio (ACR) in patients with hypertension. The study population included 3067 subjects of Thailand. The authors found that urine dipstick at the trace positive cut-off has modest sensitivity for detecting ACR of 30mg/g or more. The authors conclude that urine dipstick test cannot be used to screen albuminuria in hypertensive patients. Overall the manuscript is well-written. However, there are a set of issues to be addressed in the study.\nMajor comments:\nAbstract\nAuthors state that “there is little evidence” about the diagnostic accuracy of dipstick testing for albuminuria. This information is not accurate since the authors themselves cite studies addressing this issue in the Introduction and Discussion. The conclusion reads” Using the dipstick for screening albuminuria among hypertensive patients should not be recommended due to its low sensitivity. In response to high PPV, a trace threshold of the dipstick may be used to indicate presence of albuminuria.” After having stated that dipstick testing should NOT be recommended, it is redundant to suggest a ‘trace’ threshold for albuminuria.\n\nIntroduction\nAlthough ACR is the ideal test for albuminuria, urine dipstick analysis finds mention as part of initial work up of hypertension in existent guidelines on hypertension (ISH CPG 2020, ESH 2018). Hence in the Introduction authors should elaborate more on the advantages and disadvantages of using the dipstick in comparison to ACR. The references 3-5 do not seem to be appropriate as evidence of recommending ACR for measuring albuminuria. In the last paragraph of Introduction, it is given whether urine dipsticks “confirm” the presence of albuminuria which is not conforming to the ideas in the previous paragraph where it is mentioned as a tool for “screening”.\n\nMethods\nIt is mentioned under Participants that “Laboratory results from the last visit were used if multiple results of a urine dipstick and ACR on the same day were presented within the same patient.” It is not clear whether some patients had both the tests repeated on multiple days or multiple results on the same day were available. How did authors exclude patients with UTI? It is not clear whether the urine dipstick reading was done by visual inspection or automated analyzer. An internet search for EH 2080 reveals it is a urine sediment analyzer. Why was this instrument used for interpreting dipsticks? For calculating BMI and eGFR data on height and weight and serum creatinine were collected. Was it done on the same visit as the urine tests or within any given time frame?\nResults\nThe authors have not stated what proportion of the study population was detected to have albuminuria in the text. The information has to be deducted from Table 1. Also, it will be more informative if the albuminuria estimated by ACR is quantified into meaningful groups like moderately or severely increased albuminuria. How the dipstick performed in these two groups can also be compared; for example, as given in Reference no.19. It is not clear why statistical tests of significance were used for comparison of 2 ACR groups in Table1. It seems like proportion of diabetic patients with albuminuria is less compared to those without diabetes. The same for CKD also. If the authors want to point out any specific finding through these comparisons it has to be stated in the text. Likewise, comparison of dipstick results between the two groups conveys no extra information. The information provided in Table 2 is not clear. Terms like “≥Negative” and “≥trace” are confusing. A sensitivity of 100% and PPV of 40% for a negative test?\nDiscussion\nAlthough the authors have compared their results, especially the low sensitivity of dipstick, with other studies in literature, they have not interpreted their study results thoroughly. The trade-off between the low sensitivity and the good PPV have to be interpreted in terms of population in which the dipstick testing will be employed. According to this study, a trace positive result has 98% PPV in this particular hypertensive population where 70% were diabetic too. The authors could discuss whether the test is still worthy of use from the cost-benefit point of view. If the authors however are maintaining that the test should not be recommended because of low sensitivity, then suggesting a trace threshold for interpretation of test result is superfluous. The ‘variation in calibration’ as a cause for differing diagnostic performance characteristics across populations is not clear enough. Does this apply only to automated analyzer results? The authors state that proteinuria will be over-diagnosed with trace threshold. This point is also not substantiated since a test with a PPV of 98-100% will not have a high false-positivity rate.\n\nConclusion\nAs mentioned previously in the comments on Abstract, the conclusion has to be re-written without ambiguities.\n\nMinor comments:\nReferences 3, 5, 14, 15 & 16 are not correctly cited. The figshare dataset shows age and BMI represented with almost 10 decimal points precision. I would suggest approximating it 2 or 3 decimal points for better representation. The figshare dataset does not give the results of dipstick testing\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6875", "date": "12 Jul 2021", "name": "Win Techakehakij", "role": "Author Response", "response": "Abstract We added “in Thailand” in the text to clarify the scarce of evidence in this area of research. The sentence, “Using the dipstick for screening albuminuria among hypertensive patients should not be recommended due to its low sensitivity”, interprets the implication of the sensitivity of the test, in which it may not be appropriate to apply for mass screening. In this regard, we added “for mass screening” in the text to improve clarity of the content. On the other hand, the sentence, “In response to high PPV, a trace threshold of the dipstick may be used to indicate presence of albuminuria”, reflects another practical point of using of PPV result when the patients already presenting with results from dipsticks. Unlike the suggestion for mass screening, the PPV suggests that a trace result yields a rather high possibility of the presence of albuminuria and thus may be recommended for use in clinical practice. Introduction The use of ACR as the gold standard for detecting albuminuria is recommended by the Renal Association, with supportive evidence. All the evidence was available in reference 3-5. The pros and cons of using dipsticks in comparison with ACR were discussed in the latter 2 paragraphs with supportive references 6-11. To figure whether urine dipsticks “confirm” the presence of albuminuria does conform the ideas of this paper, which is to explore diagnostic accuracy of the test. Results from diagnostic accuracy can be applied to recommendations for both public screening and clinical diagnosis, as demonstrated in this paper. Methods This means in case of that the patients had multiple results on the same day, as mentioned in the previous sentence, “The study included patients aged 20 and over who were diagnosed with hypertension, ICD10 code “I10-14”, with the results of urine dipstick and ACR from random single spot urine being examined in the same day at least once.” Results of urine dipsticks presenting with red blood cell or white blood cell to the diagnostic criteria of urinary tract infection were construed as urinary tract infection. The urine dipstick reading was done by the automated analyzer. The EH2080 was removed. BMI and eGFR were calculated on the same visit. Results We added “Approximately 39.8% of the samples presented with albuminuria” to describe the proportion of samples with albuminuria. Concerning the demonstration of albuminuria level, we decidedly omitted this information as this is considered out of the scope of this paper. Statistical analyses shown in Table1 are deemed a compliment to the demonstration of the samples’ characteristics. The terms, e.g., “≥Negative” and “≥trace”, are used when applied the cutoffs for diagnostic test is considered. “≥Trace”, for instance, means that any results with albuminuria higher than the trace level, trace,1+,…,4+, were counted. A 100% sensitivity when applying the negative threshold is explained by the fact that the negative result is the lowest possible outcome of dipsticks. Using the negative threshold, all the samples, with or without albuminuria, would be identified as having albuminuria, resulting in a 100% sensitivity. Discussion For the purpose of mass screening in the population level, it may be appropriate to consider only sensitivity, which exhibits the test ability to uncover all diseased patients from the population. Narrative about this was described in the discussion. In addition, issues about high PPV were also provided in the discussion. The issues about difference in interpretation of sensitivity and PPV were above-mentioned. The ‘variation in calibration’ as a cause of the difference in diagnostic performance could apply to not only automated analyzer, but also the visual analyzer. Albuminuria will be over-diagnosed with trace threshold because of PPV of 86.5%. This means that only 865 of 1,000 patients with trace-or-above results will really have albuminuria. Approximately 13.5% will thus be wrongly/overly diagnosed with albuminuria. Conclusion Explanation was described above. Minor comments: References were re-checked respecting to the Vancouver style. Information in the dataset was provided with details for the users. Results of the dipsticks were provided with labels." } ] }, { "id": "86938", "date": "21 Jun 2021", "name": "Polathep Vichitkunakorn", "expertise": [ "Reviewer Expertise 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\nThe authors examined the diagnostic accuracy of urine dipstick on albuminuria among hypertensive patients in Thailand. This study tried to reveal the sensitivity, specificity, positive predictive value, and negative predictive value of the tool. This is very useful for urine dipstick in many health care settings.\nThe manuscript is clear and well documented. The rationale is well established. The authors applied appropriate methods for data analysis and the results were convincing.\nHowever, to improve paper readability, minor changes are required. I suggest the following:\nThe introduction did not discuss the heterogeneity of various accuracy of urine dipstick. The authors selected the laboratory results from the last visit. It would be great if you can provide the rationale for this selection method. The logic for calculating the sample size is missed in this manuscript. The authors may add the calculation of sample size or power analysis. Table 1: Regarding rounding decimals, the “63.52” should be “63.5”. Figure 1: Please explain or discuss the outlier (i.e., ACR ~ 5000+ in the Negative group). Table 2: the “95%CI” can be moved to the first row of the table. For discussion, the limitations and implications for this research should be mentioned for the readers.\nThank you for the opportunity of reading and evaluating this paper.\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? Yes", "responses": [ { "c_id": "6876", "date": "12 Jul 2021", "name": "Win Techakehakij", "role": "Author Response", "response": "The heterogeneity of dipstick results was discussed in details in the discussion. In case that there are more than one laboratory results within the same person, we decided to include only one result from each sample to reduce the bias from individuals who had undertaken the tests many times. Results from the last visit were decidedly selected in order to obtain the most recent outcomes in this regard. From the Bujang and Adnan (2016), with the prevalence of approximately 40%, the minimum sample size of 408 would yield the conventionally acceptable power of 0.804. However, this study contained a lot more samples of 3,067, which far exceeds the minimum to reassure the accuracy and reliability of the analysis. Decimal points were rounded to one digit for percentage and 3 digits for p-value. False negative results from dipsticks, including some outliers, ordinarily exist to indicate imperfect predictability of the index test. Possible explanation was described in the discussion part “Likewise, false negative results may have occurred with excessive hydration before collecting the urine specimen, which leads to a decrease in concentration of urine albumin and subsequently a smaller chance of detecting albuminuria.”. Owing to the limited space available in the table, we moved the 95%CI to the second line to improve readability for the audiences. The main limitation of this research is the externality of the results. This is because the samples were chosen from only one hospital in the northern Thailand, which may not well represent the diagnostic accuracy of the test in other populations, particularly with differential prevalence of albuminuria. This limitation is genuinely the gaps and rationale of this research, which was mentioned in the introduction. Concerning the implication, this research mentioned the interpretation and applicability of the results in the discussion part: “results from this study demonstrated that the dipstick has such low sensitivity in detecting albumin in urine. These results suggest that the urine dipstick not be recommended for screening urine albumin in patients with hypertension. In contrast, results of trace or higher yields high PPV, which indicates a very high possibility of the presence of microalbuminuria.”. Reference Bujang MA, Adnan TH. Requirements for minimum sample size for sensitivity and specificity analysis. Journal of clinical and diagnostic research: JCDR. 2016 Oct;10(10):YE01." } ] } ]	1	https://f1000research.com/articles/9-1244
https://f1000research.com/articles/10-964/v1	24 Sep 21	{ "type": "Brief Report", "title": "Comparison of hospitalizations and deaths from COVID-19 2021 versus 2020 in Italy: surprises and implications", "authors": [ "Alberto Donzelli", "Marco Alessandria", "Luca Orlando", "Marco Alessandria", "Luca Orlando" ], "abstract": "Data from the Istituto Superiore di Sanità (ISS) emphasized by the media indicate that COVID-19 vaccination reduces related infections, hospitalizations and deaths. However, a comparison showed significantly more hospitalizations and intensive care unit accesses in the corresponding months and days in 2021 versus 2020 and no significant differences in deaths. The combination of non-alternative hypotheses may help explain the discrepancy between the results in the entire population and the vaccination’s success claimed by the ISS in reducing infections, serious cases and deaths:\na bias: counting as unvaccinated also \"those vaccinated with 1 dose in the two weeks following the inoculation\", and as incompletely vaccinated also \"those vaccinated with 2 doses within two weeks of the 2nd inoculation\". a systematic error: counting as unvaccinated also \"vaccinated with 1 dose in the two weeks following the inoculation\", and as incompletely vaccinated also \"vaccinated with 2 doses within two weeks of the 2nd inoculation\".\nMany reports show an increase in COVID-19 cases in these time-windows, and related data should be separated\nlevels of protective effectiveness in vaccinated people, often considered stable, actually show signs of progressive reduction over time, which could contribute to reducing the overall population result unvaccinated people show more severe disease than in 2020, supporting also in humans the theory of imperfect vaccines, which offer less resistance to the entry of germs than the resistance later encountered inside the human body. This favors the selection of more resistant and virulent mutants, that can be spread by vaccinated people. This damages first the unvaccinated people, but ultimately the whole community.\nAn open scientific debate is needed to discuss these hypotheses, following the available evidence (as well as to discuss the inconsistent theory of unvaccinated young people as reservoirs of viruses/mutants), to assess the long-term and community impact of different vaccination strategies.", "keywords": [ "COVID-19 vaccinations", "trend of COVID-19 infections", "hospitalizations", "intensive care admissions", "deaths", "effectiveness of COVID-19 vaccinations strategies at the community level" ], "content": "Introduction\n\nThe Integrated Surveillance Bulletins of the Istituto Superiore di Sanità (ISS), emphasized by the national media, declare that there has been a sharp decline in infections and contagions, hospitalizations and deaths for this pathology, thanks to the vaccination. The ISS Bulletin of April 30, 2021 states: “The decrease in cases in the older age groups is attributable to the increase in the vaccination coverage in such groups. Starting from the second half of January there is a decreasing trend in the number of cases in healthcare workers and in subjects aged 60 to ≥80 years, probably attributable to the vaccination campaign.”\n\nThe campaign began on the 27th December, 2020. As of 28th April, 2021 (the update of the above-mentioned Bulletin), 18,957,365 doses had been administered (13,372,589 first and 5,584,776 second doses).1 On the 7th July, the doses administered had quadrupled, reaching 56,713,862: approximately 91% of >80 year olds in Italy received at least one dose and more than 88% completed the two doses. In the 70–79 years age group, more than 85% has received at least one dose of the vaccine.2 As of the 7th July, 56.7% of the general population had received at least one dose and 37.7% had received full doses.3\n\n\nObjective\n\nThe aim of this study was to compare the data relating to hospitalizations, access to intensive care unit (ICU) and deaths from COVID-19 in the same period (1st March–7th July) of 2020 and 2021, to highlight the possible impact of COVID-19 vaccine on these outcomes.\n\n\nMethods\n\nWe downloaded the daily bulletins of the Civil Protection Department (CPD)4; the data are continuously updated (every day positive hospitalized and deceased cases added or removed and corrected on a regional basis).\n\nFor the statistical comparison of the data between 1st March–7th July 2020 and 2021 a non-parametric analysis (Mann–Whitney test for independent samples) was used, after performing the Shapiro–Wilk normality test, inasmuch all the data of the variables considered (“Hospitalized with symptoms”, “Intensive care unit” and “Deaths”) do not show a normal distribution. The data were processed using GraphPad Prism 5 software (GraphPad Software, Inc., USA; RRID:SCR_002798), JASP (RRID:SCR_015823) is an open-access alternative software. The significance level “p” was fixed at <0.05.\n\n\nResults\n\nNo significant variations between 2020 and 2021 were observed in the Deaths variable (Figure 1).\n\nA. Trend of the Deaths variable; B. Significant differences between two periods, with p < .05; C. Mean, Standard deviation, p value.\n\nSignificant variations between 2020 and 2021 were observed in the Hospitalized with symptoms variable (p = 0.0139; Figure 2).\n\nA. Trend of the Hospitalized with symptoms variable; B. Significant differences between two periods, with p < .05; C. Mean, Standard deviation, p value.\n\nSignificant variations between 2020 and 2021 were observed in the Intensive care unit variable (p < 0.0001; Figure 3).\n\nA. Trend of the Intensive care unit variable; B. Significant differences between two periods, with *p < .05; C. Mean, Standard deviation, p value.\n\n\nDiscussion\n\nThe aim of this study was to compare the data of 2020 and 2021 of three variables used by Italian authorities to evaluate the trend of Covid-19 epidemic, to understand the impact of the vaccination.\n\nData on Deaths (Figure 1) show that, apart from the mortality peak recorded between the end of March and the beginning of April 2020, the curve from 5th May onwards practically overlaps over the two years, without significant variations.\n\nFor Hospitalizations (Figure 2) the data show, from the end of February to 2nd April, a worse situation in 2021. From the 3rd to the 27th April the two curves had a similar trend, first with fewer beds occupied in 2021, then with a worsening compared with 2020. From 28th April to the end of June, the trend of the curves favored first 2021, then favored 2020 from the 21st June until 7th July, the time limit of the graph. The statistical analysis has shown a statistical variations between two periods.\n\nAlso ICU data (fig. 4) showed significant variations between the two periods, with more cases admitted to ICU in 2021 compared with 2020, both from 1st to 21st March and from 10th April to 7th July. The 2020 is worse than 2021 only between 21th March and 10th April: 20 days out of the approximately 120 examined.\n\nThe data show a global decrease from the beginning of April, but no improvement in 2021 versus 2020. 2021 shows many more accesses in COVID-19 hospital wards than in 2020, without any decrease in specific mortality.\n\nHowever, for all the three considered variables, the increased access to hospitals in 2021 was more spread over time.\n\nThe 2020 restrictions may have had an impact on the distribution of the curve, but it is unlikely that this could have affected the total numbers as well. In fact, restrictions to movement occurred also in 2021: in 2020 they were concentrated in about 2 months (from March to the end of April, up to almost total reopening on 15th June,5 while in 2021 were spread over time, with a mild beginning at the end of October and subsequent tightening and reopening between 26th April and 1st July.6 Moreover, in 2021 there were patchy closures on Italian territory.\n\nThe distribution of the two curves is different: the curve of 2020 is distributed over a shorter time period with a slightly higher (and narrower) peak than that of 2021, that appears “spread” over a longer period. Furthermore, the comparison between groups (Mann–Whitney test for independent samples) leaves no doubt about the greater impact of the pandemic in 2021.\n\nDisaggregated data are not available, therefore it is not possible to be more precise from a statistical point of view.\n\nA much better situation could have been expected in 2021, because of the experience in care (including early and home care), and of the vaccination campaign (in the first week of July over a third of the general Italian population received the complete vaccination cycle and over 50% at least one dose.\n\nThe decrease in hospitalizations and deaths since the beginning of April was recorded in the same period also in 2020, in a rather faster way, which suggests the contribute of a seasonal effect.\n\nFrom these observations, the vaccination campaign does not seem to have influenced COVID-19 hospitalizations or deaths overall, in the examined period.\n\nHow to reconcile this finding with the ISS Integrated Surveillance Bulletin publications of June 16th and following7 about the success of vaccination in avoiding infections and especially symptomatic, serious cases and deaths? We put forward three hypotheses, which can coexist and explain this apparent paradox.\n\nA) A systematic calculation error may contribute to some extent both to the benefits attributed to the vaccinated and to the harms attributed to the unvaccinated. In fact, the ISS (see e.g. the Integrated Surveillance Bulletin from 24th June to 4th July)8 consider “unvaccinated” to be equivalent to “those vaccinated with 1 dose in the two weeks referred to the study” (which according to our calculations amount approximately to 13.5% of the total), and “vaccinated with an incomplete cycle” to be equivalent to “vaccinated with the 2nd dose performed in the two weeks referred to the study” (about 31% of the total). This would not be relevant if the trend of COVID-19 cases in these time windows coincided with the one preceding the inoculations. However, there are many reports that in the days following the inoculations there is an excess of Covid-19 cases. For example, the BMJ9 reported a Public Health England study of vaccination in over 70s, which found a “noticeable\" increase in COVID-19 infections immediately after receiving the AstraZeneca vaccine.10\n\nAlso, a study reported in February on the vaccination program in Israel found a similar spike in cases in newly vaccinated, in this case with Pfizer vaccine,11 with an approximate doubling of the daily incidence after vaccination until about day 8.\n\nThe original randomized controlled study (RCT) of Pfizer12 on adults shows in fact a similar effect, with an increase of 40% of cases of “suspected COVID-19” (409 versus 287) in the first week in the intervention arm compared with the placebo arm. There is documentation of transient immunosuppression after vaccinations.13 Vaccination with the Pfizer vaccine causes transient drop in lymphocytes over the next 3 days.14 Phase 2 RCTs of AstraZeneca also showed a drop in neutrophils;15 other vaccinations have also shown a fall of neutrophils16 and lymphocytes.17\n\nTherefore, vaccinated people can temporarily develop more cases and infect more, for at least a week after the inoculation and, in that week, the virus multiplies in vaccinated people who develop COVID-19.\n\nIf there is an increase in COVID-19 cases in the time window of 14 days following an inoculation and they are counted among the “unvaccinated”, the latter are burdened with an undue excess of cases, simultaneously relieved by “vaccinated”. Therefore, the ISS calculations should be redone, at least keeping the subjects who have received a dose of vaccine in the previous two weeks as separate from the “unvaccinated\" and “incompletely vaccinated”.\n\nB) The vaccine effectiveness after the aforementioned 14 days were believed to be quite stable and durable, while now there is evidence of a progressive deterioration in the protective effectivenss of vaccines. This seems plausible, in view of the marked decline in antibody levels measured at various time intervals (14–20 days, 21–41, 42–55, 56–69 and 70 or more) after the second vaccination,18 and as reported by the Israeli Government,19 finding a significant decline in the vaccination effectiveness over the following months.\n\nThis could help to explain the results of the overall comparison between hospitalizations and ICU accesses in 2020 versus 2021. In fact, the ISS comparisons show important differences in the outcomes between unvaccinated and vaccinated, but do not exclude a progressive reduction of protective effectiveness also in the vaccinated, influencing the overall result on the whole population.10\n\nThe ISS data show that symptomatic serious cases would largely be ascribed to unvaccinated (or vaccinated contracting the disease within 14 days of inoculation of the first dose), and it is unlikely that these results can change substantially even applying the corrections suggested in point A). This observation outlines a scenario in which the infection would have moved from a pool of subjects potentially capable of developing severe COVID-19 corresponding to the majority of the Italian population (excluding those who have overcome the infection) to the smaller pool of the “unvaccinated”. If this was true, it would mean that the latter, having reached absolute numbers of deaths comparable to those of 2020, and even greater numbers of hospitalized and ICU patients, would get Covid-19 with more severity than 2020. If so, the hypothesis that the vaccine can protect both those who receive it and others should be questioned: instead, it could pose additional risks in the unvaccinated. This may be more than just an hypothesis, already advanced in 2001,20 confirmed experimentally in 2015 for other vaccines21 and proposed again today with possible reference to SARS-CoV-2 vaccines.25 It is a problem occurring if vaccines are leaky,21,22 when they block the virus entrance door less than they fight the virus inside the body, as in fact is the case with today SARS-CoV-2 vaccines; it is likely that the natural infection (starting from the respiratory tract and not with an injection) helps even better to reduce subsequently the entry of viruses. As a result, the virus multiplies to some extent inside the organism, vaccine antibodies neutralize the vast majority of the viruses but, as unintended effect, they favor precisely those random mutations (and variants) that give the virus greater ability to escape from antibodies or to resist their attack, becoming more virulent. Consequently, some of the vaccinated people could spread more virulent viruses, harming the remaining community (and in the long term probably even the vaccinated, as immune protection tends to decline). Experiments with chickens vaccinated and infected with Marek virus (not deliberately replicable in humans, but which could be replicated in farmed mammals susceptible to SARS-CoV-2, such as ferrets)21 have shown clearly this effect, which harmed mostly the unvaccinated members of the farm, who developed a more serious disease.\n\nFirst of all, it should be accepted to discuss it seriously, comparing the scientific evidence available for or against this explanatory hypothesis. If this hypothesis is confirmed, a discussion should be opened urgently about how to improve strategies at the population level.\n\nOf course, a possible strategy is that pursued today in Italy and in most of the world: to accelerate the push for universal vaccination, using a mix of obligations, incentives and penalties, up to the complete discrimination of those who do not adhere, hoping to end the pandemic, as well as to protect those at risk. This strategy is what the national authorities have chosen so far and of which the majority of the population is convinced. However, part of the population do not accept this strategy (also because dubious constitutional legitimacy). Against such strategy there are:\n\n• the costs, including organizational ones, of universal immunization (worldwide it would involve 8 billion people, in addition to the management of the animal reservoirs already documented23); and many authorities now hypothesize to use periodic boosters;\n\n• the total and severe adverse reactions already known, of the extent of which there is, however, little awareness24,25; and the serious adverse effects known or still unknown;\n\n• the highly questionable relationship between expected benefits and risks of vaccinating young people and children, who are offered moreover a vaccine calibrated on the original viral strain, while the new variants are sometimes already called “another virus”;\n\n• the enormous loss of opportunity-cost for the equivalent amount of resources diverted from other health uses.\n\nAn alternative could be to focus vaccination on those who can benefit most (elderly, multi-pathological subjects or workers at high professional risk), and, after having protected them, not to promote vaccination towards those who have little to earn from it. Note, for example, that in Italy in 2020 the 0–49 years old population showed a paradoxical reduction of 8.5% in mortality, compared to the average of the five-year period 2015–2019.26 In the first months of 2021 the mortality reduction under the age of 50 years was even more pronounced compared to the five-year reference period (January 2021: −12%; February: −17%, March: −10.8%).27\n\n• Even more so a focused vaccination should apply to young people 0−19, who show a prevalence of asymptomatic cases >50%, paucisymptomatic >30%, and mild cases <20% in the update of mid-July.28 They can help mitigate the epidemic,a progressively transforming it into a milder endemic,35,36 by developing a natural immunity, which seems robust and lasting, as far as we know.29–32\n\nChildren and adolescents in particular have been shown37 to be less infected and infectious, to have asymptomatic infections much more often than adults and, even when asymptomatic, to develop robust humoral immune responses, persisting for at least one year.37 The argument that, before being naturally immunized, they would constitute a “dangerous reservoir” of viruses seems to have little scientific and logical support.a\n\n\nConclusions\n\nRepeating that this year “in Italy the overall drop in hospitalizations, IT and deaths is due to the vaccine” is not supported by the available data, since the respective trend was even better in 2020, without the vaccine.\n\nThe interpretation of this finding should be subject to an open and uncensored scientific debate, aimed at understanding the impact of vaccination strategies also at the global level of the population and in long-term scenarios, as a prerequisite for rational health policy choices, that optimize health outcomes (and sustainability) for the individuals and the community.\n\n\nData availability\n\nhttps://github.com/italia/covid19-opendata-vaccini - Open Data on delivery and administration of COVID-19 vaccines in Italy.\n\nhttps://www.epicentro.iss.it/coronavirus/bollettino/Bollettino-sorveglianza-integrata-COVID-19_7-luglio-2021.pdf - COVID-19 epidemic National update July 7, 2021-12 noon (I.S.S.).\n\nhttps://github.com/pcm-dpc/COVID-19/blob/master/dati-andamento-nazionale/dpc-covid19-ita-andamento-nazionale.csv - Protezione Civile data table.csv used for the development of the comparison graphs 2020 versus 2021.\n\nhttps://covid19.zappi.me/table/ - Protezione Civile data table (simple format, same data as the previus.csv file) used for the development of the comparison graphs 2020 versus 2021.\n\nhttps://www.ilpandacentrostudio.it/vaccini.html - Calculations by Antonio Caramia on data from the Extraordinary Commissioner Covid-19 (Dashboard Vaccines).\n\nhttps://opendatadpc.maps.arcgis.com/apps/dashboards/b0c68bce2cce478eaac82fe38d4138b1 - Dashboard COVID-19 Protezione Civile.\n\nhttps://www.epicentro.iss.it/coronavirus/bollettino/Bollettino-sorveglianza-integrata-COVID-19_16-giugno-2021.pdf - COVID-19 epidemic National update June 16, 2021-12 noon (I.S.S.).\n\nhttps://www.epicentro.iss.it/coronavirus/bollettino/Bollettino-sorveglianza-integrata-COVID-19_23-giugno-2021.pdf - Epidemia COVID-19 COVID-19 epidemic National update June 23, 2021-12 noon (I.S.S.).\n\nhttps://datadashboard.health.gov.il/COVID-19/general - Corona virus in Israel – general situation Dashboard.", "appendix": "References\n\nOpen Data su consegna e somministrazione dei vaccini anti COVID-19 in Italia - Commissario straordinario per l’emergenza Covid-19.\n\nTask force COVID-19 del Dipartimento Malattie Infettive e Servizio di Informatica, Istituto Superiore di Sanità: Epidemia COVID-19. Aggiornamento nazionale: 7 luglio 2021.\n\nMonitoraggio Vaccini.\n\nCovid-19 situazione Italia - Dati forniti dal Ministero della Salute - Elaborazione e gestione dati a cura del Dipartimento della Protezione Civile.\n\nDecreto del presidente del consiglio dei ministri 17 maggio 2020 - Disposizioni attuative del decreto-legge 25 marzo 2020, n. 19, recante misure urgenti per fronteggiare l’emergenza epidemiologica da COVID-19, e del decreto-legge 16 maggio 2020, n. 33, recante ulteriori misure urgenti per fronteggiare l’emergenza epidemiologica da COVID-19. (20A02717) (G.U. Serie Generale, n. 126 del 17 maggio 2020).\n\nTimeline Decreti-legge emanati durante il periodo di emergenza epidemiologica Covid-19.\n\nTask force COVID-19 del Dipartimento Malattie Infettive e Servizio di Informatica, Istituto Superiore di Sanità. Epidemia COVID-19. Aggiornamento nazionale: 16 giugno 2021. Disponibile all’indirizzo.\n\nTask force COVID-19 del Dipartimento Malattie Infettive e Servizio di Informatica, Istituto Superiore di Sanità. Epidemia COVID-19. Aggiornamento nazionale: 23 giugno 2021. Disponibile all’indirizzo:\n\nDay M: Covid-19: Stronger warnings are needed to curb socialising after vaccination, say doctors and behavioural scientists. BMJ . 2021; 372: n783. Publisher Full Text .\n\nLopez Bernal J, Andrews N, Gower C, et al.: Early effectiveness of COVID-19 vaccination with BNT162b2 mRNA vaccine and ChAdOx1 adenovirus vector vaccine on symptomatic disease, hospitalisations and mortality in older adults in England. MedRxiv. 2021 (preprint). Publisher Full Text\n\nHunter PR, Brainard J: Estimating the effectiveness of the Pfizer COVID-19 BNT162b2 vaccine after a single dose. A reanalysis of a study of “real-world” vaccination outcomes from Israel. MedRxiv. 2021 (preprint). Publisher Full Text\n\nEmergency Use Authorization (EUA) for an Unapproved Product Review Memorandum.\n\nCraig C: Covid-19: Stronger warnings are needed to curb socialising after vaccination, say doctors and behavioural scientists. BMJ . 2021; 372: n783. Publisher Full Text\n\nWalsh EE, Frenck RW Jr, Falsey AR, et al.: Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N. Engl. J. Med. 2020; 383: 2439–2450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFolegatti PM, Ewer KJ, Aley PK, et al.: Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet. 2020; 396: 467–478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuturi-Kioi V, Lewis D, Launay O, et al.: Neutropenia as an Adverse Event following Vaccination: Results from Randomized Clinical Trials in Healthy Adults and Systematic Review. PLoS One. 2016; 11: e0157385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunyer TP, Mangi RJ, Dolan T, et al.: Depressed lymphocyte function after measles-mumps-rubella vaccination. J. Infect. Dis. 1975; 132: 75–78. Publisher Full Text\n\nShrotri M, Navaratnam AMD, Nguyen V, et al.: Spike-antibody waning after second dose of BNT162b2 or ChAdOx1. Lancet. July 15, 2021; 398: 385, 387. Publisher Full Text\n\nCorona virus in Israele - situazione generale: Disponibile all’indirizzo.Reference Source\n\nGandon S, Mackinnon MJ, Nee S, et al.: Imperfect vaccines and the evolution of pathogen virulence. Nature . 2001 Dec 13; 414(6865): 751–756. Publisher Full Text\n\nRead AF, Baigent SJ, Powers C, et al.: Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens. PLoS Biol. 13(7): e1002198. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKennedy DA, Read AF: Monitor for COVID-19 vaccine resistance evolution during clinical trials. PLoS Biol. 18(11): e3001000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaurin M, Fenollar F, Mediannikov O, et al.: Current Status of Putative Animal Sources of SARS-CoV-2 Infection in Humans: Wildlife, Domestic Animals and Pets. Microorganisms. 2021; 9(4): 868. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShimabukuro T: COVID-19 vaccine safety update. Advisory Committee on Immunization Practices (ACIP). March 1, 2021.\n\nChapin-Bardales J, Gee J, Myers T: Reactogenicity Following Receipt of mRNA-Based COVID-19 Vaccines. JAMA. 2021; 325: 2201–2202. PubMed Abstract \| Publisher Full Text .\n\nIstat-ISS: Impatto dell’epidemia COVID-19 sulla mortalità totale della popolazione residente.Anno 2020. 5° Rapporto congiunto, 10 giugno 2021.\n\nIstat-ISS: Impatto dell’epidemia COVID-19 sulla mortalità totale della popolazione residente.Anno 2020 e gennaio-aprile 2021. 6° Rapporto congiunto, 10 giugno 2021.\n\nTask force COVID-19 del Dipartimento Malattie Infettive e Servizio di Informatica, Istituto Superiore di Sanità: Epidemia COVID-19. Aggiornamento nazionale: 14 luglio 2021.\n\nWang Z, Mueck F, Schaefer-Babajew D, et al.: Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year. bioRxiv preprint.\n\nAlfego D, Sullivan A, Poirier B, et al.: A population-based analysis of the longevity of SARS-CoV-2 antibody seropositivity in the United States. EClinicalMedicine. 2021; 36: 100902. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTurner JS, Kim W, Kalaidina E, et al.: SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Nature. 2021; 595: 421–425. PubMed Abstract \| Publisher Full Text .\n\nShrestha NK, Burke PC, Nowacki AS, et al.: Necessity of COVID-19 vaccination in previously infected individuals. medRxiv preprint. 2021. Publisher Full Text\n\nCevik M, Tate M, Lloyd O, et al.: SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. Lancet Microbe. 2021; 2: e13–e22. PubMed Abstract \| Publisher Full Text \| Free Full Text .\n\nTönshoff B, Müller B, Elling R, et al.: Prevalence of SARS-CoV-2 Infection in Children and Their Parents in Southwest Germany. JAMA Pediatr. 2021, 175, 586, 593. PubMed Abstract \| Publisher Full Text \| Free Full Text .\n\nLavine JS, Bjornstad O, Antia R: Immunological characteristics govern the transition of COVID-19 to endemicity. Science. 2021; 371: 741–745. PubMed Abstract \| Publisher Full Text \| Free Full Text .\n\nLavine JS, Bjornstad O, Antia R: Vaccinating children against SARS-CoV-2. Hard to justify right now for most children in most countries. BMJ . 2021; 373: n1197. Publisher Full Text\n\nRenk H, Dulovic A, Matthias Becker M, et al.: Typically asymptomatic but with robust antibody formation: Children’s unique humoral immune response to SARS-CoV-2. medRxiv preprint. posted July 22, 2021. Publisher Full Text\n\n\nFootnotes\n\na The issue continuously put forward that unvaccinated children and young people are “a reservoir” of “viruses that continue to circulate” and that “consequently mutate”, actually has limited scientific evidence (and the situation of the vaccinated may not be so different from unvaccinated). This can be explained in three steps:\n\n1) The contagiousness of adults who become infected lasts on average 7 days from the onset of symptoms plus ~2 days before symptoms. A systematic review of 79 studies 33 never found live, viable viruses after the 9th day in any of the studies, even if the emission of RNA fragments could sometimes lasts weeks.\n\nIn children, often asymptomatic or paucisymptomatic, the duration of contagiousness seems lower than that of adults: 33 therefore it can be estimated in about a week (or a little more)\n\n2) There are 52 weeks in a year. If a child is not infected, she/he is never contagious, nor can be a “reservoir”. If infected, she/he can infect (usually less than adults 34) for about a week, and for the other 51 weeks of the year the child has become immune, so not constituting any “reservoir”.\n\n3) The hypothesis A, explained in the discussion above, reports many examples where the vaccinated, in the days following the inoculation, developed more cases of COVID-19 than in the previous period, so resulting more contagious, with a temporary viral multiplication (due to infection pre-inoculum kept under control by innate defenses? Or to infection acquired because of relaxed precautions? Or by temporary immunosuppression?), with some analogies with what follows to natural infection. If the latter is symptomatic, however, the patient becomes aware of it and can isolate himself, while the vaccinated may lack the awareness of a transient potential greater infectivity." }	[ { "id": "98994", "date": "08 Nov 2021", "name": "Herald Walach", "expertise": [ "Reviewer Expertise psychology", "study design", "epidemiology", "research methods" ], "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\nReview of F1000 MS by Donzelli, Alessandria & Orlando “Comparison of hospitalizations and deaths from COVID-19 2021 versus 2020 in Italy: surprises and implications” Harald Walach\nThis is an interesting secondary analysis of publicly available data in Italy on deaths by Covid19, hospitalizations and ICU usage due to this disease, comparing years 2020 and 2021 during the same time window (end of February to beginning of July). The data were compared with robust and very simple statistical methods (non-parametric tests) and yielded significant differences for hospitalizations and ICU usage. The fact that a comparatively robust and insensitive statistical model found such strong effects shows that these effects are indeed substantial.\nThis result contradicts official statements that the vaccination campaign in 2021 has reduced the case load, mortality and hospitalizations. The authors produce a succinct argument explaining this discrepancy: it is likely due to the definition used by authorities (actually copied from pivotal regulatory trials) that counts people as vaccinated only after a certain time window after first and second vaccinations. The authors of this paper make a point that it is precisely during this time that vaccinated people are vulnerable or more likely to develop the disease, which would escape the official statistics due to the definition employed.\nThe data speak a clear language, and the authors do a good job interpreting and explaining the finding and I think this paper is essentially a valid representation of the situation. The authors miss one potentially important point in their explanation: if the Covid-19 vaccines are not as safe as claimed and reports about the side effects as published by Rose (1), which are likely on the low side, are true, then some of the deaths and hospitalizations might be also induced by the vaccination campaign. I think this is a potentially additional factor, but I leave it at the discretion of the authors, whether they want to include this. Otherwise, I think this paper is fit for publication. Below are a few points for clarification that I would recommend to make in a revised version: Major Points: Since “Death from Covid19” (and hospitalization and ICU usage) is one of the major endpoints of this study, it would be good for readers to know:\nHow this is defined in Italy and\n\nWhether this definition has changed between 2020 and 2021,\nJust to make sure that any changes potentially visible are not due to changes in definition.\nMinor Points: Abstract: The term “non-alternative hypotheses” is not clear. Perhaps explain or change. Abstract: the points “bias” and “systematic error” repeat the same information; perhaps it would be better to say “bias or systematic error” and then bring the information just once. Discussion 3rd para: “The statistical analysis has shown a statistical variations between two periods.” should read “variation” (sing.). Discussion, p. 5, middle: “which suggests the contribute of a seasonal effect.” Should read … contribution…. Discussion, p 5, penultimate para: reformulate; this is a bit awkward (for instance: “Vaccinated people are more liable to develop the disease during a short period of time and are more likely to infect others for at least a week…” Discussion, p5, last para: “the latter are burdened with an undue excess of cases, simultaneously relieved by “vaccinated”“ This is confusing. Please reformulate, for instance: “… more cases are unduly registered in the group of the “vaccinated”. At the same time, the same number of cases is arithmetically deducted from the group of the vaccinated. This yields the impression that more cases are seen in the group of the unvaccinated than in the group of the vaccinated, which is in fact an error due to case definition.” Or something similar that makes the underlying meaning clearer.\nConsider putting the information hidden in note a) into the text, if no editorial policy or limit in word length speaks against it.\nReferences and Data-Availability: Perhaps put the links of the data sources behind the references so that it is clear which data-source refers to what point mentioned in the text. “Statistic Analysis” should be “Statistical Analysis” Figure 1: in the legend, and perhaps in the text, make clear that this is “Death due to Covid19”; the same for the other variables and Figures: make clear that this is Hospitalizations due to Covid19” Results: I am not sure that “variation” is the right term here. What was observed was not only a change in variation, but a change of overall magnitude in the number of hospitalization and ICU treatments\nReference Rose, J. (2021). A report on the U.S. vaccine adverse events reporting system (VAERS) on the Covid-19 messenger ribonucleic acid (mRNA) biologicals. Science, Public Health Policy, and the Law, 2, 59-80.\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": "97925", "date": "16 Nov 2021", "name": "Marco Cosentino", "expertise": [ "Reviewer Expertise Medicine", "Pharmacology", "Immunology", "Neuroscience" ], "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\nBy use of publicly available data, this study examines SARS-CoV-2 hospitalizations, ICU use and deaths in Italy in 2020 (before mass vaccinations campaign took place) and in 2021, after most of the Italian population received the COVID-19 vaccines. Conclusions challenge the official version that COVID-19 vaccination reduces related infections, hospitalizations and deaths. Indeed, with a very simple and effective approach the study shows that between 2020 and 2021 deaths are substantially the same, while hospitalizations and ICU use are substantially increased.\nThe authors provide as possible explanation for the bias in counting as unvaccinated also \"those vaccinated with 1 dose in the two weeks following the inoculation\", and as incompletely vaccinated also \"those vaccinated with 2 doses within two weeks of the 2nd inoculation\", and the systematic error due to counting as unvaccinated also \"vaccinated with 1 dose in the two weeks following the inoculation\", and as incompletely vaccinated also \"vaccinated with 2 doses within two weeks of the 2nd inoculation\".\nIt is however unclear how, and to what extent, misclassification of vaccinated/unvaccinated people would affect results which regard the whole population.\nIndeed, in 2020 it can be assumed that no one was vaccinated, which in 2021, an increasing fraction of people got the vaccine. Would it be possible to separate hospitalizations, ICU use and deaths thus comparing unvaccinated and vaccinated? Anyway, it is my opinion that even without this distinction deaths are the same and are therefore expected to have been not so much affected by mass vaccination campaign. On the other side, increased hospitalizations in early 2021 and increased ICU use in both early and late 2021 m9ght possibly suggest any detrimental effect of vaccine of health status leading to hospitalization.\nFinally, what about criteria for linking hospitalizations, ICU use and deaths to COVID-19? Were they the same throughout all the study? Or did they change and in case to what extent?\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/10-964
https://f1000research.com/articles/10-963/v1	24 Sep 21	{ "type": "Research Article", "title": "Antecedents of generation Z towards digitalisation. A PLS-SEM analysis", "authors": [ "Kar Hoong Chan", "Tuan Hock Ng", "Lee Ying Tay", "Chiu Yu Teh", "Tuan Hock Ng", "Lee Ying Tay", "Chiu Yu Teh" ], "abstract": "Background: The Covid-19 pandemic has forced businesses in the direction of technology development. In particular, financial institutions have started the digital transformation, embracing the usage of artificial intelligence. In this respect, consumers’ willingness to adopt artificial intelligence in finance, appears to have relevance to current efforts to enhance the efficiency and effectiveness of the financial system. This study aims to better comprehend the antecedents towards the intention to adopt artificial intelligence in financial services among Generation Z, with the use of the Technology Acceptance Model. Methods: In this study, questionnaires were used to collect data from 150 male and female Malaysian undergraduates. Partial Least Squares-Structural Equation Modelling was employed to analyse the data. Results: Perceived ease of use and attitude, positively influenced the adoption of artificial intelligence in financial services. Conclusion: The results have suggested the improvement in user interface, information and activities to encourage generation Z to adopt artificial intelligence in financial services.", "keywords": [ "Technology Acceptance Model (TAM)", "intentions to adopt artificial intelligence in financial services (INT)", "perceived usefulness (PU)", "perceived ease of use (PEoU)", "attitude (ATT)" ], "content": "Introduction\n\nDigital transformation is seen extensively across many industries worldwide, partly triggered by prolonged lockdowns in response to the Covid-19 pandemic. According to The Star (2020) stated that artificial intelligence (AI) is gaining even more traction in driving customers experience. To a certain extent, financial institutions, for example, have begun to embark on the AI journey, embracing AI chatbot into the delivery process of banking and financial services. Likewise, massive investments have been made in AI-driven predictive analytics to improve efficiency and enhance business forecasts in areas like sales, operations, risk management, etc. Specifically, it also aims to assist its customers in financial consultancy and financial needs (New Straits Times, 2018).\n\nIn a recent survey conducted by Alam (2018), 93% of respondents from the wealth management industry emphasise the need for AI in handling many and various financial services, as a sharp increase in AI adoption in the financial industry is anticipated. While these represent crucial insights from the perspective of industry players, equally significant is whether customers are ready to embrace the use of AI in the financial industry. This is an intriguing yet often overlooked question. Thus, this study appears to be essential to investigate the antecedents influencing consumers' willingness to adopt AI in financial services. Generation Z (Gen Z), aged between 18 to 25 years as of 2020, is the respondents for this survey. Their intention about AI adoption is important to this research as Gen Z is a true digital native, as claimed by Gaidhani et al. (2019). Among the antecedents, perceived usefulness (PU), perceived ease of use (PEoU), and attitude (ATT) are hypothesised to positively influence Gen Z's intention to adopt AI-based financial services (INT).\n\n\nLiterature review\n\nDavis et al. (1989) suggested that TAM determines the antecedents of technology and system acceptability. The antecedents, namely PU, PEoU and ATT from the mentioned model, are adapted for this study to examine their influence on consumers' intention to adopt AI-based financial services. Venkatesh and Davis (2000) also pointed out that these antecedents affect each other. For example, PEoU will affect PU of using a new technology. When the particular system is easy to use, it is more valuable to the users to improve their work execution. Besides, PU will also affect ATT and individual's intention to accept the system's technology.\n\nBehavioural intention defines the degree to which a person performs a particular behaviour after conscious planning or self-directed motivation (Mark & Paul, 2005). According to Chuang, Liu and Kao (2016) and Cham et al. (2018), TAM is used to investigate a person's behaviour and the results show that PU, PEoU and ATT may be used to predict a person's readiness to adopt new technology.\n\nPU is a crucial factor to determine the acceptance of using AI-based financial services. PU means the extent to which an individual perceived a system that improves their job efficiency (Davis et al., 1989). Pai and Huang (2011) found that users' willingness to accept AI is related to the system's PU. Another study also demonstrates that a more positive attitude is related to a higher willingness to use AI products (Lunney et al., 2016). Likewise, Pillai and Sivathanu (2020) studied the impact of using AI-powered customer service technology in the tourism industry. The result shows that users are willing to use AI devices to plan for their trip due to the easy and interactive booking process and service.\n\nTo master or use a system or technology without much effort is identified as perceived ease of use (Davis et al., 1989). Hamid et al. (2016) found that PEoU is positively related to the adoption of e-government system. It indicates that the respondents are keen on using the new system if the technology seemed easily understood. Furthermore, Patil and Mugdha (2019) found a significant positive relationship between PeoU and adoption of chatbot services in financial services. This is because the system is a simple drag-and-drop interface and hence respondents are more willing to use it.\n\nAzjen (1991) defined attitude as the degree of a user's assessment and expression of the behaviour's execution. Favourable or unfavourable attitude emerges from the personnel's beliefs on behavioural outcomes (Cordano & Frieze, 2000). The user's attitude toward AI will affect their willingness to accept AI in financial services (Schepman & Rodway, 2020). Previous studies discovered a positive relationship between attitudes and intentions to use the system (Chang, Vitell, & Lu, 2019; Le & Kieu, 2019; Prendergast & Tsang, 2019; Tweneboah-Koduah, Adams, & Nyarku, 2019).\n\n\nMethods\n\nResearch framework (Figure 1) is shown in the proposed research framework.\n\n\nData collection\n\nA set of self-administered questionnaires is distributed online via Google Form (See underlying data) (Chan et al., 2021). Purposive sampling is used in this study because the respondents must first be knowledgeable about the AI system so they can fully comprehend the questions in the survey (Tongco, 2007). It means that the target respondents have to be technology savvy and well versed with financial terminology.\n\nThe cover page of the questionnaire stated the disclaimers, and the respondents would have to agree to participate in the survey voluntarily before they respond to the questionnaire. They also had given the written consent to publish their responses. The sample size is determined via G*Power analysis. Since there are only three predictors in this study, the minimum acceptable sample size to surpass the sufficient statistical power of 0.8 is 74 responses. This study has collected 150 usable responses from undergraduate students with the age range of 18 to 25 years to represent Gen Z in Malaysia (See underlying data) (Chan et al., 2021). The Partial Least Squares Structural Equation Modelling (PLS-SEM) method was employed to analyse the collected data. The software used is Smart PLS 3.0 and SPSS version 23.\n\n\nResults\n\nThe Cronbach's alpha test is used to determine the questionnaire's reliability. Additionally, the SPSS software is used to evaluate the reliability analysis with 30 respondents. Results shown in Table 1 indicates that Cronbach's alpha's value is greater than 0.7 for all the analysed variables, and thus, the questionnaire is reliable.\n\nTable 2 tabulates the descriptive analysis. Among 150 respondents, 44% are male, while 56% are female respondents. According to Dolot (2018), individuals born after 1995 are referred to as Gen Z. As a result, respondents are between the ages of 20 and 22 (55.3%), between 23 and 25 (22.0%), under 20 (14.0%), and above 25 (8.7%) years.\n\nThis study performs a measuring model to evaluate the elements of a path model, which contains indicators and their relationships with the constructs. The minimum threshold of 0.708, 0.70, and 0.50 are assessed on factor loadings, composite reliability and average variance, respectively. They are evaluated on the validity and reliability of the constructs (Hair et al., 2019). Hence, PU1, PU2, PEOU1, ATT1, ATT3, INT1 and INT5 were removed because their respective loadings were less than 0.708. The factor loadings, composite reliability, and average variance extracted for the final measurement model met the minimum threshold, which the values are tabulated in Table 3. The results also suggested that the measurement model was reliable and had adequate convergent validity. Subsequently, a discriminant validity assessment was performed because the content and substance of the constructs had to be identified. Therefore, this study employed the Heterotrait-Monotrait Ratio (HTMT) (Henseler et al., 2015). Firstly, the construct - the intention had failed to meet the HTMT 0.9 criteria. Upon checking with the cross-loading, INT3 was removed. Table 4 shows that all the values are lower than 0.90, which is the required threshold suggested by Gold et al. (2001). Hence, the findings shows that the proposed hypotheses are accepted and verified by the value of the discriminant validity.\n\nTable 5 presents the hypotheses statements in this study. The structural model is presented in Figure 2. These findings revealed that the Coefficient of Determination (R2) value of the model is 0.467, which suggested a moderate predictive model (Chin, 1998). It also means that 46.70% of the INT can be explained by PU, PEoU and ATT.\n\nBootstrapping was applied to determine the significant level of the constructs. The path coefficient and corresponding t-values were gathered with 5,000 resamplings. As a result, PEoU positively influenced the INT, and ATT positively influenced the INT. Hence, H2 (t-value = 2.683) and H3 (t-value = 5.408) were supported. Nonetheless, PU and INT were insignificant in this study. Thus, H1 (t-value = 0.552) was not supported. Figure 2 presented the structural model, and Table 6 summarises the hypotheses testing.\n\n\nDiscussion\n\nThese findings suggest that both PEoU and ATT are positively related to INT. In other words, H2 and H3 are supported in this study which is consistent with previous studies (Schepman & Rodway, 2020; Patil & Mugdha, 2019; Hamid et al., 2016).\n\nIndividuals will intend to use the AI-based financial services when they believe that it would eliminate obstacles such as making the right investment decision or process the handling of their financial matters much faster. (Hong et al., 2021). The Edge Markets (2017) further elaborated that AI technology allows financial service providers to engage customers more intuitively and offers real-time support, thereby transforming complicated banking processes into more simple tasks. Additionally, ATT also plays a vital role in influencing an individual's intention to use AI-based financial services. TAM model states that PEOU is one of the motivators of consumer attitude towards using new technology (Venkatesh & Davis, 2000). Individuals will develop a positive attitude when they perceive that using AI is effortless in the AI-based financial service context.\n\nMeanwhile, PU is not significant in this study. Thus, H1 is not supported. Jahangir and Begum (2008) mentioned that it would not be enough by just offering AI-based financial services to the general public and expect it to utilised. Financial service providers must establish the conviction that AI technology can improve the efficiency, security, and ease of use in financial services.\n\n\nConclusion\n\nThe main purpose of this study was to identify the factors influencing Gen Z's intention to use AI-based financial services. The results confirm that PEoU and ATT positively influenced the INT among Gen Z in Malaysia. This indicates that Gen Z is ready for the digital transformation, and they are willing to accept and use AI-based financial services.\n\nThis study draws a few implications for financial institutions/financial service providers: Financial institutions are suggested to focus on designing a user-friendly interface that provides quick access to the standard features. The AI-based financial services would be an easy tool for the consumers to use, and the system would not require much training and technical skills.\n\nAdditionally, financial service providers should make sure that their websites portray clear, concise, and intelligible information. This helps customers understand that AI-based financial services are always simple to use.\n\nFinally, financial institutions are encouraged to get involved in campaigning and frequent collaboration with the universities to create awareness among the Gen z community regarding the importance and ease of using AI tools in financial services. This will help Gen Z build a positive attitude towards AI-based financial services.\n\n\nEthical approval\n\nThis study is in accordance with the university's ethical standard, and ethical approval (number EA0432021) was obtained.\n\n\nData availability\n\nData archiving and networked services (DANS): Survay of Malaysian view on the roles of artificial intelegence for the future changes in the financial services industries\n\nDOI: https://doi.org/10.17026/dans-2xr-x6wm (Chan et al., 2021).\n\nThis project contains the following underlying data:\n\nFile 1: The blank questionnaire used in this study.\n\nFile 2: Responses to the survey of the Malaysians’ view on the roles of artificial intelligence for the future changes in the financial services industry.\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\nAuthor contributions\n\nKar Hoong Chan and Chiu Yu Teh presented the idea and background of this manuscript. Tuan Hock Ng developed the framework and the adapted theory. Chiu Yu Teh also collected the data for this study. Lee Ying Tay performed the data analysis and discussion of the study. All authors discussed and contributed to the final manuscript.", "appendix": "Acknowledgments\n\nWe would like to express our gratitude to Multimedia University, Malaysia for the financial support. Also, we would like to thank the editor and reviewers for their constructive feedbacks and insightful comments.\n\n\nReferences\n\nAlam N: AI in financial services — digital transformation or disruption. News Straits Times. July 22, 2018. Reference Source\n\nCham TH, Low SC, Lim CS, et al.: Preliminary Study on Consumer Attitude towards FinTech Products and Services in Malaysia. Int J Eng Tech. 2018; 7 (2.29), 166–169. Publisher Full Text\n\nChang HH, Vitell SJ, Lu LC: Consumers' Perceptions Regarding Questionable Consumption Practices in China: The Impacts of Personality. Asia Pacific J Marketing Logistics. 2019. Publisher Full Text\n\nChan KH: Survey of Malaysian' view on the roles of Artificial Intelligence for the future changes in the financial services industry. DANS. 2020. Publisher Full Text\n\nChin WW: The Partial Least Squares Approach for Structural Equation Modeling. In: Marcoulides GA (Ed.), Modern Methods Business Res. 1998; 295–336. Mahwah, New Jersey: Lawrence Erlbaum Associates.\n\nChuang LM, Liu CC, Kao HK: The Adoption of Fintech Service: TAM Perspective. Int J Management Administrative Sci. 2016; 3(07): 01–15. Publisher Full Text\n\nCordano M, Frieze IH: Pollution Reduction Preferences of U.S. Environmental Managers: Applying Ajzen's Theory of Planned Behavior. Academy Management J . 2000; 43(4): 627–641. Publisher Full Text\n\nDavis FD, Bagozzi RP, Warshaw PR: User Acceptance of Computer Technology: A Comparison of Two Theoretical Models. Management Sci. 1989. Publisher Full Text\n\nDigital News Asia: AI to nearly double the rate of innovation in Malaysia by 2021. Reference Source\n\nDiscriminant Validity in Variance-based Structural Equation Modeling. J Academy Marketing Sci. 43(1), 115–135.\n\nDolot A: The characteristics of Generation Z. E-Mentor . 2018: 44–50.\n\nGaidhani S, Arora DL, Sharma BK: Understanding the Attitude of Generation Z Towards Workplace. Int J Management, Technology Eng, IX. 2019; 1: 2804–2812.\n\nGold AH, Malhotra A, Segars AH: Knowledge management: An organisational capabilities perspective. J Management Information Syst. 2001; 18(1): 185–214. Publisher Full Text\n\nHair JF, Risher JJ, Sarstedt M, et al.: When to Use and How to Report the Results of PLS-SEM. Euro Business Rev . 2019; 31(1): 2–24. Publisher Full Text\n\nHamid AA, Razak FZA, Bakar AA, et al.: The Effects of Perceived Usefulness and Perceived Ease of Use on Continuance Intention to Use E-Government. Procedia Economics Finance . 2016; 35: 644–649. Publisher Full Text\n\nHenseler J, Ringle CM, Sarstedt M: A New Criterion for Assessing. 2015.\n\nHong X, Zhang M, Liu Q: Preschool Teachers' Technology Acceptance During the COVID-19: An Adapted Technology Acceptance Model. Front Psychol. 2021; 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJahangir N, Begum N: The role of perceived usefulness, perceived ease of use, security and privacy, and customer attitude to engender customer adaptation in the context of electronic banking. African J Business Management . 2008; 2(2): 032–040.\n\nLai MC, Yap SF: Technology Development in Malaysia and The Newly Industrialising Economies: A Comparative Analysis. Asia-Pacific Development J . 2004; 11(2): 53–80. Publisher Full Text\n\nLe TD, Kieu TA: Ethically Minded Consumer Behaviour in Vietnam: An Analysis of Cultural Values, Personal Values. Asia Pacific Journal of Marketing and Logistics: Attitudinal Factors and Demographics. 2019.\n\nLunney A, Cunningham NR, Eastin MS: Wearable Fitness Technology: A Structural Investigation into Acceptance and Perceived Fitness Outcomes. Computers Human Behav . 2016; 65: 114–120. Publisher Full Text\n\nMark C, Paul N: Predicting Health Behaviour: Research and Practice with Social Cognition Models.2nd ed. New York: Open University Press; 2005.\n\nTimes NS: AI in financial services - digital transformation or disruption.2018. Reference Source\n\nPai FY, Huang KI: Applying the Technology Acceptance Model to the Introduction of Healthcare Information Systems. Technological Forecasting Social Change . 2011; 78(4): 650–660. Publisher Full Text\n\nPatil K, Mugdha SK: Artificial Intelligence in Financial Services: Customer Chatbot Advisor Adoption. Int J Innovative Technology Exploring Engineering (IJITEE) . November, 2019; 9(1): 2278–3075. Publisher Full Text\n\nPillai R, Sivathanu B: Adoption of AI-Based Chatbots for Hospitality and tourism. International Journal of Contemporary Hospitality Management, 0959-6119.2020.\n\nPrendergast GP, Tsang ASL: Explaining Socially Responsible Consumption. J Consumer Marketing . 2019; 36(1): 146–154.\n\nSchepman A, Rodway P: Initial validation of the general attitudes towards Artificial Intelligence Scale. Computers Human Behav Rep. 2020; 1: 100014. Publisher Full Text\n\nSubramaniam P: Malaysia Digital Economy Blueprint: MyDigital Blueprint to Drive Malaysia's High-Income Nation Aspirations. The Edge Malaysia. 2021, March 01. Reference Source\n\nTaherdoost H: A Review of Technology Acceptance and Adoption Models and Theories. Procedia Manufacturing . 2017; 22: 960–967. Publisher Full Text\n\nThe Edge Markets: Cover Story: The future of AI in financial services. 2017. Reference Source\n\nThe Star: How technology will help to shape the future of work in 2021 and beyond.2020. Reference Source\n\nTongco MDC: Purposive Sampling As a Tool for Informant Selection. Ethnobotany Res Appl. 2007; 5: 147–158.\n\nVenkatesh V, Davis F: Theoretical Extension of the Technology Acceptance Model: Four Longitudinal Field Studies. Management Sci . 2000; 46(2): 186–204. Publisher Full Text" }	[ { "id": "100547", "date": "20 Dec 2021", "name": "Darmawan Napitupulu", "expertise": [ "Reviewer Expertise Information System", "Information Technology", "Technology Adoption", "Technology Acceptance", "e-Government" ], "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\nSome recommendations:\nThe problem of this research was not clear. What is the matter with Gen Z? If they are true digital natives, they should not have a problem with adopting technology, including AI. Besides, the TAM (Technology Acceptance Model) model has been applied in various studies. What is the significant of this study? The contribution of this study was also not clear.\n\nThis study lacks the literature to build the hypothesis. The author should add and discuss other theories of technology acceptance or adoption such as UTAUT (Unified Theory of Acceptance and Use of Technology), TPB (Theory of Planned Behavior), Delon IS Success Model, etc. Why did the author use the TAM model more than others?\n\nThe data collection and data analysis method were not explained. For example: Did the author use an online or offline questionnaire? How did they do it? Why did the author use Smart PLS?\n\nPU (Perceived Usefulness) was not significant and there is a lack of explanation of what the possible reason could be to best understand this result?\n\nThe conclusion should restate the hypothesis, the contribution, the limitations and any further research needed.\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?\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": [] }, { "id": "245496", "date": "05 Mar 2024", "name": "Sayed Fayaz Ahmad", "expertise": [ "Reviewer Expertise Engineering Management" ], "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 a lot for giving me a chance to review the paper. Overall the work is good, yet i have some suggestions for the improvement of the paper.\n1. The literature review is very short. Please make it more rich with recent literature. 2. Please write the implications in a separate section. 3. Add limitation and future work of the study. 4. Add at least 30 % more references from 2023-24.\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": [] } ]	1	https://f1000research.com/articles/10-963
https://f1000research.com/articles/10-217/v1	17 Mar 21	{ "type": "Research Article", "title": "Polymorphisms of the genes ABCG2, SLC22A12 and XDH and their relation with hyperuricemia and hypercholesterolemia in Mexican young adults", "authors": [ "Juan Manuel Vargas-Morales", "Martha Guevara-Cruz", "Celia Aradillas-García", "Lilia G. Noriega", "Armando Tovar", "Jorge Alejandro Alegría-Torres", "Juan Manuel Vargas-Morales", "Martha Guevara-Cruz", "Celia Aradillas-García", "Lilia G. Noriega", "Armando Tovar" ], "abstract": "Background: Hyperuricemia is a pathological condition associated with risk factors of cardiovascular disease. In this study, three genetic polymorphisms were genotyped as predisposing factors of hyperuricemia. Methods: A total of 860 Mexicans between 18 and 25 years of age were genotyped for the ABCG2 (rs2231142), SLC22A12 (rs476037), and XDH (rs1042039) polymorphisms, as predisposing factors of hyperuricemia. Biochemical parameters were measured by spectrophotometry, while genetic polymorphisms were analyzed by real-time PCR. An analysis of the risk of hyperuricemia in relation to the variables studied was carried out using a logistic regression. Results: Male sex, being overweight or obese, having hypercholesterolemia or having hypertriglyceridemia were factors associated with hyperuricemia (p ≤ 0.05). The ABCG2 polymorphism was associated with hyperuricemia (OR = 2.43, 95% CI: 1.41-4.17, p = 0.001) and hypercholesterolemia (OR = 4.89, 95% CI: 1.54-15.48, p = 0.003), employing a dominant model, but only in male participants. Conclusions: The ABCG2 (rs2231142) polymorphism increases the risk of hyperuricemia and hypercholesterolemia in young Mexican males.", "keywords": [ "ABCG2", "SLC22A12", "XDH", "hyperuricemia", "hypercholesterolemia" ], "content": "Introduction\n\nHyperuricemia is an abnormal metabolic trait defined as serum uric acid levels above 6 and 7 mg/dL for women and men, respectively (Bardin & Richette, 2014), and is associated with other cardiometabolic risk factors such as obesity, diabetes, hypertension, and dyslipidemia (Martínez-Quintana et al., 2016; Zuo et al., 2016). Similarly, hyperuricemia can be a predictor of impaired kidney functions as well as kidney disease progression (Galán et al., 2018; Hsu et al., 2009, and Pérez-Navarro et al., 2020). Serum uric acid levels depend on many factors including diet, sex, lifestyle, and alcohol consumption, as well as genetic heredity. In fact, some genes involved in the metabolism of purines or urates have versions that appear to predispose to a hyperuricemic condition. Some of these are the ABCG2 gene that encodes a membrane transporter, which exports urates to the kidney and intestine (Woodward et al., 2009); and the SLC2A9 gene that encodes a kidney protein called GLUcose Transporter 9 (GLUT9), an important flow regulator of urates in the proximal tubules (Caulfield et al., 2008). In the same way, the XDH gene gives rise to an enzyme denominated xanthine dehydrogenase, which breaks down purines from nucleic acids, specifically the hypoxanthine‒xanthine‒urate conversion pathway (Harrison, 2002).\n\nRecent studies have shown that genetic polymorphisms in ABCG2 and SLC2A9 genes are prevalent in the Mexican population and may contribute to an abnormal condition of hyperuricemia, even at young ages (Macías-Kauffer et al., 2019; Rivera-Paredez et al., 2019). Interestingly, there appears to be no reports to date in the literature on XDH gene polymorphisms in the Mexican population. Therefore, the aim of this study was to genotype the polymorphisms of the genes ABCG2 (rs2231142), SLC22A12 (rs476037), and XDH (rs1042039) in 860 young Mexican volunteers aged between 18 and 25 years of age as predisposing factors of hyperuricemia associated with risk factors of cardiovascular disease.\n\n\nMethods\n\nA cross-sectional design and a covencence sample of 860 subjects was coducted in the City of San Luis Potosí, México. All participants were college applicants during the spring period of 2017. Data collection was carried out according to the recruitment protocols of the University of San Luis Potosí, since applicants receive a clinical evaluation as part of the admission process. This project was approved by two Bioethics Committees: the former at the National Institute of Medical Sciences and Nutrition Salvador Zubirán (Approval # FNU-669-13-15-2), and the latter at the Faculty of Chemistry of the University of San Luis Potosí (Approval # CEID2017105-S). All participants provided written informed consent.\n\nA total of 860 applicants to the state University of San Luis Potosí, Mexico in 2017, were included in this study (448 men and 412 women). All participants were approached to take part in the study by phone and email by means of an open invitation explaining the goals of the study. Even though the invitation was open, only some accepted to participate. Participants were not paid to take part. All individuals who provided written informed consent and met the inclusion criteria were included. The inclusion criteria which were as follows: aged between 18–25 years; born in the Mexican state of San Luis Potosí; and provided written informed consent.\n\nSince all participants at the University of San Luis Potosí, Mexico receive a clinical evaluation as part of the admission process, we accessed the university medical records. The weight in kilograms and height in meters of each participant was obtained for anthropometry using a digital scale (UM-081 model; Tanita, Tokyo, Japan) and a stadiometer (Seca 213, 2009; Seca, Hanover, MD, USA), respectively. From this data, the Body Mass Index (BMI) was calculated (kg/m2). Systolic and diastolic blood pressure were taken as the mean of two readings at a 5-min interval after 5 min in a seated position, employing the Omrom model HBP-1300 portable meter (Omron Healthcare, Inc., IL, USA). Also, 6 mL of blood was obtained by venipuncture after a 12-h fast; serum was obtained by centrifuging at 1,000 x g for 10 min using a laboratory centrifuge Z306 Benchmark Scientific (NY, USA), and processed inmmediately. An addition, 3 mL of blood was collected and stored in ethylenediaminetetraacetic acid (EDTA) tubes (Vacutainer®) for subsequent DNA purification. All samples were maintained at -20°C until their analysis.\n\nSerum was used to measure uric acid, glucose, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides by spectrophotometry utilizing chemistry reagents (Paramedical S.r.l., Italy), specially developed to work with Mindray BS 300 Auto Chemistry Analyzer (Mindray, Shenzhen, China), with the following catalog numbers: uric acid (PDIBS200040), glucose (PDIBS200020), total cholesterol (PDIBS200030), LDL-C (PDIBS200170) HDL-C (PDIBS200160), and triglycerides (PDIBS200060).\n\nDNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The quality of DNA samples was verified by spectrophotometry using NanoDrop™ 2000. Polymorphisms of the genes ABCG2 (rs2231142), SLC22A12 (rs476037), and XDH (rs1042039) were measured by allelic discrimination real-time polymerase chain reaction (PCR) with the Taqman probe (Applied Biosystems®). Briefly, the sequences of the probes were the following:\n\nABCG2: GAACT(G/T)TAAGTTTTCTCTCACCGTCAGAGTG;\n\nSLC22A12: CCCTCCAGGCCTCAGCCACCTGCCC(A/G)CCACATCCTCTGCCTGCTGTCCCCT, and XDH: TTTATTCTTTCCAATGATTCAAAGA(C/T)GGTTAGAGGATTTAACCTTACCCCA.\n\nAll PCR reactions were carried out using an ABI Prism 7900 HT detection system in 96-well plates (Applied Biosystems®). Reactions were adjusted to a final volume of 10 µL including Master mix probes and 50 ng/µL of genomic DNA, with the following amplification protocol: denaturation 94° for 9 min; followed by 50 cycles of denaturation at 95°C for 30 s, and annealing and extension at 68°C for 11 min. Negative controls and duplicated samples were included to check the accuracy of the genotyping.\n\nAfter analyzing the data distribution, comparison of medians between groups (normal uricemia vs. hyperuricemia) was carried out with the Mann‒Whitney U test for continuous variables. Allelic and genotypic frequencies as well as the Hardy‒Weinberg equilibrium were calculated for each polymorphism utilizing the chi-square test. Likewise, a logistic regression analysis was used to calculate the risk of hyperuricemia in relation to the variables studied and the influence of the different genotypes on uric acid levels in blood through a dominant model. The level of significance was set at p <0.05. Data were analyzed using SPSS version 19.0 statistical software.\n\n\nResults\n\nIn total, 860 participants were included in this study: 52% men and 48% women (Alegría-Torres, 2021). Participant characteristics, as well as the allelic and genotypic frequencies of the ABCG2 (rs2231142), SLC22A12 (rs476037) and XDH (rs1042039) gene polymorphisms, are presented in Table 1. Medians and genotypic distribution between hyperuricemic and normouricemic participants were analyzed using the Mann-Whitney U test, finding differences for sex, BMI, systolic and diastolic pressure, triglycerides, total cholesterol, and LDL-C (p = <0.001 for all), as well as a marginal distribution difference for the ABCG2 (rs2231142) polymorphism corresponding to G/T alleles (p = 0.056), therefore other genetic models were tested later. All the genotypes analyzed were in Hardy‒Weinberg equilibrium: ABCG2 (rs2231142) X2 = 1.82X10-6, p = 0.9; SLC22A12 (rs476037) X2 = 0.03, p = 0.84; and XDH (rs1042039) X2 = 0.08, p = 0.76. Both allelic and genotypic distributions of the three polymorphisms studied did not differ significantly between men and women (Table 2).\n\nAbbreviations: BMI, body mass index; ABCG2, gene that encodes an ATP-binding cassette transporter subfamily G member 2; SLC22A12, gene that encodes an urate transporter 1 gene; XDH, gene that encodes a xanthine dehydrogenase.\n\naChi-square test.\n\nbMann‒Whitney U test.\n\ncSerum uric acid ˃6 and 7 mg/dL was considered as hyperuricemia for women and men, respectively.\n\nData are shown as median and 25th and 75th percentile range (p25–p75).\n\nThe level of significance was set at p <0.05.\n\nAbbreviations: ABCG2, gene that encodes an ATP-binding cassette transporter subfamily G member 2; SLC22A12, gene that encodes an urate transporter 1 gene; XDH, gene that encodes a xanthine dehydrogenase.\n\naChi-square test.\n\nThe level of significance was set at p <0.05.\n\nConsidering the statistically significant differences among the variables, a multivariate logistic regression analysis was performed, including sex, BMI, hypercholesterolemia, and hypertriglyceridemia as predictors of hyperuricemia (Figure 1). A predisposition to abnormal serum uric acid levels was found in some conditions as follows: male sex (odds ratio (OR): 2.14, 95% confidence interval (CI): 1.41-3.24, p = ˂ 0.001); overweight or obese (OR: 2.6, 95% CI: 1.71-3.88, p = ˂0.001); having hypercholesterolemia (OR: 2.8, 95% CI: 1.47-5.46, p = 0.002); or having hypertriglyceridemia (OR: 1.7, 95% CI: 1.04-2.69, p = 0.033).\n\nThe cutoff criteria were based on the World Health Organization and the National Cholesterol Education Program Adult (ATP III). Overweight: body mass index ≥ 25, Obesity: body mass index ˃30; hypercholesterolemia: fasting serum cholesterol levels ˃ 200 mg/dL; hypertriglyceridemia: fasting serum triglycerides levels ˃ 150 mg/dL.\n\nIn Table 3, an analysis by genotype is shown for the ABCG2 (rs2231142), SLC22A12 (rs476037), and XDH (rs1042039) polymorphisms, employing a dominant model for all three. With respect to the ABCG2 (rs2231142) polymorphism, statistically significant differences were found between the GT+TT genotypes vs. GG for serum uric acid (p = 0.003) total cholesterol (p = 0.005) and HDL-C levels (p = 0.003). GA+AA genotypes vs. GG of the SLC22A12 (rs476037) polymorphism only showed to influence LDL-C levels (p = 0.043). Finally, no statistically significant differences were found by genotype for the XDH (rs1042039) polymorphism (p > 0.05).\n\nAbbreviations: BMI, body mass index; ABCG2, gene that encodes an ATP-binding cassette transporter subfamily G member 2; SLC22A12, gene that encodes an urate transporter 1 gene; XDH, gene that encodes a xanthine dehydrogenase.\n\naMann‒Whitney U test. The level of significance was set at p <0.05.\n\nWhen the OR and the association between genotypes for the ABCG2 (rs2231142) and SLC22A12 (rs476037) polymorphisms and cardiovascular risks were calculated by sex, some significant results were found utilizing a dominant model. Table 4 reports that GT+TT genotypes for the ABCG2 (rs2231142) polymorphism significantly statistically increases the risk of hyperuricemia (OR = 2.43, 95% CI: 1.41-4.17, p = 0.001) and hypercholesterolemia (OR = 4.89, 95% CI: 1.54-15.48, p = 0.003), but only in male participants. On the other hand, no significant results were found for the genotypes of the SLC22A12 (rs476037) and XDH (rs1042039) polymorphisms.\n\nAbbreviations: BMI, body mass index; SP, systolic pressure; DP, diastolic pressure. LDL-C, LDL cholesterol; HDL-C, HDL cholesterol; ABCG2, gene that encodes an ATP-binding cassette transporter subfamily G member 2; SLC22A12, gene that encodes an urate transporter 1 gene; XDH, gene that encodes a xanthine dehydrogenase.\n\naThe cut-off criteria were according to The World Health Organization and the National Cholesterol Education Program Adult (ATP III). Overweight was defined as BMI ≥25, while Obesity was BMI ˃30; Hyperuricemia was defined as serum uric acid ˃6 and 7 mg/dL for women and men, respectively; hypertriglyceridemia as fasting serum triglycerides ˃150 mg/dL, hypercholesterolemia as fasting serum cholesterol ˃200 mg/dL, and low HDL-C as a serum concentration of ˂50 mg/dL and 40 mg/dL for women and men, respectively.\n\nMultivariate logistic regression analysis: data are shown as odd ratio (95% Confidence Interval). The level of significance was set at p <0.05.\n\n\nDiscussion\n\nA total of 860 college applicants between the ages of 18 and 25 years of both sexes were included in this study, an important age range to identify early disorders. Indeed, we found a 15% prevalence of hyperuricemia, being higher among males (19.6%) than females (10%), as previously observed in another study with comparable age groups in Mexico (Alegría-Díaz et al., 2018). Regarding uric acid levels, similar blood concentrations have also been reported in Mexican young persons (Pérez-Navarro et al., 2020). The subgroup with hyperuricemia was prone to higher levels of BMI, blood pressure, triglycerides, total cholesterol, and LDL cholesterol (Table 1). In fact, the association between serum uric acid levels and the traits of metabolic syndrome has been previously analyzed (Lin et al., 2006; Zhang et al., 2020a), although some authors found no causal evidence of uric acid levels being associated with metabolic syndrome and its components (Wang et al., 2020). According to our results, being a man, being overweight or obese, or having dyslipidemia are related to high uric acid levels (Figure 1). Men have a lower capacity to eliminate urate via the kidney compared with females due to a deficiency of estrogen and progesterone (Hak et al., 2010). Likewise, the involvement of uric acid in lipid metabolism can lead to hyperuricemia, a condition considered as predictor factor of dyslipidemia (Kuwabara et al. 2020; Lima et al., 2015; Son et al., 2016) and can subsequently alter blood pressure (Teng et al., 2011; Zhang et al., 2020a). In this way, our results summarized in Figure 1 show that being male, being obese and having dyslipidemia increases the risk of hyperuricemia (Liu et al., 2020a).\n\nThe study of the genetic influence on blood uric acid levels has included the search for single nucleotide polymorphisms (SNP) in genes involved in purine metabolism and urate removal. Here, we analyzed three polymorphisms in three different genes, including ABCG2 (rs2231142), SLC22A12 (rs476037), and XDH (rs1042039). The first corresponds to the exchange of a glutamine for a lysine at position 141 of an adenosine triphosphate (ATP)-binding cassette transporter subfamily G member 2 (ABCG2). This exchange predisposes individuals to hyperuricemia (Nakashima et al., 2020; Wrigley et al., 2020). A frequency of 0.25 for the risk for T allele was found, a slightly lower prevalence than that reported in Asian and New Zealand populations (Kim et al., 2015; Liu et al., 2020b; Toyoda et al., 2019). The distribution of the T allele was marginal between hyperuricemic and normouricemic groups, being more frequent in the hyperuricemic group (p = 0.056). When a dominant model was carried out, the risk for the T allele was associated with hyperuricemia and hypercholesterolemia (total and HDL cholesterol). This association was confirmed only in men, showing that the ABCG2 (rs2231142) polymorphism increases the risk for hyperuricemia 2.43 times (95% CI: 1.41-4.17, p = 0.001) and hypercholesterolemia 4.89 times (95% CI: 1.54-15.48, p = 0.003) in a dominant model. Other studies have also found a greater influence of this polymorphism in men (Narang et al., 2019) although, under some conditions, the ABCG2 (rs2231142) polymorphism could contribute to increased uric acid in women (Guo et al., 2020; Roman et al., 2020). Although initially our work focused on hyperuricemia in young people, the increase in serum cholesterol in men associated with the ABCG2 (rs2231142) polymorphism was evident. There are still few studies linking the ABCG2 gene and cholesterolemia. A relevant fact is that mRNA expression levels of ABCG2 appear to be higher in individuals with hypercholesterolemia (Rodrigues et al., 2009); likewise, the activity of ABCG2 has been associated with cholesterol levels both in vitro and in vivo (To et al., 2014).\n\nThe SLC22A12 (rs476037) polymorphism comprises the transition from guanine to adenine in the 3´-UTR region in the urate transporter 1 gene; this transition appears to modify uric acid levels (Flynn et al., 2013; Köttgen et al., 2013). In this study, the frequency of the minor allele was 0.32; however, there was no statistically different distribution of this between hyperuricemic and normouricemic groups. Although no differences in uric acid levels were associated with this polymorphism, lower LDL cholesterol levels were observed in the group of carriers of the A allele when data were analyzed in a dominant model (p = 0.043), suggesting a protective effect of this allele (Simon et al., 2014). Since the SLC22A12 (rs476037) polymorphism is located at a potential miRNA binding site (Flynn et al., 2013), the epigenetic mechanism involved in the regulation of uric acid levels needs to be studied.\n\nWith respect to the XDH (rs1042039) polymorphism, we studied the xanthine dehydrogenase polymorphism located in the 3´-UnTRanslated (UTR) region of the XDH gene. The replacement of T by C is considered a risk factor related to hypertension (Wu et al., 2015). We found that the minor allele frequency was 0.37 for the C allele, contrary to what was reported in a Chinese population, where the C allele had a higher frequency, in addition to its being associated with hypertension (Wu et al., 2015). However, the link between the XDH (rs1042039) polymorphism and hypertension was not demostrated in Taiwanese women (Lee et al., 2019). In the present study, we did not find a statistically significant different distribution of the C allele between the hyperuricemic and normouricemic groups, and the risk allele was also not associated with any component of the metabolic syndrome.\n\nFinally, hyperuricemia is an undesirable condition that has been seen as a minor trait of metabolic syndrome, cardiovascular risk, as well as other types of disorders such as psoriasis and alopecia, which have been associated with high levels of blood uric acid (Guo et al., 2020; Ma et al., 2020; Talebi et al., 2020; Zhang et al., 2020a). Even though our criterion for hyperuricemia were defined as blood uric acid >6 mg/dL for women and 7 mg/dL for men, cut-off points could be reconsidered according to the considerations made by Alegría-Díaz et al. (2018). In this regard, a recent study considers uric acid levels below 5 mg/dL for men and below 2-4 mg/dL for women to be optimal for a lesser risk of cardiometabolic diseases in a Japanese population (Kuwabara et al., 2020). Although there are modifiable factors related to lifestyle, genetic inheritance plays a decisive role in the control of uricemia.\n\nThis study has two main limitations: i) only one polymorphism was genotyped for every gene, analysis of multiple SNPs by haplotypes could be more appropriate; and ii) new cut-off values for hyperuricemia have been suggested (Alegría-Díaz et al., 2018); however the conservative criteria of >6 mg/dL for women and 7 mg/dL for men were considered in this study.\n\n\nConclusions\n\nIn this study, we found that the ABCG2 (rs2231142) polymorphism increases the risk of hyperuricemia as well as of hypercholesterolemia in young Mexican males. Since the ABCG2 (rs2231142) polymorphism can modify the efficacy of statins in reducing cholesterol (Zhang et al., 2020b), carriers of the risk allele represent a vulnerable group of interest for future pharmacogenetic research. Some considerations for future studies are including lifestyle and diet factors, the monitoring of the study population, and exploring more polymorphisms in the ABCG2, SLC22A12, and XDH genes, as well as studying haplotypes.\n\n\nData availability\n\nData mendeley: Polymorphisms of the genes ABCG2, SLC22A12 and XDH and their relation with hyperuricemia and hypercholesterolemia in Mexican young adults. http://dx.doi.org/10.17632/243ft29b7m.1 (Alegría-Torres, 2021).\n\nThis project contains the following underlying data:\n\n- DATABASE ART ARCHIVES OF PHYSIOL AND BIOCHEM.sav (spreadsheet of participant data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAlegría-Díaz A, Valdez-Ortiz R, Murguía-Romero M, et al.: Clinical significance of serum uric acid levels in Mexican young adults. Contrib Nephrol. 2018; 192: 125–134. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "81558", "date": "25 Mar 2021", "name": "Ilham Akbar Rahman", "expertise": [ "Reviewer Expertise Metabolic syndrome", "hyperuricemia", "insulin resistance", "endocrinology", "internal medicine", "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 study assessed the relationship of ABCG2, SLC22A12, and XDH genes polymorphism on their relationship with hyperuricemia and risk factors of cardiovascular disease. It seems that the authors have provided a significantly huge amount of effort and cost in conducting this study. This polymorphism study could provide potential implications in the future on the prevention, diagnosis, and treatment of hyperuricemia associated with cardiovascular risk factors. The authors display the knowledge on good writing skills including grammar and word choice, sentence structure, and paragraph development. However, several points need to be addressed:\nThe study has provided a significant relationship on ABCG2 polymorphism gene in the risk of hyperuricemia on specific age of 18-25 years old. Detailed explanation has been provided for the reasons of relationship between ABCG2 polymorphism on hyperuricemia. However, are there any specific relations on the ABCG2 on specific young adults of age? Why perform the study in specific young adult age groups? Is it better to examine the relationship in a more general group?\n\nIs there any consideration of excluding preexisting disease such as cardiovascular, liver, kidney, and secondary hyperuricemia (leukemia, myeloma, drugs induced, hypothyroidism, etc) in this study?\n\nIs there any specific reason to choose the rs2231142 SNPs from all polymorphisms? The study should also describe the reasons to choose this rs2231142 SNP.\n\nIt has been revealed in the study that gender, obesity/overweight, hypercholesterolemia, and hypertriglyceridemia are associated with the risk of developing hyperuricemia. Therefore, in the analysis of the relationship of ABCG2 gene polymorphism and hyperuricemia, it is better to perform the analysis of other subgroups other than gender (such as BMI, cholesterol levels, and triglyceride)\n\nThe author should earlier define the diagnostic criteria used for hyperuricemia and its cut off value in the methods section.\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": "6526", "date": "06 Apr 2021", "name": "Jorge Alegría", "role": "Author Response", "response": "Regarding the five points discussed, we respond to each of them: 1. Although examining the relationship in a more general group could provide more information, this study included college applicants due to the facilities to be recruited. In addition, we directed the study to recognize risk factors in young population, an age group less likely to have metabolic diseases. 2. In fact, participants with pre-existing diseases were not excluded. As mentioned in the methods section: “All participants were approached to take part in the study by phone and email by means of an open invitation explaining the goals of the study”. 3. The ABCG2 (rs2231142) polymorphism is one of the most studied and it seems to have the greatest influence on uric acid levels in both men and women (Guo et al., 2020; Narang et al., 2019; Roman et al., 2020). Likewise, this polymorphism could be influencing both the mRNA expression levels and activity of ABCG2 modulated by cholesterol levels (Rodrigues et al., 2009; To et al., 2014), as this was mentioned in the discussion. In relation to the first point, most studies of polymorphisms focus on older patients or hemodialysis patients; however, we studied young people. 4. In order to summarize and present the most relevant results, the data were shown taking into account the genotype as a grouping criterion (Table 3). When the data was grouped and analyzed by another criterion (data not shown), there were no relevant findings. 5. Serum uric acid ˃6 and 7 mg/dL was considered as hyperuricemia for women and men, respectively (Table 1). We considered it more appropriate to include this information where the study group data are shown." } ] }, { "id": "91797", "date": "24 Aug 2021", "name": "Jing He", "expertise": [ "Reviewer Expertise Molecular 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\nIn the current study, the authors investigate the association between three polymorphisms in the ABCG2, SLC22A12, and XDH genes and hyperuricemia risk by including 860 subjects. They found the ABCG2 rs2231142 polymorphism increases the risk of hyperuricemia and hypercholesterolemia in young Mexican males.\nI have some comments that should be addressed by the authors:\nIn the Abstract, it's better provide the number of cases and controls, as well as the nucleotide alterations for the polymorphisms they chose.\n\nThe authors only chose one polymorphism for each gene, that was not rationalised. It's better chose at least two potentially functional SNPs for each gene.\n\nIn the Materials and Methods, there was no need to provide the sequences of the probes. The authors should provide the function of the selected polymorphisms.\n\nFalse-positive report probability and statistical power should be calculated as the positive findings for the samples included were so small (see the following articles, in which I have been involved with: He et al. Mol Carcinog. 2013; 52 Suppl 1: E70-91 and He et al. Mol Ther Nucleic Acids. 2018; 11: 1-82).\n\nThe authors found the ABCG2 (rs2231142) polymorphism increases the risk of hyperuricemia and hypercholesterolemia in young Mexican males. It is lacking functional validation for the function of ABCG2 (rs2231142) polymorphism. It’s better to perform genotype-based mRNA expression analysis to further explore the potential role of the significant polymorphism (He et al. Mol Ther Oncolytics, 2021; 20: 199-2083; Zhuo et al. Mol Ther Nucleic Acids, 2020; 22:17-264). From these two references, which I have been involved with, the authors can perform genotype based mRNA expression analysis to further explore the potential role of the ABCG2 (rs2231142) polymorphism.\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": [ { "c_id": "7172", "date": "24 Sep 2021", "name": "Jorge Alegría", "role": "Author Response", "response": "Regarding the five points discussed, we respond to each of them: 1. The number of the cases and controls as well as the description of sequence variants were included in the abstract. 2. According to the introduction section, we selected three genes involved in the metabolism of purines or urates, which have versions that appear to predispose to a hyperuricemic condition; the ABCG2 gene that encodes a membrane transporter, which exports urates to the kidney and intestine (Woodward et al. 2009); and the SLC2A9 gene that encodes a kidney protein called GLUcose Transporter 9 (GLUT9), an important flow regulator of urates in the proximal tubules (Caulfield et al. 2008). In the same way, the XDH gene gives rise to an enzyme denominated xanthine dehydrogenase, which breaks down purines from nucleic acids, specifically the hypoxanthine‒xanthine‒urate conversion pathway (Harrison 2002). We chose polymorphisms in different genes for the following two reasons: Previous reports have shown the combined effect of genetic variants (specifically SLC2A9 and ABCG2) are able to explain 3-4% of hyperuricemia, modulated by gender and body mass index (Brandstätter et al., 2010; Köttgen et al., 2013; Huffman et al., 2015 and Merriman et al., 2015;). Further, the ABCG2 (Q191K) and SLC22A12 (517G>A) polymorophisms have been studied in Mexican population showing a high prevalence (Macías-Kauffer et al., 2019 and Rivera-Paredez et al. 2019). Besides, since the XDH (518T>C) polymorphism it has been poorly studied in Mexicans, we wanted to explore if it plays a role in hyperuricemia. We only analyzed polymorphisms with a minor allele frequency >5%, being: ABCG2 (Q191K) 25%, SLC22A12 (517G>A) 32%, and XDH (518T>C) 37%; as well as that the polymorphisms were in Hardy-Weinberg equilibrium. References: Brandstätter A, Lamina C, Kiechl S, Hunt SC, Coassin S, Paulweber B, et al. Sex and age interaction with genetic association of atherogenic uric acid concentrations. Atherosclerosis 2010; 210:474-8. Huffman JE, Albrecht E, Teumer A, Mangino M, Kapur K, Johnson T, et al. Modulation of Genetic Associations with Serum Urate Levels by Body-Mass-Index in Humans. Crawford DC, editor. PLoS One 2015; 10:e0119752 Köttgen A, Albrecht E, Teumer A, Vitart V, Krumsiek J, Hundertmark C, et al. Genome-wide association analyses identify 18 new loci associated with serum urate concentrations. Nat Genet 2013; 45:145-54. Merriman TR. An update on the genetic architecture of hyperuricemia and gout. Arthritis Res Ther 2015; 17:98. 3. The sequences of the probes was deleted from the Materials and Methods section. The function of the selected polymorphisms is described in the discussion section: The ABCG2 (Q191K) polymorphism corresponds to the exchange of a glutamine for a lysine at position 141 of an adenosine triphosphate (ATP)-binding cassette transporter subfamily G member 2 (ABCG2). This exchange predisposes individuals to hyperuricemia (Nakashima et al. 2020, Wrigley et al. 2020). The SLC22A12 (517G>A) polymorphism comprises the transition from guanin to adenine in the 3´-UTR region in the urate transporter 1 gene; this transition appears to modify uric acid levels (Flynn et al. 2013, Köttgen et al. 2013). With respect to the XDH (518T>C) polymorphism, the replacement of T by C is considered a risk factor related to hypertension (Wu et al. 2015). 4. In fact, our results are impacted by the small sample size and the compromised statistical power. This point is discussed and included as a limitation of this study. 5. This point is argued in the discussion section: There are still few studies linking the ABCG2 gene and cholesterolemia. A relevant fact is that mRNA expression levels of ABCG2 appear to be higher in individuals with hypercholesterolemia (Rodrigues et al. 2009); likewise, the activity of ABCG2 has been associated with cholesterol levels both in vitro and in vivo (To et al. 2014). Therefore, perform genotype-based mRNA expression analysis to further explore the role of the ABCG2 (Q191K) polymorphism in hyperuricemia and hypercholesterolemia should be contemplated." } ] } ]	1	https://f1000research.com/articles/10-217
https://f1000research.com/articles/9-580/v1	09 Jun 20	{ "type": "Research Article", "title": "Topical ozonated virgin coconut oil improves wound healing and increases HSP90α, VEGF-A, EGF, bFGF and CD34 in diabetic ulcer mouse model of wound healing", "authors": [ "Renni Yuniati", "Prasetyowati Subchan", "Wibi Riawan", "Matthew Brian Khrisna", "Maryam Restiwijaya", "Niken Safitri Dyan Kusumaningrum", "Muhammad Nur", "Prasetyowati Subchan", "Wibi Riawan", "Matthew Brian Khrisna", "Maryam Restiwijaya", "Niken Safitri Dyan Kusumaningrum", "Muhammad Nur" ], "abstract": "Background: Diabetes is a disease that affects people worldwide, including in Indonesia. The prevalence of diabetes in Indonesia is increasing from year to year. One of the most devastating complications of diabetes mellitus is diabetic ulcers, which is a limb-threatening complication. Over the past few decades, ozone generated using plasma medical technology has been investigated as an agent that helps wound healing. This study aims to evaluate the effects of topical ozonated virgin coconut oil (VCO) in a diabetic wound mouse model. Methods: This study was an experimental study with a post-test control design. An ulcer wound model was made in 50 diabetic male Wistar mice, divided into five groups, and a control group of 10 non-diabetic mice. The control groups were given conventional therapy only and the treatment groups were also given topical ozonated VCO with different flow durations (0 min, 90 min, 7 h, 14 h). Macroscopic appearance and wound contraction were observed. HSP90β, VEGF-A, EGF, bFGF and CD34 levels were measured from the immunostained slices of wound margins. Results: The reduction of wound length was proportionally related to the duration of ozone flow. Ozonated VCO with a longer duration of ozone flow healed the wound more quickly and had the shortest wound length. VCO with ozone flow for 14 hours (16837.10 µm) had the biggest reduction in wound length compared to other groups. The wounds treated with ozonated VCO showed an increase in HSP90β, VEGF-A, EGF, bFGF and CD34 levels that correlated to improved wound healing. A longer period of treatment resulted in higher levels of wound healing biomarkers compared to shorter therapeutic durations. Conclusions: Topical ozonated VCO improved the wound healing process in a diabetic ulcer mouse model by improving macroscopic wound appearance and increasing levels of wound healing biomarkers.", "keywords": [ "diabetic ulcer", "ozone", "virgin coconut oil", "full-thickness wound", "delayed wound healing", "HSP90α", "VEGF-A", "EGF", "bFGF", "CD34" ], "content": "Introduction\n\nDiabetes is a non-communicable disease that has become a worldwide burden. In 2014, the number of people with diabetes in the world reached 422 million (8.5% of the total world population). According to a WHO report, the prevalence of diabetes has kept increasing steadily over the course of the last three decades, especially in low-income and middle-income countries such as Indonesia1. In 2013, the prevalence of diabetes mellitus among productive-age citizens of Indonesia was 4.6%2. In line with the WHO report, the International Diabetes Federation found that in 2017, the prevalence of diabetes mellitus among adults in Indonesia had increased to 6.7% (10,276,100 people).\n\nDiabetes is a risk factor and cause of several other diseases, including blindness, kidney failure, diabetic neuropathy, diabetic vasculopathy, and diabetic ulcers. The International Diabetes Federation estimates that by 2030, the total number of diabetic patients worldwide will reach 366 million people. From those diabetic patients, 15% of them will develop a diabetic ulcer. A diabetic ulcer is an open wound on the skin that extends down into the dermis and usually occurs on the soles of the feet. If the diabetic ulcer is not managed properly, it can develop into gangrene, thus increasing the likelihood of amputation. 12–24% of patients who have diabetic ulcers end up requiring amputation3–6.\n\nDiabetic ulcers are a serious complication in patients with diabetes7. A diabetic ulcer is defined as the presence of a full-thickness lesion located distal relative to the ankle. Diabetic ulcers are caused by a combination of several risk factors, including neuropathy, peripheral arterial occlusive disease, and previous ulcerations7,8. Of all diabetic patients worldwide, 6.3% are affected by diabetic ulcers9. Plantar ulcers account for nearly twice the amount of dorsal ulcers7. Diabetic ulcers are a limb-threatening condition that can potentially result in the amputation of a limb1,7. This condition also causes a reduction in the quality of life of the diabetic patients10. Research conducted in Jakarta found that 28.6% of diabetic patients had a diabetic ulcer. Most of the patients enrolled in that research had a fifth degree diabetic ulcer11,12.\n\nThe management of diabetic ulcers needs a holistic approach, taking into consideration systemic conditions, complications of neuropathy or atherosclerosis, and classification of injuries. Treatment of diabetic ulcer wounds involves three stages, consisting of cleansing (washing), debridement, and dressing (using proper wound dressing). In addition, pressure control and infection control are also needed. Ulcers can become an area of bacterial growth that must be treated with antibiotics according to the results of bacterial culture13–15.\n\nVirgin coconut oil (VCO) is a product of coconut that is produced by processing fresh coconuts at a low temperature. This processing method keeps important substances in the coconut oil intact and ensures they do not degrade. VCO mainly contains 90% saturated fatty acids and 10% unsaturated fatty acids. VCO contains antioxidants such as tocopherol and betacarotens, and has properties to enhance moisturization of the skin16. VCO has been shown to increase wound contraction rate in chronic wounds that exhibited delayed wound healing, and has the potential to increase wound healing biomarker levels in a full-thickness wound mouse model17,18.\n\nOzone is an inorganic triatomic molecule composed of three oxygen atoms and is highly soluble19,20. Ozone is known to have a role in protecting the ecological balance of the earth and can interact at a basic level with industrial pollutants. Ozone also has a unique biological ability that has the potential to be used in the medical field, which has been widely investigated in recent years. Medical uses of ozone use ozone dissolved in water (ozonated water), ozonated oils, and ozone in gas form21. One of the early uses of ozone in the medical field started in the first world war era, where ozone was used to treat anaerobic Clostridium infections in soldiers with gas gangrene22. Christopher Frieddrich Schoonbein was one of the first people to unleash the potential of ozone to treat wounds and infections caused by anaerobic bacteria23. Previous studies found that ozone acts as an agent that helps ulcer wound healing in patients with diabetes mellitus. Ozone interactions with skin tissue can: cause inactivation of bacteria, viruses and fungi; stimulate antioxidant production; reduce blood and plasma viscosity; increase erythrocyte membrane fluidity; loosen tissue; stimulate hemoglobin activity and increase oxygen absorption and release; improve blood circulation to tissues; induce tissue collagen formation; activate formation of granulation tissue; accelerate epithelialization (growth of epidermal cells); and increase the phagocytic activity and activation of fibroblasts. Ozone also supports the treatment of diabetes mellitus through decreasing blood sugar levels and increasing oxygen supply into the tissues24–26.\n\nHeat shock protein 90 (HSP90) is a protein chaperone released and upregulated from pancreatic beta cells under inflammatory conditions27. HSP90 is a member of the heat shock protein superfamily and itself consists of HSP90α and HSP90β28. HSP90 is excreted when normal cells are injured and heat shock proteins, especially HSP90α, are essential for the early phase of wound healing because they promote keratinocyte differentiation, re-epithelialization, dermal cell recruitment, and also protect tissues from further injury29–31. Vascular endothelial growth factor (VEGF) has an important role in the coordination of wound healing32. VEGF-A exhibited mis-regulation in diseases with microvascular disorders33. Diabetes causes reduced VEGF signaling, alongside impaired angiogenesis and decreased collateral blood vessel formation34. Besides causing microvascular and macrovascular disorders, this impaired angiogenesis also has a significant role in the pathogenesis of diabetic wound healing35. Increased VEGF-A has been shown to be beneficial in diabetic wound healing by increased angiogenesis, vessel diameter, re-epithelialization and wound closure36. Epidermal growth factor (EGF) plays an important role in wound healing through stimulation, proliferation and migration of several types of cells, such as keratinocytes, epithelial cells, endothelial cells and fibroblasts, thus facilitating dermal regeneration37,38. EGF is synthesized in the pancreas, and its levels are decreased in diabetic animal models38. Increased EGF levels might have several benefits for wound healing in diabetic wounds and ulcers by increasing granulation tissue formation in diabetic wounds39–41. Basic fibroblast growth factor (bFGF) aids in wound healing42. bFGF influences chemotaxis, cell differentiation, cell proliferation, and tissue regeneration, and was found to increase epithelial wound healing in an early study43,44. bFGF also improves the appearance of healed wounds by improving scar quality and regeneration45. CD34+ is a biomarker that can be used to predict the healing of diabetic wounds. CD34+ levels are decreased in subjects with diabetes compared to subjects with normal glucose tolerance, and lower levels of CD34+ are also correlated with a significantly higher risk of diabetes46,47. Levels of sphingosylphosphorylcholine (SPC) in the bloodstream can be used to predict the healing of diabetic neuropathic wounds, which might show its significance in improving the healing of diabetic wounds48.\n\nCurrently, there are limited studies of ozone treatment in animal models of diabetic ulcers and a lack of clinical trials concerning the efficacy of ozone treatment for ulcers in patients with diabetes mellitus, especially in Indonesia. We used VCO as the ozone carrier liquid in this study. This study aims to evaluate the effect of topical ozonated VCO using a diabetic wound mice model, assessed by macroscopic wound improvement and immunohistochemical staining parameters.\n\n\nMethods\n\nThis study is an experimental study with a post-test control design. This research was approved and declared ethically feasible by the Ethics Commission of the Public Health Faculty, Diponegoro University Semarang with ethical clearance number 078/EC/FKM/2018. All efforts were made to ameliorate harm to animals by administering anesthesia to all of the study animals before full-thickness wound model creation, keeping the animals in a well-maintained cage, and ensuring graceful termination of animals before we took tissue samples for histopathology examination.\n\nThe subjects of this study were male adult laboratory mice (Wistar strain). The mice were aged three months with a mean weight of 250 ± 50 grams. We obtained the mice from a local laboratory-grade mice breeder, with a pure-breeding status. The mice included in this study fulfilled the inclusion criteria for healthy conditions (active movement), and exhibited no signs that met our exclusion criteria: behavioral changes (activities seemed weak and lazy). The choice of mice strain and size is related to the wound model creation; the large size of Wistar mice enables us to have an ample area on the mice back to create the full-thickness wound model. Mice were kept in stainless-steel laboratory-grade mouse cages at a constant room temperature of 28.0±2.0°C, which was monitored with a digital thermometer. The mice were kept in our laboratory mouse room in cages with wood shavings as the bedding material, which was changed once a week. Each cage was inhabited by two to three mice according to the cage size. We did not mix mice from different groups in one cage. Fluorescent lighting was turned on for 12 hours per day between 9.00 AM to 9.00 PM, with adequate ad libitum food supply (BioFeed pellets, manufactured by Karunia Kasih Abadi, Klaten, Indonesia) and water supply (clean drinkable water). We routinely assessed the clinical and behavioral status of the mice every day during routine checks to prevent any clinical or behavioral changes in the laboratory mice. No mice were found dead, having decreased appetite/growth, or exhibited behavioral changes during the study period.\n\nIn the diabetic group, we induced type 1 diabetes in the mice by injecting a single dose of 40mg/kg body weight streptozotocin, dissolved in a buffer solution of 50 mM citrate with pH of 4.5 to obtain a final concentration of 40 mg/ml. We assessed the blood glucose level before streptozotocin injection as a baseline value and repeated 10 days after streptozotocin injection to determine whether the diabetic induction was successful or not. The measurements of blood glucose level were done using glucose oxidase phenol 4-aminoantipyrine (GOD-PAP) obtained from Diagnostic Systems International. The diagnosis of type 1 diabetes was made if blood glucose levels reached more than 200 mg/dl (11.1 mmol/liter) 10 days after streptozotocin injection, or if blood glucose levels reached twice the baseline blood sugar noted at the time of injection.\n\nThe manufacture and testing of ozonated VCO were carried out at the Plasma Research Center (PRC), Diponegoro University, Indonesia. The tools used for making ozonated oil were ozone generators (ozone generator manufactured in-house by Plasma Research Center, Diponegoro University) and magnetic stirrers. The ozone outlet is connected to an anti-oxidation hose with a diffuser, which served to increase the effectiveness of ozone absorption in the oil. Magnetic stirrers were used to facilitate the ozone dissolving process into the oil. The oil used in this study was VCO. Ozone was dissolved into VCO with a volume of 100 cc in each cycle and an oxygen flow rate of 0.1 liters/minute with an ozone concentration of 3360 ppm. We varied the duration of the ozonation process to create several concentrations of ozonated oil. The duration of ozonation was 90 minutes, 7 hours, and 14 hours.\n\nThe full-thickness wound model, treatment application, and observation were done simultaneously on all study groups; however, the diabetic induction was conducted 10 days earlier before the wound model creation to make sure all mice were ready to undergo wound model creation simultaneously in their final state (diabetic or non-diabetic according to the group). The research process began by inducing the diabetic ulcers in the Wistar mice in the diabetic group. We soaked cotton with 0.05ml of chloroform and put the cotton into a glass beaker containing a mouse. This allowed us to carry out aerosol administration of anesthesia, which we applied for two minutes. The administration of anesthesia was carried out directly before we excised the wounds, at our laboratory mouse surgery table, which was cleaned and sanitized before the process began to minimize the risk of contamination from the surgical field or surgical apparatus. After hypoesthesia was achieved (measured by loss of muscular tone, loss of righting and palpebral reflexes, decreased rate of respiration, and loss of response to painful stimulation), the hair in the region of the spine at the top of the back (thoracolumbar area) was shaved49. Then we made four excisions, each with a diameter of 6 mm, separated by the median line. The full-thickness skin excision was done using punch biopsy with a depth of 1 mm.\n\nA total of 60 mice were used in this research. The sample was then divided into six groups randomly (achieved by simple randomization), where each group consisted of 10 mice. The minimum sample size for this study was obtained according to the Frederer sample size calculation formula: (t-1) (n-1) ≥ 15; where t = number of groups and n = number of replications/number of minimum samples per group. There are six groups in this study; therefore, the formula was (6-1) (n-1) ≥ 15, which results in 5n ≥ 20. We added more mice (twice the sample amount) due to the need to obtain three mice every histopathology examination, which was done three times during the course of the study (explained in the next section). These groups were: the negative control group (C-), a control group of mice with negative diabetes mellitus status that received conventional treatment such as washing the wound with normal saline and gentamycin antibiotic ointment (Kimia Farma pharmaceutical industry, Indonesia); the positive control group (C+), a control group with positive diabetes mellitus status that received similar conventional treatment to the negative control group, such as washing the wound with normal saline and gentamycin antibiotic ointment (Kimia Farma pharmaceutical industry, Indonesia); the first treatment group (P1), which received normal saline wound wash and non-ozonated VCO treatment; the second treatment group (P2), which received normal saline wound wash and 90-minute ozonated VCO oil therapy; the third treatment group (P3), which received normal saline wound wash and 7-hours ozonated VCO oil therapy; and the fourth treatment group (P4), which received normal saline wound wash and 14-hours ozonated VCO oil therapy.\n\nWe applied the oils (ozonated VCO or regular VCO according to the group) to the wound once a day in the morning (approximately 8 a.m or 9 a.m) for fourteen consecutive days, thinly covering the entire wound surface with each treatment. The mice were first taken out of the cage, received the treatment at our mouse surgical table (which was cleaned and sanitized beforehand), and then returned back to the cage. The wound was left open and not covered with any kind of covering. For the macroscopic wound contraction measurement, we measured the wound contraction length using a digital measurement caliper (0,01 mm measurement scale, 150mm maximum length) on the first day, third day, fifth day, seventh day and fourteenth day before we applied the ozonated VCO each day, and recorded it in a follow-up table for further analysis.\n\nFor the immunohistochemistry staining, we analyzed the wound three times during the study period: on the first day, on the third day and on the seventh day. We sacrificed three mice each period according to the standard operational procedure for immunohistochemistry staining50. The anesthesia was administered by aerosol chloroform, similar to the method we used earlier when we created the wound model. We did not use injectable anesthesia due to our concern that the injectable anesthesia might disturb the histopathological appearance of the tissues. The mice was first anesthetized using ether gas before we took samples from the wound margin. Afterwards, we fixed the slices using 10% neutral buffered formalin for 24 hours, embedded it to a paraffin block and thinly sliced it. We then we measured the immunohistochemistry levels using specific staining for each of the biomarkers. The immunohistochemistry interpretation was done by pathology experts and was done blindly to make sure there were no subjectivity bias. The inclusion of each mouse in each immunohistochemistry period was randomized to prevent any bias. The remaining mice were terminated according to animal ethics by cervical dislocation.\n\nData analysis was done using SPSS version 16.0, using a single animal as the unit of analysis. We used one-way ANOVA to compare means between the control group and the diabetic group. Data were considered as significant if p <0.05.\n\n\nResults\n\nDuring the research, none of the 60 mice dropped out or died before the research process ended and there were no observed adverse effects in the animals used in this study.\n\nThe measurements of wound length on day 1 to 14 (Table 1) as an indicator of wound healing shows a better wound healing process for the VCO with a longer duration of ozone flow. The reduction of wound length is directly proportional to the duration of ozone flow. The greatest to smallest wound length reduction is found in the VCO with ozone flow for 14 hours group (P4), followed by the 7 hours ozonated VCO group (P3), 90 minutes ozonated VCO group (P2), non-ozonated VCO group (P1), and the diabetic wound without VCO group (C-). The P4 group, which received VCO with ozone flow for 14 hours shows the greatest reduction in wound length (16837.10 µm)51. The smallest reduction in wound length was found in the C+ group (6370.77 µm), as expected. The results can be seen in Table 1 and Figure 1.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hour ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nVCO, virgin coconut oil.\n\nFigure 2 shows the macroscopic wound morphology comparison of wound healing between the study groups. As shown in the picture, the wounds in the treatment groups healed much faster compared to the positive control group without any treatment. The fastest wound healing was found among the P4 group, which had almost closed completely on the 14th day, where at the same time the wound on the positive control group had still barely healed. The P3 group had a similar wound healing result, although the wound length is still greater than that of the P4 group, signifying that the P4 group achieved the best wound healing result compared to all other therapeutic groups. Therefore, these results suggest that topical application of ozonated VCO can improve wound healing in diabetic mice, as shown by the improved wound contraction length compared to the diabetic positive control group without any treatment.\n\nThe wound for the 14-hours ozonated VCO group healed faster and better compared to the control group.\n\nVCO, virgin coconut oil.\n\nDescriptive measurements of HSP90Α levels are shown in Table 2. The lowest HSP90Α levels were obtained in the C+ group and the highest HSP90Α levels were obtained in the P4 group throughout the measurement period. The histopathological images of HSP90a-stained cells are shown in Figure 3. One-way ANOVA showed a significant difference for all three measurement days. For the first day measurement, both the P3 group and P4 group exhibited significantly higher HSP90Α levels compared to the C+ group. No group showed significantly higher HSP90A levels as compared to the C- group for the first day measurement. For the third day measurement, all therapeutic groups exhibited significantly higher HSP90Α levels compared to the C+ group, with P2, P3 and P4 groups showing significantly higher HSP90Α levels compared to the C- group. Results for the seventh day measurement were similar to the third day measurement, where all therapeutic groups exhibited significantly higher HSP90Α levels than to the C+ group, with P3 and P4 groups showing significantly higher HSP90Α levels compared to the C- group.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hour ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nTop row, from left to right: negative control group with negative diabetic status; positive control group with positive diabetic status; non-ozonated VCO therapeutic group. Bottom row, from left to right: 90-minute ozonated VCO therapeutic group; 7-hour ozonated VCO therapeutic group; 14-hour ozonated VCO therapeutic group. Size bar indicates 20 µm.\n\nVCO, virgin coconut oil.\n\nDescriptive measurements of VEGF-A levels are shown in Table 3. The lowest VEGF-A levels were obtained in the C+ group and the highest VEGF-A levels were obtained in the P4 group throughout the measurement period. The histopathological images of VEGF-stained cells are shown in Figure 4. One-way ANOVA showed a significant difference for all three measurement days. For the first day measurement, significantly higher VEGF-A levels were found for P2, P3 and P4 groups as compared to the C+ group, with P3 and P4 groups showing significantly higher VEGF-A levels compared to the C- group. For the third day measurement, P2, P3 and P4 groups exhibited significantly higher VEGF-A levels compared to the C+ group, with only the P4 group showing significantly higher VEGF-A levels compared to the C- group. Results for the seventh day measurement shows similar results to the first and third day measurements, where P2, P3 and P4 groups exhibited significantly higher VEGF-A levels compared to both C+ and C- groups.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hour ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nTop row, from left to right: negative control group with negative diabetic status; positive control group with positive diabetic status; non-ozonated VCO therapeutic group. Bottom row, from left to right: 90-minute ozonated VCO therapeutic group; 7-hour ozonated VCO therapeutic group; 14-hour ozonated VCO therapeutic group. Size bar indicates 20 µm.\n\nVCO, virgin coconut oil.\n\nDescriptive measurements of EGF levels are shown in Table 4. The lowest EGF levels were obtained in the C+ group and highest EGF levels were obtained in the P4 group throughout the measurement period. The histopathological images of EGF-stained cells are shown in Figure 5. One-way ANOVA showed a significant difference for all three measurement days. For the first day measurement, significantly higher EGF levels were found for P2, P3 and P4 groups compared to the C+ group, with no group showing significantly higher EGF levels compared to the C- group. For the third day measurement, all therapeutic groups exhibited significantly higher EGF levels compared to the C+ group, with the P3 group showing significantly higher EGF levels compared to the C- group. Results for the seventh day measurement show that all therapeutic groups exhibited significantly higher EGF levels compared to the C+ group, with P2, P3 and P4 groups showing higher EGF levels compared to C- group.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hour ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nTop row, from left to right: negative control group with negative diabetic status; positive control group with positive diabetic status; non-ozonated VCO therapeutic group. Bottom row, from left to right: 90-minute ozonated VCO therapeutic group; 7-hour ozonated VCO therapeutic group; 14-hour ozonated VCO therapeutic group. Size bar indicates 20 µm.\n\nVCO, virgin coconut oil.\n\nDescriptive measurements of bFGF levels are shown in Table 5. The lowest bFGF levels were obtained in the C+ group and highest bFGF levels were obtained in the P4 group throughout the measurement period. The histopathological images of bFGF-stained cells are shown in Figure 6. One-way ANOVA showed a significant difference for all three measurement days. For the first day measurement, significantly higher bFGF levels were found for P2, P3 and P4 groups compared to the C+ group, with only P4 showing significantly higher bFGF levels compared to the C- group. For the third day measurement, all therapeutic groups exhibited significantly higher bFGF levels compared to the C+ group, with P2, P3 and P4 groups showing significantly higher bFGF levels compared to the C- group. Similar to the third day, results for the seventh day measurement show that all therapeutic groups exhibited significantly higher bFGF levels compared to the C+ group, with P2, P3 and P4 groups showing higher bFGF levels compared to the C- group.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hour ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nTop row, from left to right: negative control group with negative diabetic status; positive control group with positive diabetic status; non-ozonated VCO therapeutic group. Bottom row, from left to right: 90-minute ozonated VCO therapeutic group; 7-hour ozonated VCO therapeutic group; 14-hour ozonated VCO therapeutic group. Size bar indicates 20 µm.\n\nVCO, virgin coconut oil.\n\nDescriptive measurements of CD34 levels are shown in Table 6. The lowest CD34 levels were obtained in the C+ group and highest CD34 levels were obtained in the P4 group throughout the measurement period. The histopathological images of CD34-stained cells are shown in Figure 7. One-way ANOVA showed a significant difference for all three measurement days. For the first day measurement, significantly higher CD34 levels were only found for the P4 group compared to both C+ and C- groups. For the third day measurement, all therapeutic groups exhibited significantly higher CD34 levels compared to the C+ group, with P2, P3 and P4 groups showing significantly higher CD34 levels compared to the C- group. Similar to the third day, results for the seventh day measurement show that all therapeutic groups exhibited significantly higher CD34 levels compared to the C+ group, with only the P4 group showing higher CD34 levels compared to the C- group. Therefore, the P4 group most consistently had higher levels of all growth factors and biomarkers measured in this study.\n\nC-, negative control group with negative diabetic status; C+, positive control group with positive diabetic status; P1, non-ozonated VCO therapeutic group; P2, 90-minute ozonated VCO therapeutic group; P3, 7-hour ozonated VCO therapeutic group; P4, 14-hours ozonated VCO therapeutic group; VCO, virgin coconut oil.\n\nTop row, from left to right: negative control group with negative diabetic status; positive control group with positive diabetic status; non-ozonated VCO therapeutic group. Bottom row, from left to right: 90-minute ozonated VCO therapeutic group; 7-hour ozonated VCO therapeutic group; 14-hour ozonated VCO therapeutic group. Size bar indicates 20 µm.\n\nVCO, virgin coconut oil.\n\n\nDiscussion\n\nVCO can be produced from fresh coconut or coconut milk, which is rich in medium-chain triglycerides and lauric acid. Lauric acid is a precursor of monolaurin, which can modulate immune cell proliferation. If a wound occurs, the inflammation process commences and immune cell activity increases52,53. Previous research found that VCO caused a significant reduction in ear edema, claw edema and granuloma formation in Sprague Dawley rats54. VCO has been shown to have significant antioxidant effects including increased levels of the superoxide dismutase enzyme in the wound tissue of normal mice17,53. In the case of chronic human skin conditions such as xerosis and atopic dermatitis, VCO shows a significant healing effect55. VCO is also efficacious as a therapy for wound healing and angiogenesis. VCO showed a significant effect on the wound healing of diabetic mice through increased wound closure rate, total protein content, increased collagen synthesis and re-epithelialization. VCO proved to be significantly better than silver sulphadiazine cream in healing diabetic wounds56.\n\nThe results of this study show that the application of ozone helps speed up the healing process of a diabetic ulcer. These results are similar to several previous studies. Izadi et al. found that ozone therapy shortened the time needed for diabetic wounds to heal32. A systematic review of several studies reported that ozone therapy reduces the ulcer area and shortens the length of hospitalization compared to control therapies. Ozone therapy was associated with a greater reduction of ulcer area compared to antibiotic therapy26.\n\nReis et al. reported that ozone provided by a high-frequency device might potentially be useful in the treatment of ulcers, thus contributing to the healing process57. This might be promoted by a decrease in bacterial infection, fibroplasia activation and keratinocyte proliferation. Ozone is a powerful oxidant; if ozone comes into contact with body fluids, it will result in the formation of reactive oxygen molecules and lead to several biochemical events that influence cellular metabolism, tissue repair and microbial infection. Ozone has the potential to promote the activation of transcription factor NF-kB, regulating inflammatory responses, and release of platelet-derived growth factor and transforming growth factor β1 from platelets22,58,59.\n\nThe previous study conducted by Kim et al. found an increased intensity of collagen fibers and a greater number of fibroblasts in an acute cutaneous wound for the ozone group60. Furthermore, during a systematic review of the therapeutic use of ozone in wounds, the authors discovered that most of the studies analyzed in their research found stimulation of the healing process (62.2%), followed by improvement in wound appearance (43.5%) and a decrease in pain (17.4%). The mechanism of how ozone improved wound healing might be related to its effect on the growth factors, activation of the antioxidant system and activation of superoxide dismutase61,62. Besides its effect on the wound healing process, ozone also improves the diabetic condition itself by improving glycemic control, preventing oxidative stress and normalizing organic peroxides, aside from activation of superoxide dismutase as explained above61. This condition might be beneficial to the wound healing process due to the improved condition of the diabetes itself. The authors concluded that ozone could be an important treatment option for wounds and may bring numerous benefits to patients61.\n\nIn terms of immunohistochemistry parameters, our study found that 14-hour ozonated VCO treatment increased HSP90α, VEGF-A, EGF, bFGF and CD34 levels as measured in our study. Ozone has the potential to increase wound healing by reducing the wound area infection, controlling wound contamination and reducing wound healing time20,63. Ozone has also been found to increase granulation tissue formation, which also increases wound healing speed22. Zhang et al. showed that ozone increases the expression of several wound healing biomarkers, such as VEGF, transforming growth factor-β and platelet-derived growth factor. They also found that ozone improves wound healing in diabetic foot ulcers with a higher wound size reduction in the ozone-treated group25.\n\nAs far as we know, there have been no other studies that show any therapeutic modalities, except direct application of HSP90, that alters the wound healing result64. HSP90α helped to accelerate wound healing by recruiting both epidermal and dermal cells, promoting dermal cell migration, and was able to override the inhibitory effects of hyperglycemia on cell migration in diabetes65. Several studies have found that topically applied HSP90α protein improves wound healing in several kinds of wounds, especially diabetic wounds31,64–66. The main pathology of diabetes is chronic hyperglycemia, which causes destabilization of a key regulator of the HSP90α protein, hypoxia-inducible factor 1 -alpha (HIF-1α)30,64. The HIF-1α pathway is involved in wound healing by helping human dermal fibroblast migration; this is one of the causes of diabetic wound pathology64,66. HSP90α improves wound healing by mediating hypoxia-stimulated human dermal fibroblast motility under normal glycemic conditions and “jumpstarts” cell migration under hyperglycemia conditions31,64. Therefore, HSP90α helps diabetic wound healing by by-passing downregulation of HIF-1α and helps to ‘rescue’ the migration of hyperglycemic cells that otherwise do not respond properly to the environmental hypoxia64. Cheng et al. found that HSP90α can promote wound healing in diabetic mice in a more effectively manner65. The middle domain of HSP90α contains the F5 fragment, which acts as a pro-motility factor that affects all three human skin cell types. This HSP90α fragment promotes dermal and epidermal cell migration through LRP-1 or CD91 surface receptors64,65.\n\nMost complications that arise from diabetes are related to vascular alterations caused by diabetes. The pathology of diabetic wound healing is tightly associated with insufficient angiogenesis and reduced VEGF signaling34,35. Seitz et al. found that diabetic mice have decreased VEGF-A levels compared to normal mice67. The low VEGF tissue levels in diabetes patients might be caused from the inability of diabetes patients to appropriately upregulate VEGF expression in response to hypoxia68,69. Galiano et al. found that VEGF-A treated diabetic mice showed accelerated cutaneous wound closure compared to untreated mice. The mechanism of how VEGF facilitates tissue repair is by both increasing vascular permeability, which allows inflammatory cells to enter into the site of injury, and also increasing the migration and proliferation of pre-existing endothelial cells. In the VEGF-A treated diabetic wound model, there is a lot of abundantly vascularized granulating tissue on the wound bed, which causes the wound bed to become very hyperemic. When the VEGF-A treated wounds are almost completely healed, the control wounds were just starting to grow granulating tissue, and the granulation itself did not exhibit hyperemia and vascularization as the VEGF-A treated diabetic wounds did. VEGF treatment also induces neovascularization and cell proliferation in the diabetic wounds68.\n\nDiabetes also causes EGF synthesis to be diminished, as shown in diabetic animal models38. EGF is an important factor for wound healing, as it increases the stimulation, proliferation, and migration of several cells related to wound healing, such as keratinocytes, endothelial cells and fibroblasts, facilitates better dermal regeneration, and accelerates the level of wound contraction related to collagen deposition and proliferation of myofibroblasts37,70. Degradation of several growth factors, including EGF, might result in refractory diabetic ulcer wounds. Several studies have shown benefits of EGF therapy in diabetic wound models. Zhang et al. found that EGF improved diabetic wound healing by: increasing the formation of granulation tissue, collagen uniformity and the extracellular matrix; increasing the number of fibroblasts and improving fibroblast morphology; and also increasing the number of capillaries in the wound. EGF can also induce the migration of inflammatory cells away from the wound, which results in improvement of the wound microenvironment and tissue nutritional status39,39. EGF also has the potential to enhance wound healing and reduce wound healing time, as found by Tsang et al. and Singla et al.40,41\n\nThis study found an increase in bFGF levels in wounds treated with ozonated oil compared to nontreated wounds. Re et al. found that ozone increases the basal concentration of bFGF in ozone-incubated platelet rich plasma71. An early study showed that bFGF improves diabetic wound healing in a diabetic mice model by increasing the rate of cellular infiltration and capillary ingrowth, which might be related to the ability of bFGF to induce angiogenesis in wound healing42,72. bFGF is the primary promoter for cell proliferation73. bFGF contributes to the wound healing by inducing angiogenesis, fibroblast proliferation and endothelial cell migration74. A recent study using bioinspired hydrogels with bFGF done by Zhang et al. found that bFGF enhances cell proliferation, re-epithelialization, collagen deposition, and wound contraction in the full-thickness wound healing model. Chronic wound healing is also benefited by the increase of bFGF73.\n\nThis study found an increase of CD34 levels in wounds treated with ozonated oil compared to nontreated wounds. CD34 has been used to improve wound healing in diabetic wound models using a CD34 injection direct to the wound itself or systemic injection of CD34 cells. Sivan-Loukianova et al. found that CD34 cells decreased diabetic wound size, accelerated epidermal healing, and rapidly accelerated neovascularization in diabetic wounds, as shown by an increase in blood vessel numbers and diameter75. Cil et al. found that direct application of human umbilical-derived CD34 to the wound margins improves diabetic wound healing by decreasing inflammatory reactions and increasing wound revascularization76. A recent study conducted by Kanji et al. also found that systemically-injected CD34 cells accelerate wound healing in streptozotocin-injected mice. CD34 cells improved re-epithelialization, increased neovascularization, improved granulation tissue formation and increased collagen and myofibroblasts deposition77. Diabetic wounds have decreased angiogenesis and increased matrix metalloproteinases (MMPs), which inhibits proper wound healing in patients with diabetes35. CD34 cells decreased the pro-inflammatory activity of NF-kB, which therefore also reduced the level of MMP-1 expression by inhibiting the recruitment of NF-kB to the MMP-1 promoter site. Therefore, CD34 can reverse the delayed wound healing factors in patients with diabetes. Besides healing effects, the measurement of CD34 cells in the wound margin can predict the healing of diabetic wounds. Thom et al. found that the number of CD34 cells in stained epidermis taken from the wound margin of diabetic wounds from diabetic patients was positively correlated with wound healing itself. Therefore, CD34 measurement in the early weeks of the diabetic wound provides insight into how well the diabetic wound will respond and can be used to predict the healing result of diabetic wounds48.\n\nThere are several limitations in this study. First, we only measured macroscopic wound contraction length without analyzing the histological and biochemistry aspects of the wounds itself. The comparison of wound healing histological events and biochemistry profiles between diabetic control mice and therapeutic group mice might enable further explain on a mechanistic basis how ozone improved wound healing in diabetic wounds. Moreover, we only analyzed the wounds until the seventh day and did not measure the events that happened until the wound successfully closed, as the end-result of the wound healing is restoration of the wound itself. In addition, insufficient literature about bFGF and CD34 in wound healing, especially diabetic wound healing, also lead to difficulty in determining the exact action of bFGF and CD34 in diabetic wound healing and the relation between its increase and its effect in the wound healing process. These could be considered to seek a better understanding of how ozone improved the wound healing process.\n\nAs ozonated VCO treatment can increase the levels of several wound healing biomarkers, such as HSP90α, VEGF-A, EGF, bFGF and CD34 levels, its increase might potentially aid the wound healing process. This study is a preliminary study in animals (in this case Wistar mice). Mice can be used as an animal model for several skin diseases, including wound healing. Therefore, we hope that this research can be utilized as a basis for further research on the usage of ozonated VCO in human wounds78.\n\n\nConclusions\n\nOzonated VCO has the potential to increase immunohistochemistry wound healing parameters in a diabetic wound mice model, as shown by the increase of HSP90α, VEGF-A, EGF, bFGF and CD34 levels. The improvement of the wound healing process was proportional to the duration of ozone flow in VCO. A longer treatment period and a longer VCO ozonation duration resulted in higher levels of biomarkers. Further research should be done to improve the application of ozonated oil treatment for diabetic ulcers in everyday practice.\n\n\nData availability\n\nOpen Science Framework: Topical ozonated virgin coconut oil improves wound healing and increases HSP90α, VEGF-A, EGF, bFGF and CD34 in diabetic ulcer mouse model of wound healing. https://doi.org/10.17605/OSF.IO/89FQC51.\n\nThis project contains the following underlying data:\n\n- Data Tables (raw data in XLSX format)\n\n- Images (original unedited histopathology image files in JPG format)\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 would like to express our gratitude to the Department of Dermatology and Venereology, and Center Plasma Research (CPR), Diponegoro University, Semarang.\n\n\nReferences\n\nWorld Health Organization: Global Report on Diabetes. World Health Organization. 5. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSivan-Loukianova E, Awad OA, Stepanovic V, et al.: CD34+ Blood Cells Accelerate Vascularization and Healing of Diabetic Mouse Skin Wounds. J Vasc Res. 2003; 40(4): 368–77. PubMed Abstract \| Publisher Full Text\n\nÇil N, Oğuz EO, Mete E, et al.: Effects of Umbilical Cord Blood Stem Cells on Healing Factors for Diabetic Foot Injuries. Biotech Histochem. 2017; 92(1): 15–28. PubMed Abstract \| Publisher Full Text\n\nKanji S, Das M, Joseph M, et al.: Nanofiber-expanded human CD34+ Cells Heal Cutaneous Wounds in Streptozotocin-Induced Diabetic Mice. Sci Rep. 2019; 9(1): 8415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAvci P, Sadasivam M, Gupta A, et al.: Animal Models of Skin Disease for Drug Discovery. Expert Opin Drug Discov. NIH Public Access; 2013; 8(3): 331–55. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77529", "date": "19 Jan 2021", "name": "Gamal Badr", "expertise": [ "Reviewer Expertise 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\nIn the present manuscript entitled: Topical ozonated virgin coconut oil improves wound healing and increases HSP90α, VEGF-A, EGF, bFGF and CD34 in diabetic ulcer mouse model of wound healing, the authors focused to investigate the effect of ozonated VCO on the healing process of diabetic wounds using STZ-induced a diabetic mouse model. Several points should be seriously taken in consideration for the following reasons:\nThe authors are advised to extensively revise the manuscript against English language errors that are present throughout the manuscript. The manuscript was poorly written and can not be accepted in the current version.\n\nThe authors should respect the use of abbreviations throughout the manuscript. The abbreviation must be defined at the first appear in the text and then the authors should use the abbreviations throughout the manuscript.\n\nThe introduction section was too long and should be improved.\n\nThe most critical point of this study is that the authors used 6 animal groups (10 mice per group) but they investigated the IHC expression of HSP90, VEGF, CD34, FGF and EGF on at 1, 3, 5 and 7 days post-wounding. However, the mice in the 14 days post-wounding time point were terminated. The authors must investigate all the parameters at all time points post-wounding.\n\nThe statistical analysis should be revised, and a multiple comparison test must be performed.\n\nAll the data in the tables must be presented in bars figures under the main figures.\n\nFigure 1 lacks legend on the X- and Y-axis and also lacks symbols for significance.\n\nThe authors must present the histopathological alterations throughout the interval time points using H&E staining.\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?\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": [ { "c_id": "6547", "date": "06 May 2021", "name": "Matthew Brian Khrisna", "role": "Author Response", "response": "Dear Prof. Gamal Badr, Thank you very much for your review. In response to your reservations, we have uploaded a newer version of this article. Kindly review these new versions as the revised version of this article. Thank you. Regards, Authors" } ] } ]	1	https://f1000research.com/articles/9-580
https://f1000research.com/articles/10-257/v1	30 Mar 21	{ "type": "Method Article", "title": "The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells", "authors": [ "Rachel Tanner", "Emily Hoogkamer", "Julia Bitencourt", "Andrew White", "Charelle Boot", "Claudia C. Sombroek", "Stephanie A. Harris", "Matthew K. O'Shea", "Daniel Wright", "Rachel Wittenberg", "Charlotte Sarfas", "Iman Satti", "Frank A.W. Verreck", "Sally A. Sharpe", "Helen A. Fletcher", "Helen McShane", "Emily Hoogkamer", "Julia Bitencourt", "Andrew White", "Charelle Boot", "Claudia C. Sombroek", "Stephanie A. Harris", "Matthew K. O'Shea", "Daniel Wright", "Rachel Wittenberg", "Charlotte Sarfas", "Iman Satti", "Frank A.W. Verreck", "Sally A. Sharpe", "Helen A. Fletcher", "Helen McShane" ], "abstract": "The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.", "keywords": [ "3Rs", "refinement", "non-human primate", "macaque", "mycobacterial growth inhibition assay", "tuberculosis", "vaccines" ], "content": "\n\nPotential to expedite the development of a much-needed effective TB vaccine through rapid down-selection of candidates at an early stage.\n\nTractable system for the exploration of immune mechanisms underlying the control of mycobacterial growth.\n\nOpportunity to biologically validate the direct PBMC MGIA through correlation with protection from in vivo M.tb challenge on an individual animal basis.\n\nRefining early efficacy testing of TB vaccine candidates by using the MGIA in place of in vivo infection with pathogenic M.tb.\n\nReducing the number of NHPs used in TB vaccine testing and associated immunology studies by down-selecting the number of candidates going forward to in vivo testing and by allowing the testing of multiple conditions using cells from a single group.\n\nBridging of the assay to use in target species including humans to replace the use of preclinical models in some settings.\n\nMeasures of vaccine efficacy obtained more rapidly than in vivo M.tb challenge studies (2 weeks vs. 12 weeks routinely required for in vivo challenge). Quantification using the BACTEC MGIT system also more rapid than conventional colony counting on agar.\n\nNegates the need for high containment animal facilities required for in vivo M.tb challenge.\n\nMore cost-effective, much lower resource requirement and less technically challenging than in vivo M.tb challenge studies in NHPs.\n\nAssessing the BCG vaccine-induced response as a benchmark and comparing between different routes of administration37.\n\nComparing outcomes with levels of protection from in vivo M.tb or BCG challenge to determine biological validity37.\n\nApplying to other aspects of TB research, such as assessing ability to control mycobacterial growth following M.tb infection and comparisons between species36.\n\nExploring underlying immune mechanisms including associations between growth inhibition and various cell type frequencies, specific antibodies, and baseline characteristics35, [Tanner R, unpublished data].\n\nAssessing protective efficacy of novel TB vaccine candidates.\n\nUnderstanding associated immune mechanisms of protection.\n\nMeasuring vaccine potency, lot-to-lot consistency and stability.\n\nAdaptation for use with other pathogens (e.g. S. aureus).\n\n\n1.0 Introduction\n\nApproximately 1 in 4 people globally are infected with tuberculosis (TB), with 10 million new infections and 1.4 million deaths reported in 20191. This serious public health threat is further exacerbated by the spread of multi- and extensively-drug resistant strains of the causative agent, Mycobacterium tuberculosis (M.tb)2. An efficacious vaccine is widely acknowledged to be the most effective intervention strategy. The Bacillus Calmette-Guérin (BCG) vaccine, first introduced in 1921, remains the only currently-licenced TB vaccine. Although protective in infants against severe forms of TB disease, BCG affords extremely variable levels of protection against the most common and infectious form of TB, pulmonary disease, in adults3. BCG-induced protection against pulmonary TB is lowest in regions close to the equator such as sub-Saharan Africa and India where an effective vaccine is most desperately needed3. However, development of a successful TB vaccine is severely hampered by the lack of a validated correlate or biomarker of protection4. It remains unclear which aspects of the immune response confer protection from TB disease, and therefore which parameters to target with a vaccine and to assess as a reliable measure of protective efficacy.\n\nIn the absence of a validated immune correlate of protection from TB, the only currently available approach to early efficacy testing of TB vaccine candidates is the use of preclinical ‘challenge’ (infection) models. Animals used typically include mice, guinea pigs and non-human primates (NHPs). In vivo testing offers the obvious and unparalleled advantage of modelling the complexities of biological systems (the immune system representing one of the most complex and systemic of all) in the context of their natural microenvironment over time. NHPs are considered the most representative model for human TB due to their anatomical and physiological similarities, natural susceptibility to M.tb infection and comparable pathological and clinical outcomes5. Rhesus and cynomolgus macaques in particular are widely used in TB vaccine studies as BCG vaccination offers partial and quantifiable protection against M.tb challenge in these species6–10. There has been recent emphasis on the use of NHPs as the ‘gatekeeper’ for progression of TB vaccine candidates to clinical trials, and the numbers used in the field are increasing11.\n\nIn order to evaluate the protective efficacy of a candidate TB vaccine, animals must be exposed to virulent M.tb in a ‘challenge’ experiment following vaccination. Infection with M.tb results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure12. Welfare considerations include the infection process itself, disease symptoms, and the definition of humane endpoints. A study assessing the lifetime experience of macaques found that the combined welfare assessment score increased from <10 to >50 following M.tb challenge, reflecting a decline in procedural, physical, psychological and environmental welfare13. Other limitations of the NHP challenge model in TB vaccine testing include the long and costly nature of such experiments, and have been discussed further elsewhere14.\n\nOne potential alternative or complementary tool for assessing vaccines is functional in vitro assays such as growth inhibition assays (GIAs) as a potential surrogate measure of vaccine efficacy. Such assays aim to provide unbiased read-outs of the combined effects of the host immune response, strain virulence and influences of interventions. They have been applied with some degree of success to a range of other disease models including HIV, malaria and meningitis15–17. A number of mycobacterial GIAs (MGIAs) for TB have been previously described in the literature, including the use of reporter strains in whole blood18,19 and primary or secondary lymphocyte/monocyte co-cultures in humans20,21, bone marrow macrophage/splenocyte cultures in mice22,23, and cattle peripheral blood mononuclear cell (PBMC)24,25. These have been comprehensively reviewed elsewhere26. However, in all cases such assays are technically challenging and limited follow-up work has been conducted to qualify an MGIA that could be transferred across laboratories using a standardised, reproducible method.\n\nWe have previously worked to optimise and standardise a simplified MGIA (known as the ‘direct MGIA’) for use in humans and mice, adapted from methods originally described by Wallis et al., using the BACTEC MGIT mycobacterial quantification system27–29. Applying this approach, we have demonstrated a BCG vaccine-induced effect in these species30–32, and an association with in vivo protection from mycobacterial challenge has also been described33,34. Importantly, preclinical MGIAs represent a potential alternative to the in vivo M.tb challenge step in early TB vaccine testing: a major refinement which is particularly important for NHPs due to the additional welfare and behavioural considerations that apply when using these species in medical research. Furthermore, the NHP model represents a unique opportunity for biological validation of the assay against direct measures of in vivo protection, as discussed below, permitting bridging to use in other species including humans. We present a protocol for the first example of an NHP MGIA using in vitro cell co-culture, adapted from our direct MGIA methods described in humans and mice, with the aim of refining and expediting early TB vaccine testing35–37.\n\n1.2.1 Refinement. The main 3Rs objective of the direct NHP MGIA is to provide a potential refinement to the process of early TB vaccine testing in NHP models through offering a functional in vitro assay as an alternative to in vivo infection with pathogenic M.tb. If the MGIA were applied in place of in vivo M.tb infection, animals would still be required for vaccination, but blood samples could be taken before and at various time-points after vaccination, and ability to control growth of mycobacteria assessed in vitro without the need for M.tb infection of the animals. Lifetime experience would be improved and the severity rating for the experiment would be downgraded from ‘Moderate’ to ‘Mild’ under EU legislation12,13,38.\n\n1.2.2 Reduction. A successful, validated MGIA could be used to test and down-select experimental TB vaccine candidates at an early stage of development, reducing the number going forward to virulent M.tb challenge experiments and therefore the number of animals used. It would also reduce the number of animals required as multiple conditions (such as control of different mycobacterial strains or contribution of different immune parameters) could be tested using samples from a single group of vaccinated animals. It is increasingly acknowledged that the level of protection conferred by a TB vaccine candidate may be influenced by the M.tb strain/s prevalent in the geographical region in which it is being tested39,40. Furthermore, preclinical testing of TB vaccine candidates generally use standardised (and sometimes attenuated) laboratory strains of M.tb as challenge agents. It is prudent to test vaccine efficacy against a range of M.tb strains or clinical isolates to ensure widespread applicability. Using an in vivo challenge model, this would necessitate an additional group of experimentally infected animals for each strain. The NHP MGIA allows samples from a single group of animals to be assessed for ability to control multiple mycobacterial strains, particularly as there are no restrictions on the inoculum, unlike for assays which use reporter strains for example.\n\nThe MGIA also offers a tractable model for the exploration of underlying immune mechanisms involved in the control of mycobacterial growth. Cell types of interest may be depleted, purified and added back at different concentrations, pathways interrupted, receptors blocked and so forth to elucidate those of importance. The ability to conduct such experiments in vivo, for example adoptive transfer, is limited and requires large numbers of animals. Using the MGIA, multiple conditions can be explored with a sample set from a single group of animals, in contrast to the additional groups required for equivalent in vivo experiments with the associated impacts on disease severity. To illustrate, a hypothetical in vivo experiment designed to test a novel vaccine candidate would require minimum group sizes of 8 macaques to detect a nine-AU reduction in total pathology given a group standard deviation of 5.8 with a power of 80% and an α of 0.05. Including a naïve control group, a BCG-vaccinated group as a benchmark, and a group for the novel vaccine candidate, testing efficacy against three strains of M.tb would require three groups for each condition (as each animal can only be challenged with one strain) = 72 animals vs. 24 for in vitro assessment using the NHP MGIA (Table 1). This represents a 3-fold reduction in the number of animals used.\n\n1.2.3 Replacement. As described, our direct MGIA method has also been optimised for use with human cells29. However, the biological relevance of the direct MGIA, as for any potential correlate of protection, can only be confirmed by demonstrating an association with in vivo protection from either controlled or natural infection or disease. While controlled infection with BCG may be used as a potential surrogate in human challenge studies, virulent M.tb cannot ethically be used33. The NHP model provides an opportunity to validate the assay against protection from M.tb as well as BCG infection, allowing greater confidence in the relevance of the human assay such that preclinical models such as mice, guinea pigs and NHP may ultimately be replaced with human samples in some settings. Furthermore, the MGIA could be applied in the measurement of vaccine potency, lot-to-lot consistency and stability as an alternative to in vivo infection experiments. The 3Rs relevance of the NHP MGIA is summarised in Figure 1.\n\nThe potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This currently includes groups in Europe such as the UK, Sweden and the Netherlands; Pittsburgh, Chicago, Boston, California, New Orleans, Texas and Seattle in the USA; Osaka, Japan; Wuhan, China; and the Philippines among others41. Peña et al. reviewed the major NHP TB studies published between 2001 and 2014. During this period, the mean number of publications increased from 1 per year in 2001–2007 to 4 per year in 2007–2014. The mean number of animals used per publication was 14 for rhesus macaques (range 3–32) and 20 for cynomolgus macaques (range 2–44). For TB vaccine-related studies specifically, the mean number of animals used was 20 (range 12–32)41. Conducting a literature search for the time-period 2019–2020 using the Google Scholar search terms ‘tuberculosis’ and ‘macaque’, and excluding results relating to TB diagnostics or drugs, and/or SIV coinfection, we identified 21 publications reporting NHP studies of TB vaccines and/or TB immunology employing a mean of 27 animals (range 6–75), the majority of which involved challenge with M.tb. This illustrates a trend towards increased use of NHPs in the field and larger group sizes. Widespread adoption of a validated NHP MGIA could significantly reduce the number of animals undergoing infection with virulent M.tb and potentially the overall numbers used.\n\n\n2.0 Methods\n\nThe reagents and equipment required for the direct NHP MGIA are described in Table 2 and Table 3 respectively. It is not essential to use a specific supplier of reagents or equipment unless it is specified in the table.\n\n2.2.1 NHP samples. Stored samples used in the optimisation experiment shown in Figure 6 were collected from n=7 female rhesus macaques of Indian genotype aged 14–15 years as part of a study of BCG vaccination conducted at Public Health England (PHE) in the UK. Design and procedures of the original study were approved by the Public Health England Animal Welfare and Ethical Review Body and authorized under an appropriate UK Home Office project license. Animals were housed in compatible social groups in accordance with the Home Office (UK) Code of Practice for the Housing and Care of Animals Used in Scientific Procedures (1989) and the National Centre for Refinement, Reduction and Replacement (NC3Rs) Guidelines on Primate Accommodation, Care and Use, August 2006 (NC3Rs, 2006). They were provided with enrichment in the form of food and non-food items on a daily basis; animal welfare was monitored daily. Animals were captive-bred for research purposes, were obtained from established breeding colonies at PHE, were healthy and had not been used previously for experimental procedures. Animals were sedated by intramuscular (IM) injection of ketamine hydrochloride (Ketaset, 100 mg/ml, Fort Dodge Animal Health Ltd, Southampton, UK; 10 mg/kg) for procedures requiring removal from their housing. Animals were weighed, had rectal temperature measured and were examined for gross abnormalities whenever procedures (vaccination, blood sample collection) were conducted. There were no adverse events, and no humane endpoints for this study as it did not involve M.tb challenge.\n\n2.2.2 Human samples. Samples used in the optimisation experiments shown in Figure 5 and Figure 7 were obtained from volunteers at the Jenner Institute, Oxford, in accordance with University of Oxford policy. All human samples were collected in accordance with the ethical principles set forth in the Declaration of Helsinki as agreed by the World Medical Association General Assembly (Washington 2002), ICH Good Clinical Practice (GCP) and local regulatory requirements; volunteers gave written informed consent.\n\nFor the experiments shown in Figure 5, the experimental unit was a co-culture containing 1 × 106 cells from a single volunteer to ensure all variables were constant apart from the one under investigation (treatment of mycobacterial stock). The sample sizes were duplicate co-cultures for Figure 5A as this was a time-course with repeated measures of a single condition, and (n=6) replicate co-cultures for Figure 5B as this was a group-wise comparison of different stock conditions. For the experiments shown in Figure 6 and Figure 7, the experimental unit was an individual macaque (n=7) or an individual volunteer (n=6) respectively each tested in duplicate; these were group-wise comparisons of co-cultures containing different serum/plasma conditions. Samples were not selected but were used according to availability and recovery of a sufficient number of PBMC post-thawing and a sufficient volume of serum/plasma. No data points were excluded from the analysis. Minimum sample size (n=6) for these experiments was calculated based on the effect size of 0.2 log10 CFU (colony-forming units) observed in previous MGIA experiments considered to be biologically relevant (given matched measures of in vivo efficacy) and estimates of variability within a group with a power of 80% and an α of 0.05. Cells were allocated to conditions by pipetting to mix and adding to conditions in repeated sequence where relevant (Figure 5B). Operator blinding was not possible because the comparisons required operator interventions in the laboratory and the BCG status of the animals was not relevant to these experiments. In all cases, the outcome measured was effect of co-culture condition (stock treatment, plasma vs. serum, or collection/treatment of serum) on mycobacterial growth over the 96 hour co-culture period, as measured by MGIT time-to-positivity (TTP) and/or converted to log10 CFU normalised to the direct-to-MGIT inoculum control. Statistical analysis was conducted using GraphPad Prism v.7, and data was analysed using non-parametric tests due to the small sample sizes; multi-group data was corrected for multiple comparisons using Dunn’s test (all conditions vs. all other conditions). Following confirmation of normality in the distribution of differences between paired measurements, the Bland-Altman method was used to compare MGIA outcomes between serum and plasma in Figure 6. 95% confidence intervals for the Bland-Altman limits of agreement were calculated using the methods described by Carkeet42.\n\n2.4.1 PBMC preparation. Cryopreserved PBMC were rapidly thawed in a water bath at 37°C until a small amount of frozen material remained. Samples were gradually added to 10ml RPMI (containing 10% foetal calf serum and 2mM L-glutamine) using a Pasteur pipette. The cryovial was rinsed using 1ml of fresh medium and added to the corresponding tube, which was then centrifuged at 350 g for 7 min. Supernatants were removed by inversion and cells resuspended at an approximate concentration of 2 × 106 cells per ml of RPMI (containing 10% foetal calf serum and 2mM L-glutamine) and 2µl/ml of 25 U benzonase added to each tube. Cells were rested at 37°C for 2 h with 5% CO2 before counting using an automated CASY cell counter.\n\n2.4.2 MGIA. For the human MGIA experiments shown in Figure 5 and Figure 7, 600µl RPMI (containing 2mM L-glutamine and 25mM HEPES) seeded with 1 × 106 PBMC and ~100 CFU (Figure 5) or ~500 CFU (Figure 7) BCG Pasteur was added to duplicate 2ml screw-cap tubes. The co-cultures were incubated on a 360° rotator at 37°C for 96 hours, after which time tubes were microcentrifuged at 15,300 g for 10 minutes and the supernatant carefully removed by pipetting. Cells were lysed with the addition of 500µl sterile water and the tubes pulse-vortexed at 0, 5 and 10 minutes. For the NHP direct PBMC ‘in-plate’ MGIA, shown in Figure 6, 3 × 106 PBMC and ~500 CFU BCG Pasteur in a total volume of 480μl RPMI (containing 2mM L-glutamine and 25mM HEPES), plus 120μl autologous serum or plasma matched to animal were added per well of a 48-well plate (total volume 600μl per well). Co-cultures were incubated at 37°C for 96 hours with CO2 and then transferred to 2ml screw-cap tubes and centrifuged at 15,300 g for 10 minutes. During this time, 500μl sterile water was added to each well to lyse adherent monocytes and release intracellular mycobacteria. Supernatants were carefully removed from the 2ml screw-cap tubes by pipetting, and water from the corresponding well added to the remaining pellet. In all cases, tubes were pulse vortexed and lysates transferred to a BACTEC MGIT tube supplemented with PANTA antibiotics (polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin) and OADC enrichment broth (Becton Dickinson, UK) before being placed on the BACTEC 960 machine (Becton Dickinson, UK) and incubated at 37°C until the detection of positivity by fluorescence. On day 0, duplicate direct-to-MGIT control tubes were set up by inoculating supplemented BACTEC MGIT tubes with the same amount of mycobacteria as the samples. The TTP read-out can be converted to log10 CFU using stock standard curves of TTP against inoculum volume and CFU. ‘Normalised mycobacterial growth’ is equal to (log10 CFU of sample – log10 CFU of growth control).\n\n\n3.0 Detailed protocol for the NHP direct PBMC MGIA\n\nNote: All work must be performed under sterile tissue culture conditions in a Class II biological safety cabinet and filter tips should be used throughout.\n\n3.1.1 Thaw one vial of BCG Pasteur (or other desired mycobacterial strain) at room temperature.\n\nNote: We recommend a standardised stock of BCG Pasteur produced by Aeras specifically for use in the direct MGIA for consistency. Other stocks may be used but clumping may compromise assay reproducibility and sensitivity; if this is the case, vortexing with ~50 × 1mm borosilicate glass beads for 2 minutes prior to inoculation is recommended to reduce clumping.\n\nNote: If M.tb strains are used, all work should be conducted in an appropriate high biosafety containment laboratory.\n\n3.1.2 Prepare 6 sterile 2ml screw-cap tubes by adding 1.35ml sterile PBS to each tube and labelling 1 to 6.\n\n3.1.3 Add 150µl neat BCG Pasteur stock to tube 1, mix by pipetting up and down, and then take 150µl from this tube and add to tube 2, and so on to make a 1:10 dilution series.\n\nNote: The contents of each tube should be mixed thoroughly by pipetting up and down several times before adding to the next tube in the dilution series.\n\n3.1.4 Prepare MGIT supplement medium by pouring one bottle of OADC growth enrichment into one bottle of lyophilised PANTA. Mix by inverting several times until fully dissolved.\n\nNote: Enrichment media should be used on day of reconstitution.\n\n3.1.5 Add 800µl of supplement medium to each of 14 BACTEC MGIT tubes (2 per standard curve dilution).\n\nNote: Tubes are oxygen-enriched and time without caps should be minimised.\n\nNote: MGIT tubes should be used on day of supplementation and should not be stored following supplementation.\n\n3.1.6 Add 500µl of neat BCG stock directly to each of two supplemented MGIT tubes and invert to mix.\n\n3.1.7 Add 500µl from each of dilution tubes 1–6 to MGIT tubes in duplicate and invert to mix.\n\nNote: After addition of BCG, MGIT tubes should be capped immediately and inverted to mix.\n\n3.1.8 Scan MGIT tube barcodes on the BACTEC MGIT machine and place in the indicated slots.\n\nNote: The machine will generate an alarm when tubes reach a predefined level of fluorescence (indicating that mycobacteria have utilised the oxygen previously quenched to the fluorochrome). Positive tubes will be indicated by flashing lights and can be scanned out of the machine and the corresponding TTP recorded.\n\n3.1.9 Divide four 7H10 agar plates into quadrants and spot 3 × 20µl from the neat vial and each dilution into a quadrant on each of two plates. Leave plates to dry in the Class II cabinet before sealing with parafilm and placing in a CO2 incubator at 37°C. Plates should be checked after 2 weeks and daily henceforth; as soon as colonies are visible they should be counted and the number of spots recorded for each dilution and averaged across the 3 replicates.\n\n3.1.10 Generate a standard curve by plotting TTP against CFU for each input volume and use regression analysis to obtain an equation for the curve. CFU should be fitted using a semi-log line, and log10 CFU with a linear regression. Solve the equation describing the line for X: Y = AX+B -> X = (Y-B)/A where A = slope and B = y-intercept). By inserting the TTP (=Y) for any given sample, the corresponding number of CFU (or log10 CFU) can now be calculated. Further information including a sample MGIT read-out and standard curve have previously been provided by Zelmer et al.43.\n\n3.2.1 Thaw cryopreserved cells by holding the lower portion of the vial in a 37°C water bath.\n\nNote: Vials should be removed from the water bath when a small amount of frozen material is still visible and the outside of the vial should be cleaned with 70% ethanol.\n\n3.2.2 Pipette cells up and down gently using a Pasteur pipette and gradually add to 10ml RPMI (with L-glutamine and sodium pyruvate but NO antibiotics (pen/strep)).\n\nNote: Prepare labelled tubes with 10ml media before beginning the thawing process.\n\n3.2.3 Rinse out the cryovial contents with 1ml of fresh medium and add the remaining cells.\n\n3.2.4 Centrifuge at 350 g for 7 minutes.\n\n3.2.5 Pour off supernatant and resuspend cells at approximately 2 – 3 × 106 cells per ml of RPMI (with L-glutamine and sodium pyruvate but no antibiotics (pen/strep)).\n\n3.2.6 Rest for 2 hours with loosened caps in a 37°C incubator with 5% CO2.\n\n3.2.7 Count viable cells using standard methods (such as a haemocytometer or automated cell counter) and resuspend at 10 × 106 cells per ml of RPMI (with 2mM L-glutamine and 25mM HEPES but NO antibiotics (pen/strep)).\n\n3.2.8 Place 300μl of cell mix (containing 3×106 PBMC) into labelled wells of a 48-well plate.\n\nNote: Replicate cultures should be performed if sufficient cells are available, but replicates have been demonstrated to be consistent (coefficient of variation (CV) <10%)37 such that a single culture is acceptable where cell availability is limiting.\n\nNote: Do not use wells on the outside rows/columns of the 48-well plate for cultures. These should contain 600μl of RPMI medium only.\n\n3.2.9 Add 120µl of non-heat inactivated autologous serum or plasma matched to the animal and time-point (to give a final concentration of 20%).\n\nNote: Serum should be kept sterile or syringed through a 0.2µM cellulose acetate filter prior to use.\n\nNote: 10% filtered pooled human AB serum may be used if autologous serum is not available, but will not capture the influence of serum factors such as antibodies on control of mycobacterial growth.\n\nNote: Autologous plasma may be used if serum in unavailable (see section 4.1.2.3); if plasma is viscous, it may be warmed in a 37°C incubator.\n\nNote: Ensure serum/plasma is mixed well (for example by briefly vortexing) before adding.\n\n3.2.10 Thaw BCG stock at room temperature and prepare to the correct concentration in RPMI (with 2mM L-glutamine and 25mM HEPES but NO PEN/STREP). The appropriate dilution factor will depend on the particular stock, but should be calculated using the standard curve generated in section 3.1 to give a concentration of 500 CFU (equivalent to a TTP of approximately 8.5 days) per 180µl of media for each co-culture well required.\n\nNote: If stock is highly concentrated, the stock should be diluted in several steps (e.g. serial 1:10 dilutions) to avoid pipetting very small volumes.\n\nNote: If other mycobacterial species or strains are used, the optimum multiplicity of infection (MOI) for each strain should be determined prior. For an example MOI optimisation experimental design, please refer to Zelmer et al.32.\n\n3.2.11 Add 180µl (containing 500 CFU) of the BCG final preparation to each sample well.\n\n3.2.12 Incubate the 48-well plates in a CO2 incubator at 37°C for 96 hrs (4 days).\n\n3.2.13 Supplement one MGIT tube with 800µl PANTA/enrichment to produce supplemented Middlebrook 7H9. Decant the contents into a fresh falcon tube for use in step 3.2.15.\n\n3.2.14 Supplement 2 further MGIT tubes with 800µl PANTA/enrichment. These are the direct-to-MGIT inoculum controls.\n\n3.2.15 Add an equal volume (180µl) of diluted BCG Pasteur prepared in step 3.2.10 to each of the 2 direct-to-MGIT controls. Using the extra supplemented Middlebrook 7H9 produced in step 3.2.13, make up the added volume to 500µl (so if 180µl of BCG preparation is added, add an additional 320µl of supplemented Middlebrook 7H9). Invert to mix, scan the barcode and place on the BACTEC MGIT machine. Refer to section 3.1.8 and section 3.1.10 for obtaining results.\n\nThe effect of A) time from thawing to inoculation and B) de-clumping method for mycobacterial stock on BACTEC MGIT time to positivity (TTP) were determined using in-tube co-cultures of human PBMC and BCG Pasteur. For A), points represent the mean of n=2 duplicate co-cultures with the standard error of the mean (SEM). For B), points represent n=6 individual replicate co-cultures, boxes indicate the median value with the interquartile range and whiskers indicate the minimum and maximum values. A Kruskal-Wallis test was performed with a Dunn’s multiple comparisons test, where indicates a p-value of <0.05, indicates a p-value of <0.01, and * indicates a p-value of <0.001.\n\nThe NHP PBMC direct MGIA was conducted using either autologous serum or plasma for n=7 macaques. Co-cultures were performed in duplicate where sufficient numbers of cells were recovered. The solid green line indicates the mean difference between measurements and the dotted red line indicates the upper and lower limits of agreement (mean difference ± 1.96 standard deviation of the difference) with red vertical bars showing the 95% confidence intervals for the limits of agreement.\n\nThe effect of heat inactivating (HI) pooled human serum or of collecting blood in EDTA tubes was assessed using in-tube co-cultures for n=6 human PBMC. Points represent individuals with the mean of two replicate co-cultures, boxes indicate the median value with the interquartile range and whiskers indicate the minimum and maximum values. A Friedman test with Dunn’s correction for multiple comparisons was used where ** indicates a p-value of <0.01. TTP = MGIT time to positivity, HI = heat inactivated.\n\n3.3.1 Before harvesting co-cultures, supplement 1 MGIT tube per culture well with 800μl PANTA enrichment and label.\n\n3.3.2 Pipette the cultures in the well up and down three times, collect the liquid and transfer to a 2ml screw-cap tube.\n\n3.3.3 Microcentrifuge tubes at 15,300 g for 10 minutes.\n\n3.3.4 Add 500μl of sterile, tissue culture-grade water to each well, and incubate at room temperature for at least 5 minutes.\n\n3.3.5 Remove 500μl of supernatant from the 2ml tubes, ensuring the pellet remains intact. Supernatant can be discarded unless required for later cytokine analysis.\n\nNote: Pellets appear as a small ‘smudge’ and are easily disturbed; particular care should be taken during this step to avoid disturbing the pellet.\n\n3.3.6 Pipette the water in the wells up and down ~8 times to detach monocytes that have attached to the bottom of the well (avoid forming bubbles as far as possible) and completely remove the water from the well, transferring it to the corresponding tube containing the cell/BCG pellet.\n\n3.3.7 Pulse vortex for 1–2 seconds, and add all of the sample from the 2ml tube to the corresponding MGIT tube. Use some media from the MGIT tube to rinse the 2ml tube and add back to the same MGIT tube.\n\n3.3.8 Invert all MGIT tubes to mix and place on the BACTEC MGIT instrument until positivity is reached (see section 3.1.8).\n\n3.4.1 Record TTP for control and sample MGIT tubes and convert to log10 CFU values using the corresponding stock standard curve generated in section 3.1.\n\nNote: Stored samples should be batched as far as possible; if more than one batch or experiment is to be directly compared, each sample read-out should be normalised to its corresponding direct-to-MGIT control (by subtracting the log10 CFU of the control from the log10 CFU of the sample) to account for differences in input inocula between assay runs.\n\n\n4.0 Results\n\nA range of optimisation and characterisation studies were conducted during the development of the direct MGIA. While some of these were performed using human cells (where specified) for reasons of ethics and sample availability, outcomes have informed the development of the macaque assay protocol.\n\n4.1.1 Mycobacterial stock. In order to minimise the variability associated with low-titre mycobacterial inocula, two stock parameters were assessed: a) time from thawing to inoculation and b) de-clumping methods. Mycobacteria were thawed and added to duplicate human PBMC co-cultures every hour for 5 hours after resting on the bench at room temperature. Mycobacterial viability showed a progressive, albeit modest, decrease for the first 3 hours, before beginning to recover at 4 hours (Figure 5A). Six methods of de-clumping were compared using 6 replicate in-tube co-cultures containing cells from the same human sample for each method: 1) vortexing for 5 minutes on the highest speed, 2) standing on the bench for 5 minutes to allow clumps to settle and then removing only the top fraction, 3) centrifuging at a low speed to bring clumps down and then removing only the top fraction, 4) sonicating for 2 minutes, 5) vortexing with 1mm borosilicate solid-glass beads for 2 minutes, and 6) syringing through a 5µM cellulose acetate filter.\n\nMycobacterial recovery was highest using the glass beads method, while other methods (particularly centrifuging and filtering) resulted in some loss of mycobacteria. BCG growth was significantly higher (lower TTP) following vortexing with glass beads compared with centrifuging or filtering (p=0.0002, Δ mean TTP = 90 hours; and p=0.008, Δ mean TTP = 83 hours respectively; Kruskal Wallis with Dunn’s multiple comparisons test, p=0.0002, Figure 5B). Reproducibility between replicates was greatest for glass beads and filtering (coefficient of variation, CV = 2.2% and 1.2% respectively), and poorest for vortexing (CV = 13%). Based on these findings, we recommend that mycobacterial stocks suffering from clumping should be vortexed with sterile 1mm borosilicate glass beads (Sigma Aldrich, UK) for 2 minutes prior to inoculation, and that inoculation should be conducted as soon after thawing as possible.\n\nM.tb is the pathogen of interest and may be used as the mycobacterial inoculum in the direct NHP MGIA; indeed we have demonstrated a BCG-vaccine induced effect and a correlation with protection from in vivo mycobacterial challenge using whole blood from macaques co-cultured with M.tb H37Rv37. However, a similar MGIA kinetic was observed whether BCG or M.tb was used as the inoculum, with a correlation between the two measures37. Such an association has also been reported in the human direct MGIA28,44. In the NHP direct MGIA, we observed improved intra-assay reproducibility using M.tb compared with BCG which may have improved ability to detect a correlation with in vivo protection. However, using BCG increased sensitivity to observe a vaccine response (post-vaccination growth – baseline growth), and it was this measure that correlated most consistently with in vivo protection in our studies37. On balance, we chose to pursue assay development using BCG to aid transferability by negating the need for high containment level laboratory facilities.\n\n4.1.2 Co-culture conditions\n\n4.1.2.1 Whole blood vs. PBMC\n\nWhile whole blood may represent the most ex vivo sample, we previously reported a correlation between mean corpuscular haemoglobin (Hb) and mycobacterial growth in the human direct MGIA35. Furthermore, addition of either Hb or ferric ammonium citrate to both human and macaque PBMC MGIA co-cultures enhanced mycobacterial growth, whereas the addition of the iron chelator deferoxamine reduced it35. Taken together, these data indicate an association between Hb/iron and mycobacterial growth, likely via the heme iron uptake pathway45. This effect is particularly pertinent in preclinical models such as the macaque, where blood collections can perturb Hb levels. Indeed, while levels remained within the normal range for the species, we observed a significant decrease in Hb concentration at 4 and 8 weeks relative to baseline following a fortnightly blood collection regimen in rhesus macaques35. Thus, while the direct whole blood MGIA may be appropriate to studies that use infrequent longitudinal sample collections or where variation in clinical parameters including Hb levels form part of the overall response, it may confound measures of vaccine-induced control of mycobacterial growth and reduce sensitivity to detect a vaccine response.\n\nIn humans, the direct PBMC MGIA demonstrated a stronger primary vaccine effect and greater reproducibility over repeated baseline bleeds compared with whole blood, most likely due to the evaluation of longitudinal PBMC samples in one batch30. Ability to batch samples in this way also improves logistical feasibility and transferability, particularly to institutes without immediate access to a BACTEC MGIT instrument. Furthermore, the use of cryopreserved cells enables additional retrospective studies of samples from historical NHP vaccine studies for validation and exploratory work, thus reducing the number of animals used. We therefore focussed our assay development efforts on the PBMC compartment. Importantly, we previously reported a significant influence of penicillin-streptomycin (P/S) antibiotics on mycobacterial growth if included in the culture medium during thawing and the subsequent cell rest period for human cells30. Optimisation experiments using 1×106 cells from n=3 volunteers in duplicate and a BCG inoculum of ~800 CFU in the in-tube MGIA confirmed a pronounced inhibitory effect of P/S if present in the culture medium post-thawing but not pre-freezing (mean TTP with P/S pre-freezing and post-thawing = 262 hours, STDEV = 26; P/S post-freezing only = 281 hours, STDEV = 7.4; P/S pre-freezing only = 113 hours, STDEV = 4; no P/S = 109 hours, STDEV = 3.6). Although penicillin has no reported activity against mycobacteria, streptomycin is a broad-spectrum bactericidal drug used as a first-line treatment for TB46. Uptake of streptomycin into human cells does occur, where it is sequestered in lysosomes and redistributed into the cytosol and concentrated47. Therefore it is likely that, despite washing cells following the cell rest, residual or retained streptomycin remained present in the co-culture. We therefore recommend the use of P/S in the pre-freezing medium only.\n\n4.1.2.2 Multiplicity of infection\n\nWe previously demonstrated that reducing the MOI by increasing cell number rather than reducing mycobacterial inoculum increases ability to detect a BCG vaccine induced response using the NHP direct MGIA37. This was consistent with findings using both the mouse and human direct MGIAs29,32. We also showed that repeatability and ability to detect a vaccine induced response is improved by co-culturing in static 48-well plates compared with rotating screw-cap tubes37, again reflecting findings in other species29,34. Based on these observations, the limitations of cell availability, and to ensure consistency with the equivalent human assay29, we recommend the conditions of 3 × 106 cells co-cultured in 48-well plates with 500 CFU BCG as described in section 3.0. However, an alternative protocol using 1 × 106 cells in sealed, rotating 2ml screw-cap tubes has been successfully applied in humans, and used in the NHP model to demonstrate improved control of mycobacterial growth following M.tb infection29,36. Some researchers consider that the in-tube protocol may be applied where cell number is limiting and biological effects strong, and can be used to further dissect the mechanism of mycobacterial growth control29,48. Details of this alternative method and the associated protocol may be found in the report of optimisation and standardisation of the human direct MGIA29.\n\n4.1.2.3 Serum\n\nWe recommend the addition of autologous time-point matched serum to co-cultures to resemble ex vivo conditions as closely as possible and ensure that any effects of vaccination mediated by serum factors are taken into account. We recently demonstrated that the addition of autologous serum contributes to improved control of mycobacterial growth following BCG vaccination in the human direct PBMC MGIA [Bitencourt et al. submitted49]. Using autologous serum also has the 3Rs benefit of not using foetal bovine serum (FBS) which has ethical implications50. We titrated the serum concentration using in-tube human PBMC co-cultures (n=4), and found that mycobacterial growth was similar when adding 5, 10 or 20% serum (mean TTP = 285, 257 and 316 hours respectively), but increased when serum was at a concentration of 30% (TTP = 180 hours). While 5–20% is a standard serum concentration for cell culture, 30% may be detrimental to cell viability, allowing mycobacteria to proliferate unchecked.\n\nDue to limitations regarding the maximum blood volume permitted for collection from macaques, plasma may be a more feasible alternative to serum. As specific antibodies are likely the main component of serum contributing to control of mycobacterial growth in the MGIA, we compared levels of PPD-specific IgM, IgG and IgA between serum and plasma from matched animals at baseline. In all cases there was a strong correlation, although serum contained modestly but significantly higher levels of specific antibodies at most time-points measured [Bitencourt et al. submitted49]. We therefore compared the use of autologous serum vs. autologous plasma in the direct NHP MGIA co-culture (n=7 animals), in which other components such as complement factors may also contribute to functional control of mycobacterial growth, and observed an intraclass correlation coefficient (ICC) of 0.58 (moderate agreement). As shown by Bland-Altman analysis relating the difference between paired measurements to the mean of the pair, there was minimal bias between the two methods (mean bias = 0.025). Furthermore, all samples were within the 95% limits of agreement (the interval of 1.96 standard deviations of the measurement differences either side of the mean difference), which extended from -0.20 (95% CI, -0.50 to -0.13) to 0.25 (95% CI, 0.18 to 0.55) log10 CFU (Figure 6). Although the sample size was small and there is some in inherent intra-assay variability, this suggests that plasma may be substituted where serum is unavailable or limited in volume, but we do not recommend using the two samples interchangeably within a single experiment or direct comparison.\n\nThe effect of heat inactivating serum was assessed by measuring mycobacterial growth at the end of in-tube n=6 human PBMC co-cultures. Mycobacterial growth was lower when co-cultures contained serum that had been heat-inactivated compared with serum that had not been heat inactivated, but this was not statistically significant by Friedman with Dunn’s correction for multiple comparisons (Δ mean TTP = 24 hours; Friedman with Dunn’s correction for multiple comparisons, Figure 7). It has been reported that heat inactivation of serum decreases uptake of mycobacteria into monocytes due to the destruction of complement51. As monocytes provide the target host cell for mycobacterial survival and replication, a decrease in monocyte invasion may lead to decreased mycobacterial growth. Finally, we compared serum/plasma separated from blood collected in either serum clot-activator or Ethylenediaminetetraacetic acid (EDTA) vacutainers. Adding plasma separated from an EDTA vacutainer to the MGIA co-culture resulted in significant inhibition of mycobacterial growth (p=0.003, Δ mean TTP = 68 hours; Friedman with Dunn’s correction for multiple comparisons, Figure 7). EDTA has been shown to have anti-tubercular activity and has even been suggested for potential use in treatment of drug-resistant TB52. Based on these findings, we recommend that autologous serum/plasma should be added to a final concentration of 20%, should not be heat-inactivated and should not be collected in vacutainers containing EDTA.\n\n4.1.3 Day 4 processing. At the end of the 96-hour co-culture period, cells are lysed to release intracellular mycobacteria. We previously compared mycobacterial recovery under 5 different cell lysis conditions using the human in-tube direct PBMC MGIA: 1) none, 2) sterile water, 3) PBS with Tween 20, 4) 0.2% Saponin, and 5) 0.067% Sodium Dodecyl (lauryl) Sulfate (SDS) across three different sites. BCG recovery was comparable across conditions at all sites29. While the cell lysis step can thus be omitted for the in-tube protocol, it must be included in the recommended 48-well plate protocol to ensure that mycobacteria are released from monocytes that have adhered to the well surface; we suggest the use of sterile water to maximise transferability.\n\n4.1.4 Characterisation of intra- and inter-assay reproducibility. We previously characterised the repeatability of the direct NHP MGIA at 3 different sites. The median CV between replicate co-cultures was 2.69% (range 0.59 to 6.12%, n=8), 1.67% (range 0.78 to 8.52%, n=5) and 2.71% (range 0 to 7.33%, n=5) at sites 1, 2 and 3 respectively. The ICC values were 0.90 (‘almost perfect’ agreement), 0.34 (‘fair’ agreement) and 0.95 (‘almost perfect’ agreement) respectively37. A single sample set (n=8) was assayed on two separate occasions at the same site to assess inter-assay precision. The median CV between assay runs was 6.83% (range 2.13 to 7.76%) with an ICC value of 0.80 (‘substantial’ agreement). While there was a strong consistency agreement, mycobacterial growth was systematically higher (indicated by a shorter TTP) in run 2. The most likely cause is a difference in inoculum due to differences in titre or viability between mycobacteria stock vials. However, as shown by Bland-Altman analysis, the bias was not fully compensated by normalising growth using the direct-to-MGIT control (mean bias = 0.39). We thus recommend assaying all samples from different treatment groups or across a longitudinal time-course in a single batch. It should be noted that all samples between the two runs were within the 95% limits of agreement, which extended from 0.12 (95% CI, -0.19 to 0.21) to 0.66 (95% CI, 0.58 to 0.97) log10 CFU37, but further work is required to achieve absolute agreement.\n\nThe biological relevance of the MGIA as a surrogate measure of vaccine efficacy can only be confirmed by comparing outcomes with levels of protection following in vivo mycobacterial challenge or infection. Similar assessments have been conducted of the malaria growth inhibition assay in relation to protection from controlled malaria infection in NHPs and humans15,53–56. This has previously been achieved for the human and murine direct MGIAs at the group level22,31,33,57. However, validation at an individual level would be more stringent given the variability in BCG-induced protection between individuals and animals58,59. We recently described an association between mycobacterial growth in the direct PBMC MGIA and outcome of in vivo intradermal BCG infection at the individual level in humans33. BCG was used in this study as a potential surrogate challenge agent for virulent M.tb, which cannot ethically be used in human infection studies60. The NHP model provides an opportunity to validate the assay against direct measures of protection from M.tb as well as BCG infection, allowing greater confidence in the relevance of the human assay such that preclinical models may ultimately be replaced in some settings.\n\nAs previously reported, we used samples from BCG vaccinated NHPs across four different studies to evaluate biological validity of the NHP MGIA37. In the first study, there was a significant correlation between M.tb growth in the whole blood MGIA at the peak of response and the number of BCG CFU recovered from the axillary lymph nodes following in vivo BCG challenge. There was a more pronounced association between MGIA vaccine response (post-vaccination growth – baseline growth) and lymph node CFU. MGIA vaccine response at the peak time-point also correlated with multiple measures of protection following in vivo M.tb challenge in a further two studies37. This suggests that the magnitude of vaccine response relative to baseline (which is akin to fold change and captures more information in a single measure) is a more representative measure of in vivo protection than absolute inhibition at a given time-point. This correlation between MGIA outcome and measures of protection from in vivo challenge with either BCG or M.tb at an individual animal level affords confidence that the assay is measuring a biologically meaningful response, although further validation is required alongside ongoing in vivo studies.\n\n\n5.0 Discussion\n\nOne of the objectives when developing the direct MGIA was to provide an assay that was, technically and logistically, as simple as possible to maximise reproducibility and transferability28. In the absence of a validated correlate of protection, we also chose not to include stimulation or expansion steps to avoid biasing, or over-representing certain aspects of, the immune response. We previously sought to transfer and harmonise the protocol defined here to ensure that the 3Rs impact is maximised and that comparable information can be extracted from ongoing and future studies of different preclinical vaccine candidates across organisations. As recommended by Smith et al., we conducted side-by-side operator training at end-user institutes61, and then assessed reproducibility (variation between multiple determinations of a single sample analysed at different laboratories or sites62) by conducting inter-site comparisons between sites 1 and 2 and sites 1 and 3 using two shared sample sets. Between sites 1 and 2, the median CV was 14.19% (range 11.57 to 17.29%, n=7) with an intraclass correlation coefficient (ICC) value of 0.57 (‘moderate’ agreement). Between sites 1 and 3, the median CV was 3.17% (range 0.39 to 8.62%, n=8) with an ICC of 0.83 (‘almost perfect’ agreement)37. The comparison between sites 1 and 2 resulted in lower inter-site reproducibility, which may have been due to the more homogeneous sample set used which had similar levels of growth control across animals. We therefore selected a sample set with a broader dynamic range for the comparison between sites 1 and 3, and observed a close mirroring in the pattern of control37.\n\nOur reproducibility values were comparable to those reported for the human PBMC MGIA29 and were well within the 50% limit of acceptable variation suggested by Tuomela et al. for the measurement of a bacterial target in a cell-based assay62. However, we did observe a systematic difference in the site 1–2 comparison. Again, normalising growth values using the corresponding direct-to-MGIT control did not fully compensate for this bias and further work is required to achieve absolute agreement37. However, all samples were within the 95% limits of agreement, which extended from -0.61 (95% CI, -0.89 to -0.54) to -0.21 (95% CI, -0.27 to 0.07) log10 CFU for the site 1–2 comparison and -0.26 (95% CI, -0.70 to -0.15) to 0.49 (95% CI, 0.37 to 0.93) log10 CFU for the site 1–3 comparison. Importantly the delta between the highest and lowest values was consistent between sites, and given that the magnitude of vaccine response (post-vaccination growth – baseline growth) appears to be the most relevant measure as a surrogate of protective efficacy, systematic differences may be less problematic37. The delta between baseline and post-vaccination time-points, or between vaccinated and unvaccinated animals, should thus be considered in comparisons of vaccine efficacy measured at different sites rather than absolute growth values.\n\nThe main barrier to uptake of this assay by other potential end-users is the requirement for a BACTEC MGIT machine and the cost of associated reagents. While we recommend this quantification system as a faster, simpler, more sensitive and more objective alternative to CFU plating on solid agar, Kolibab et al. have demonstrated a highly significant linear inverse correlation between BACTEC MGIT TTP and CFU on solid agar following a 7 day MGIA using mouse splenocyte co-cultured with bone marrow macrophages63. It may therefore be possible to use traditional colony counting in resource-limited settings. That said, the BACTEC MGIT machine is a widely-used TB diagnostic tool available in most hospitals worldwide and many academic medical research groups have indirect access. Furthermore, using cryopreserved PBMC permits the batching of samples which improves logistical feasibility for those with limited MGIT access compared with whole blood assays, which must be run in real-time at multiple time-points. An additional potential barrier to uptake of MGIAs is access to high containment level facilities for the handling of virulent M.tb. For this reason, we focussed our optimisation work around the use of BCG as a surrogate agent of in vitro infection as discussed in section 4.1.1.\n\nThe relative simplicity of the direct MGIA method described makes it highly translatable across host species and compartments28. We have demonstrated optimisation and application of the assay using splenocytes31,32,43 and, more recently, lung cells64 from mice. Applying the assay in place of M.tb challenge experiments locally has downgraded the severity of many of our murine TB vaccine studies from ‘Moderate’ to ‘Mild’ as vaccination is the only in vivo procedure required. Other groups have also reported use of the murine assay34,57. Attempts to adapt the assay for use in the bovine model have, however, been less successful65, suggesting that translation may not be straightforward in all cases. In humans, we have optimised and harmonised the direct MGIA as part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium29, applied it to demonstrate a BCG vaccine effect30, and validated it against protection from in vivo experimental BCG infection33. Studies by our group and others demonstrate how the direct MGIA may be employed to address different aspects of TB research including clinical studies of TB patients44,48,66,67, coinfections68, and underlying immune mechanisms of protection48,69–73. Indeed, the direct NHP MGIA has also been applied to demonstrate improved control of mycobacterial growth following M.tb infection36, consistent with findings in recently M.tb-infected humans44,48. Beyond TB, we have recently adapted the assay for use with other pathogens including S. aureus and K. pneumoniae to explore the potential non-specific effects of BCG vaccination in humans [Wilkie M and Tanner R, unpublished data].\n\nBased on our experience of standardisation and harmonisation of the NHP MGIA, we recommend that repeatability between replicate co-cultures and precision between different runs of the same samples should be below 10% CV and above 0.5 ICC. Ideally an inter-site comparison between the developer and end-user site should be conducted using a shared sample set, with a reproducibility cut-off of below 15% CV and above 0.5 ICC. Bland-Altman analyses for both inter-assay and inter-site comparisons allowed us to define limits of agreement (as reported in section 4.1.5 and section 5.1), which may be considered estimates of population parameters, although it should be noted that the systematic biases described will influence these values. Comparisons of standard curves from a common stock between sites would also aid confidence in initial assay transfer. The ultimate test of acceptance is conducting the NHP MGIA alongside one or more in vivo mycobacterial infection stud(ies) and demonstrating a significant association between outcomes. As BCG is currently the only licenced TB vaccine, ability to detect a BCG vaccine-induced response is the benchmark for assessing correlates of protection, and could be used in this context (using samples where BCG is known to have conferred protection in vivo). A more stringent measure would be correlating MGIA outcomes with measures of in vivo protection mediated by BCG and other TB vaccine candidates at an individual animal level, as we have previously described for BCG vaccination37.\n\n5.4.1 Scientific benefits. Broadly speaking, a reliable and validated MGIA for use with samples from immunised NHPs would permit high-throughput cost-effective evaluation of vaccine candidates, and down-selection of those going forward into in vivo efficacy testing; this would ultimately expedite the development of a much-needed effective TB vaccine. The direct MGIA also provides a tractable system for the assessment of immune mechanisms underlying the control of mycobacterial growth; manipulation of immune parameters in this way (e.g. cell depletions) is often not logistically or ethically feasible in vivo. Findings may further inform our understanding of protective immunity from TB and thus direct improved vaccine design as well as development of diagnostic and therapeutic tools. The NHP MGIA in particular offers the opportunity to biologically validate the assay through correlation with direct measures of protection from in vivo M.tb challenge on an individual animal basis. This is not possible using mice (where animals must be euthanised for the splenocyte MGIA and can therefore only be correlated by group) or humans (where M.tb challenge is not ethically viable). Such validation allows bridging to use in target species including humans where direct measures of protection cannot be obtained.\n\n5.4.2 3Rs benefits. The process of early testing of TB vaccine candidates in NHP models could be refined by using the MGIA in place of in vivo infection with pathogenic M.tb. Furthermore, the number of NHPs used in TB vaccine testing and associated immunology studies could be reduced, as the MGIA allows:\n\na) Testing of multiple conditions (for example different mycobacterial clinical isolates and immunological mechanisms) using cells from a single group, rather than requiring multiple groups of animals.\n\nb) Down-selection of vaccine candidates at an early stage of development such that fewer go forward to in vivo efficacy testing.\n\nUltimately, biological validation in NHPs allows bridging of the assay to use in target species including humans which may replace the use of preclinical models in some settings.\n\n\nData availability\n\nFigshare: NHP MGIA methods optimisation experiments, https://doi.org/10.6084/m9.figshare.14040074.v274.\n\nFigshare: ARRIVE checklist for ‘The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells’, https://doi.org/10.6084/m9.figshare.14040074.v274.\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 Aeras for supporting the early MGIA development work and providing and distributing the standardised BCG Pasteur stock.\n\n\nReferences\n\nWHO: World Health Organisation Global tuberculosis report 2020. 2020. 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PubMed Abstract \| Publisher Full Text\n\nKolibab K, Yang A, Parra M, et al.: Time to detection of Mycobacterium tuberculosis using the MGIT 320 system correlates with colony counting in preclinical testing of new vaccines. Clin Vaccine Immunol. 2014; 21(3): 453–455. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPainter H, Prabowo SA, Cia F, et al.: Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells. Sci Rep. 2020; 10(1): 3311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPepponi I, Khatri B, Tanner R, et al.: A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development. Tuberculosis (Edinb). 2017; 106: 118–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaguma R, Penn-Nicholson A, Smit E, et al.: Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population. PLoS One. 2017; 12(9): e0184563. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee H, Kim J, Kang YA, et al.: In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection. Front Immunol. 2019; 10: 896. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO'Shea MK, Fletcher TE, Muller J, et al.: Human Hookworm Infection Enhances Mycobacterial Growth Inhibition and Associates With Reduced Risk of Tuberculosis Infection. Front Immunol. 2018; 9: 2893. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaranbhai V, Fletcher HA, Tanner R, et al.: Distinct transcriptional and anti-mycobacterial profiles of peripheral blood monocytes dependent on the ratio of monocytes: lymphocytes. EBioMedicine. 2015; 2(11): 1619–1626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZelmer A, Stockdale L, Prabowo SA, et al.: High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis [version 2; peer review: 2 approved]. F1000Res. 2018; 7: 296. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcNeill E, Stylianou E, Crabtree MJ, et al.: Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin. Nat Commun. 2018; 9(1): 5409. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen T, Blanc C, Eder AZ, et al.: Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction. J Infect Dis. 2016; 214(2): 300–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith SG, Zelmer A, Blitz R, et al.: Polyfunctional CD4 T-cells correlate with in vitro mycobacterial growth inhibition following Mycobacterium bovis BCG-vaccination of infants. Vaccine. 2016; 34(44): 5298–5305. PubMed Abstract \| Publisher Full Text\n\nTanner R: NHP MGIA methods optimisation experiments. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14040074.v2" }	[ { "id": "82506", "date": "15 Apr 2021", "name": "Amanda J. Gibson", "expertise": [ "Reviewer Expertise Innate Immunology", "Mycobacteriology" ], "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 in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells by Tanner et al. describes the method for the use of a direct MGIA assay using NHP cells and autologous serum acquired from vaccination studies. Particular highlights are the use of the described assays to reduce the number of NHPs used in the TB vaccine research pipeline as well as negating the use of virulent M.tb challenge (thereby improving lifetime welfare of NHPs) with the added benefit of investigating the intricacies of immune cell interactions in post-vaccination responses. Method is well described, both in preceding rationale and in a technical stepwise fashion such that the reader may easily replicate. To enhance transferability of the method, the authors suggest alternative components to support completion of the method in more limited laboratory settings. Key points within the method that enhance reproducibility are also highlighted with extensive notes to assist the reader in reproducing the method with repeatable results.\nWhile the method is well described and clear such that replication by others would be possible, there are some minor improvements (listed below) that may be considered to further support the reader in adopting the MGIA within another laboratory environment.\nTable 2 – indicate glucose supplementation level of RPMI 1640 formulation and ensure formulation number is correct for both RPMI entries.\n\nSection 2.4 – take care to include spaces between numbers and units e.g. 2mM should read 2 mM throughout the method sections, in addition please take care to remain consistent with time notation; use of min, minutes, hr, hours, h are used interchangeably throughout the methods sections and graphical representations. Should addition of sodium pyruvate be included here as in Section 3.2?\n\nSection 3.1.2 – specifies sterile PBS, but Figure 2 describes PBS-Tw80.\n\nSection 3.1.9 – this describes using Miles and Misra spotting for the determination of CFU – Table 3 specifies the use of 60 mm petri dishes, is this correct? 3 x 20 ul spots on 60 mm plates is technically challenging without pre-drying of agar plates to ensure robust surface tension of dilution spots to prevent spreading. Guiding the reader to an appropriate reference of this CFU technique may assist those not familiar with Miles and Misra spotting for CFU counting.\n\nFigure 2 – please describe the significance or meaning of the red asterisk after BCG.\n\nSection 3.2.2 – please specify the concentrations used for L-glutamine and sodium pyruvate.\n\nSection 3.2. 7 – 25mM HEPES in underlined.\n\nFigure 3 – ensure centrifuge speeds are consistent with manuscript text, define R10 media.\n\nSection 3.2. 10 – PEN/STREP is capitalised.\n\nSection 3.2. 15 – should the added volume be made to 600 ul not 500 ul to remain consistent with other controls and samples?\n\nFigure 4 – ensure centrifuge speed notation is consistent with manuscript text.\n\nSection 4.0 Results – second sentence use of “outcomes” is not clear.\n\nSection 4.1.2.1 – is there a word missing between most and ex vivo in the first sentence e.g. used?\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": [ { "c_id": "7148", "date": "23 Sep 2021", "name": "Rachel Tanner", "role": "Author Response", "response": "Many thanks for the very helpful comments, which have resulted in a corrected and improved version of the paper. Table 2 – indicate glucose supplementation level of RPMI 1640 formulation and ensure formulation number is correct for both RPMI entries. The glucose supplementation level of RPMI-1640 (1.8-2.2 g/l) has now been added and the formulation number has been corrected to RPMI-1640 for both entries. Section 2.4 – take care to include spaces between numbers and units e.g. 2mM should read 2 mM throughout the method sections, in addition please take care to remain consistent with time notation; use of min, minutes, hr, hours, h are used interchangeably throughout the methods sections and graphical representations. Should addition of sodium pyruvate be included here as in Section 3.2? Spaces between numbers and units have now been added and time notation made consistently longhand throughout the text and figures. Sodium pyruvate and its concentration has now been added where relevant. Section 3.1.2 – specifies sterile PBS, but Figure 2 describes PBS-Tw80. Tween 80 has been removed from Figure 2 and the reagents list, as this should read sterile PBS only. Section 3.1.9 – this describes using Miles and Misra spotting for the determination of CFU – Table 3 specifies the use of 60 mm petri dishes, is this correct? 3 x 20 ul spots on 60 mm plates is technically challenging without pre-drying of agar plates to ensure robust surface tension of dilution spots to prevent spreading. Guiding the reader to an appropriate reference of this CFU technique may assist those not familiar with Miles and Misra spotting for CFU counting. Table 3 has been corrected to specify 100mm petri dishes, and more information on the plating method including a reference to Miles and Misra spotting has been added to section 3.1.9. Figure 2 – please describe the significance or meaning of the red asterisk after BCG. This asterisk has been removed to prevent confusion. Section 3.2.2 – please specify the concentrations used for L-glutamine and sodium pyruvate. The concentration for L-glutamine (2 mM) and sodium pyruvate (1 mM) have now been added. Section 3.2. 7 – 25mM HEPES in underlined. This has been corrected to remove the underlining. Figure 3 – ensure centrifuge speeds are consistent with manuscript text, define R10 media. These have been amended to ensure consistency between figures and text in the unit of speed (g), and the reference to R10 in the figure has been removed (it now reads ‘Media + HEPES’). Section 3.2. 10 – PEN/STREP is capitalised. All cases of capitalised PEN/STREP have been amended to lower-case. Section 3.2. 15 – should the added volume be made to 600 ul not 500 ul to remain consistent with other controls and samples? This has now been amended accordingly. Figure 4 – ensure centrifuge speed notation is consistent with manuscript text. This has been amended accordingly to ensure all speeds are in ‘g’. Section 4.0 Results – second sentence use of “outcomes” is not clear. This has been changed to ‘results’ for clarity. Section 4.1.2.1 – is there a word missing between most and ex vivo in the first sentence e.g. used? This has been reworded for clarity." } ] }, { "id": "82511", "date": "04 May 2021", "name": "Delia Goletti", "expertise": [ "Reviewer Expertise Infectious diseases specialist. Scientist studing the immune response to pathogens." ], "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'The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells' by Tanner et al. describes the method for the use of a direct MGIA assay using NHP cells and autologous serum acquired from vaccination studies. The manuscript is well written and the figures are very clear.\n\nThe authors emphasize the use of the reported tests to reduce the number of animal models used in the TB vaccine research pipeline as well as negating the use of virulent M.tb challenge (thereby improving lifetime welfare of the animal) with the added benefit of investigating the intricacies of immune cell interactions in post-vaccination responses.\nThe methodology section is very accurate in a way that the reader may easily perform the test. The authors add key points within the method to enhance the reproducibility.\nThe manuscript is complete.\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": [] } ]	1	https://f1000research.com/articles/10-257
https://f1000research.com/articles/9-261/v1	16 Apr 20	{ "type": "Opinion Article", "title": "Does standard cosmology really predict the cosmic microwave background?", "authors": [ "Hartmut Traunmüller" ], "abstract": "In standard Big Bang cosmology, the universe expanded from a very dense, hot and opaque initial state. The light that was last scattered about 380,000 years later, when the universe had become transparent, has been redshifted and is now seen as thermal radiation with a temperature of 2.7 K, the cosmic microwave background (CMB). However, since light escapes faster than matter can move, it is prudent to ask how we, made of matter from this very source, can still see the light. In order for this to be possible, the light must take a return path of the right length. A curved return path is possible in spatially closed, balloon-like models, but in standard cosmology, the universe is “flat” rather than balloon-like, and it lacks a boundary surface that might function as a reflector. Under these premises, radiation that once filled the universe homogeneously cannot do so permanently after expansion, and we cannot see the last scattering event. It is shown that the traditional calculation of the CMB temperature is flawed and that light emitted by any source inside the Big Bang universe earlier than half its “conformal age”, also by distant galaxies, can only become visible to us via a return path. Although often advanced as the best evidence for a hot Big Bang, the CMB actually tells against a formerly smaller universe and so do the most distant galaxies. While standard cosmology has additional deficiencies, those disclosed here defy rationality and therefore make a more well-founded cosmology indispensable.", "keywords": [ "cosmic background radiation", "cosmology theory", "concordance cosmology", "big bang cosmology" ], "content": "Introduction\n\nIn 1964, Penzias & Wilson (1965) serendipitously discovered the cosmic microwave background (CMB), a thermal radiation with a temperature of 2.7 K. Prior to this, the presence of a cosmic heat bath with a temperature of a few K had already been conjectured by several researchers on various grounds unrelated to the Big Bang (Assis & Neves, 1995). Based on absorption lines of interstellar CN-molecules, McKellar (1940) had suggested a maximum temperature of interstellar space of no more than 2.7 K. Alpher & Herman (1948) and Alpher et al. (1967), who were contemplating thermonuclear reactions in the expanding universe (for historical perspectives see Naselsky et al. (2006) and Alpher (2012), expected a thermal radiation with about 5 K as a residual of a hot Big Bang. In this, they built on Tolman’s studies (Tolman, 1931; Tolman, 1934) of model universes filled with blackbody radiation as a thermodynamic fluid, so that “The model of the expanding universe with which we deal, then, is one containing a homogeneous, isotropic mixture of matter and blackbody radiation” (Alpher & Herman, 1975). They did not really discuss and clarify under which conditions such a state is sustainable in Big Bang models.\n\nWhen Penzias & Wilson (1965) were bothered by the presence of unexpected radiation, another group of scientists (Dicke et al., 1965) did expect it in a hot Big Bang model and was developing an experiment in order to measure it. After asking whether the universe could have been filled with black-body radiation from its possible high-temperature state, they say “If so, it is important to notice that as the universe expands the cosmological redshift would serve to adiabatically cool the radiation, while preserving the thermal character. The radiation temperature would vary inversely as the expansion parameter (radius) of the universe.” This is also what Tolman (1934) said.\n\nDicke et al. (1965) were initially in favor of a model in which the universe expands, slows down and contracts to a minimal size (not necessarily a singularity), for a new cycle to begin, but they concluded that “with the assumption of general relativity and a primordial temperature consistent with the present 3.5°K, we are forced to adopt an open space, with very low density.” (Dicke et al., 1965). They had expected the temperature to exceed 30 K in a closed space.\n\nIn subsequent Big Bang models, the universe expanded from a very dense and opaque initial state in which it was filled with a hot and dense plasma consisting of protons, electrons and photons colliding with these. When the plasma had cooled sufficiently by the expansion of the universe, electrons and protons combined into H atoms. This event is still referred to as “recombination”, although cyclic models had lost support in the late 1990s, when an accelerated expansion suggested itself (within the Big Bang paradigm) in the redshift-magnitude relation of supernovae (Perlmutter, 2012; Riess, 2012; Schmidt, 2012) instead of an expected decelerated one. Only after recombination and decoupling, when the charged particles had been neutralized, the photons could move freely.\n\nIt is now commonly estimated that the universe became transparent about 380,000 years after the Big Bang (Smoot, 2007), when it had cooled to about 3000 K. The thermal radiation is said to have been emitted from a “last scattering surface” (LSS) and to have retained its blackbody spectrum because it expanded adiabatically. Due to the ever continuing expansion, which uses to be ascribed to “space”, the light waves were stretched and their energy density decreased. The wavelength at which the radiation is strongest, which according to Wien's displacement law is inversely proportional to temperature, would have become roughly 1100 times longer since the radiation was emitted (Bennet et al., 2003), while the temperature decreased to the present 2.7 K. Since the 1970s, the presence of this radiation has routinely been advanced as the strongest piece of evidence for a hot Big Bang.\n\nThe idea that the CMB comes directly, although redshifted, from a last scattering surface emerged only after 1965. The followers of Tolman (1934) may still have assumed that the CMB photons we receive have been further scattered on their way to us. In any case, they did not raise the questions of what constitutes the confinement of an expanding universe and which difference the motion or absence of a boundary surface would make. The problem we are concerned with here arose at the latest when these questions were still not raised when the assumption of a directly viewed LSS had made them crucial.\n\nIf one considers the following question, one can easily see that Big Bang cosmology requires the universe to be suitably confined or curved in order for radiation from the LSS to become visible at all.\n\nIf the CMB originated at the last scattering surface\n\nand all matter originated within the region enclosed by this surface,\n\nwhile light escaped from there at c, maintaining this velocity for eons,\n\nand the matter of which we consist left the same region more slowly,\n\nthen, how can it be that we can see the light?\n\nWith these premises, we cannot reasonably be ahead of the light. The ‘flash’ of light from the LSS had a substantial duration, but it must have passed our place very long ago. Now, it could only become visible at our place if the light had been reflected back to us or taken a curved return path of the right length. In a model, this needs to be specified. Before turning to the standard model, which will be shown to be inconsistent, let us first consider a non-reflective “flat” model and then briefly also reflective versions and a positively curved model.\n\nModel 1. In a non-reflective flat Big Bang model (curvature 0), light will escape from the expanding material universe and proceed farther at velocity c. The material universe will be surrounded by an expanding empty region inside a spherical shell that contains radiation, perhaps also cosmic rays, but no ordinary matter. In such a universe, the conditions assumed by Tolman (1931); Tolman (1934) and presupposed by his followers are not permanently retained after last scattering. However, the belief that radiation from a past epoch, named “relic radiation” or “residual radiation”, could permanently fill the whole volume of an expanding, formerly smaller universe even in the absence of a reflective boundary surface or a suitable “curvature” was inherent in the reasoning by Alpher & Herman (1948); Alpher et al. (1967) and Dicke et al. (1965), and it has remained so in the more recent literature, e.g. Peebles et al. (1991) and Peebles (1993). Alpher & Herman (1975) described their expanding universe in retrospect as “one containing a homogeneous, isotropic mixture of matter and blackbody radiation”. This can and should be read as a warning against uncritical adoption, since the authors did not reason about how such a state could maintain itself over time, given the speed difference between radiation and matter. Dicke et al. (1965) stated that “The radiation temperature would vary inversely as the expansion parameter (radius) of the universe”. Their calculation presupposes the radiation to fill their expanding universe permanently. Likewise, Peebles et al. (1991) wrote: “In the standard model, … space was (and is) filled with black-body radiation, the cosmic background radiation”, but the “(and is)” qualifies as a non-sequitur. Correctly and transparently reasoned,\n\nradiation from a past epoch fills, at each instant, only the volume that is traversed by the rays or “future light cone” from that epoch.\n\nAbscissa: comoving distance χ in Glyr. Ordinate: conformal time η in Gyr. V-like golden band: future light cone of last scattering surface (LSS, red horizontal dash close to zero-point, visible directly only from within golden band). Blue Λ-like trace: our past light cone – we at its peak, not in golden band. Region beyond golden band (dotted extension of blue trace) has not come into existence. In standard cosmology, galaxy GN-z11 and fictitious LSS placed in this region nevertheless (the latter at χ ≈ ±46 Glyr). Between dashed vertical lines: confined universe that co-expands with material universe (co-moving diameter constant and equal to that of LSS, mentioned under model 2). So confined, LSS remains permanently visible. Place of GN-z11 in this model and zigzag path to us via 17 reflections also shown. Dotted horizontal lines: see Table 1. Last scattering at η ≈ 0.95 Gyr, t ≈ 0.38 Myr; last visibility of LSS and blackbody conditions at η ≈ 1.9 Gyr, t ≈ 1.95 Myr.\n\nValues based on 5-year WMAP data and ΛCDM model computed using WolframAlpha®.\n\nModel 2. In a flat Big Bang universe that is surrounded by a boundary surface, light can be reflected there. Complete reflection occurs if the impedance of space becomes infinite (or zero) there. If space just loses its existence at an “edge”, the impedance becomes undefined, which is problematic, but the location of the reflective surface is also problematic. In order for the CMB to become visible, the reflection must occur at a certain distance from us, within the future light cone of the LSS. If the reflection occurred at a constant distance from us, this could work in our epoch, but the CMB would not have been visible between our epoch and the time when the direct view of the LSS was lost. If the reflection formerly occurred at a smaller distance, the CMB may have been visible then, but this would have blocked any later view from a larger distance. An elaborate model that avoids this problem and/or describes a view via repetitive reflections at opposite surfaces does not appear to have been proposed.\n\nThe present standard model is in basic respects equivalent to model 2. In it, the expansion is described by the scale factor a(t) = (1 + z)-1, which is applied to co-expanding structures in three dimensions and also to the dimension of time, while\n\nit is disregarded that radiation not only expands in these four dimensions but also escapes from its origin at c\n\nModel 3. In a positively curved Big Bang model (curvature +1), which, reduced by one dimension, can be imagined as the surface of an inflating balloon, the LSS could be visible because these models allow a return path of light. This visibility can be expected to evolve with the expansion factor of the universe from continuous to periodic before finally being lost. Here, we shall not delve into the question under which premises it could be permanent or be lost entirely, because it would require assumptions that are not made in standard cosmology. Instead, when analyses of high-resolution maps of the CMB were found to be compatible with a flat universe (Davis et al., 2007; de Bernardis et al., 2000) rather than with a positively curved one, the flat universe became adopted as the standard. This flatness came unpredicted and posed a “coincidence problem” (Debono & Smoot, 2016). Recently, based on CMB data from the Planck mission, a positive curvature has been argued for (Di Valentino et al., 2019), but this is not a feature of the present standard model.\n\nModel 4. The present standard model (Ryden, 2016; Smoot, 2007) is a confused version of model 1, in which the universe is flat and non-reflective, although the CMB radiation density is calculated as if the material universe was surrounded by a reflective sphere that co-expanded with the LSS, as in model 2. Despite this, which already involves the blunder mentioned there, the reasoning is further confused by accepting without further ado that the CMB we actually see originates from a different place, namely from a spherical surface or shell around the observer’s, i.e., our position, from which it has taken the photons 13.7 Gyr to reach us directly by now. This is distinctly problematic because a flat Big Bang universe in which no reflection occurs contains no points from which it would take so much time.\n\nAs can be seen in Figure 1, the points of origin are located outside the space that was brought into existence by the Big Bang, whose future light cone, from another point of view referred to as the “particle horizon”, delimits this space. In this Figure, “conformal time” (presently η = 46.7 Gyr) and “comoving distance” (in Glyr) have been chosen as coordinates for the clearest illustration of the crucial aspects. Both of these coordinates are nonlinearly expanded, but they give us the same constant light cone slopes of ±1 lyr/yr as in a flat and static spacetime. The apparent places of origin of the CMB, which define a fictitious LSS, are maximally remote, in comoving distance about ±45.7 Glyr farther away from the original LSS, at which the temperature is calculated to have been 3000 K at decoupling, i.e., at t = 380 kyr. In terms of comoving distance, the extension of this surface had then already grown to almost ±1 Glyr, but no more than that. Note that the use of ordinary, unexpanded coordinates would make the place-discrepancy much smaller, but it would not make any difference to what is inside and outside the Big Bang universe.\n\nThe statement that it takes a CMB photon 13.7 Gyr to reach us here and now is just an estimate of the time that has elapsed since its emission. While CMB photons may actually require this time to reach us from their source, and the Universe may well be flat and infinite, a flat and reflection-free Big Bang universe does not provide the spacetime that would be necessary in order to accommodate a ray (a null-geodesic) of the corresponding length. If a ray of this length is to end at us, it must have its origin outside the Big Bang universe. This may well be so, but if this is accepted, the very idea of a Big Bang is untenable and must be rejected. It is irrational to calculate the properties of the CMB on the basis of its origin at a LSS inside a Big Bang universe and simultaneously to admit its origin at a maximally remote place outside the said universe, where the conditions are very different if ascertainable at all. The custom of referring also to this place by the attributes of “last scattering”, “decoupling” etc., which apply only in an expanding universe, is deceptive.\n\nFigure 1 illustrates the relevance of the problem to other observables than the CMB as well: in a flat geometry,\n\nour direct view is limited to events that happened after the universe had attained half its present age in conformal time (at η ≈ 23.35 Gyr).\n\nIn stark contrast to what is traditionally claimed, the CMB actually tells against a formerly smaller universe and so do the most distant galaxies. The visibility of these is hard to reconcile with the idea of a Big Bang. As for the visibility of the CMB, the self-contradiction might disappear if we actually saw mirror images [as in model 2], but in order for galaxies to be seen in this way and the actual isotropy of the CMB to be obtained, the reflector would need to be of all too spectacular stability and flatness - like that required in a telescope of giga-lightyears in length. As mentioned previously, the presence of a reflective confinement would also be incompatible with the cosmological principle and put us not only at a special time but also at a special place in the universe.\n\n\nDiscussion\n\nBecause of the inherent inconsistency of the standard ΛCDM concordance cosmology, here represented by model 4, it does not come as a surprise that “misconceptions and confusions have long been common in papers on cosmology, also in many by renowned authors”, as reported by Davis & Lineweaver (2004). These authors deserve credit for having paid attention to those. However, they did not either notice that early events cannot be seen directly. In proceeding without considering reflections (last passage of their section 3.3), they mistook the intersection between our past light cone and the future light cone of the LSS [where a reflection would need to occur] for “the points from which the CMB was emitted” (Davis & Lineweaver, 2004, p. 101). Although this is not yet beyond the particle horizon of the Big Bang, it would still be off target by half as much as model 4). The confusion arose by equating this particle horizon with the surface of last scattering, which the authors refer to as “our effective particle horizon” (Davis & Lineweaver, 2004). It also disagrees with the caption of their Figure 1, which presupposes model 4 as such.\n\nWhen Tolman (1931) considered “the highly idealized model of a non-static universe filled with black-body radiation as a thermodynamic fluid”, he did not discuss the implications of the large size of the universe and the possible absence of a reflective confinement or its equivalent. It deserves to be noted that the time required for cavity radiation to attain a desired degree of homogeneity (after a sufficient number of reflections) increases in proportion to the linear size of the cavity. In a Big Bang universe, this will even with modest demands take much longer than its age. If there is no boundary surface other than one that recedes at c, we have seen that any old radiation will eventually disappear from view. In a flat and non-reflective Big Bang universe [model 1) above and 4) before confusion], this must happen to the radiation from the original LSS, which, thus, cannot remain visible. The CMB must have a different source, whose identification exceeds the scope of this paper.\n\nWhile the irrationality of the assumption about the visibility of radiation from a past epoch in a Big Bang universe, which was disclosed in The problem, can be clearly seen in a spacetime diagram such as Figure 1, it may be missed if the ordinary coordinates of time and distance are used, especially if a past light cone is shown (in these coordinates shaped like an avocado seed) that continues below t = 1.7 Gyr down to the origin, while it is not made evident that the region it traverses there lies outside the Big Bang universe. For examples see the “avocado seeds” in Davis & Lineweaver (2004), more detailed in Whittle and without any scale under “Manipulating Space-Time Diagrams” in Wright.\n\nThe fact that the irrationality has remained unnoticed by professionals is an instance of the ordinary uncritical passing down of human culture, of languages, myths, etc. from generation to generation. In this wider cultural context, science stands out as an exceptional, more critical endeavor that requires practitioners not simply to accept and adopt what they were taught, but to check the relevant assumptions and doctrines for consistency and tenability and to recheck them when premises and/or relevant knowledge change. This may sometimes fail to happen, especially in cases like this, where the presence of an inconsistency became potentially clear only gradually, here after 1965, when a teaching practice had already established itself since Tolman (1934). This practice appears to have prevented the disclosure of the irrationality, which would likely have become obvious after a fresh look at the facts. It is in line with this that the rejection of the idea of a Big Bang has been blocked in model 4, although the evidence that requires the rejection has been accepted. Such blockage fosters chimera and absurd excuses (“inflation” and a plurality of universes), which have been left out of consideration here. While scientific journals often tolerate speculative ideas like these, it is unfortunate that most of them refuse through prejudice to publish any paper that discredits the currently accepted doctrine within their field from inside. For editors, it is rational to reject such papers right away: these might threat their reputation if later shown to be erroneous. Also for reviewers who lack a critical attitude against the established practice and doctrine, it is a priori inconceivable that the whole community of well-educated professionals, here mainstream cosmologists, could have made the same cardinal blunder. This holds also in cases like the present one, in which the presence of at least one inconsistency is obvious to the uncommitted.\n\nAlthough the deficiencies disclosed here can be judged as completely unacceptable, other ones need to be addressed as well (López-Corredoira, 2017; Merritt, 2017; Spergel, 2015; Traunmüller, 2014; Traunmüller, 2018). Just consider that both Λ (dark energy) and CDM (cold dark matter) have remained in the imaginary realm and so merely represent mythical factors or immunizing tactics (also called “conventionalist stratagems”) that protect a doctrine from empirical falsification (Merritt, 2017). Approaches that rely on such factors are at least excessively speculative, but inconsistencies such as the two revealed here must be desisted from in any discipline that is meant to qualify as rational. In order to progress, one should preferably eliminate any old deficiencies instead of suggesting some fancy new physics that might hide them. One should rather strive for well-foundedness in the physical principles (Traunmüller, 2018) than merely for a rationalized mythology, but it is, of course, even more fundamental to respect reason at all.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nAlpher RA, Gamow G, Herman R: Thermal Cosmic Radiation and the Formation of Protogalaxies. Proc Natl Acad Sci U S A. 1967; 58(6): 2179–2186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlpher RA, Herman RC: On the Relative Abundance of the Elements. Phys Rev. 1948; 74(12): 1737–1742. 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Publisher Full Text" }	[ { "id": "62422", "date": "29 Apr 2020", "name": "Indranil Banik", "expertise": [ "Reviewer Expertise Modified Newtonian Dynamics (MOND) I felt I was able to assess all aspects of the article", "as I work on a non-mainstream cosmological model and need to pay close attention to issues like the cosmic microwave background radiation and cosmology", "which remains a little uncertain in MOND" ], "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 following article claims to raise serious conceptual problems with the standard cosmological model:\n\nI have to recommend that the article be rejected. First of all, the discussion section is offensive - as a researcher working on non-mainstream ideas, I can understand the sometimes difficult struggle when challenging the mainstream paradigm. But to suggest that I am recommending rejection in order to protect my career is extremely offensive, when my career in fact relies on challenging the mainstream view. The referee might like to know this before dismissing my rejection as a sign of anything other than scientific invalidity of his ideas. But I agree that it is occasionally possible for articles to be rejected which are actually correct, because the referee is protecting personal interests. This is certainly not as common as the author makes out, and indeed I have had generally respectful discussions with mainstream cosmologists despite viewing the Universe very differently. For such a (hopefully) polite discussion, the author may like to watch this debate:\n\nhttps://www.eso.org/sci/meetings/2020/Cosmic-Duologues.html\nRegarding the article itself, the main problem is the author has not understood the basics of the Big Bang model in which there is not an explosion in space, but an expansion of space. In this model, the universe is infinite and almost homogeneous at early times. In co-moving co-ordinates, this expansion is cancelled out and you would just see a static Universe. Suppose a flash of light is emitted from every location at the same time. Photons in some region would of course be moving at c, thus leaving through the boundary - but other photons would enter. The number density of photons would thus remain the same in co-moving co-ordinates. There is no inconsistency here. It explains why we expect to measure the same co-moving number density of primordial photons today as there was at the time of last scattering.\n\nRegarding the issue of where the surface of last scattering is, the author should simply consider a photon that has been travelling at c for a Hubble time in a straight line. The result is at some distance, independent of direction - so the surface is a sphere. However, this is not a real surface - it is just the locus of points from which photons will later hit the Earth exactly 13.8 Gyr later. At the time of emission, nothing whatsoever is special about material in this surface. The whole sphere may well be much larger than 380 kly, which is somewhat non-intuitive since the age of the universe was (in this model) only 380 kyr then.\n\nThe author raises an important point about how the Universe was in thermal equilibrium at early times. This is related to the horizon problem, which - as the author points out - is thought to be resolved by inflation. Briefly, the idea is that the Universe was small for an extended period of time, during which it was in causal contact and thus reached thermal equilibrium. The particle horizon then expanded faster than c due to a period of accelerated expansion similar to what we are experiencing now, causing that photons reaching us from different sides of the sky have the same temperature. The author then goes on to unscientifically attack the hypothesis of inflation - indeed, it is well known that this is not confirmed. However, the author does not raise any substantive arguments against the inflation model, and does not propose his own ideas.\n\nOf course, if the mainstream idea was that the Universe was 380 kly wide at some time 13.8 Gyr ago and that every location within it emitted photons at that moment (almost) equally in all directions, then it is clear that these photons would be unobservable now - depending on what happens at the edge. However, the Universe is not thought to have any such edge, and is considered to be infinite. It is certainly the case that light emitted from close to us would no longer be observable, so what we see as the cosmic microwave background (CMB) must have originated rather far away, on the surface of last scattering. The longer we wait, the further away this surface is - though it always corresponds to the same moment in the history of the universe. There will never be a moment when there is no CMB altogether (barring absorption of all its photons), because one can always imagine reversing the arrow of time and integrating a trajectory moving away from us at c in a contracting universe. The photon will have some well-defined location at the moment of last scattering, so this point will be part of the surface of last scattering to such an observer. No special reflective surface is required, and indeed no new assumptions are needed at all to explain the observability of the CMB. Of course, explaining its detailed properties remains a challenge given other constraints e.g. the Hubble tension. But the particular criticism of the standard cosmological model raised by the author is completely erroneous. Therefore, this manuscript must be rejected - even though I do consider that the same is true for the standard cosmological model as a whole, which does indeed contain many hypothetical ingredients that I am not at all a fan of & have publicly spoken against on several occasions e.g. here:\n\nhttps://www.youtube.com/watch?v=PYVC0VtmpDg\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? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [ { "c_id": "5507", "date": "03 Jun 2020", "name": "Hartmut Traunmüller", "role": "Author Response", "response": "Reviewer's comment: I have to recommend that the article be rejected. First of all, the discussion section is offensive - as a researcher working on non-mainstream ideas, I can understand the sometimes difficult struggle when challenging the mainstream paradigm. But to suggest that I am recommending rejection in order to protect my career is extremely offensive, when my career in fact relies on challenging the mainstream view. The referee might like to know this before dismissing my rejection as a sign of anything other than scientific invalidity of his ideas. Author's response: This review (there is no separate referee) emphasizes something other on the preceding lines. It has probably to do with my mentioning, in the next to last passage of the article, of a good reason editors might have for rejecting certain manuscripts prior to review. In the same passage, I suggest a milder, equally rational explanation - one that applies beside editors also to reviewers and definitely also to myself in many analogous situations. Reviewer's comment: But I agree that it is occasionally possible for articles to be rejected which are actually correct, because the referee is protecting personal interests. This is certainly not as common as the author makes out, Author's response: This is common in certain cases, also self-censorship. To make it clear in which cases, I have inserted a reference to Lakatos, who had observed that the “hard core” of “research programmes” (such as mainstream cosmology) is beyond criticism. Reviewer's comment: the main problem is the author has not understood the basics of the Big Bang model in which there is not an explosion in space, but an expansion of space. Author's response: In the next to last passage of the Introduction, in which I summarize the characteristics of a Big Bang model that are essential here, I told that the expansion uses to be ascribed to “space”, i.e., to an expansion of space. An expansion in space was nowhere implied, but at the end of the second passage under Model 4, I have now inserted a parenthetic phrase “or at least empty” after “non-existent” only for telling that this related distinction makes no difference in this context. Author's response to the Reviewer's remaining comments: These have brought me to underline the fact that, in a Big Bang universe, it is not the case that blackbody radiation lost from a region is balanced by an equal amount gained from outside and, above all, to extend the description and analysis of the present standard model (new text under Model 4) as summarized in the \"Update text\". I kindly ask the reviewer for a new evaluation." } ] } ]	1	https://f1000research.com/articles/9-261
https://f1000research.com/articles/9-303/v1	28 Apr 20	{ "type": "Research Article", "title": "Influence of socio-demographic and environmental factors on childhood diarrhea in Cambodia", "authors": [ "Vong Pisey", "Pannee Banchonhattakit", "Vong Pisey" ], "abstract": "Background: Diarrhea is still the leading cause of childhood death worldwide, as well as a major cause for concern in developing countries. This study was conducted to investigate the factors related to childhood diarrhea in Cambodia. Methods: A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014. A generalized linear mixed model was used to analyze the determinant factors of childhood diarrhea. Results: The surveys included 2,828 children, aged 12 to 35 months. The prevalence of diarrhea was 16.44% (95% CI: 14.72%-18.31%). Factors with statistically significant associations with childhood diarrhea in Cambodia were: maternal unemployment, compared with being in employment (AOR = 1.43; 95% CI: 1.14-1.78); the child being male (AOR = 1.25; 95%CI: 1.02-1.53); the presence of unimproved toilet facilities (AOR = 1.17; 95%CI: 1.05-1.31) compared with improved toilet facilities; and unhygienic disposal of children’s stools (AOR = 1.32; 95%CI: 1.06-1.64) compared with hygienic disposal of children’s stools when controlling for other covariates. Both maternal age (one year older; AOR = 0.85; 95%CI: 0.78– 0.93) and child age (one month older; AOR = 0.86; 95%CI: 0.78-0.94) had significant negative associations with the occurrence of childhood diarrhea. Conclusion: Childhood diarrhea remains a public health concern in Cambodia. The probability of diarrhea occurring is shown to be increased by maternal unemployment, the sex of the child being male, lack of provision of improved toilet facilities, and the unhygienic disposal of children’s stools; whereas increasing maternal age and child’s age were associated with a reduced chance of diarrhea occurring. On the basis of these results, we recommend provision of programs focusing on reducing diarrhea through the construction of improved toilet facilities and the promotion of behavior to improve hygiene, specifically targeting younger mothers.", "keywords": [ "Socio-demographic", "environmental", "childhood diarrhea", "generalized linear mixed model", "Cambodia" ], "content": "Introduction\n\nDiarrhea is defined as the passage of loose or watery stools, three or more times each day, or more frequent passage than is normal for an individual1. Diarrhea remains a leading cause of child mortality and morbidity in the world, with an estimated 1.7 billion cases of childhood diarrhea and 525,000 deaths of children under five caused by diarrhea each year. Diarrhea is the second leading cause of death in children under the age of five years1,2. 88% of diarrhea cases globally are attributable to poor water, poor sanitation or poor hygiene3. There is not just the one single factor associated with childhood diarrhea but multiple factors, including unimproved drinking water sources4–7, untreated water8–10, unimproved toilet facilities6,8,9,11, unhygienic disposal of children’s stools12–14, lack of hand washing facilities15,16, type and location of residence11,16, the child’s age4,13,16, the child’s sex (male)13, maternal illiteracy12,13,17, the mother’s occupation9,12, maternal age14,18, wealth index4,19, and whether or not the child is breastfed10,15.\n\nIn 2014, Cambodia still had one of the highest prevalence levels of diarrhea among children under the age of five amongst countries in South-East Asia, at 12.8%20. By comparison, Myanmar had a prevalence of 10.4% in 2015–1621, Malaysia 4.4% in 20167, Laos 6.5% in 201722, Philippines 6.1% in 201723, and Indonesia 14.1% in 201724. According to 2016 data from UNICEF, Cambodia had 5,947 total neonatal deaths, of which 20 were due to diarrhea; 5,248 post-neonatal deaths, of which 672 were due to diarrhea (13%); and 692 deaths of children under five due to diarrhea (6%)25. High rates of diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia, and an estimated 10,000 deaths overall each year26. This demonstrates that diarrhea is the most common cause of death in Cambodian children. According to the Cambodia Demographic and Health Survey (CDHS) 2014, the prevalence of diarrhea among children aged 12 to 35 months was high, which is known to affect for child development and growth20.\n\nIt is of great importance to understand the factors related to the prevalence of diarrhea among children aged 12 to 35 months. There are no existing studies on the association in this age group, and no national studies on childhood diarrhea in Cambodia have yet been published.\n\n\nMethods\n\nThis research project received approval from the Khon Kean University Ethics Committee in Human Research (HE632097). This study uses existing CDHS data and re-analysis was done under the original consent provided by the participants.\n\nThe CDHS 2014 collected data nationally across the country, which is subdivided into 19 province domains. Its sampling frame consisted of 28,455 eligible enumeration areas (EAs), which comprised the 2008 Cambodian General Population Census (GPC). The sample considered any domain with a power allocation, including both urban and rural areas. The stratified sample was selected in two stages. In the first stage, a fixed number of EAs were chosen using probabilities weighted proportional to the size of the EA. In the second stage, 24 and 28 households were picked up from every urban cluster and rural cluster, respectively, through a systematic sampling process with equal probability weighting. 15,825 households, 17,578 women, and 5,190 men were interviewed between the 2nd June and the 12th December 2014; further details can be found in the CDHS 2014 report20. The final sample size comprised 2,828 children aged 12 to 35 months, providing a suitable degree of power (0.9627, 0.9682).\n\nTwo raw CDHS 2014 datasets, comprising household data and children’s data were combined for use in this study. All entries and variables in these datasets were included in this study.\n\nThe prevalence of diarrhea is the dependent variable considered in this study. This is referred to the questionnaire thus: “Has (NAME) had diarrhea in the last 2 weeks?” The dichotomous variable childhood diarrhea can take values “1” representing a response of “yes” or “0” representing “no” and “don’t know” responses.\n\nSocio-demographic characteristics take the form of continuous variables such as maternal age, child’s age, and number of household members and categorical variables such as maternal education (no education/primary/secondary/higher), maternal occupation (employed/unemployed), mother’s knowledge of oral rehydration salts (ORS) (good/poor)27, exposure to media (yes/no)28, sex of the child, breastfeeding (ever/never), deworming (yes/no)27, vaccination (ever/never), residence (urban/rural) and wealth index (poorest/poorer/middle/richer/richest)27. CDHS data were organized in 19 province domains, which we regrouped into four regions: Central Plain; Tonle Sap; Coastal and Sea; and Plateau and Mountains29. Environmental characteristics were also treated as categorical variables, including drinking water source (improved/unimproved)30, whether or not the same source of drinking water was used during wet and dry seasons (same/different), whether or not water was treated before drinking (always/no), type of toilet facility (improved/unimproved)30, hygiene (adequate/inadequate)30, and disposal of children’s stools (sanitary/unsanitary)31.\n\nStatistical data analyses were performed using STATA/SE 14.032 as follows.\n\nCategorical data were analyzed to provide frequency and percentage. Continuous data were treated as means, standard deviations, and ranges for analysis. A weighting variable was used in the form of the woman’s individual sample weighting. Cross-tabulations were run with the appropriate sample weights to provide nationally representative results19. The svyset command was used to test for complex survey sampling methods used in the original surveys, in order to adjust for differences in the probabilities of sample selection and to avoid using over-sampled strata within the survey data27.\n\nThe prevalence of diarrhea was estimated as a percentage. The numerator was the number of living children aged 12 to 35 months with an occurrence of diarrhea during the two weeks preceding the interview (i.e. an answer “yes” to, “Has (NAME) had diarrhea in the last 2 weeks?”) and the denominator was the number of living children aged 12 to 35 months.\n\nA bivariate analysis with simple logistic regression was performed using the svyset (svy command). A linearity test was conducted between the continuous variable and dependent variable. Any independent variables significant at p<0.25 were entered into the initial model33,34. Multicollinearity was performed with the independent variables-variance inflation factor (VIF)35. Finally, a multivariate analysis was performed using a generalized mixed linear model with four regions picked as ‘random effects’ corresponding to the various clusters in the sampling design36. The backward stepwise procedure was applied as the model fitting strategy. Statistical significance was considered at a threshold of p<0.05 and the adjusted odds ratio (AOR) with 95% confidence intervals (CI) was considered as the magnitude of the effect.\n\n\nResults\n\nA total of 2,828 children were included in the study. The majority of the children (84.12%) lived in rural areas. Nearly half (44.03%) lived in Central Plain and one third (33.32%) in Tonle Sap. The average maternal age was 28.27±5.89 years old. More than half the mothers (51.08%) attended primary school. Three quarters (75.10%) of the mothers were employed and the average number of household members was five. More than half (51.18%) of the children were male and the mean age was 23.33±6.79 months. Almost all (96.17%) children had been breastfed; 59.60% had received deworming treatment; and 77.97% of them had never been vaccinated. Out of 2,828 households, more than half (54.07%) always had treated water to drink; 57.97% had an unimproved toilet facility; while 68.01% used adequate hygiene; and 70.25% used sanitary disposal of children’s stool (Table 1).\n\nSD, standard deviation.\n\nFactors with a significant association with childhood diarrhea (p<0.05) were maternal age, maternal occupation, the child’s age, available toilet facilities, and the method of stool disposal (Table 2). Further, the factors of the child’s sex, their vaccination history, the number of household members, wealth index, source of drinking water during dry season, whether or not the same source of drinking water is used during wet and dry seasons, and the treatment/non-treatment of drinking water did not reach significance but did meet the pre-determined threshold of p<0.25 for inclusion in the initial model. Finally, region (p<0.25) also met the criteria for inclusion in the initial model and was used as a random effect. As such, the multivariate analysis was conducted using a generalized mixed linear model with each of the four regions of Cambodia treated as random effects.\n\nCOR, crude odds ratio; CI, confidence interval.\n\nThe multivariate analysis (Table 3) showed that as maternal age increased by a year, the odds of the child suffering from diarrhea decreased 15% (AOR = 0.85; 95%CI: 0.78– 0.93; p=0.001). The odds of suffering from diarrhea was 43% higher (AOR = 1.43; 95% CI: 1.14-1.78; p=0.002) in children whose mother was unemployed compared to employed. As the child’s age increased by a month, the odds of the child suffering from diarrhea decreased 14% (AOR = 0.86; 95%CI: 0.78-0.94; p=0.001). The odds of suffering from diarrhea was 25% higher (AOR = 1.25; 95%CI: 1.02-1.53; p=0.031) in males compared to females. The odds of suffering from diarrhea was 17% higher (AOR = 1.17; 95%CI: 1.05-1.31; p=0.004) in children living in a household with unimproved toilet facilities compared with those with improved toilet facilities. The odds of suffering from diarrhea was 32% higher (AOR = 1.32; 95%CI: 1.06-1.64; p=0.011) in children whose stools were disposed of unhygienically compared to children whose stools were disposed of hygienically.\n\nAOR, adjusted odds ratio; CI, confidence interval.\n\n\nDiscussion\n\nThis is the first study to report factors associated with diarrhea in children aged 12 to 35 months at the national level in Cambodia. Younger maternal age, maternal unemployment, younger child age, being male, lack of unimprovement to toilet facilities, and unhygienic disposal of children’s stools were found to be associated with childhood diarrhea.\n\nSocio-demographic characteristics such as maternal age were significantly associated with reduced incidence of diarrhea, in line with studies conducted in Brazil and Tanzania14,18, and perhaps due to the mother having more experience in childcare and feeding. The association of maternal unemployment with the incidence of diarrhea is consistent with a study in Senegal9. The child’s age had a significant, negative association with incidence of diarrhea, in line with many studies in Ethiopia and Tanzania4,14,16, and potentially due to the development of the immune system throughout childhood. Males were more likely to suffer from diarrhea than females, which may simply reflect a natural predisposition of males to develop diarrhea more frequently than females37, but is also supported by a previous study conducted in India13.\n\nEnvironmental characteristics such as the lack of improvements to toilet facilities were significantly associated with the incidence of diarrhea, consistent with many studies including a systematic review4,6,8,11. Finally, disposal of children’s stools was significantly associated with the incidence of diarrhea, consistent with previous studies in Ethiopia, India, and Tanzania12–14. These findings demonstrate that the quality of sanitation facilities strongly influences the prevalence of childhood diarrhea in Cambodia.\n\nA limitation of this research study was that it used a cross-sectional design with just one outcome measure (diarrhea prevalence) taken as a snapshot at a given point in time. Future longitudinal studies may improve on this. The CDHS 2014 was not fully comprehensive in that it did not cover the WASH factors of hand washing before preparing meals and after defecating. The inclusion of these questions in the survey would give a more comprehensive analysis of hygiene practices in the population. Further, the CDHS 2014 captured data by household, rather than by individual person, which may introduce a confound in that it has a tendency to under-estimate the quality of both drinking water source and sanitation facility available.\n\n\nConclusion\n\nDiarrhea still remains a public health concern among children in Cambodia. The probability of developing diarrhea is strongly associated with maternal unemployment, being male, not having access to improved toilet facilities, or practicing hygienic disposal of children’s stools. Conversely, increasing maternal and child age is associated with a reduction in the probability of developing diarrhea.\n\n\nRecommendations\n\nBased on this finding, the authors provide the following recommendations.\n\nNational level: The WASH program managed by the Ministry of Rural Development in Cambodia should focus on delivering guidance to younger mothers with younger children and also to unemployed mothers. Guidance should include sanitary disposal of children’s stools in addition to water treatment, sanitation and health. Health policy decision makers should design appropriate intervention programs focusing on reducing diarrhea through the construction of improved toilet facilities in addition to promoting hygienic behaviors.\n\nCommunity level: Younger mothers should be encouraged to enroll in health education for the prevention of childhood diarrhea. Improvements should be made to the general community sanitation facilities as well as the construction of additional toilet facilities.\n\nFuture study: Longitudinal studies on childhood diarrhea need to be conducted in order to measure the impact of interventions.\n\n\nData availability\n\nOur study used raw children’s and household data from DHS, Cambodia 2014. Data are free to access for research purposes and can be obtained through the DHS Program after registering and obtaining an approval letter from the Inner City Fund (ICF) (https://dhsprogram.com/data/Access-Instructions.cfm).", "appendix": "Acknowledgements\n\nThe authors would like to express sincere thanks and appreciation to:\n\nDr. Kavin Thinkhamrop, Health and Epidemiology Geoinformatics Research (HEGER), Faculty of Public Health, Khon Kaen University; Dr. Wilaiphorn Thinkhamrop, Data Management and Statistical Analysis Center (DAMASAC), Faculty of Public Health, Khon Kaen University for their statistical support; and Rebecca S Dewey, University of Nottingham for language editing.\n\n\nReferences\n\nWorld Health Organization: Diarrhoeal disease. 2017. Reference Source\n\nLiu L, Johnson HL, Cousens S, et al.: Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet. 2012; 379(9832): 2151–61. 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Reference Source" }	[ { "id": "64014", "date": "15 Jun 2020", "name": "Siyan Yi", "expertise": [ "Reviewer Expertise Epidemiology", "community-based intervention and evaluation", "infectious diseases" ], "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 used data from Cambodia Demographic and Health Surveys to identify risk factors associated with diarrhea in children aged 12 to 35 months. Overall, the study findings are interesting and may contribute to the literature in this area given the scarcity of data in low- and middle-income countries. The analyses appeared appropriate. The quality of the writing is acceptable, although more careful proofreading is required before the paper can be published.\nHere are my specific comments:\nTitle:\nThe term 'influence' may not be appropriate for this study as it can only tell the associations of variables, not causal relationships.\n\nAbstract:\nMethods: The first sentence \"A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014\" is hard to understand. The authors may want to make it clear that this study used secondary data from CDHS. What are the two data sets?\n\nWhat was the time frame for the prevalence of diarrhea - past month or lifetime?\n\nIntroduction:\nPlease provide a reference for the statement in the second sentence, paragraph 1.\n\nAvoid starting sentences with numbers.\n\nIt would be helpful for readers if authors could define some terminology; e.g., neonatal deaths, post-neonatal deaths, under-five deaths, etc.\n\nParagraph 2: - \"...and 692 deaths of children under five due to diarrhea (6%).\" What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia.\" Please clarify these. - \"High rates of diarrhea alone account for...' Diarrhea alone? - It is confusing that this study used data from CDHS 2014, but also cited the prevalence of diarrhea in the same population and from the same data, while claiming that no national studies on childhood diarrhea in Cambodia have yet been published. - The rationale of the study needs improvement.\n\nMethods:\nWhat 'province domains' mean?\n\nWhat does this mean: '...,which comprised the 2008 Cambodian General Population Census (GPC)?'\n\n'The sample considered any domain...' is not understandable.\n\nAlthough the CDHS 2014 was referred to, some variables require a clear definition; e.g., improved/unimproved water sources, toilet facilities, adequate/inadequate hygiene, sanitary/unsanitary disposal of children's stools, etc.\n\nData analyses: - It is not accurate to state this \"Continuous data were treated as means, standard deviations, and ranges for analysis.\" Perhaps something like 'For continuous variables, mean and standard deviations were calculated...\" - I am not sure what authors wanted to tell by this \"A weighting variable was used in the form of the woman’s individual sample weighting.\" - Any independent variables significant at p<0.25 in bivariate analyses were entered into the initial model. -Multicolinearity assessment was performed...\n\nResults:\n...and one third (33.32%) in Tonle Sap region?\n\nIt should be mean (SD xx).\n\n\"More than half the mothers (51.08%) attended primary school.\" Did this include mothers who had no education?\n\nAny details to define the breastfeeding - duration, exclusivity...?\n\nPlease check this data: \"...and 77.97% of them had never been vaccinated.\" This could be very wrong as the immunization coverage in Cambodia has been globally recognized as very high.\n\nDiscussion:\nThis section can be improved by extending more in-depth literature in this area and link to the policy implication of the findings.\n\nFurther limitations of the study should also be included (e.g., self-reporting measures, recall bias...).\n\nConclusions and recommendations can be combined.\n\nRecommendations can be summarized.\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": "5669", "date": "03 Jul 2020", "name": "PISEY VONG", "role": "Author Response", "response": "Review1 Title: The term 'influence' may not be appropriate for this study as it can only tell the associations of variables, not causal relationships. A: Agree with the reviewer. As suggested, we have revised the term to “The association”. See title, para 1 on page 1 Abstract: 1. Methods: The first sentence \"A cross-sectional study was conducted using the combination of two datasets from the Cambodia Demographic and Health Survey 2014\" is hard to understand. The authors may want to make it clear that this study used secondary data from CDHS. What are the two data sets? A: Taken care. \"A cross-sectional study of the secondary data from the Cambodia Demographic and Health Survey (CDHS) 2014 was conducted using the combination of household data and children’s data. See Abstract, 1st sentence, para 2 on page 1 2. What was the time frame for the prevalence of diarrhea - past month or lifetime? A: Taken care. The authors added the text “in the last 2 weeks”. See Abstract, 2nd sentence, para 3 on page 1 Introduction: 1. Please provide a reference for the statement in the second sentence, paragraph A: Taken care. “1,2”. See 2nd sentence, para 1 on page 3. 2. Avoid starting sentences with numbers. A: Taken care. Globally, 88% of diarrhea cases are attributable…… See 4th sentence, para 1 on page 3. 3. It would be helpful for readers if authors could define some terminology; e.g., neonatal deaths, post-neonatal deaths, under-five deaths, etc. A: We are sorry, we do not agree with the peer reviewer on this. Authors have retained our original because we think it is not much helpful to add terminology of neonatal deaths, post-neonatal deaths, under-five deaths. 4. Paragraph 2: - \"...and 692 deaths of children under five due to diarrhea (6%).\" What is the denominator of the 6%? In the following sentence, the authors stated that 'diarrhea alone account for one fifth of the deaths of children under the age of five in Cambodia.\" Please clarify these. - \"High rates of diarrhea alone account for...’Diarrhea alone? - It is confusing that this study used data from CDHS 2014, but also cited the prevalence of diarrhea in the same population and from the same data, while claiming that no national studies on childhood diarrhea in Cambodia have yet been published. - The rationale of the study needs improvement. A: The denominator of the 6% is the “number of under five children”, however in the data from UNICEF, they do not put it. They just put only “under five deaths due to diarrhea: 692”; and “% underfive deaths due to diarrhoea: (6%)”. According to calculation by the authors, denominator of (6%) is about 11,533. A: Please, see the reference on number 26 which mentioned as “High incidences of diarrhoeal diseases alone account for one fifth of the deaths of children age five and under in Cambodia, and an estimated 10,000 overall deaths annually, largely owing to lack of sanitation and poor hygiene practices”. A: Agree with the reviewer. As suggested, we have revised the text to “There are no existing studies on the association in this age group, and no national studies on the associated factors with childhood diarrhea in Cambodia have yet been published”. See 2nd sentence, para 3 on page 3. Methods: 1.What 'province domains' mean? A. Province domains means the 19 sampling domains of provinces. There are 24 provinces in Cambodia in CDHS 2014, of which fourteen individual provinces (Banteay Meanchey, Kampong Cham, Kampong Chhnang, Kampong Speu, Kampong Thom, Kandal, Kratie, Phnom Penh, Prey Veng, Pursat, Siem Reap, Svay Rieng, Takeo, and Otdar Meanchey); and five groups of provinces (Battambang and Pailin, Kampot and Kep, Preah Sihanouk and Koh Kong, Preah Vihear and Stung Treng, and Mondul Kiri and Ratanak Kiri) 2. What does this mean: '..., which comprised the 2008 Cambodian General Population Census (GPC)?' A. It means “which used the 2008 Cambodian General Population Census” That has been mentioned in CDHS 2014 as “The sampling frame used for the 2014 CDHS was derived from the list of all enumeration areas (EAs) created for the 2008 Cambodia General Population Census (GPC), provided by NIS 3. The sample considered any domain...' is not understandable. A: Taken care. We have revised the text to “The sample was allocated into urban and rural in each domain with a power allocation preventing oversample urban, and can represent Cambodia is mainly rural. See 3rd sentence, para 3 on page 3. 4. Although the CDHS 2014 was referred to, some variables require a clear definition; e.g., improved/unimproved water sources, toilet facilities, adequate/inadequate hygiene, sanitary/unsanitary disposal of children's stools, etc. A: Agree with the reviewer. As suggested, we have added the text “World Health Organization (WHO) guidelines on water, sanitation and hygiene (WASH) were used to classify WASH as either improved or unimproved according to the WHO/UNICEF Joint Monitoring Programme (Table1 and Table 2). See 3rd sentence, para 2 on page 4; and we also added the Table 1 and Table 2. See on page 11 and 12. 5. Data analyses: - It is not accurate to state this \"Continuous data were treated as means, standard deviations, and ranges for analysis.\" Perhaps something like 'For continuous variables, mean and standard deviations were calculated...\" - I am not sure what authors wanted to tell by this \"A weighting variable was used in the form of the woman’s individual sample weighting.\" - Any independent variables significant at p<0.25 in bivariate analyses were entered into the initial model. -Multicolinearity assessment was performed... A: Agree with the reviewer. As suggested, we have revised the text “Categorical variable were analyzed to provide frequency and percentage. Continuous variable were calculated as means, standard deviations, and ranges”. See 1st sentence, para 3 on page 4. A: Because it is survey data, by providing national representative, we used Woman’s individual weighting because child data was accessed by asking for their mother. A: Yes, please see in the “Result” on 2nd sentence, para 2 on page 6. A: Taken care “Multicolinearity assessment was performed...” Results: ...and one third (33.32%) in Tonle Sap region? A. Taken care. We have revised by adding “lived”. See on 2nd sentence, para 1 page 5 2. It should be mean (SD xx). A. Taken care. 3.\"More than half the mothers (51.08%) attended primary school.\" Did this include mothers who had no education? A: No, It did not include mothers who had no education 4. Any details to define the breastfeeding - duration, exclusivity...? A: No, in the data showing only “ever breastfed (not currently breastfeed, never breastfed, still breastfeeding”; so we group as “ever and never”. 5. Please check this data: \"...and 77.97% of them had never been vaccinated.\" This could be very wrong as the immunization coverage in Cambodia has been globally recognized as very high. A: Agree with the reviewer. As in the data of 2828 showed that 2.15% was no, 22.03% was yes, 0.27% was don’t know, and 75.55 was missing. The authors request to delete this variable since it is too much missing, moreover according to literature review this variable is not related with childhood diarrhea, only rotavirus vaccination is associated. Discussion: 1. This section can be improved by extending more in-depth literature in this area and link to the policy implication of the findings. A: Agree with the reviewer. However, we do not extending more in-depth literature. We have retained our original. 2. Further limitations of the study should also be included (e.g., self-reporting measures, recall bias...). A. Taken care. See 4th sentence, para 3 on page 7. 3. Conclusions and recommendations can be combined. A: Authors have retained our original because it will be a good idea to separate the limitation, recommendations and conclusion. Moreover, conclusion is an important part of the paper. 4. Recommendations can be summarized. A: Taken care. As suggested, the authors have summarized the text in Recommendations part. See page 8." } ] } ]	1	https://f1000research.com/articles/9-303
https://f1000research.com/articles/10-959/v1	22 Sep 21	{ "type": "Research Article", "title": "University-Industry R&D Alliances – An Exploration of Alliance Capabilities Measures", "authors": [ "Nabilah Kamaruzaman", "Arnifa Asmawi", "Kok Wai Chew", "Nabilah Kamaruzaman", "Kok Wai Chew" ], "abstract": "Background: Alliance capabilities studies have long emerged since the 1990s, focusing mainly on firm-to-firm collaboration. However, research on university-industry alliances only emerged from the 2000s. Alliance capabilities are portrayed as a crucial condition to achieve the targeted collaboration outcomes and sustainable relationships. As most alliance capabilities studies focus on firm-to-firm collaboration, research on university-industry research and development (R&D) alliance is still scarce. Thus, the measurement items for alliance capabilities in the university-industry R&D context are still under-developed. Thus, to investigate how alliance capabilities affect university-industry R&D performance in Malaysia, the relevant measures must first be defined. This paper intends to properly define the measurement items for alliance capabilities in the context of university-industry R&D alliances. Methodology: The alliance capabilities measures are adapted from various literature to accommodate both university and industry perspectives. In finalizing the measurement, in-depth pre-testing was conducted by five strategic management subject matter experts in ensuring face and content validity. Results: There are three alliance capability dimensions. The first dimension is alliance management capability which includes goal setting, process configuration, alliance structure, coordination, management support, and alliance evaluation. The second dimension is alliance integration capability which incorporates relational capabilities, inter-organizational communication, relational capital, and project team effectiveness. The third is alliance learning capability which measures alliance experience, knowledge articulation, knowledge sharing, knowledge codification, internalization, and relationship learning. Although this study successfully develops a set of measurement items for alliance capabilities in university-industry R&D, further statistical analysis is required to test this scale. Conclusion: To date, quantitative measurement items for alliance capabilities in the context of university-industry R&D alliances are still at the infancy stage. Although the measurements are yet to be statistically analyzed, they can be used as a benchmark for future university-industry R&D alliances studies.", "keywords": [ "alliance capabilities", "university-industry", "alliance management capability", "alliance learning", "alliance relationship", "R&D" ], "content": "Introduction\n\nIndustry 4.0 has been the main agenda for many countries,1 including Malaysia.2 Industry 4.0 is the technological evolution and digitalization of the manufacturing processes to increase productivity and efficiency.3 However, it is observed that Malaysia is relatively slow in picking up the pace in transitioning into Industry 4.0.3 Hence, various Malaysian policies such as the National Transformation 2050 Policy, underscore the role that innovation and R&D can play to ensure a smooth transition into Industry 4.0.4 These policies clearly suggest that stronger alliances between universities and industries are crucial in accelerating R&D and innovation capabilities.5,6\n\nHowever, successful alliances between universities and industry are not without their own set of challenges.7 A glance at the strategic management study, particularly on alliance capabilities at the micro-level processes, opens the need to further investigate the level of alliance capabilities in Malaysian university-industry R&D alliances.8 The research on alliance capabilities, specifically at the micro-level processes (which include the ability to manage, integrate and learn from the alliance relationship), is crucial to achieve strategic benefits and sustainability.8,9\n\nAlthough research on alliance capabilities existed since the 1990s, extant literature primarily focuses on firm-to-firm collaboration.10 Studies on university-industry alliances emerged in the 2000s while specific research on knowledge-based alliance capabilities only started in 2008.11 To date, studies on the university-industry alliance capabilities, primarily on quantitative analysis, are still limited. Thus, causing a hurdle for researchers in finding the measurement items on alliance capabilities that can fit both the university and industry R&D context.\n\nHenceforth, this paper purports to develop a set of measurement items on micro-level processes of alliance capabilities in the context of university-industry R&D alliances. Based on the framework adapted from Kohtamäki et al.,8 alliance capabilities refer to the firm's ability in managing, integrating, and learning from the alliance relationship. Figure 1 illustrates the alliance capabilities dimensions used in this study.\n\nAlliance management capability is the ability of university-industry R&D alliances to set targets, implement tasks, and perform alliance evaluation.8 Alliance target setting refers to the ability of a firm to establish goals,12,13 configure processes,14 and developing alliance structures.15 On the other hand, alliance task implementation denotes the ability of a firm to coordinate with strategic alliance partners16 and rally the support of the management team.17 Meanwhile, the third aspect of alliance management capability is the evaluation of the alliance tasks and activities.18\n\nAlliance integration capability looks at how R&D alliance partners develop their alliance capabilities through social and structural integration.8 The ability of alliance partners in utilizing relational capability,19 inter-organizational communication,20 and relational capital13 are measured under social integration. Whereas structural integration can be measured through the firm’s ability to achieve project team effectiveness.21\n\nAlliance learning capability is the third dimension of alliance capabilities. It evaluates R&D partners’ ability to create, assimilate, and internalize knowledge from their relationship.8 In knowledge creation, the firm will assess how they can generate knowledge from experience,22 articulate and share the knowledge.23 Knowledge assimilation, on the other hand, is measured through knowledge codification and combination.23 While knowledge internalization can be assessed through the firm’s ability to internalize knowledge23 and acquire relationship learning.24\n\n\nMethods\n\nMeasurement items or scales are normally used to measure actions, behaviors, or feelings that cannot be measured using a single item or variable.25,26 This paper presents the measurement items for alliance capabilities in the context of Malaysian university-industry R&D alliances. Based on the item development method by Boateng et al.,26 the first step is specifying the variable, dimensions, and constructs in measuring alliance capabilities. In this study, measurement items are carefully chosen from intensive review of existing literature. Due to limited alliance capabilities measures in the context of university-industry alliances, measurement items are adapted from various inter-firm collaboration studies to suit the context of this study.27 All scales are measured in a 5-point Likert scale (1-Strongly disagree to 5-Strongly Agree). Once the measurement items were finalized, an in-depth pre-testing was performed with five subject matter experts in the field of Strategic Management. This process is necessary to ensure content validity and face validity of the measurement items.26\n\nThe data for these measures will be collected through a survey. When investigating alliance capabilities in university-industry R&D alliances, the appropriate unit of analysis will be dyads. The survey forms will be distributed to university researchers and their respective industry partners who are working or have worked together in an R&D project. Reputational and snowball sampling will be used due to the inclusion criteria of the respondents. Subsequently, the data will be analyzed using the Multi-Group Analysis (MGA) technique using PLS-SEM.\n\nIn addition, as this type of study involves human participation, consent from the respondents will be acquired. Due to the Covid-19 pandemic, this study will be conducted using an online survey platform (Google Form). Hence, a statement of consent will be included on the first page of the survey information sheet. Respondents can opt to provide their consent by answering the survey (i.e., clicking the ‘Next’ button) and vice versa.\n\n\nResults\n\nUpon completion of the measurement development, and pre-testing for face validity and content validity,28 the measurement items for alliance capabilities in Malaysian university-industry R&D alliances are presented below.\n\nGoal setting is the ability of R&D alliance partners to align their common goals. Differences in respective organizational goals will impede the alliance success.7 Therefore, it is important for both parties to achieve common understanding and agree on shared project objectives and requirements prior to the establishment of the R&D alliance. Figure 2 shows the measurement items used in measuring goal setting.\n\nProcess configuration measures the ability of R&D alliance partners in combining internal and external factors that are important for successful project execution and developing relational capability.29 Specifically, process configuration evaluates the R&D alliances’ ability in configuring resources (planning tools, standard operating procedures, and technological expertise), team members qualification in problem-solving, and task fulfillment (the input of R&D alliance partners is also considered in completing the project).14 Measurement items for process configuration adapted from Jacob14 are as shown in Figure 3.\n\nAlliance structure measures the readiness of dedicated supporting units and individuals in managing the R&D alliance relationship.15 The availability of dedicated functions or individuals in managing the alliance relationship is argued to be one of the crucial aspects in managing the collection of knowledge and alliance practices.30 Figure 4 illustrates the measurement items used in measuring alliance structure.\n\nCoordination evaluates how R&D alliance partners achieve mutual project goals through individual roles and activities’ specifications.31 As part of the control processes, coordination between alliances enables them to streamline their R&D activities, enhancing learning capability, and increase their communication quality.32 Figure 5 illustrates the items to measure coordination.\n\nManagement support focuses on the leading capability of the R&D alliances’ respective organizations in inspiring individuals to achieve the designated R&D outcomes.33 Through this mechanism, researchers who are involved in the R&D alliance will understand the strategic importance of the alliance to the organization and are aware of the management commitment to this collaborative arrangement.34 The measurement items for management support are as shown in Figure 6.\n\nAlliance evaluation is one of the control processes in alliance capabilities. Through alliance evaluation, R&D alliance partners will be aware if there are any problems in the relationship that may affect the alliance performance.18 This controlling and monitoring activity is essential to make sure the tasks were executed as planned to achieve the targeted outcomes.35 Figure 7 illustrated the measurement items for alliance evaluation.\n\nRelational capabilities are part of the social integration in the alliance capabilities framework. They focus on the relationship quality of the R&D alliance partners.19 The high relational capability will impact knowledge transfer intensity which also signals trust development.36 Therefore, relational capability can be explained as the readiness of a firm in forming and sustaining the R&D alliance relationship with its partners.37 Measurement items for relational capabilities are as shown in Figure 8.\n\nInter-organizational communication measures the effectiveness of information sharing and communication in both formal and informal ways between R&D alliance partners.38 As R&D alliances are formed between two or more different entities, the ability to create effective communication and interaction will result in loyalty, enabling knowledge exchange, and sustaining the relationship.20 Figure 9 shows the measurement items used in measuring inter-organizational communication.\n\nRelational capital in R&D alliance relationships is portrayed as the ability to create a sense of shared destiny, trust, and open interaction.39 The ability to develop relational capital between R&D alliance partners is crucial mainly in the innovation value chain, as it promotes effective knowledge integration and innovative activities.13,40 Figure 10 illustrates the measurement items for relational capital.\n\nProject team effectiveness investigates how the joint involvement of alliance partners in the R&D project positively influences the project outcomes.21 Petersen et al.41 has precisely emphasized project team effectiveness, enhancing problem-solving, decision making, and team functionality. Project team effectiveness measurement items are as shown in Figure 11.\n\nAlliance experience is argued as a crucial knowledge generator in any alliance relationships.42 Apart from technical knowledge, experience gained from previous alliances will enhance the competency in managing future similar relationships.43 Based on Emden et al.,22 learning from alliance experience can be evaluated through the organizational ability in acquiring, analyzing, and distributing knowledge gathered from the alliance relationship. Figure 12 shows the measurement items in measuring alliance experience.\n\nKnowledge articulation focuses on R&D partners’ ability in externalizing knowledge gathered throughout the alliance relationship into a format that is accessible to others.23 In an alliance relationship, knowledge can be garnered through the individuals' interaction with alliance partners. This type of knowledge is categorized as tacit knowledge, and thus, the ability to document this knowledge for others to access may increase the ability for others to learn from it.44 Measurement items for knowledge articulation are as shown in Figure 13.\n\nKnowledge sharing, on the other hand, measures the efficiency of knowledge distribution between individuals through personal interaction.23 The ability to share knowledge among individuals who are involved in an R&D alliance will lead to better project performance.44 Figure 14 presents the measurement items for knowledge sharing.\n\nKnowledge codification is the merging of explicit knowledge into a systematic and meaningful document that can be used as a guide in managing future R&D alliances.23 The codification of valuable knowledge into a systematic documentation can assist alliance partners in knowledge assimilation and facilitate decision making.16,23 Measurement items for knowledge codification are as shown in Figure 15.\n\nKnowledge internalization in an R&D alliance aims to assist individuals to better understand the needs and requirements of their partners.45 Knowledge internalization focuses on developing the ability to internalize knowledge gathered from the alliance through proper training activities.46 Figure 16 illustrates the measurement items for knowledge internalization.\n\nRelationship learning focuses on joint activities between R&D alliance partners to improve the relationship through information sharing, creating common learning platforms, and modifying their teamwork behaviors.12,24 These joint activities are further interpreted and integrated into relationship-specific memory that can influence their behaviors and routines.47 Through relationship learning, both R&D alliance partners will be able to re-evaluate and re-adjust their routines to achieve the expected outcomes.45 Figure 17 shows the measurement items for relationship learning.\n\n\nDiscussion\n\nIn summary, quantitative measurement items for alliance capabilities in the context of the university- industry R&D alliances are still at an infancy stage. However, based on extant research on alliance capabilities in various inter-organizational contexts,8,10 it is possible to deploy these measures after appropriate content validity and face validity assessment at the pre-testing stage. Therefore, this paper has successfully developed a comprehensive set of measurements items to determine the extent of alliance capabilities in the context of university-industry R&D alliances.\n\nThese measures will be deployed in a quantitative study which intends to develop a framework on how micro-level alliance capabilities affect university-industry R&D alliance success. This study will examine how alliance capabilities function at the micro-processes and relational level. This research will analyze the dyadic relationship between individual university researchers and their industry partners (dyads). It is hoped that this study will be able to develop a composite model to explain the interplay of the alliance capability dimensions and their influence on R&D alliance success.48\n\n\nConclusions\n\nThis study aims to develop the measurement items for alliance capabilities which consist of managing, integrating, and learning from the alliance relationship. From the item development process, this study managed to produce a set of measurement items for alliance capabilities relevant in the context of Malaysian university-industry R&D. We would like to emphasize that these scales have not been statistically analyzed. Thus, further assessment on construct validity such as construct discriminant validity, convergent validity, and other reliability tests is required.28\n\n\nData availability\n\nFigshare. Alliance Capabilities Measurement items and framework.pdf. DOI: https://doi.org/10.6084/m9.figshare.14872140.v1.49\n\nThis project contains the following data: This document contains the measurement items for alliance capabilities in the context of university-industry R&D.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC BY 4.0 Public domain dedication).\n\n\nEthical approval\n\nThis research received an ethical approval from the Research Ethics Committee (REC) of Multimedia University (Approval Number: EA1302021).", "appendix": "Acknowledgments\n\nWe would like to thank the three (3) Subject Matter Experts (SMEs) in the Strategic Management field from two universities in Malaysia that contributed to the pre-testing of the measurement items discussed in this paper.\n\n\nReferences\n\nUN General Assembly: Transforming our world: the 2030 Agenda for Sustainable Development.2015. Reference Source\n\nMalaysia Ministry of International Trade and Industry: Industry4WRD.2019. 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British Academy of Management 2020 Conference in the Cloud: Innovating for a Sustainable Future. 2020.\n\nAsmawi A, Kamaruzaman N, Chew KW: Alliance Capabilities Measurement items and framework.pdf. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "101482", "date": "07 Dec 2021", "name": "Antonio Messeni Petruzzelli", "expertise": [ "Reviewer Expertise Innovation Management" ], "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 deals with a very interesting and fascinating topic. However, despite the potential relevance of its contribution, I believe that it presents some issues that need to be tackled.\nIn the following, you can find some suggestions and comments that I hope may be useful to improve the contribution of your study.\nFirst, research gap and contributions need to be better theoretically discussed in the introduction.\n\nSecond, I suggest to develop an ad hoc section discussing the theoretical background of the study.\n\nThird, I suggest to consider recent and relevant contributions on the topic (For example these articles by the present reviewer: Messeni Petruzzelli, 20111; Messeni Petruzzelli and Rotolo, 20152; Messeni Petruzzelli and Murgia, 20203, 20214).\n\nFourth, the methodology needs to be theoretically justified.\n\nFifth, the concluding section needs to be enriched by discussing the main theoretical and practical implications, as well as the main limitations and directions for future research.\n\nSixth, the paper may benefit from a professional copyediting.\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?\nNot applicable\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? No", "responses": [] }, { "id": "100922", "date": "02 Feb 2022", "name": "Ainurul Rosli", "expertise": [ "Reviewer Expertise University-Industry Collaboration" ], "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 was a great pleasure to have had the opportunity to review this paper, focusing on developing the measurement items for alliance capabilities which consist of managing, integrating and learning from alliance relationships. There is a decent amount of prior contributions on this topic which the author(s) missed, and this need to be reflected in the literature cited. Nevertheless the title entices me to read further so somehow it illustrates it’s potential. However, as I read through the manuscript, I found some sections or areas that require substantive improvement, with greater clarity to make the contribution clearer.\nThe introduction can be written in a much more convincing manner. Some statements in this section left me unclear about what to expect in the rest of the paper. The research gap hasn’t been outlined, and it is not clear. Why is a new measurement needed? Is it due to the rise of Industry 4.0? Or perhaps because the framework of Kohtamaki et al. didn’t include a clear measurement? More explanation is required on the need to focus on the development of the measurement. What is the problem or limitation of the current way of measurement? With limited information addressing these issues, readers are left wondering why the need for this paper.\n\nThe methods section is a bit misleading. The focus here should be about the methods of developing the measurement tool. Somewhere in the paper, it was mentioned that two specialists in the area reviewed the scale. This should be included in the section on the development of the measurement variables. What has been done to ensure the quality of the work? Perhaps the focus here is on the steps taken to ensure the validity and reliability of such data collection tools used to support the conceptualisation of alliance capabilities measurement items.\n\nThe conclusion is too simplistic. In fact, when the reader completes reading the conclusion, it then makes sense that this is a measurement development paper and not a full empirical paper. It should not happen that way. Despite the confusion (due to the very limited argument in the introduction and literature section), the conclusion should include some indication of the importance/contribution of the work. My suggestion is for you to draw from the various paper on how your approach to the development of the measurement differs from others and how it can bring a new perspective to understanding the topic/area.\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-959
https://f1000research.com/articles/10-957/v1	22 Sep 21	{ "type": "Research Article", "title": "Hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi, Kenya", "authors": [ "Holi Kevin", "Lucy Njue", "George Abong", "Lucy Njue", "George Abong" ], "abstract": "Background: Poor milk handling practices due to lack of knowledge or deliberate adulteration compromises the safety of milk and that of the consumer. With the increase of milk vending machines in informal settlements, the knowledge and practices of vendors concerning milk hygiene are critical in determining milk safety. The operation of milk vending machines is guided by strict regulations that aim at ensuring proper practices among milk handlers, but the level of implementation among milk vendors is questionable. This study sought to establish hygienic knowledge and handling practices of vendors operating milk vending machines in Nairobi’s informal settlements of Kibra and Dagoretti North. Methods: Using a cross-sectional study design, 37 milk vendors were interviewed at the study setting with the aid of questionnaires. Data was then recorded in datasheets and analyzed using frequencies, correlation, and t-test with the aid of Statistical Package for Social Sciences software. Results: The mean percentage score for knowledge and practices was 68.83% and 54.05%, respectively. The knowledge and practices of vendors significantly improved with the increase in their level of education (p=0.04 and p=0.02). There was a significantly positive correlation between knowledge and practices (r=0.626, p=0.000). Conclusions: There is a low level of hygienic knowledge and poor practices among vendors operating milk vending machines in the study area. The efforts by the regulator to ensure proper implementation of the regulations was also not sufficient. Training is required to improve the hygienic knowledge and practices of milk vending machine handlers in informal settlements. Frequent inspections should also be carried out in the informal settlements to ensure proper implementation of regulations on the operation of milk vending machines.", "keywords": [ "Milk handlers", "knowledge", "practices", "milk vending machines", "hygiene" ], "content": "Introduction\n\nIn most developing countries, low incomes, weak government structures, and poor enforcement of regulations have led to the rapid growth of the informal sector. This sector has grown over time to being recognized as an important source of employment and contributes to the growth of the economy (Grace et al., 2019). In terms of food safety, the informality of the informal sector is attributed to the ease of escaping systemic sanitary inspection resulting in retail practices being carried under unhygienic conditions. Due to poor enforcement of regulations in the informal sector (Brown et al., 2018), it has made it accessible to anyone, both the skilled and the unskilled, to engage in any form of retail business. This is risky when it comes to the retail of milk which requires a high level of consciousness and proper understanding and adherence to milk hygiene standards.\n\nPost-harvest handling of milk plays a major role in determining milk safety (Godfrey, 2013). Milk handling practices entail the day-to-day activities that are carried out along the milk value chain, from production, during transportation, and at the retail stage. Unhygienic handling of milk is a leading cause of hazards in milk especially in developing countries (Grace, 2015). Studies have previously shown that milk safety is highly compromised at the retail point due to poor handling and storage practices (Alonso et al., 2018). The knowledge of vendors on milk hygiene is critical in guiding their daily practices. A study assessing knowledge and practices of milk traders around Nairobi showed that only 30% of the respondents were aware of regulations on milk retail, 51% of them were not sure about some regulations while 19% of them said they are not aware of any existing regulations (Brown et al., 2018).\n\nInformal settlements tend to face extraordinary challenges when it comes to general hygiene, this is a result of inadequate resources and hygienically poor environmental surroundings that characterize these settlements (Grace et al., 2019). The increase of milk vending machines in informal settlements raises concerns about the proper handling of the machines. Practices of milk vendors in these areas are critical in determining the safety of milk at the retail point, poor handling practices either due to lack of knowledge or negligence could result in post-pasteurization contamination of milk (Sadhu, 2018). Prolonged storage of milk in tanks at the retail points at temperatures that favor microbial growth could increase the bacterial load in milk hence affecting its safety (Paludetti et al., 2018).\n\nIn Kenya, the dairy industry is regulated by the Kenya Dairy Board which is a state corporation under the ministry of agriculture and was established through an Act of Parliament, CAP 336 of the Laws of Kenya (Kenya Dairy Board [KDB], 2018). Kenya Dairy Board’s regulatory mandate entails licensing, inspection, and surveillance of milk for both the local and export market. The Kenya Bureau of Standards on the other hand is responsible for assuring quality standards for milk and dairy products sold in the formal domestic market as per the code of hygienic practice for milk and milk products KS1552:2016 (Kenya Bureau of Standards, 2016).\n\nThe wide usage of milk vending machines has necessitated legislation on their operation, this is aimed at standardizing vendors’ operations to ensure the safety and suitability of milk from the vending machines. These regulations are aimed at addressing the possibility of quality breaches by milk vending machine handlers, which may include the use of sub-standard milk dispensers, stocking of raw instead of pasteurized milk, breaking the cold chain during storage of milk, poor location of the dispensers, limited skills by the operators as well as poor hygiene standards (Raposo et al., 2015). Despite the existence of these regulations, there have been constant breaches from the operating standards of milk vending machines by handlers.\n\nThis study sought to establish the hygienic knowledge and practices of vendors handling automatic milk dispensing machines in Kibra and Dagoretti North informal settlements of Nairobi, Kenya.\n\n\nMethods\n\nThe study was carried out in the informal settlements of Nairobi County, Kenya. Nairobi serves as the capital city of Kenya and has 17 sub-counties with a population of over four million people according to the 2019 census report (Kenya National Bureau of Statistics, 2019). The majority of these people are low-income earners who live in informal settlements distributed across these sub-counties. Two sub-counties were purposively selected for this study, which include Kibra and Dagoretti North. The areas were purposively chosen since they contain some of the largest informal settlements in the county with a high population density.\n\nFigure 1 shows the locations of Kibra and Dagoretti North. Kibra is located to the southwest of the city of Nairobi, it is approximately 12.1km² and has a population of around 178,000 people. Dagoretti North is located to the west of Nairobi city, it is approximately 29 km² with a population of around 181,000 people. These two regions have areas characterized by unplanned settlements, poor quality of housing, inadequate access to safe water and sanitation, and poor drainage systems among others.\n\nA cross-sectional design was used to carry out the study. This design was appropriate for the study since it made it possible to estimate the extent of the outcome of interest which, in this case, is the level of hygienic knowledge and practices of vendors and also enabled the assessment of many study outcomes within a reasonable time (Kesmodel, 2018).\n\nThe informal settlements of Kibra and Dagoretti North represent the setting where the study was conducted. In Kibra, four wards were purposively selected for the study, these include Makina, Laini Saba, Lindi, and Sarang’ombe. In Dagoretti North, two wards were purposively selected, which include Kawangware and Gatina wards. These areas are characterized majorly by informal settlements and structures along which milk vending machines are stationed. The vending machines are randomly distributed in the areas without any specific order.\n\nThe study population comprised of all milk handlers operating milk vending machines located in the informal areas of Kibra and Dagoretti North. A pre-visit was conducted at the study setting to establish the total number of milk vending machine handlers available and in operation. A representative from the Kenya Dairy Board, a regulatory body in the dairy industry, also formed part of the study population, the representative officer was invited to participate in the study through a formal request to the regulatory institution.\n\nAn exhaustive sampling of the available milk vending machine handlers was carried out in the study areas, a total of 37 milk vendors operating milk vending machines were identified, 20 of them were from Dagoretti North while 17 from Kibra. All the vendors identified were included in the study.\n\nThe sampling of milk vending machine handlers for interviews was conducted within a period of one month, in October 2020; this began with Dagoretti North then followed by Kibra. Respondents were physically invited for the interviews through one-on-one engagement by explaining the nature and purpose of the interview. The interviews were conducted daily at the respondents’ milk vending machine premises with informed consent obtained from the vendors before an interview by explaining to them the objectives of the study and assuring them of the confidentiality of their data. All the available milk vending machine handlers within the study setting were interviewed individually using semi-structured questionnaires with every interview taking roughly 20 minutes to 30 minutes.\n\nInterviews were conducted by the corresponding author at the study site after undertaking relevant training on conducting research interviews. A questionnaire containing 26 questions was first pilot tested on a sample of the respondents and modified according to the observations from the pilot test before the actual interview. Vendors were interviewed on a face-to-face basis to validate the accuracy of the responses, with the interviews being conducted either in English or Swahili (through translation) using semi-structured questionnaires. The questionnaire was divided into three sections with the first part of it capturing the socio-demographic characteristics of the respondents with details about their gender, age, and level of education. The second part entailed 15 questions with the choices of True, False, and Don’t Know to assess the hygiene knowledge of the respondents. The third part of the questionnaire had 21 questions covering the practices of handlers with choices of Yes, No, or a structured answer which the interviewer interpreted to be either the right or wrong practice. A copy of the questionnaire can be found in the Extended data. A representative from the Kenya Dairy Board was also interviewed separately at the institution’s premises following official appointment request to conduct the interview by the corresponding author. The officer was interviewed on a one-on-one basis on measures the institution is putting in place to ensure that regulations governing the operation of milk vending machines are adhered to. A copy of the questionnaire can be found in the Underlying data (in the “Holi Regulator's Questionnaire.docx” which also contains the answers). No follow-ups were conducted after the interviews.\n\nResearch tools used to collect the data include questionnaires for conducting interviews, pencils, and erasers for recording feedback from respondents on copies of the questionnaires by the corresponding author, datasheets, a camera for taking photos of the study area and the nature of its surroundings, and a stationery bag to store the data collection materials. Results from the interviews were then recorded on a datasheet for analysis. This data was stored in sealed envelopes under lock and key for security purposes and labeled “confidential,” only to be opened during analysis.\n\nData was analyzed using Statistical Package for Social Sciences software (IBM SPSS v25). Each study question was classified and assigned points, responses on questions assessing the hygiene knowledge of respondents were categorized as either correct or incorrect. For every correct answer, one point was awarded while an incorrect response was awarded zero points. On questions assessing the hygiene practice of respondents, one point was awarded for the right practice while a bad practice was awarded zero points. All the scores for handling knowledge and practices of respondents were tallied and calculated as a percentage. Individual scores for questions on knowledge and practices were summarized using frequencies and descriptive statistics. Pearson’s correlation coefficient was used to establish an association between knowledge and practices of respondents. Scores above 75% were considered to be indicative of high knowledge and good practice with statistical significance set at p<0.05. Responses from the Kenya Dairy Board’s representative were assessed based on the milk vending machine quality manual from the institution and presented descriptively by stating the regulatory measures being put in place and the existing gaps.\n\nPotential sources of bias were addressed by involving the participating authors to independently interpret the data for comparison and verifying the findings with other similar data sources supporting the findings. The findings and conclusions were also shared with peers for review.\n\n\nEthical approval\n\nThis study was approved by the National Commission for Science, Technology and Innovation of Kenya under license number: NACOSTI/P/20/5347\n\n\nConsent from participants\n\nBefore every interview, respondents were informed that the interview was for research purposes and were asked to sign a consent form indicating the purpose and confidentiality of the data.\n\n\nResults\n\nOut of 37 milk vendors who were enrolled in the study as respondents, 56.8% were male and 43.2% were female. Out of the total male respondents, 61.9% were from Dagoretti North while 38.1% from Kibra. Out of the total female respondents, 43.8% were from Dagoretti North while 56.2% of them from Kibra. From these respondents, the youngest was 20 years and the oldest 45 was years with their mean age being 26 years. There was no significant difference (p=0.51) between the ages of males and that of females. In Kibra, the mean age of the respondents was 26.18 while in Dagoretti North it was 26.05, the difference in the average age of the two study areas was, however, not significant (p=0.95).\n\nThe majority of the respondents (64.9%) had attained secondary education with only 21.6% having tertiary education and 13.5% primary education. In Dagoretti North, 5% of the respondents had primary education, 70% secondary education, and 25% with tertiary education. In Kibra, those with primary education were 23.6%, 58.8% having secondary education, and 17.6% tertiary education (Table 1).\n\nOut of the total percentage of males (56.8%), 28.6% had attained tertiary education, and 66.6% had secondary education, while only 4.8% had primary education. Among the females (43.2%), 12.5% had attained tertiary education, 62.5% had secondary education, and 25% had primary education (Table 2). Differences in the level of education between males and females as calculated using Fisher’s exact test was however not significant (p=0.2).\n\nTable 3 shows hygiene knowledge responses by milk vending machine handlers for this study. Milk vending machine handlers had a low level of knowledge on milk hygiene with a mean percentage score of 68.83±11.2. Male respondents registered a higher knowledge score of 68.88±11.02 compared to females with 68.74±11.86, but the difference was not statistically significant (p=0.97). The mean percentage score of vendors’ knowledge in Dagoretti North was 72.66±11.2 while in Kibra it was 64.31±9.7 with the difference being significant (p=0.02). Milk hygiene knowledge scores increased with the level of education; vendors with college education showed a high hygiene knowledge on milk handling (76.67±11.8), followed by those with secondary education (67.78±10.5), and lastly those with primary education (61.33±7.3), with the difference in their knowledge scores being significant (p=0.04 and F=3.632).\n\nThe majority of the respondents (89.25%) correctly answered that only pasteurized milk should be stored and sold from the vending machines with all the respondents (100%) not knowing that pasteurized milk, despite being safe for consumption, can be contaminated by microorganisms. More than half (75.7%) of the respondents thought that any detergent and disinfectant can be used to clean milk vending machines with 100% of them believing that water used for cleaning milk vending machines should be one that is also safe for drinking. Additionally, only 43.2% of the respondents believed that milk vendors should undergo medical checkups to ensure they are free from contagious diseases. About 75.7% of the vendors believed that milk should only be purchased from licensed suppliers. The majority of the respondents (81.1%) believed that milk storage containers should not be used to store other liquid materials. About 35.1% of vendors believed that milk can be stored in the vending machines at any temperature while 56.8% of them stated that milk can be kept in the vending machines for more than 24 hours. More than half of the respondents (64.9%) did not know that there are laws and regulations that guide the selling of milk using milk vending machines.\n\nTable 4 shows vendor practices responses by milk vending machine handlers for this study. Milk vending machine handlers in the current study demonstrated poor milk handling practices with a mean percentage score of 54.05±11.5. Male respondents demonstrated better practices (54.20±12.5) as compared to female respondents (53.87±10.5), but the difference was not significant (p=0.93). The mean percentage score of vendors’ practices in Dagoretti North was 54.52±11.2 while in Kibra it was 53.50±12.2; the difference was, however, not significant (p=0.79). Milk safety practices increased with the level of education; respondents with tertiary education showed better practices (61.9±11.9) as compared to those with secondary education (53.57±11.3) and primary education (43.8±8.5), with their mean score difference being quite significant (p=0.02 and F=4.641).\n\nThe majority of the respondents (91.9%) stated that the milk they sell using the vending machines was pasteurized, which is a practice that is compliant with the regulation. A good percentage of them (83.8%) confirmed that they use detergents while cleaning milk vending machines but none of them stated that the detergent was food grade, which is against the regulations. Most of the vendors (94.6%) stated that they can access adequate clean water for cleaning the milk vending machines. About 86.5% of the respondents said that they use tap water in cleaning the vending machines. Only 78.4% of them indicated that they clean the machines at least twice (morning and evening) daily with 21.6% of them only cleaning the vending machines once, which goes against the regulations.\n\nMore than half of the vending machines in the study (78.4%) displayed a temperature monitoring device; out of these, only 51.4% indicated that the milk was being stored at 0℃-4℃ while a big percentage of them (48.6%) being not sure about the temperature of storage or stored milk beyond 4℃ against the regulation. None of the vendors kept temperature monitoring records of daily temperature fluctuations in the vending machine as per the regulations. Only 8.1% of them had some cleaning and sanitation records. The majority of the respondents in this study (70.3%) were not transferring milk directly from the supplier’s original container to the dispenser as required. Regarding the duration for which milk is kept in the vending machines, only 73% of them stored milk in the machines for less than 24 hours as per the regulation with 27% of them not being compliant.\n\nOnly 18.9% of the respondents admitted to having undertaken medical tests for contagious diseases with 81.1% stating that they had not been medically examined to be free from contagious diseases. A good percentage of the respondents (64.9%) stated that they had not been trained for handling and operating milk vending machines and that they were relying on transferred knowledge on basic operations of the milk vending machines from the owners or previous operators. About 81.1% of the vending machine handlers had a valid operational license, however, 54.1% stated that their premises are not inspected periodically.\n\nThere was a positive correlation between milk hygiene knowledge and practices in this study. From Pearson’s correlation analysis (Table 5), a strong positive correlation (r=0.626) existed between hygiene knowledge and practices, and it was highly significant (p=0.000).\n\nThe correlation between knowledge and practices of milk vending machine handlers is illustrated in Figure 2. The dots inside the tables represent knowledge and practices scores. From the distribution of the dots as illustrated, a close association between the scores on knowledge and practices is indicted by their alignment towards a possible line of best fit showing that the knowledge of vendors closely influenced their practices.\n\nFigure 3 shows variations of the mean percentage scores for hygiene knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi. The green bars represent the variation in hygiene knowledge of handlers in Dagoretti North and Kibra; the blue bars represent variation in safety practices between handlers in the two study areas while the orange bar represents the overall hygiene knowledge and the red bar shows overall safety practices.\n\nA Kenya Dairy Board representative, a regulatory officer, interviewed in this study stated that the institution offers licensing for milk vending machines. However, the exact statistics of how many vending machines have been licensed within Nairobi County at the time of the study was not provided. The regulatory officer also admitted to the availability of unlicensed milk vending machines being used in the market and that they constitute those machines that had been fraudulently obtained. The officer also stated that the institution does not have a structured means of training vendors on the operation of milk vending machines but a published quality document on regulatory requirements that vendors are expected to adhere to exists. The officer confirmed that they usually conduct inspections on milk vending machines to establish vendors’ operations and practices but that this has not been carried out intensively in the informal settlements, with the frequency of these inspections being described as routinely. According to the officer, measures taken on unlicensed vendors and those who do not comply with regulations include withdrawal of license and shutting down of business. The regulatory officer admitted to being aware of reported concerns on the quality of milk sold using milk vending machines. The officer, however, stated that Kenya Dairy Board has not yet begun carrying out regular and intensive surveillance tests to ascertain the safety of milk from milk vending machines and that the institution is in the process of building capacity to carry out these checks.\n\n\nDiscussion\n\nThe level of knowledge on hygiene among milk vending machine handlers in this study was relatively low at a mean percentage of 68.83. In comparison with similar studies (Mandefero & Yeshibelay, 2018), milk handlers had poor scores on overall food safety knowledge, personal hygiene, and temperature control. This is attributed to a lack of adequate training on milk hygiene knowledge among handlers. The level of hygiene knowledge among handlers in this study increased with increase in the level of education, this could be because one’s general understanding of hygiene and possible compromises to food safety improves as one progresses with education, and these findings are in tandem with those of other studies (Blackmore et al., 2015), which found the knowledge of handlers being influenced by the level of education. The belief that pasteurized milk is safe cannot be contaminated by microorganisms alludes to a lack of enough knowledge on potential milk hazards among the respondents, a finding that is consistent with that of Lindahl et al. (2018) which revealed that only 9.1% of the respondents in that study believed that diseases can be transmitted by milk. In another study, Galičič et al. (2015) stated that one of the reasons milk vending machines have been highly embraced is the perception that the milk sold from these machines is of good quality and safe for consumption. While a majority of the vendors believe that milk storage facilities should always be kept clean using portable water, most of them do not know the type of detergents to use and could end up compromising the safety of the milk stored. While respondents understand that milk and milk vending machines should be purchased from licensed suppliers, however, the lack of awareness among vendors on the appropriate milk storage temperature is a major threat to milk safety. It is stated in the regulation that milk stored in the vending machine should be cooled and maintained at not higher than 4℃ (KDB, 2018), and the control and monitoring of milk storage parameters such as temperature is critical in determining the level of microbial load in milk (O’Connell et al., 2016). The majority of the milk handlers do not know that there are laws and regulations that guide the selling of milk using milk vending, which is a challenge that could be attributed to a lack of enough training and sensitization.\n\nMilk safety is highly dependent on the handling practices of vendors, and several cases of milk contamination are attributed to poor handling practices (Angelidis et al., 2016). Milk vending machine handlers in the current study demonstrated poor handling practices as indicated by the mean percentage score of 54.05. This finding is similar to other studies (Abunna et al., 2019) which found the level of practices among milk vendors to be poor. The majority of the vendors in this study sold pasteurized milk via vending machines in line with the Regulatory Act on milk vending machines in Kenya (KDB, 2018). The finding, however, contrasts with that of another study by Abunna et al. (2019), which found out that 81.5% of the vendors sold raw milk. Despite observing appropriate practices during cleaning of the vending machines, record keeping of routine hygiene and handling operations was a major challenge among handlers, which makes it hard for traceability in the event of safety concerns due to milk contamination (Ndambi et al., 2020).\n\nVendors are required to routinely monitor temperature fluctuations and conduct periodic calibration of the vending machines while keeping these records as evidence during inspections. However, none of the vendors in this study kept monitoring and calibration records while some were not carrying out periodic monitoring and calibration. Efforts being put in place to reduce the possibility of cross-contamination in milk are not satisfactory, this is seen through the practice by 70.3% of the vendors storing milk in intermediate containers instead of transferring it directly from the supplier’s container to the vending machine. The lack of practices among the majority of the vendors to undertake medical tests for contagious diseases puts the consumer at risk of contracting these diseases through contaminated milk. Despite the need for vendors to be trained before they are issued with a license (KDB, 2018), the majority of the vendors (64.9%) both with or without a license stated that they had not received any training and did not know that it existed, which is a drawback on the part of the regulator.\n\nThe level of practices among milk handlers increased with an increase in the level of education. This can be attributed to the increased hygiene knowledge among vendors, which is also influenced by education level, and has statistically been proven by the positive correlation between knowledge and practices of milk vending machine handlers in the current study. The overall poor milk handling practices can be attributed to a lack of training among a majority of the milk vending machine handlers as indicated in this study, which is a finding consistent with that of similar studies (Amentie et al., 2016). The poor level of compliance with regulations by the milk vending machine handlers in this study can be attributed to the complexity of some regulatory standards that are seen to be either unclear to the vendors or out of touch with the reality of the informal settlements which are characterized by poor infrastructure and inadequate capital, therefore discouraging compliance (Blackmore et al., 2015).\n\nOne limitation of this study is that it was not done over a longer follow up period to ascertain the exact impact of hygiene knowledge on practices of milk vending machine handlers. Another limitation is that some information obtained from the interviews on hygiene practices by respondents is prone to respondent bias since some may not have presented the true position on their milk handling practices due to personal fears hence compromising the authenticity of the study findings.\n\n\nConclusion\n\nMilk vending machine handlers in the informal settlements of Nairobi have a low level of knowledge and poor practices on milk hygiene and safety and are not adequately adhering to regulations governing the operation of milk vending machines. This is attributed to a lack of proper training on milk hygiene and handling practices. While efforts are being put in place by handlers to keep milk in good condition, critical omissions by vendors still threaten the safety of milk and consumers’ health at large.\n\nVendors’ awareness of milk hygiene and regulations relating to the operation of milk vending machines is inadequate especially in informal settlements. Increasing vendor awareness on matters of milk hygiene and correct practices through training and sensitization is crucial for improving regulatory compliance. Proposed changes to the Dairy Act should include routine inspections of milk vending machine premises in both the formal and informal settlements to assess the practices of vendors and possible gaps that exist, which could guide corrective actions. Penalties for non-compliance through poor practices by handlers should be strengthened but only after adequate training and corrective action measures have been provided to the vendors.\n\n\nData availability\n\nZenodo: Data on hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi Kenya. https://doi.org/10.5281/zenodo.5204466 (Holi, 2021a).\n\nThis project contains the following underlying data:\n\n-Dataset (sav)\n\nZenodo: Questionnaire on hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi Kenya. https://doi.org/10.5281/zenodo.5508163 (Holi, 2021b).\n\nThis project contains the following underlying data:\n\n- Holi Regulator's Questionnaire.docx (a copy of the questionnaire for the regulator including the answers)\n\nZenodo: Questionnaire on hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi Kenya. https://doi.org/10.5281/zenodo.5508163 (Holi, 2021b).\n\nThis project contains the following extended data:\n\n- Holi Knowledge and Practices Questionnaire.docx (a copy of the questionnaire for vendors)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgment\n\nThe authors express gratitude to all the milk vending machine handlers who volunteered to take part in this study.\n\n\nReferences\n\nAbunna F, Tasew N, Ragassa F, et al.: Handling Practices, Quality and Safety of Milk along the Dairy Value Chains in Selected Sub Cites of Addis Ababa, Ethiopia. Biomed J Sci Tech Res. 2019; 13(1). Publisher Full Text\n\nAlonso S, Muunda E, Ahlberg S, et al.: Beyond food safety: Socio-economic effects of training informal dairy vendors in Kenya. Glob Food Sec. 2018; 18: 86–92. Reference Source\n\nAmentie T, Guya M, Mekasha Y, et al.: Milk postharvest handling practices across the supply chain in Eastern Ethiopia. J Adv Vet Ani Res. 2016; 3(2): 112–126. Publisher Full Text\n\nAngelidis AS, Tsiota S, Pexara A, et al.: The Microbiological Quality of Pasteurized Milk Sold By Automatic Vending Machines. Lett Appl Microbiol. 2016; 62(6): 472–479. PubMed Abstract \| Publisher Full Text\n\nBlackmore E, Alonso S, Grace D: Legitimising informal markets: A case study of the dairy sector in Kenya. 2015. Reference Source\n\nBrown LH, Alonso S, Lindahl J, et al.: Regulatory Compliance in the Kenyan Dairy Sector: Awareness and Compliance among Farmers and Vendors. IFPRI : International Food Policy Research Institute, 2018. Reference Source\n\nGaličič A, Kanjec N, Kibiš A, et al.: Consumer’s attitude and manipulation of raw milk from milk vending machines. International Journal of Sanitary Engineering Research. 2015; 9(1). Reference Source\n\nGodfrey K: Milk Quality and On-Farm Factors Leading To Milk Spoilage In Bugaaki Sub County- Kyenjojo District. 2013; 72. Reference Source\n\nGrace D: Food Safety in Low and Middle Income Countries. Int J Environ Res Public Health. 2015; 12(9): 10490–10507. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrace D, Dipeolu M, Alonso S: Improving food safety in the informal sector: Nine years later. Infect Ecol Epidemiol. 2019; 9(1): 1579613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoli K: Data on hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi Kenya [Data set]. Zenodo. 2021a. http://www.doi.org/10.5281/zenodo.5204466\n\nHoli K: Questionnaire on hygienic knowledge and practices of milk vending machine handlers in the informal settlements of Nairobi Kenya. Zenodo. 2021b. http://www.doi.org//10.5281/zenodo.5508163\n\nKDB: The Dairy Industry Regulations. 2018; 114. Reference Source\n\nKEBS: Code of hygienic practice for milk and milk products/KS 1552: 2016—Full Catalogue Listing. 2016. Reference Source\n\nKesmodel US: Cross-sectional studies - what are they good for? Acta Obstet Gynecol Scand. 2018; 97(4): 388–393. PubMed Abstract \| Publisher Full Text\n\nKNBS: Kenya National Bureau of Statistics—Humanitarian Data Exchange. 2019. Reference Source\n\nLindahl JF, Deka RP, Asse R, et al.: Hygiene knowledge, attitudes and practices among dairy value chain actors in Assam, north-east India and the impact of a training intervention. Infect Ecol Epidemiology. 2018; 8(1): 1555444. Publisher Full Text\n\nMandefero D, Yeshibelay G: Assessment of community knowledge, attitude and practice on milk borne zoonoses disease in Debre- Birhan town, north Shewa, Ethiopia. J Public Health Epidemiol. 2018; 10(4): 123–131. Publisher Full Text\n\nNdambi A, Njiru R, van Knippenberg C, et al.: A Review of Interventions and Parameters Used to Address Milk Quality in Eastern and Southern Africa. Food Control. 2019; 116: 107300.\n\nO’Connell A, Ruegg PL, Jordan K, et al.: The effect of storage temperature and duration on the microbial quality of bulk tank milk. J Dairy Sci. 2016; 99(5): 3367–3374. PubMed Abstract \| Publisher Full Text\n\nPaludetti LF, Jordan K, Kelly AL, et al.: Evaluating the Effect of Storage Conditions on Milk Microbiological Quality and Composition. Irish J Agric Food Res. 2018; 57(1): 52–62. Publisher Full Text\n\nRaposo A, Carrascosa C, Pérez E, et al.: Vending machines: Food safety and quality assessment focused on food handlers and the variables involved in the industry. Food Control. 2015; 56: 177–185. Publisher Full Text\n\nSadhu SP: Effect of cold chain interruptions on the shelf-life of fluid pasteurised skim milk at the consumer stage. Braz J Food Technol. 2018; 21. Publisher Full Text" }	[ { "id": "100363", "date": "06 Jan 2022", "name": "Victor Kuri", "expertise": [ "Reviewer Expertise Food Safety", "Food Operation Management", "Pathogen Control", "Food Science", "Food Product 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\nThe manuscript reports an interview survey carried out on milk vending machine handlers in Kenya, which included some questions to probe knowledge on some specific aspects of the regulations, hygiene, and their understanding and practices.\n\nIt also includes a relation of an interview to an officer of a related board, and moves on to recommend changes on legislation.\nSome aspects of the discussion don't seem to be evidence-based, and aspects of the protocol may not be fully reproducible.\nAbstract - results: \"Knowledge and practices of vendors significantly improved with....\" They did not improve... a sub-sample received 'higher scores' than other.\nIn the study setting, authors state that: \"The vending machines are randomly distributed in the areas without any specific order.\" ... If no test for randomness was carried out, please remove this as it may be false, patterns of accessibility, transport, pockets of population transit or availability of location may have influenced distribution... an unknown order does not mean that there is no order. What does an \"exhaustive sampling\" mean? it would be better to report the numbers available against the number of volunteers included.\nData collection: An interview to one officer may provide valid background information or knowledge, but no research data to be bundled with other data.\nThe resulting assessment may be suited for an opinion paper, but not as part of a the research report; the isolated statements of a handful of individuals (interviewee and author(s) providing appraisal) are not reproducible.\n\nHK Question 2 is confusing... Authors state that pasteurized milk, despite being safe for consumption, can be contaminated by microorganisms; The question state that \"Pasteurized milk is safe for consumption AND cannot be contaminated by microorganisms\" Would term 'cannot' be always clearly discriminated from 'could not', 'should not', 'must not' or 'ought not to be' by the target sample cohort?\nAlso the link between both components of the question is problematic. Are both premises necessary for the statement to be correct? What if there are no organisms but pasteurized milk is unsafe for consumption because there is a toxin or perhaps a physical risk by foreign body? A suggestion is that results linked to this question are eliminated. Table 4, Q 8... the reference to Q5 seems to be incorrect. When describing Table 4, authors state: \"....that the detergent was food grade, which is against the regulations.\" Review writing, as this seems to be incorrect. What precisely is against the regulations ?\nTable 5. Not all information listed is relevant, remove table, relevant value already included within the text? Detail also could be integrated within Figure 2.\nFigure 3 appears redundant. This could be best shown on a table or explained within the text.\nUnder last heading on result section, this is unclear: \"....vendors are expected to adhere to exists. \"\nSection labelled: \"Role of regulators in ensuring vendor compliance to regulatory practices\" Some of the information here fits better within the introduction, and will ideally use citations to mentioned regulations, laws, or KDB reports. Other information could be used in the discussion to provide context and possibly to suggest explanations, linked to 'personal communication' citations. Otherwise, remove section, as this is not research data.\n\nDiscussion: the variable: level of knowledge on hygiene is not clear enough to refer to it as a percentage. This is linked to a set survey... refer to the survey then.\nConclusion: Some of this makes sense, but it is not related directly to the evidence presented or to the data obtained. Personal and professional opinions have a place in technical literature, but this is mixed up with a research report, which is unacceptable.\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "115485", "date": "28 Jan 2022", "name": "Puja Dudeja", "expertise": [ "Reviewer Expertise food safety" ], "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 congratulate the authors for undertaking research on this relevant topic. However, some observations are as under:\nThe sample size is very small. further, the authors have converted them into percentages.\n\nThe strength of correlation between knowledge and practice though significant is not strong. it is only a fair correlation and hence should not be commented upon.\n\nSince the sample size is small the authors should restrict presenting their findings as descriptive statistics only and not apply statistical tests as done for analytical studies.\n\nA line about milk-borne infections and its prevalence in the area of study would add value to the relevance of current work\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": [] } ]	1	https://f1000research.com/articles/10-957
https://f1000research.com/articles/10-378/v1	12 May 21	{ "type": "Research Article", "title": "Nutrient loading and farm characteristics of giant gourami fish aquaculture systems in Lake Maninjau, Indonesia: basic knowledge of production performance", "authors": [ "Hafrijal Syandri", "Azrita Azrita", "Eni Sumiarsih", "Elfiondri undefined", "Azrita Azrita", "Eni Sumiarsih", "Elfiondri undefined" ], "abstract": "Background Aquaculture systems for giant gourami, Osphronemus goramy Lacepède (1801), have significantly improved fish production yields and food security in Indonesia. However, these systems also cause serious problems in terms of eutrophication in waterbodies. This study analysed the nutrient loading and farm characteristics of giant gourami in floating cages in Lake Maninjau. Method A total of 20 floating cages were used to record these nutrients in feed supply, female and male juvenile fish, dead fish and harvested fish to estimate nutrient loading. Data on the harvested fish, production cycle, stock number and cage capacity were used to estimate the stocking density, feeding rate, feed efficiency, and net fish yield, and the relationship between feed supply and nutrient loading and farm characteristics was analysed by least squares regression methods. Results A total of 20 floating cages released nutrients into waterbodies at an average rate of 236.27±60.44 kg/cycle for C, 84.52±20.86 kg/cycle for N and 8.70±3.63 kg/cycle for P. On average, fish production for each floating cage (±SD) was 1226±282 kg wet weight/cycle, and the net fish yield was 12.63±2.82 kg/m3/cycle. Survival rates ranged from 86.33 to 95.27%/cycle. The production cycles varied from 160 to 175 days with feed conversion ratios between 1.60 and 1.75, feed conversion efficiencies were between 0.58 and 0.63. The production parameters that had strong relationships with the net fish yield were feed supply (r2=0.960), stocking rates (r2=0.924) and feeding rates (r2=0.961). In contrast, the length of the production cycle was not strongly related to the net fish yield (r2=0.187). Conclusion Nutrient loading from the supplied feed was greater than that from the harvested fish, juvenile fish and dead fish. Increasing the net fish yield in floating cages was better predicted by the stocking densities and feeding levels than by the other factors.", "keywords": [ "Lake Maninjau", "giant gourami culture", "floating cage aquaculture", "nutrient loading", "farm characteristics." ], "content": "Introduction\n\nFish are a source of protein, lipids, carbohydrates, vitamins and essential minerals.1–3 Therefore, fisheries production is very important to increasing food security3,4 through capture fisheries and aquaculture sectors.5 To increase the global production of aquaculture, freshwater can be provided in a variety of aquaculture systems, such as freshwater ponds, tanks and floating cages.6–9\n\nCage aquaculture is expanding in tropical lakes and has been ongoing for a long time.9–11 Lake Maninjau in Indonesia has used cage aquaculture since 1992 (Nazarudin-Sepakat Aquaculture’s farm manager, pers. comm.). Some authors have reported that the dominant species being cultured in tropical lakes is tilapia, and commercial feed pellets are used.9,11–15 In the past five years, fish farmers in Lake Maninjau have also conducted giant gourami fish farming activities in floating cages with commercial feed pellets because it is an economically important species for food security in Indonesia, and most of the giant gourami that have been consumed for decades have been produced by aquaculture in freshwater ponds.7,16,17\n\nEnvironmental impacts of tilapia aquaculture operations that have been recorded in tropical lakes have also been reported in Lake Malawi,11 Lake Taihu,18 Lake Victoria,11,15,19 and Lake Kariba.9 In contrast, Syandri et al.20 reported that in a small lake, i.e., Lake Maninjau in Indonesia, tilapia aquaculture is approximately 17 km long by 8 km wide and has mean and maximum depths of approximately 112 and 178 m, respectively. Many studies have been carried out to evaluate nutrient loading, such as C, N and P loadings, and the growth performance of tilapia farms in lakes and reservoirs.11,12,18 However, no data are available for nutrient waste loads from feed, juvenile fish, dead fish and harvested fish, including data on the characteristics of farming giant gourami in floating cages, such as stocking density, total stock weight, feed conversion ratio, production cycle, harvest size, feeding level and specific growth rate. To address these issues, the present study was conducted to evaluate the C, N and P nutrient loads of giant gourami fish in floating cages and the operational characteristics to determine the relationship between production and cultivation efficiency to provide basic knowledge about production performance for the future.\n\n\nMethods\n\nIn the present study, no permits from the Government of the Republic of Indonesia were needed to record data on feed supply, initial weight, stocking density, fish production, fish mortality and production cycle of giant gourami in 20 floating cages in Lake Maninjau from 2019 to 2020. The study included collecting sediment and fish and killing as many as three giant gourami in each floating cage to analyse the chemical composition of carbon, nitrogen and phosphorus from the carcasses. This research was recommended by the Research and Community Service Universitas Bung Hatta with sponsorship from the Indonesian Education Management Institution, Ministry of Finance Republic of Indonesia, through a competitive grant called Productive Innovative Research 2019 with contract number PRJ-99/LPDP/2019. Ethical approval was granted by the Ethics Commission for Research and Community service at Universitas Bung Hatta (098/LPPM/Hatta/X-2019).\n\nThe research was conducted in Lake Maninjau, located in the Agam District, West Sumatera Province, Indonesia, at an altitude of 463 m above sea level with a surface area of 97.37 km2, a water volume of 10.4 km3, a water retention time of 24.5 years, and a catchment area of 13.26 km2. Since 1973, lake water has been used for electric power generation with a capacity of 64 MW, and starting in 1992, the lake has also been used floating-cage fish farming activities.\n\nA total of 200 floating cages using for giant gourami aquaculture by fish farmers in Lake Maninjau (Nazaruddin-Sepakat Aquaculture farm manager, personal communication). A total of 20 floating cages for giant gourami culture were used as samples. The sample was determined by simple random sampling using an ordinal method.21 The data recorded were stock size (g), stock number (fish), total stock weight (kg), mortality (fish), feed supply (kg), total harvest weight (kg) and production cycle (days). Each floating cage had a capacity of 75 m3 (5×5×3 m) and was constructed using a 10 mm mesh sieve. Each floating cage was combined with other resources, such as a buoy, a feeding lodge and cage pathways.\n\nThe chemical compositions carbon (C), nitrogen (N) and phosphorus (P) of the feed, fish and faeces were analysed. For the feed nutrient analysis, the feed samples were floating commercial feed (pelleted). The approximate composition of the feed was 12% moisture, 29% crude protein, 6% crude lipid, 12% crude fibre and 6% crude ash. The fish were sampled from 10 floating cages (3 fish/cage) that were cultured for 150 days, and the fish weighed between 235 and 250 g/fish. Carbon (C) and nitrogen (N) concentrations (as % of dry weight) of the feed and fish were determined by the standard methods of the Association of Official Analytical Chemists.22 The phosphorus (P) concentrations were determined using a spectrophotometer (Shimadzu UV-160 UV160 UV-Vis-NIR Spectrophotometer in Hayward, CA, USA) and the molybdate–ascorbic acid method indicated by the Association of Official Analytical Chemists22 at the Chemistry Laboratory of Universitas Bung Hatta Padang. To complement the data, we also analysed the waste material of cultured giant gourami fish collected with traps under the floating cages. To collect the faeces, ten giant gourami were kept for 3 days in an aquarium with a capacity of 0.48 m3 (2×0.6×0.4 m), and then, the faeces were deposited on the bottom of the aquarium. Furthermore, the deposited faeces were sucked into a clean bowl and dried. Waste material and faeces were analysed by the AOAC method.22\n\nThe C, N and P loadings from feed, juvenile fish, dead fish and harvested fish were estimated according to the method described by23. The following parameters with their corresponding equations were analysed:\n\nC (loss, kg) = (F × CDF + J × CDJ) − (H × CDH + M × CDM)\n\nN (loss, kg) = (F × NDF + J × NDJ) − (H × NDH + M × NDM)\n\nP (loss, kg) = (F × PDF + J × PDJ) − (H × PDH + M × PDM)\n\nwhere F, J, H and M are the dry weight (kg) of the supplied feed, stocked juvenile fish, harvested fish and total dead fish in floating cages, respectively. The data were recorded at the end of each production cycle from the 20 floating cages. CDF, CDJ, CDH and CDM are the carbon contents in dry feed (DF), dry juvenile (DJ), dry harvest (DH) and dry mortality (DM), respectively.\n\nThe farm characteristic parameters were analysed using the following formulas:\n\nSpecific growth rate (%/day)= (Log harvests weight−Log stock weight)Culture days×100\n\nGross fish yield (kg/m3) = (Total harvest number in individual×average final fish weight in kg)Cage capacity\n\nNet fish yield (kg/m3) = (Total number of fish harvest in kg−total stock weight in kg)Cage capacity\n\nFeed conversion ratio (FCR) = Feed supply in kgTotal harvest weight in kg\n\nFeed conversion efficiency (FCE) = 1Feed conversion ratio\n\nFeeding rate (%) = Average weight gain per day in kgMean harvests size in g×100\n\nSurvival rate (%) = Total number of fish harvestedTotal number of fish stocked×100\n\nThe relationships between feed supply and nutrient load, harvested fish, production cycle and net fish yield, feeding level, feed conversion efficiency, stocking density and net fish yield were estimated by the least square’s regression method,24 and the figures were plotted using Microsoft Office Professional plus 2019.\n\n\nResults\n\nThe C, N and P contents of the feed, fish and faeces of the giant gourami in this study are presented in Table 1. Furthermore, the estimated mass balances of C, N and P of the feed, juvenile fish, dead fish and harvested fish from the 20 floating cages are summarized in Table 2.\n\nFish feed was the main factor accounting for the C, N and P nutrient loadings of the giant gourami reared in floating cages, while stocked juvenile fish and dead fish accounted for a minor amount (Table 2). The average C, N and P loadings estimated from the floating cages were 236.27 kg/cycle, 84.52 kg/cycle and 8.70 kg/cycle, respectively, while the C, N and P loadings from each floating cage of giant gourami fish are displayed in Figure 1. Feed supply and carbon, nitrogen, and phosphorus loadings had linear relationships for the giant gourami reared in floating cages as shown by C = 0.1339 × FS − 37.238 (with r2 = 0.988, Figure 2), N = 0.0455 × FS − 8.1604 (with r2 = 0.996, Figure 3), and P = 0.0048 × FS − 1.117 (with r2 = 0.991, Figure 4). The feed supply and net fish yield (kg/m3/cycle) relationship for the giant gourami reared in floating cages was shown by a net fish yield=0.0059×FS+0.7396 (with r2=0.9609, Figure 5).\n\nIn this study, a 75 m3 floating-cage capacity was used by fish farmers (5 × 5 × 3 m). The giant gourami fish stock number was between 40 individuals/m3 (3000 individuals/cage) and 106 individuals/m3 (8000 individuals/cage), with an average fish stock number of 75 individuals/m3. A mean weight of approximately 50 g for juveniles was stocked at the beginning of culture and reared from 160 to 175 days. To maximize the growth of giant gourami, all fish farmers used commercial, floating feed pellets (30% crude protein and 5% crude lipid). Based on recorded data by the fish farmers, the fish were fed daily at 09:00–10:00 h and 16:00–17.00 h. The amount of feed provided was adjusted according to temporal changes in biomass and the growth of the fish in the floating cages during the production cycle. The results of our analysis were that their feeding levels ranged from 1.24 to 3.47% of their body mass. Harvested fish weight ranged from 225 to 290 g/fish, and the gross yield of fish was 10.4 and 24.25 kg/m3/cycle, while the net fish yield was 8.17–18.92 kg/m3/cycle. The giant gourami were reared in the floating cages for each production cycle of 160 to 175 days, and the specific growth rate ranged from 0.87 to 1.04%/day. The net fish yield (kg/m3/cycle) in the floating cages was better predicted by the stocking rates (fish/m3) (r2 = 0.9246, Figure 6) than by the length of the production cycles (r2 = 0.1875, Figure 7). In addition, the supplied feed was not strongly related to the survival of the giant gourami (r2 = 0.6123). On the other hand, there was a strong linear correlation between feeding levels and the net fish yield (kg/m3/cycle) (r2 = 0.9611, Figure 8).\n\n\nDiscussion\n\nMany studies have reported that aquaculture has a negative impact on the aquatic environment,12,25–28 that is generally caused by waste loads of C, N and P from supplied feed, faeces and dead fish.29–31 In this study, the C, N and P loadings from the supplied feed were more predominant than those from the harvested fish, juvenile fish and dead fish because the content of C, N and P in the feed was higher than that in the harvested fish, juvenile fish and dead fish (Table 1). In addition, the average feed conversion ratio (FCR) of the cultured giant gourami cultured was 1.65, and the feed conversion efficiency (FCE) was 0.60 (1 kg of feed fish results in 0.60 kg of fish). This result suggests that the waste load was 0.40 kg (1 kg feed−0.60 kg of fish). These FCE values were lower than those of Nile tilapia and common carp cultured in floating cages in Lake Maninjau.13 Increasing amounts of C, N and P released into waterbodies from intensive aquaculture activities can cause or accelerate eutrophication in natural water systems.32–34 Nevertheless, accelerated eutrophication also depends on diet composition, feed characteristics, feed intake and feed quality.13,35–37 On the other hand, accelerated eutrophication in freshwater is largely determined by phosphorus. Therefore, efforts to control eutrophication in waterbodies focus mostly on phosphorus reduction. In the present study, the P load from giant gourami was 4.29 kg/tonne of feed and lower than the P load from common carp (11.45 kg/tonne of feed) and Nile tilapia (9.11 kg/tonne of feed).13 Therefore, giant gourami fish farming can be considered for long-term development based on the aquaculture carrying capacity in Lake Maninjau and other regions.\n\nTrophic food habits of fish might also affect the C, N and P was retained in the fish body because these habits are correlated with digestibility coefficients. Under natural conditions, giant gourami is an herbivorous fish.38 In comparison to other fish, herbivorous fishes have more efficient digestion of feed because their extralong intestines contain special enzymes and microbes, such as cellulose enzymes and Bacteroides and Cetobacterium.39,40 In the present study, the types of enzymes and microbial communities that were dominant in the giant gourami intestines are poorly understood. Regardless, herbivorous fish such as giant gourami release less N and P nutrients into waterbodies than omnivorous fish and carnivorous fish such as Nile tilapia, Oreochromis niloticus38 and Crimson snapper, Lutjanus erythropterus.41\n\nIn the present study, in comparison with the harvested and juvenile fish, the dead fish released only a small amount of nutrients into the waterbodies during the production cycle. Conversely, the availability of N and P in the waterbodies was significantly high after the extensive tilapia deaths due to upwelling (local namely: tubo belerang) and had a negative effect on the water quality of Lake Maninjau.33 In contrast, giant gourami did not experience extensive fish death because this species has a labyrinth organ. Many scientists have reported that the release of significant amounts of C, N and P waste loads into waterbodies from feed and extensive fish deaths has a negative environmental impact.14,42–45 In fact, feed supply and C, N and P loadings had a strong relationship with giant gourami cultured in floating cages, except in terms of fish mortality.\n\nThe 20,608 units of floating cages used for rearing Nile tilapia and common carp have exceeded the estimated aquaculture carrying capacity in Lake Maninjau over the past several years.33 This factor has had a negative impact on the water quality of Lake Maninjau, and the net yields of Nile tilapia and common carp were 14.42 and 14.11 kg/m3/cycle, respectively.13,46 In contrast, poor water quality does not have a negative impact on the growth and mortality of giant gourami because this species is resistant to poor water quality. Hence, the survival of giant gourami in floating cages ranged from 86.33 to 95.27%/cycle, and the net fish yield was as high as 18.92 kg/m3/cycle. In addition, the survival of giant gourami also depended on feeding level during the rearing period. Our analysis of the feeding level of giant gourami by fish farmers varied between 1.24 and 3.47%/body weight/day, and the majority of the fish farmers (80%) provided pellet feed at less than 3%/body weight/day. For giant gourami, a feeding level of 4-6%/body weight/day has been recommended.47 Similarly, Skov et al.48 concluded that biomass weight gain and the specific growth rate of Nile tilapia depend on feeding rate and the feed conversion ratio. In this study, the feeding rate and feed conversion ratio had a strong linear correlation with the net fish yield. Therefore, feeding levels played a significant role in increasing the net giant gourami yield. Many studies have reported that a lower feeding level might result in slow growth and inefficient aquaculture, whereas overfeeding may lead to feed waste, inefficiency and negative environmental impacts.37,49–51\n\nOn the other hand, the length of the production cycle did not have a strong linear correlation with the net giant gourami yield (kg/m3/cycle). In contrast, the stocking rate had a strong correlation with the net fish yield. In this study, the stocking rate ranged from 40 to 107 fish/m3, and the majority (70%) ranged between 40 and 80 fish/m3. Therefore, we recommended achieving a market size of 300 g/fish and a net fish yield (18.92 kg/m3/cycle) using a stocking density of 107 fish/m3 for 170 days of culture. Conversely, if the equation by Schmittou23 was applied to meet the target mean weight of 300 g/fish and net fish yield at harvest of 30.93 kg/m3/cycle, then we recommend using a stocking density of 106 fish/m3, with a length production cycle of 170 days. Therefore, to increase production performance of giant gourami in floating cages the management strategy must be to control the optimal seed stock, fish health, feed quality, feeding level, feeding time and husbandry factors. Based on current scientific knowledge, scientists strongly advocate a combination of optimal stocking density, feeding practices, rearing techniques and eco-dam system to increase fish production performance and reduce the aquaculture waste released into waterbodies.41,52,53\n\n\nConclusion\n\nThis research analysed the carbon, nitrogen and phosphorus loadings and the farm characteristics of giant gourami reared in floating cages in Lake Maninjau. There was a strong linear relationship between feed supply and nutrient loading for the reared giant gourami. Nutrient loading from feed supply was greater than that from juvenile fish, dead fish and harvested fish. Keys to increasing the net fish yield were stocking density and feeding level. The maximum target for the net fish yield and market size was achieved for 160 days. Therefore, giant gourami cultivation is an important practice to consider continuing in Lake Maninjau in accordance with the aquaculture carrying capacity because the phosphorus released into the waterbodies was very low, and this species also has a high survival rate in floating cages.\n\n\nData availability\n\nFig share: Underlying data for ‘Nutrient loading and farm characteristics of giant gourami fish aquaculture systems in Lake Maninjau, Indonesia: basic knowledge of production performance’. https://doi.org/10.6084/m9.figshare.14369999\n\nThe project contains the following underlying data:\n\nTable 1. Carbon (C), nitrogen (N) and phosphorus (P) composition (%) of the dry weight of the feed, harvested fish and faeces\n\nTable 2. Raw data carbon loss from 20 floating cages\n\nTable 3. Raw data nitrogen loss from 20 floating cages\n\nTable 4. Raw data phosphorus loss from 20 floating cages\n\nTable 5. Raw data production performance of giant gourami fish from floating cages in Lake Maninjau\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 for Rionald Silaban the Director of Indonesian Education Fund Management Institution, Ministry of Finance Republic of Indonesia, for supporting this study through the competitive grant for productive and innovative research (policy/governance) 2019. We appreciate all of the students (Puji Kurniawan and Muhammad Vajri Djauhari), fish farmers (Satria Aferi and Nazarudin) and partners (Ermanto) who helped the authors during data collection in the field.\n\n\nReferences\n\nMohanty BP, Mahanty A, Ganguly S, et al.: Nutritional composition of food fishes and their importance in providing food and nutritional security. Food Chem. 2019; 293: 561–570. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuna M, LIorente I, Cobo A: Integration of environmental sustainability and product quality criteria in the decision-making process for feeding strategies in seabream aquaculture companies. J. Clean. Prod. 2019; 17: 691–701. Publisher Full Text\n\nMungkung RJ, Aubin TH, Prihadi, et al.: Life Cycle Assessment for environmentally sustainable aquaculture management: a case study of combined aquaculture systems for carp and tilapia. J. Clean. Prod. 2013; 47: 249–256. Publisher Full Text\n\nNi Z, Wu X, Li L, et al.: Pollution control and in situ bioremediation for lake aquaculture using an ecological dam. J. Clean. Prod. 2018; 172: 2256–2265. Publisher Full Text\n\nSyandri H, Azrita, Sumiarsih E, et al.: Underlying data for ‘Nutrient loading and farm characteristics of giant gourami fish aquaculture systems in Lake Maninjau. Indonesia: basic knowledge of production performance’. Publisher Full Text" }	[ { "id": "88018", "date": "23 Jul 2021", "name": "Zahidah Hasan", "expertise": [ "Reviewer Expertise I am aquaculturist majoring in water quality" ], "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 clearly written. The literature cited are mostly up to date. The methods are mostly clearly written except for those related to the collection of dead fish and waste material.\nWaste material collection needs to be explained further. How long is the trap installed; is the trap removed periodically or left throughout the cultivation period? Is there also faeces in the waste material component?\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "92790", "date": "14 Sep 2021", "name": "Christopher Mulanda Aura", "expertise": [ "Reviewer Expertise Aquatic sciences" ], "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 have read through the paper and it is well written and at the same time, it will add value to cage culture knowledge on Ecosystem Approach to Aquaculture.\nAlthough the article is well written, there is a need to provide some data on selected physio-chemical parameters such as Oxygen, Conductivity, depth etc. that may have a role in the discussion of the results (i.e. are factors affecting nutrient loading and farm characteristics covered in terms of data to help in the discussion).\nFurthermore, it would be important if the methodology adopts a comparing scenario to offer justifiable results.\nIf the above major comments are incorporated, the paper will be in good shape.\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-378
https://f1000research.com/articles/10-146/v1	25 Feb 21	{ "type": "Study Protocol", "title": "Physical rehabilitation versus no physical rehabilitation after total hip and knee arthroplasties: Protocol for a pragmatic, randomized, controlled, superiority trial (The DRAW1 trial)", "authors": [ "Troels Mark-Christensen", "Kristian Thorborg", "Thomas Kallemose", "Thomas Bandholm", "Kristian Thorborg", "Thomas Kallemose", "Thomas Bandholm" ], "abstract": "Background: Following total hip- and knee arthroplasty (THA and TKA), post-discharge physical rehabilitation is common practice, but varies significantly regarding content, duration, intensity and mode of delivery. Recent systematic reviews have found home-based rehabilitation to be as good as outpatient rehabilitation in terms of pain and physical function. We therefore wonder if physical rehabilitation “works” at all when compared to no physical rehabilitation after THA and TKA – “no rehabilitation” defined as no prescribed therapeutic rehabilitation exercises. The purpose of this trial is to compare the effectiveness of home-based telerehabilitation, home-based rehabilitation and no physical rehabilitation following THA and TKA. Methods: This pragmatic, randomized controlled trial will include 168 patients following discharge after THA or TKA, in Bornholm Denmark. Patients will be randomized into one of the three 6-week rehabilitation strategies: home-based telerehabilitation, home-based rehabilitation or no physical rehabilitation. The trial is designed as a superiority trial to test the hypothesis that rehabilitation (home-based telerehabilitation and home-based rehabilitation) is superior to no physical rehabilitation. The primary outcome will be the hip disability and osteoarthritis outcome score (HOOS)/ the knee injury and osteoarthritis outcome score (KOOS)-subscale: function of daily living at first follow-up (end of the 6-weeks' intervention). Additional follow-ups are scheduled at 3 and 12 months. Outcome assessors and data analysts are blinded to group allocation. Conclusions: Knowledge about the effectiveness of the three investigated rehabilitation strategies will help guide the future organization of post-discharge rehabilitation after THA and TKA. Trial registration: Clinicaltrials.gov NCT03750448 (23/11/2018)", "keywords": [ "Total Joint Replacement", "Total Hip Arthroplasty", "Total Knee Arthroplasty", "Rehabilitation", "Telerehabilitation." ], "content": "Background\n\nOsteoarthritis is the leading cause of disability and pain among adults1, and when non-surgical treatment does not adequately relieve symptoms, joint arthroplasty is considered. Total hip and knee arthroplasty (THA and TKA, respectively) are considered effective for end-stage osteoarthritis in restoring function of the affected joint, relieve symptoms and improving quality of life2–4. Post-discharge physical rehabilitation following THA and TKA is widely promoted to restore function and mobility of the affected joint5,6. However, the practices of post-discharge physical rehabilitation following THA and TKA vary significantly in content, duration and intensity, where the current practices are ranging from a single session of professional advice while still in hospital to several weeks of intensive in- or outpatient physical rehabilitation or home-based interventions5–11.\n\nSeveral systematic reviews and meta-analyses of randomized controlled trials (RCTs) have examined the effectiveness of different types of post-discharge rehabilitation delivery strategies following THA and TKA5,12–14. Generally, home-based rehabilitation after initial instruction is as effective as more closely supervised outpatient rehabilitation in the long term5,7,13–16, even in patients at risk of a poor outcome after TKA17. When compared to no or minimal physical rehabilitation, physiotherapy exercises seem superior for improvements in physical function, pain and range of motion up to 3–6 months following TKA7. We recently undertook a systematic review18 of RCTs that compared physical rehabilitation strictly to a “no physical rehabilitation” comparator on patient-reported outcomes for function and pain and identified only two trials19,20. The review was inconclusive, and we – along with others7 – call for sufficiently powered trials that investigate physical rehabilitation after THA and TKA against no physical rehabilitation comparators.\n\nTraditionally, the type of physical rehabilitation exercise and degree of supervision are regarded as important factors for postoperative recovery of functional performance; however, this does not seem to be the case. Rehabilitation with little supervision has been shown to be as equally effective as rehabilitation with much more supervision following THA and TKA in terms of self-reported function, physical performance and pain5,7, as reviewed above. So, a natural next step for us is to challenge our own belief that physical rehabilitation (in itself) is important – and surely better than “no physical rehabilitation” – when it comes to enhancing postoperative recovery of functional performance after these surgeries.\n\nIn the DRAW 1 trial, we will investigate three different rehabilitation strategies after THA and TKA. We will use two rehabilitation strategies that are commonly used in the community rehabilitation setting in Denmark; home-based telerehabilitation and home-based rehabilitation after initial instruction and compare them to a strategy of no physical rehabilitation as defined in the methods section below. Our primary outcome is the hip disability and osteoarthritis outcome score (HOOS)/knee injury and osteoarthritis outcome score (KOOS)-subscale: function of daily living at the end of the 6-weeks' intervention. This is chosen to be the primary outcome as it is usually physical function that prescribed rehabilitation is targeted to improve5,7. The primary endpoint is at the 6-week follow up, as it represents current clinical practice in terms of rehabilitation duration for the municipality of Bornholm, Denmark. When we collected stakeholder input for the trial design, the municipality’s main interest in the trial was related to this time point firstly and the other time points secondarily.\n\nThe objective of the trial is to compare the effectiveness of home-based telerehabilitation, home-based rehabilitation and no physical rehabilitation following THA and TKA.\n\n\nMethod\n\nThis trial is named the “Does rehabilitation after total hip and knee arthroplasty “work”? (DRAW1 trial)”. It uses a superiority, three-arm, parallel group, RCT design with blinded outcome assessments at baseline (before intervention), at 6 weeks (end of intervention) and follow-ups at 3 and 12 months. The trial protocol is based on the PREPARE trial guide21 and the SPIRIT checklist22,23. The trial report will adhere to the Consolidated Standards of Reporting Trials (CONSORT) using the extension for non-pharmacological treatments24. The interventions will be described in details using the recommended generic template for intervention description and replication (TIDieR)25. For a detailed exercise-specific description of the intervention, the consensus on exercise reporting template (CERT)26 will be used. The trial is pre-registered at ClinicalTrials.gov (NCT03750448 on 23rd November 2018)27 and ethical approvals was obtained from the ethics committee of the Capital Region Denmark28 and the Danish Data Protection Agency29 before the first participant was recruited. The first participant was enrolment in January 2019. The research question, objective, and trial design were developed using stakeholder input from patients, orthopaedic surgeons, physiotherapists and policymakers.\n\nThe municipality of Bornholm (Bornholms Regional Municipality), where the trial takes place, has been involved in the formulation of the research question and study design. They are considering full-scale implementation of the home-based telerehabilitation-solution based on cost-benefit considerations when compared to their current practice, which is home-based rehabilitation (no tele-setup). The effectiveness of this type of technology-assisted exercise intervention has already been compared to usual care in four municipalities in Copenhagen, Denmark, where comparable effects regarding physical performance and self-reported physical function were seen in both the usual care and telerehabilitation group after a 6-week intervention30,31. While usual care consisted of supervised exercises twice weekly during 6 weeks (12 supervised exercise sessions in total), the telerehabilitation intervention received 6 supervised exercise classes during the 6-week intervention, limiting what conclusions can be drawn regarding the true efficacy of telerehabilitation. Before investing in the telerehabilitation technology, the municipality of Bornholm wants an estimate of the effectiveness of telerehabilitation during a 6-week intervention without supervised exercise sessions to explore the full potential of telerehabilitation (please see the description of the different interventions below).\n\nAll patients will be included by consecutive sampling from three outpatient rehabilitation trial sites on the isle of Bornholm, Denmark. This consecutive sampling, along with the use of few eligibility criteria, will ensure generalizability of the results. All patients, who receive a THA or TKA and reside on the island, are referred to postoperative, free-of-charge rehabilitation at our institution, reflecting the current clinical practice in Eastern Denmark for patients undergoing THA or TKA. Patients referred to our institution will receive a letter of invitation to initiate outpatient rehabilitation following their surgery23. Written trial information and the patients’ rights as a potential trial participant will be included in this letter, including the right to have a bystander present during the first consultation at our rehabilitation centre.\n\nPatients deemed eligible for study inclusion will be introduced to the trial individually by an experienced physiotherapist, serving as the intervention deliverer, during the first consultation. The consultation will take place in a room with no external interruptions, where patients will be given thorough written and oral information about the nature of the trial by the physiotherapist. Eligible patients will be informed that the trial includes random allocation to one of three intervention groups (home-based telerehabilitation group, home-based rehabilitation group and no physical rehabilitation group) with the purpose of investigating the effectiveness of the three different rehabilitation strategies following THA and TKA. Before deciding to participate in the trial, the patients will be informed about their rights as a trial participant, including the right to have timely consideration before enrolling in the trial. The patients may consider trial participation in the study up to 10 days after they have received oral and written trial information. Patients interested in participating in the trial will be asked to provide written consent according to the template provided by the Ethics Committee of the Capital Region Denmark23. Patients who are willing to participate will sign an informed consent form before any study related procedures are performed.\n\nTo prevent ascertainment bias32, eligible patients are blinded to the trial hypothesis. The physiotherapist attending the first consultation will conduct baseline assessment immediately after the patient has provided written consent and before randomization. All outcome assessors dedicated to the trial will be trained by the primary investigator to ensure standardized outcome assessment and exercise instruction. Patients will be informed that they can, without the need to provide any reason, withdraw their consent and stop their participation in the trial. If written consent is not provided, the patient will be offered usual care at our rehabilitation facility, which is home-based rehabilitation with regular follow-ups.\n\nIn order to increase the external validity and improve the generalizability of this trial, only few eligibility criteria will be used. This will furthermore reflect current clinical practice and enhance the pragmatic nature of the trial.\n\nInclusion criteria:\n\n- Patients having had primary, unilateral THA or TKA due to osteoarthritis.\n\n- Patients being referred to receive postoperative rehabilitation at our institution.\n\n- Patients being able to speak, read and understand Danish language.\n\n- Patients aged ≥ 18 years.\n\nExclusion criteria:\n\n- Patients not able to comply with exercise instructions.\n\n- Patients who are discharged to a nursing-home facility or receiving in-home rehabilitation by home care.\n\nWhen baseline assessment is completed, the patients will be randomly allocated to one of the three rehabilitation strategies (see assignment of interventions). The attending physiotherapist will then instruct the patients individually according to their allocated group assignment. Once the initial instruction is completed and the patients are comfortable with the intervention to which they are assigned, the session is completed, and no further direct supervision will be given during the 6-week intervention. All patients - no matter their allocation - will receive pamphlets where postoperative information such as recommendations regarding the return to activities of daily living, possible complications and expected discomforts is outlined.\n\nAll patients will be given similar pamphlets as mentioned above. The pamphlets will include information of encouragement to stay active as recommended by the Danish National Health Board33 and encouragements to gradually return to activities of daily living. There will be no specific exercise-encouragement given to the no physical rehabilitation group. The two other intervention groups will be prescribed specific exercises for restoring function following THA or TKA. The detailed intervention description and the exercise description are further outlined in our extended data23.\n\nHome-based telerehabilitation. Patients randomized to this group will receive interactive virtual rehabilitation using a mobile app. The home-based telerehabilitation is based on sensor technology, developed by ICURA34 (Figure 1). This technology consists of motion sensors that can measure and analyse the quantity and quality of the exercises, and a mobile application that can guide the patient with visual response. A unique feature of ICURA trainer allows the physiotherapist to remotely supervise the individual patient’s exercise adherence and progress. This technology has already been successfully implemented in several different rehabilitation facilities across Denmark, and, hence, reflects current clinical practice in these places34.\n\nHome-based rehabilitation. Patients randomized to this group will be instructed in identical exercises as patients allocated to home-based telerehabilitation. However, this group will receive a written exercise program with instructions on how to perform the exercises at home. The home-based exercise program will be created using exercise templates from Exorlive (Figure 2). Using a link provided in the exercise program, the patients will be able to see short instruction-videos of the individual exercises.\n\nNo physical rehabilitation. Patients randomized to this group will not be prescribed any therapeutic rehabilitation exercise. This means that they will not receive any physical activity or exercise designed and prescribed for restoring normal function or reducing pain cause by disease, injury or surgery. This is in contrast to usual care, where exercise instructions are given. Participants in the no physical rehabilitation group will however be encouraged to stay active and continue life as usual, gradually returning to their activities of daily living when they feel ready for it.\n\nIf any patients in any of the allocated groups develop postoperative complications (e.g. postoperative infections, deep venous thrombosis), their participation in the study will be discontinued. The patients will then receive appropriate care and offered outpatient rehabilitation as needed. Study participants will be retained in the trial (unless they withdraw their consent) to enable follow-up data collection and to prevent missing data.\n\nOnly patients who receive home-based telerehabilitation and home-based rehabilitation are given instructions to modify their allocated intervention. Should any of these patients experience severe hip- or knee symptoms, they will be instructed to lessen the intensity of the prescribed exercise intervention, until the pain has subsided. If this is not sufficient in relieving pain symptoms, the patients are instructed to reduce the volume of the exercise, until the pain has subsided. If the pain persists, the patients are instructed to stop performing the exercises, until the pain has reduced. The patients will then be instructed to gradually progress to the physical intervention to which they are assigned. Detailed, written information will be given to all patients during the first consultation where expected discomfort following THA and TKA will be explained thoroughly.\n\nDuring the first consultation, the importance of following the study guidelines and adhering to the allocated group will be highlighted. Exercise adherence will be recorded as a percentage of completed exercises (based on adherence to the prescribed exercise frequency and repetitions) during the first follow up (after the 6-week intervention) for both the home-based telerehabilitation and home-based rehabilitation groups. Exercise adherence will be taken into consideration when performing the per-protocol analysis (please see the statistical methods section).\n\nAll enrolled patients will be encouraged to contact the principal investigator directly, if experiencing problems related to their recent surgery or to their allocated group intervention. The principal investigator will then fill out a standardized form at these calls. Patients can use pain-relief products as needed, prescribed by their physician after their surgery. To avoid study-contamination, patients will be asked to adhere to the allocated rehabilitation intervention and not to seek alternative health-care services specific to their hip- or knee arthroplasty during the course of the study. Patients will be advised to contact their general practitioner or the principal investigator as needed.\n\nThis trial is designed with four outcome assessments:\n\n- At baseline (t0), during the first consultation following the patient’s THA or TKA (usually between 5 and 7 days postoperatively).\n\n- After the 6-week intervention (t1), at our outpatient rehabilitation facility.\n\n- 3 months postoperatively (t2), follow-up assessment at our outpatient rehabilitation facility.\n\n- 12 months postoperatively (t3), final follow-up assessment at our outpatient rehabilitation facility.\n\nPrimary outcome. The primary outcome is the difference between groups in mean score of HOOS/KOOS function in the daily living-subscale (ADL-subscale) at the end of the 6-week intervention. This is chosen as the primary outcome as it is usually physical function that prescribed rehabilitation is targeted to improve5,7 and therefore the best suited outcome to measure rehabilitation effectiveness. Secondly, restoration of function is often a crucial element of treatment success, which makes this subscale a suitable outcome measure to indicate the restoration of function. The ADL-subscale was identified through semi-structured patient-interviews; patients found the HOOS/KOOS subscale most relevant in order to measure postoperative progress. The outcome will not be calculated as a change score using the baseline value. We do not consider it possible to obtain a valid HOOS/KOOS ADL score before 6 weeks after surgery, because some of the items in the HOOS/KOOS ADL subscale have most often not been sufficiently addressed or experienced by the participants within the first 6 weeks after surgery.\n\nSecondary outcomes. Secondary outcome measurements include the difference between intervention groups during all follow-ups of three subscales from HOOS/KOOS (i.e. pain, symptoms, and quality of life)35,36. Patient global assessment will be evaluated using a standard question: “How would you rate your current level of function during your usual activities of daily living?” quantified on a 0–100 visual rating scale37. Performance-based assessment will be measured by the 30-s chair stand test, and 40 meters walking test as recommended by the Osteoarthritis Research Society International (OARSI)38. Furthermore, patient satisfaction39 and exercise adherence will be assessed at the end of the 6-week intervention. Current use of analgesics, current use of walking assistant devices and adverse events will be registered within each group (e.g. postoperative infections, deep venous thrombosis or arthrofibrosis) at each follow-up assessment. Outcome assessors will document the time of each individual consultation to investigate the total time of each patient’s rehabilitation and within each of the three rehabilitation strategies. The additional time spent during monitoring of the homebased telerehabilitation intervention will also be documented.\n\nData from medical records. All the above-mentioned outcome measurements will be passed on from medical record files in collaboration with the responsible physiotherapist. All data will first be recorded and passed on after the patient has provided oral and written consent to participate in the trial.\n\nOutcome measurement will adhere to OARSI clinical trials recommendations37.\n\nThe patients will complete all questionnaires at each follow up on paper and in individual consultation rooms with no external interruptions. The score of each questionnaire will be calculated by the consulting physiotherapist. The 30s chair-stand-test will be conducted in the consultation room using a test-chair, while the 40 metres walking test will be performed on a test-track near the consultation rooms. All outcome data will finally be reported in the patient’s electronically stored medical record.\n\nPrimary outcome. Hip disability and osteoarthritis outcome score (HOOS) / knee injury and osteoarthritis outcome score (KOOS) subscale: function in daily living (ADL). This patient-reported outcome measurement (PROM) has proven to be a valid, reliable and responsive outcome measurement following THA and TKA35,40–42. This subscale consists of 17 questions related to the patient’s function in activities of daily living such as “descending stairs”, “standing” and “getting in/out of car”. The subscale is scored by the degree of difficulty that the patient experienced in the last week on a 5-point Likert scale (none, mild, moderate, severe, and extreme) and calculated to a score ranging from 0 (worst/extreme difficulties) to 100 (best/ no problems). The total questionnaire takes about 10 minutes to complete. At least 50% of the questionnaire items are required to be answered to permit calculation of a mean score.\n\nSecondary outcomes. Remaining three sub-scales of HOOS/KOOS: symptoms, pain, and hip- or knee related quality of life. A fourth subscale, sport and recreation, was excluded as an outcome measurement at 6 weeks, as most of the items in the HOOS/KOOS sport and recreation subscale often will not have been sufficiently addressed or experienced by the participants within the first 6 weeks after surgery.\n\nLike the above-mentioned subscale, these subscales are scored by the degree of difficulty the patient has experienced in the last week on a 5-point Likert scale. Each subscale provides a score, which can be calculated to a total score ranging from 0 (worst/extreme difficulties) to 100 (best/ no problems). At least 50% of the questionnaire items in each subscale are required to be answered to permit calculation of a mean score. A total score of the questionnaires has not been validated and is therefore not recommended36.\n\nPatient global assessment. At baseline the patient’s perception of his or her overall functional ability will be assessed using a single numeric rating scale, as recommended by OARSI37. The patient will be asked a single question: “How would you rate your current level of function during your usual activities of daily living?” During follow-up assessment patients will be asked the same question as at baseline. Their answer will be rated on a 0–100 visual rating scale with end-points anchored as “Inability to perform any daily activities” (0) and “No problem with any daily activity” (100)37.\n\n30-s chair stand test. Performance-based assessment will be performed using the 30-s chair-stand test, which represents the sit-to-stand activity. The test counts the number of sit-to-stand repetitions the patient can perform in 30 seconds. The straight-back chairs used for testing during all outcome assessments will be of the same model, same height (approximately 43 cm) and will be placed against a wall. Any other adaptations (i.e. the use of armrests or assistive devices) will be reported. To assure understanding, two slow-paced repetitions will be practiced before the test initiates. The 30-s chair stand test has proven good reliability to measure functional performance following THA and TKA43,44.\n\n40 meters walking test. This performance-based assessment measures short distance walking activity. The patient will be instructed to walk as quickly as safely possible to a mark 10 meters away, return and repeat for a total distance of 40 meters. Each walk of 10 meters (excluding the turn time) is recorded and expressed as speed m/s by dividing distance by time in seconds. A practice walk up and back will be performed to assure understanding. Any assistive devices used will be recorded.\n\nAnalgesics. The use of analgesics will be recorded at baseline and at all follow-up assessments, using a nominal scale (yes/no) regarding the daily consumption of opioids, non-steroid-anti inflammatorily drug (NSAID) and paracetamol. If the patient is not aware of his or her use of analgesics, a medical record will be obtained to assess the use of analgesics.\n\nWalking assistant devices. The type and number (e.g. one or two elbow crutches) will be recorded at baseline and at all follow-up assessments. The need for walking assistant devices will be assessed by the number of walking assistant devices the patient uses during normal activities of daily living.\n\nPatient satisfaction. Patient satisfaction will be assessed at the end of the 6-week intervention, where patients will be asked to answer four questions to indicate their level of satisfaction. The answers will be based on a 4-Likert ordinal scale with the categories: very satisfied (100 points), somewhat satisfied (75 points), somewhat dissatisfied (50 points), and very dissatisfied (25 points). The score is the unweighted mean of the combined scores. The four questions are: “How satisfied are you with the results of your surgery and rehabilitation?”, “How satisfied are you with your surgery and rehabilitation for improving your pain?”, “How satisfied are you with the results of your surgery and rehabilitation for improving your ability to do home or yard work?” and “How satisfied are you with the results of surgery and rehabilitation for improving your ability to do recreational activities?”. The questions are modified from the self-administered patient satisfaction scale for primary hip and knee arthroplasty39 and have in its original form proven to be a valid and reliable outcome measurement for patient satisfaction following THA and TKA39.\n\nExercise adherence. Exercise adherence will be measured as a percentage (0–100), where 0 % indicates total non-adherence and 100 % indicates total adherence to the exercise prescription. Exercise adherence in the home-based telerehabilitation and home-based rehabilitation group will be measured as a self-reported outcome, where patients will be asked: “What percentage of the prescribed 42 exercise sessions (one exercise session per day for 6 weeks; corresponding to 100 %) have you completed the past 6 weeks?”\n\nAdverse events. Adverse event during enrolment of the trial will be recorded, regardless of the relation to the trial.\n\nTime usage. We wish to explore the economic resources required for each of the three rehabilitation strategies. We intend to use this data in our cost-benefit considerations compared to the current practise (home-based rehabilitation).\n\nPatients deemed eligible for study participation and provided written consent will be randomly assigned to one of the three interventions, after the completion of baseline assessment. A physiotherapist, not otherwise involved in the study, will perform a computer-generated random allocation sequence (1:1:1 allocation rate) concealed in 168 consecutively numbered, opaque, sealed envelopes determining allocation to home-based telerehabilitation, home-based rehabilitation or no physical rehabilitation. Blocked randomisation will be used to achieve balanced group allocations and to ensure concealment; the block sizes will not be disclosed.\n\nBefore intervention allocation, baseline measures will be performed and recorded. Experienced physiotherapists, who are blinded to group allocation, will record the follow-up outcome measurements (t1 + t2 + t3) at our outpatient facilities. The patients will be asked not to disclose any information that could give away the group allocation, and the physiotherapist will be instructed not to ask questions that could be used to identify the allocated group of the patient, before follow-up assessment has taken place.\n\nDuring the following outcome assessment, a different physiotherapist will record the outcome assessment to keep the outcome assessor blinded to group allocation at all times. The patients are only blinded to the trial hypothesis in order to prevent ascertainment bias. The principal investigator will not be blinded, as he does not conduct any outcome assessments, and therefore no emergency unblinding procedure will be necessary.\n\nThe outcome assessor will record data (patient demographics, use of analgesics, walking assistant devices and possible adverse events) and obtain the HOOS or KOOS questionnaire from the patient on a standardized form during the first consultation, which will take place approximately 3–7 days after discharge. The performance-based outcome assessments will be performed thereafter and also recorded on the standardized form. This procedure will be repeated at the following follow-ups by different physiotherapists to keep outcome assessors blinded for group allocation. Four questions regarding patient satisfaction will be included in the online questionnaire before the 6-week follow-up.\n\nAll original written information and case report forms will be stored in a secured room and kept for three years after the trial completion. Electronic data will be anonymized, coded and saved on a secured server in the Capital Region of Denmark, Bornholm. A complete backup of the data entries will be performed every month until trial completion.\n\nExercise data from the home-based telerehabilitation will be coded and saved on a secured server in the Capital Region of Denmark. The stored data set will be coded in a way so that group allocation is concealed, allowing for blinding of the data analyst.\n\nParticipant retention and completion of follow-up assessments are expected to be relatively high due to the comprehensive follow-up assessment of this trial. While standard of care usually includes a 3 months’ follow up, patients included in this trial are invited to follow-up assessments at 6 weeks, 3 and 12 months postoperatively, which includes a free-of-charge consultation with a physiotherapist.\n\n\nStatistical methods\n\nThe main analysis plan is outlined below, while a detailed analysis can be found in our extended data23.\n\nThe trial is designed to test the hypothesis that physical rehabilitation (home-based telerehabilitation and home-based rehabilitation combined) is superior to no physical rehabilitation following THA and TKA or if one of the physical rehabilitation strategies is superior to no physical rehabilitation. Superiority of physical rehabilitation over no physical rehabilitation will be claimed if the between-group contrast at the 6 weeks follow-up is 10 HOOS/KOOS ADL-subscale points or greater in favour of the physical rehabilitation. Superiority of an individual physical rehabilitation strategy over no physical rehabilitation will be claimed if the same criteria are met and in favour of that physical rehabilitation.\n\nThe primary outcome is the mean difference in the HOOS/KOOS subscale: function in daily living (ADL). Primary endpoint will be differences between groups at first follow up (t1), secondary endpoints being differences at second follow-up (t2) and final follow up (t3) (see extended data item 3: schedule for enrolment, intervention, and outcome assessments23).\n\nData analysis will follow the intention-to-treat principle. To allow for full data analysis, missing data will be imputed using multiple imputations.\n\nThe primary analysis will evaluate the mean difference at 6 weeks follow-up between physical rehabilitation (home-based telerehabilitation and home-based rehabilitation) and no physical rehabilitation. This will be assessed by independent two-sample t-tests if normality assumptions are acceptable, otherwise the Wilcoxon sum rank-test will be used. The same tests will also be used to compare the individual physical rehabilitations to no physical rehabilitation.\n\nThe secondary analysis will test differences in the mean score of all continuous secondary outcomes at the primary, secondary and final endpoints and will be performed in the same way as the primary analysis. Nominal outcomes will be analysed by the chi-squared test or in cases with less than five expected counts for any observation, Fishers exact test will be used.\n\nAt baseline descriptive statistics will be presented. At each endpoint, mean scores, standard deviations, between-groups mean (with corresponding 95% confidence intervals) and p-values will be reported (see extended data item 4: analysis outline for primary and secondary analysis23).\n\nWe will undertake exploratory subgroup analyses with stratification on THA or TKA similar to that outlined for the non-stratified analysis.\n\nAll analyses will follow the intention-to-treat (ITT) principle (analysed as randomized). Additionally, per protocol (PP) analysis will be done for the primary outcome. All patients with an exercise adherence of at least 80 % will be considered adherent to the rehabilitation strategy and included in the PP analysis. All patients in the no rehabilitation group will be included in the PP analysis. Missing values in both ITT and PP analysis will be imputed by multiple imputation. Imputation models will be based on all other available variables in the data.\n\nNo data monitoring committee will be composed, as the interventions pose little or no known risk for the participating patients. Likewise, no auditing procedures are expected to take place.\n\nA clinically important difference of 10 points on the HOOS/KOOS subscale function in daily living (ADL)36,45 is used as our superiority margin, with an expected standard deviation of 20 points, a power of 0.80 and a significance level of 0.05 resulting in a sample size of 50 patients in each group. With 10% loss to follow up46 this results in 56 patients in each group. This calculation will be used for each comparison of individual physical rehabilitations to no physical rehabilitation; therefore, 56 patients are needed for each of the three groups resulting in 168 patients included in total (3 groups of 56 patients). Comparison of any physical rehabilitation to no physical rehabilitation is done under the same clinically important difference and expected standard deviation as the individual comparisons. With a sample size of 100 (patients receiving one of the two physical rehabilitation strategies) and 50 (patients receiving no physical rehabilitation) the expected power is 0.89.\n\nWe expect a relatively low inclusion rate of 50%, since one of the interventions includes “no physical rehabilitation”, compared to standard rehabilitation. Furthermore, based on the expected patient flow at our trial sites and the need to conduct the trial within a realistic timeframe, we do not initially intend to stratify THA and TKA for subgroup analysis. There will not be any specific strategies for increasing participant enrolment related to this trial.\n\n\nEthics and dissemination\n\nThis trial obtained approvals from the Ethics Committee of the Capital Region Denmark28 and the Danish Data Protection Agency29 and was registered in a public trial registry before the first participant was enrolled. Written consent from each trial participant will be obtained during the first consultation at our outpatient facility by an experienced physiotherapist.\n\nAll study-related information about the participants will be stored securely on the study site. Paper forms will in stored in locked cabinets, while electronic data will be stored on a password protected drive at the study site. Only persons involved in the trial will have access to the study-related information. Individual study information will not be released outside of the study without permission of the individual participant. This study will be handled in accordance with the Data Protection Act from the Danish Protection Agency.\n\nIt is not expected that the allocated interventions will cause any harm to the participants. If any harm is caused, at the end of the 6-week intervention, the patients will be offered free-of-charge in- or outpatient rehabilitation as deemed clinically needed.\n\nThe principal investigator (TMC) and the principal supervisor (TB) will have full access to the data set. The full-anonymized data set will be made available for the journal reviewing the manuscript.\n\nThe trial is planned to be reported in four manuscripts and expected to be published in international, peer-reviewed journals. The first manuscript will be the trial protocol. The second manuscript will be the primary report on the investigated effectiveness of the three rehabilitation strategies under investigation. The third manuscript will report the result of the trial from an economic perspective, while the fourth manuscript will report exploratory analyses depending on the primary trial findings. All results, including positive, negative or inconclusive results, will be presented at relevant scientific conferences and symposiums.\n\nAll patients will be offered to receive an information letter with the results of this trial when the trial is completed and published. Trial data can be requested by contacting the principal investigator.\n\nStudy status: Active, not recruiting. Participant no. 168 was recruited on the 27th of January 2021. The final follow-up data are expected to be obtained by January 2022.\n\n\nDiscussion\n\nIn 2018, over 10,000 THAs47 and over 9,000 TKAs48 were performed in Denmark. The number of these procedures is expected to increase49,50, which puts further emphasis on the importance of finding the optimal rehabilitation strategy for this group of patients. As there are significant variations in the rehabilitation practices following THA and TKA both nationally and internationally, investigations of different rehabilitation strategies and their effectiveness are warranted.\n\nUsing the FINER-criteria51 we consider the research question feasible, interesting, novel, ethical and relevant. We believe this clinical trial to be highly useful, as we have conducted a thorough literature review of the problem base, included context placement and information gain in the background section, according to the essay published by Ioannidis in 201652. Additionally, with careful methodological considerations such as few exclusion criteria and PROM’s we have also ensured a pragmatic and patient-centred trial52. As we will pre-register the trial; use the TIDieR-framework for intervention description25 and publish the trial protocol, we consider the trial design and intent to be as transparent as possible.\n\nIn summary, the objective of this trial is to investigate the effectiveness of three different rehabilitation strategies following THA and TKA in terms of function in daily living. Knowledge about the effectiveness of the three investigated rehabilitation strategies will help guide the future organization of post-discharge rehabilitation after THA and TKA.\n\nThis trial has several strengths. Firstly, this trial will be the first trial to compare the effectiveness of two commonly used postoperative rehabilitation strategies to a “no physical rehabilitation”-intervention following THA and TKA. Secondly, the trial will be conducted at a geographically ideal setting. The Isle of Bornholm, Denmark, offers a unique opportunity to control study contamination, as our institution is the only one of this kind on the isle. Thirdly, home-based telerehabilitation and home-based rehabilitation-interventions will be given the exact same rehabilitation exercise instructions in terms of exercise type, dosage and intensity, which will make the two physical rehabilitation interventions comparable. Fourthly, randomization and the blinding of outcome assessors and data analysts will greatly decrease the risk of bias of this trial. The consecutive sampling and the relatively unrestricted inclusion criteria increase the external validity.\n\nLastly, this protocol follows the clinical recommendations from SPIRIT22,23, CERT26, and TIDieR25, allowing replication and direct clinical use of the described exercise interventions. Furthermore, the adherence to the clinical recommendations from OARSI37 will permit comparison to other trials, eventually allowing meta-analysis.\n\nA noteworthy limitation to this study is the lack of a preoperative outcome assessment. Many patients choose to have their surgery performed at a hospital not located on the island of Bornholm. This means it is not practically possible to obtain preoperative data of these patients before their surgery.\n\nDue to the nature of this trial, there is a risk of selection bias, where active patients will be more likely to participate, compared to inactive patients who are expected to more likely feel the need for supervised rehabilitation. Likewise, the use of a single centre study design represents a common limitation for the external validity of rehabilitation intervention trials. We also anticipate that patient-perceived lack of technical understanding will present some selection bias. However, the current study will nevertheless provide level-one evidence of the effectiveness of the three investigated rehabilitation strategies.\n\n\nData availability\n\nNo data are associated with this article.\n\nHarvard Dataverse: DRAW1. https://doi.org/10.7910/DVN/BUNJQV23\n\nThis project contains the following extended data:\n\n- Flowchart of enrolment, randomization, treatment and follow-up\n\n- Schedule for enrolment, intervention and outcome assessments\n\n- Analysis outline for primary and secondary analysis\n\n- Template for intervention description and replication (TIDier) checklist for trial interventions\n\n- Exercise description\n\n- Participant information sheet\n\n- Participant letter of invitation\n\n- Participant consent form\n\nHarvard Dataverse: DRAW1. https://doi.org/10.7910/DVN/BUNJQV23\n\nThis project contains the following reporting guidelines:\n\n- SPIRIT checklist\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nTrial registration: Clinicaltrials.gov: NCT03750448 (23/11/2018)\n\nTrial protocol draft version: 5 (05.01.2021)\n\nProtocol amendments: None", "appendix": "Author contributions\n\n\n\nTMC and TB conceived the study idea. TMC and TB initiated the study design. TMC drafted the study protocol. All authors contributed to the final version of the trial design and protocol. TMC conducted training of trial personnel in testing procedures. TMC will draft the manuscripts for publications with contributions from all authors.\n\n\nReferences\n\nAllen KD, Golightly YM: State of the evidence. Curr Opin Rheumatol. 2015; 27(3): 276–283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShan L, Shan B, Suzuki A, et al.: Intermediate and long-term quality of life after total knee replacement: a systematic review and meta-analysis. J Bone Joint Surg Am. 2015; 97(2): 156–168. PubMed Abstract \| Publisher Full Text\n\nShan L, Shan B, Graham D, et al.: Total hip replacement: a systematic review and meta-analysis on mid-term quality of life. Osteoarthr Cartil. 2014; 22(3): 389–406. 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PubMed Abstract \| Publisher Full Text\n\nMahomed N, Gandhi R, Daltroy L, et al.: The Self-Administered Patient Satisfaction Scale for Primary Hip and Knee Arthroplasty. Arthritis. 2011; 2011: 591253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoos EM, Toksvig-Larsen S: Knee injury and Osteoarthritis Outcome Score (KOOS) - validation and comparison to the WOMAC in total knee replacement. Health Qual Life Outcomes. 2003; 1: 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeer MA, Lane J: The Knee Injury and Osteoarthritis Outcome Score (KOOS): a review of its psychometric properties in people undergoing total knee arthroplasty. J Orthop Sports Phys Ther. 2013; 43(1): 20–28. PubMed Abstract \| Publisher Full Text\n\nThorborg K, Roos EM, Bartels EM, et al.: Validity, reliability and responsiveness of patient-reported outcome questionnaires when assessing hip and groin disability: a systematic review. Br J Sports Med. 2010; 44(16): 1186–1196. PubMed Abstract \| Publisher Full Text\n\nUnver B, Kahraman T, Kalkan S, et al.: Test-retest reliability of the 50-foot timed walk and 30-second chair stand test in patients with total hip arthroplasty. Acta Orthop Belg. 2015; 81(3): 435–441. PubMed Abstract\n\nUnver B, Kalkan S, Yuksel E, et al.: Reliability of the 50-foot walk test and 30-sec chair stand test in total knee arthroplasty. Acta Ortop Bras. 2015; 23(4): 184–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNilsdotter A, Bremander A: Measures of hip function and symptoms: Harris Hip Score (HHS), Hip Disability and Osteoarthritis Outcome Score (HOOS), Oxford Hip Score (OHS), Lequesne Index of Severity for Osteoarthritis of the Hip (LISOH), and American Academy of Orthopedic Surgeons (AAOS) Hip and Knee Questionnaire. Arthritis Care Res (Hoboken). 2011; 63 Suppl 11: S200–7. PubMed Abstract \| Publisher Full Text\n\nZelle BA, Bhandari M, Sanchez AI, et al.: Loss of follow-up in orthopaedic trauma: is 80% follow-up still acceptable? J Orthop Trauma. 2013; 27(3): 177–181. PubMed Abstract \| Publisher Full Text\n\nDanish Hip Arthroplasty Register. National Årsrapport 2019. 2019.\n\nDansk Knæalloplastikregister årsrapport 2019: Danish Knee Arthroplasty Register. 2019.\n\nCulliford D, Maskell J, Judge A, et al.: Future projections of total hip and knee arthroplasty in the UK: results from the UK Clinical Practice Research Datalink. Osteoarthr Cartil. 2015; 23(4): 594–600. PubMed Abstract \| Publisher Full Text\n\nKurtz S, Ong K, Lau E, et al.: Projections of primary and revision hip and knee arthroplasty in the United States from 2005 to 2030. J Bone Joint Surg Am. 2007; 89(4): 780–785. PubMed Abstract \| Publisher Full Text\n\nHulley SB: Clinical Research. 4th ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins; 2013.\n\nIoannidis JPA: Why Most Clinical Research Is Not Useful. PLoS Med. 2016; 13(6): e1002049. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "85823", "date": "02 Jun 2021", "name": "Michael Masaracchio", "expertise": [ "Reviewer Expertise Orthopedic and sports rehabilitation", "spinal manipulation for cervical and thoracic spine pathologies and health services 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 the opportunity to review this important manuscript. Overall, this is one of the better manuscripts I have reviewed in some time. This is an important topic and the writing is concise, clear, and easy to follow. Of course another read-through is always worth it.\n\nAs this is a protocol, my comments will be specific to the information in this manuscript, however, other comments I make can be applicable to the pending RCT that can have huge implications for physical therapy.\nFirst, I think the title of this paper needs to clearly specify that this is tele-rehabilitation or home rehabilitation and not simply say rehabilitation. I strongly suggest this as depending on the results we would want all readers to clearly know the type of rehabilitation provided up front, especially since no group is getting formal outpatient physical therapy.\nSecond, it appears that the initial PT making the introduction to the patients about the trial has no other role in the study? If this is the case, I would like to see that made more explicitly clear.\nThird, my biggest concern is two fold. First, why was the word rehabilitation used as opposed to physical therapy? Are there going to be providers who are not physical therapists in this study? If the authors want to use the word rehabilitation, I think it is important to operationally define why this was chosen rather than physical therapy. Perhaps, this can be specific to Denmark, but in the US, these terms are not synonymous and can have implications and be interpreted differently. If is specific to Denmark, and the authors feel there would be no miscommunication or confusion, my comment can be ignored. Secondly and more importantly, my concern is the term rehabilitation and how the interventions are being applied. Physical therapy/rehabilitation is not only about exercise. There are other components, such as manual therapy, and motor control/movement training that can be just as important if not more important following THA or TKA. Depending on the results it may be more appropriate to conclude that exercise and education in isolation are no better/better (yes, this needs to be determined with the RCT) than no physical rehabilitation. It is important to clarify this because in the US physical therapy is not only exercise. It is a combination of manual therapy, exercise, and movement training, along with possible modalities at times. It is the clinical reasoning that is important in how these interventions are put together and in what dosage they are prescribed at.\n\nLastly, I would suggest assessing some form of strength measure for the quadriceps and hip abductors as research continues to discuss the long term strength deficits for the quadriceps after TKA and gluteus medius following THA depending on the approach used.\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? Not applicable", "responses": [ { "c_id": "7151", "date": "22 Sep 2021", "name": "Troels Mark-Christensen", "role": "Author Response", "response": "We would like to thank the reviewer for the helpful comments provided. We found the comments to be fair and the provided input to be very valuable. Our response to the comments are as follows: Reviewer 1, comment 1: Thank you for the opportunity to review this important manuscript. Overall, this is one of the better manuscripts I have reviewed in some time. This is an important topic and the writing is concise, clear, and easy to follow. Of course another read-through is always worth it. As this is a protocol, my comments will be specific to the information in this manuscript, however, other comments I make can be applicable to the pending RCT that can have huge implications for physical therapy. I think the title of this paper needs to clearly specify that this is tele-rehabilitation or home rehabilitation and not simply say rehabilitation. I strongly suggest this as depending on the results we would want all readers to clearly know the type of rehabilitation provided up front, especially since no group is getting formal outpatient physical therapy. Author response: We initially want to emphasize that the trial was designed firstly as a superiority trial for the comparison of physical rehabilitation versus no physical rehabilitation, which is also reflected in the pre-defined plan for the analyses. This is the main focus and rationale of trial and, hence, what we want to emphasize transparently in the title. We do, however, clarify the different types of rehabilitation provided in the abstract so that anyone who does a title/abstract screen will see this. We hope the reviewer can appreciate the reasons for this choice. Action taken: None Reviewer 1, comment 2: Second, it appears that the initial PT making the introduction to the patients about the trial has no other role in the study? If this is the case, I would like to see that made more explicitly clear. Author response: We agree. The introducing PTs role is to conduct baseline assessment first and deliverer of intervention second, and should be explicitly stated. Action taken: Sentence added on page 6, line 119-121: “Baseline assessment is performed before randomization and the allocated intervention is reveal to the patient and the attending physiotherapist”. Sentence added on page 6, line 121-122: “All outcome assessors dedicated to the trial are licensed physiotherapists”. Lastly, sentence added on page 6, line 126-129: “After the baseline assessment has been performed and information about the allocated intervention has been provided, the consultation ends and the attending physiotherapist who conducted the baseline outcome assessment has no further role in the study. Reviewer 1, comment 3: Third, my biggest concern is two fold. First, why was the word rehabilitation used as opposed to physical therapy? Are there going to be providers who are not physical therapists in this study? If the authors want to use the word rehabilitation, I think it is important to operationally define why this was chosen rather than physical therapy. Perhaps, this can be specific to Denmark, but in the US, these terms are not synonymous and can have implications and be interpreted differently. If is specific to Denmark, and the authors feel there would be no miscommunication or confusion, my comment can be ignored Author response: In Denmark, hospital discharge following THA or TKA is combined with a postoperative “rehabilitation plan” which is essentially a prescription of an outpatient health care service (physical rehabilitation) to be delivered in the patient’s own municipality. In most cases, the service deliverer are physical therapists. There are, however, regional differences in how that municipality rehabilitation is organized and constructed - but it reflects the context-specific interpretation of the available scientific evidence. It typically includes both exercise and non-exercise components. Because the DRAW1 trial is a pragmatic trial, we have made no efforts to modify current clinical practice, as it represents “real (clinical) life” in one municipality in Denmark. We simply ask if the commonly used practice of a post-operative rehabilitation plan and corresponding out-patient physical rehabilitation in the patient’s own municipality is superior to no physical rehabilitation. So, to reflect this organization of health care service and the terms used to describe if, we think “physical rehabilitation” is the best fit. Action taken: We have added a section on page 4, sentence 47-51 outlining parts of the answer above: “Because the DRAW1 trial is a pragmatic trial, we have made no efforts to modify current clinical practice, as it represents “real (clinical) life” and interpretation of the available scientific evidence in one municipality in Denmark. We ask if the commonly used practice of a post-operative rehabilitation plan and corresponding out-patient physical rehabilitation in the patient’s own municipality is superior to no physical rehabilitation”. Reviewer 1, comment 4: Secondly and more importantly, my concern is the term rehabilitation and how the interventions are being applied. Physical therapy/rehabilitation is not only about exercise. There are other components, such as manual therapy, and motor control/movement training that can be just as important if not more important following THA or TKA. Depending on the results it may be more appropriate to conclude that exercise and education in isolation are no better/better (yes, this needs to be determined with the RCT) than no physical rehabilitation. It is important to clarify this because in the US physical therapy is not only exercise. It is a combination of manual therapy, exercise, and movement training, along with possible modalities at times. It is the clinical reasoning that is important in how these interventions are put together and in what dosage they are prescribed at. Author response: In our response to comment 3 above, we provide our reasoning for the choice of wording. We do not agree that particular components of physical therapy/rehabilitation or single modalities offer superior effects in the rehabilitation efforts following THA or TKA, neither on short nor long term. The supporting evidence is now quite substantial (https://pubmed.ncbi.nlm.nih.gov/24287215/, https://pubmed.ncbi.nlm.nih.gov/25886975/, https://pubmed.ncbi.nlm.nih.gov/27584144/, https://pubmed.ncbi.nlm.nih.gov/27401004/, https://pubmed.ncbi.nlm.nih.gov/25724323/). As physical rehabilitation in the form of physical activity and exercise prescription is currently recommended despite its lack of clear superiority over a range of different comparators, we have decided to investigate if such prescription is superior to no prescription of physical rehabilitation Action taken: No action taken. Reviewer 1, comment 5: Lastly, I would suggest assessing some form of strength measure for the quadriceps and hip abductors as research continues to discuss the long term strength deficits for the quadriceps after TKA and gluteus medius following THA depending on the approach used. Author response: We did consider to include some form of strength measure, but since we wanted to design a pragmatic trial with a simple outcome assessment (to increase uptake and fidelity). As patients are concerned about their functional performance, and not their strength (even though is can be used as a surrogate outcome measure), we prioritized a functional assessment. And finally, as recruitment has been concluded, inclusion of another outcome measure is not possible. Action taken: No action taken." } ] }, { "id": "85826", "date": "15 Jun 2021", "name": "Thomas W Wainwright", "expertise": [ "Reviewer Expertise Physiotherapy", "Rehabilitation", "Orthopaedics", "Exercise Therapy" ], "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 the opportunity to review this well-written study protocol. The protocol utilises reporting guidelines appropriately, and as such is thorough and transparent.\n\nThe research question and aim is valid and needed, and we agree that to our knowledge this will be the first to compare the effectiveness of physical rehabilitation (either home-based tele-rehab or home-based self guided rehab) vs no physical rehabilitation.\n\nWe have the following general points that we encourage the authors to consider when reporting the outcomes and drawing conclusions from this study (we note that patient recruitment has already finished and so the experimental procedures will not be able to be altered).\nPrimary outcome measure - Use of the KOOS/HOOS is valid and justified by the authors, but please can it's use as the primary outcome measure instead of performance based function tests (which are secondary outcome measures) be explained in light of research that shows early functional recovery post TKA/THA does not always correlate with PROMs (e.g. Luna et al., 20171).\nTime usage (or health economic) outcome measure - Within the rationale for the study, the authors explain the need for the local health service to understand the cost benefits/comparisons of the interventions. We feel that more explanation of how the economic analysis will be performed is required. Do the authors intend to include a health resource use measure in addition to comparing physio time? Such health resource questionnaires can account for total related costs of interventions, experienced by both the health service and the personal health related costs of the individual patient.\n\nThe inclusion of both THA and TKA - From a pragmatic and recruitment perspective we understand why they are grouped together. However, these are two different procedures on different joints with different rehab needs. The early functional recovery (at 6 weeks) of THA and TKA patients can differ, therefore we feel there is a strong need to include a sub-group analysis. From our reading of the text, it is not clear to us whether this is planned or not, for example on page 8 in the \"additional analysis\" section a subgroup analysis with stratification is discussed, but on page 9 in the \"sample size\" section it says that an initial subgroup analysis is not planned. Please can this be clarified?\nIn summary, this is a well-designed study, and I look forward to learning the findings, and implications for future research and clinical practice. We hope our feedback is useful.\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? Not applicable", "responses": [ { "c_id": "7152", "date": "22 Sep 2021", "name": "Troels Mark-Christensen", "role": "Author Response", "response": "We would like to thank the reviewers for the helpful comments provided. We found the comments to be fair and the provided input to be very valuable. Our response to the comments are as follows: Reviewer 2, comment 1: Thank you for the opportunity to review this well-written study protocol. The protocol utilises reporting guidelines appropriately, and as such is thorough and transparent. The research question and aim is valid and needed, and we agree that to our knowledge this will be the first to compare the effectiveness of physical rehabilitation (either home-based tele-rehab or home-based self guided rehab) vs no physical rehabilitation. We have the following general points that we encourage the authors to consider when reporting the outcomes and drawing conclusions from this study (we note that patient recruitment has already finished and so the experimental procedures will not be able to be altered)Primary outcome measure - Use of the KOOS/HOOS is valid and justified by the authors, but please can it's use as the primary outcome measure instead of performance based function tests (which are secondary outcome measures) be explained in light of research that shows early functional recovery post TKA/THA does not always correlate with PROMs (e.g. Luna et al., 20171). Author response: We agree with the reviewer’s comment, that performance-based outcomes does not always correlate with PROMS and that both outcome assessments are relevant, which is why we also use both kind of assessments. We chose to use PROM as the primary outcome measurement, as this outcome measurement was determined as the most appropriate primary outcome by the stakeholders during the planning of the trial, and we have now highlighted this in the protocol. Furthermore, the use of a PRO instrument is advised when measuring a concept best known by the patient or best measured from the patient perspective (Guidance for Industry (fda.gov). Action taken: No action taken. Reviewer 2, comment 2: Time usage (or health economic) outcome measure - Within the rationale for the study, the authors explain the need for the local health service to understand the cost benefits/comparisons of the interventions. We feel that more explanation of how the economic analysis will be performed is required. Do the authors intend to include a health resource use measure in addition to comparing physio time? Such health resource questionnaires can account for total related costs of interventions, experienced by both the health service and the personal health related costs of the individual patient. Author response: Thank you very much for bringing up this issue. The municipality intends to investigate health resource use by taking the physiotherapists time into account. No other resource use item is intended to be applied. We do, however, acknowledge the need for a more thorough economic assessment of the interventions applied, if the primary aim of this trial was in a health economic perspective. As patient recruitment is finished, no alterations can be applied to the experimental procedures of the trial. However, in hindsight, additional health resource questionnaires should have been included. Action taken: No action taken. Reviewer 2, comment 3: The inclusion of both THA and TKA - From a pragmatic and recruitment perspective we understand why they are grouped together. However, these are two different procedures on different joints with different rehab needs. The early functional recovery (at 6 weeks) of THA and TKA patients can differ, therefore we feel there is a strong need to include a sub-group analysis. From our reading of the text, it is not clear to us whether this is planned or not, for example on page 8 in the \"additional analysis\" section a subgroup analysis with stratification is discussed, but on page 9 in the \"sample size\" section it says that an initial subgroup analysis is not planned. Please can this be clarified? In summary, this is a well-designed study, and I look forward to learning the findings, and implications for future research and clinical practice. We hope our feedback is useful. Author response: Thank you for bringing this to our attention. We do agree that THA and TKA are different procedures on different joints with different rehab needs, which is why we also intent to include a subgroup analysis between THA and TKA. As the initial sample size does not allow for stratified analysis, it was our intention to double the sample size of the trial by continuing the study setup in a supplementary trial if possible. After the trial inclusion was completed and the target sample size was obtained, an opportunity to continue the trial were presented and at the same time the municipality asked for stratified data to help guide their rehabilitation practice. This first trial (DRAW1) will therefore not include stratified analyses, while a following trial (DRAW2) eventually will allow for stratified analysis. A detailed description of DRAW2 can be found here: https://clinicaltrials.gov/ct2/show/NCT04960241?term=mark-christensen&draw=2&rank=1 Action taken: Sentence deleted on page 8 (page 13, line 421-422 in MW-document): “We will undertake exploratory subgroup analyses with stratification on THA or TKA similar to that outlined for the non-stratified analysis.”" } ] }, { "id": "86733", "date": "12 Jul 2021", "name": "Bayram Unver", "expertise": [ "Reviewer Expertise Orthopedic Physiotherapy and Rehabilitation" ], "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 criticized the article called \"Physical rehabilitation versus no physical rehabilitation after total hip and knee arthroplasties: Protocol for a pragmatic, randomized, controlled, superiority trial (The DRAW1 trial)\".\nMy opinions are as follows:\nParticipants aged ≥18 are specified in the inclusion criteria, but this is a very wide age range, and accordingly, the responses of the participants to rehabilitation vary and affect the results of the study. The lack of preoperative evaluations also contributes to this variability. Therefore, I recommend performing age-related subgroup analyzes or specifying the age of the participants in the inclusion criteria.\n\nSecondly, participants' involvement in other exercise or treatment programs affects the clinical process. Therefore, 'participating in any program outside of study' should be added to the exclusion criteria.\n\nThe other thing not mentioned in the study is the discharge criteria. Whether or not the same discharge criteria are used for each participant and the details of these criteria contribute to the clarification of the clinical process. Therefore, I recommend including this in the method.\n\nSince THA and TKA are two different joints and surgeries, rehabilitation procedures and interventions are also different. In the study, these two different interventions were not clearly explained. The type, intensity and dosage of planned interventions or exercises should be detailed.\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? Partly", "responses": [ { "c_id": "7153", "date": "22 Sep 2021", "name": "Troels Mark-Christensen", "role": "Author Response", "response": "We would like to thank the reviewer for the helpful comments provided. We found the comments to be fair and the provided input to be very valuable. Our response to the comments are as follows: Reviewer 3, comment 1: I criticized the article called \"Physical rehabilitation versus no physical rehabilitation after total hip and knee arthroplasties: Protocol for a pragmatic, randomized, controlled, superiority trial (The DRAW1 trial)\". Participants aged ≥18 are specified in the inclusion criteria, but this is a very wide age range, and accordingly, the responses of the participants to rehabilitation vary and affect the results of the study. The lack of preoperative evaluations also contributes to this variability. Therefore, I recommend performing age-related subgroup analyzes or specifying the age of the participants in the inclusion criteria. Author response: We initially planned to have very wide, pragmatic inclusion criteria, but we do appreciate and acknowledge the reviewers comment on the inclusion criteria. We will therefore, as recommended, perform an age-relate subgroup analysis. Action taken: Sentence added on page 13, line 422-423: “As the age of the participants may vary and affect the results of the study, we will undertake an age-related subgroup analysis.” Reviewer 3, comment 2: Secondly, participants' involvement in other exercise or treatment programs affects the clinical process. Therefore, 'participating in any program outside of study' should be added to the exclusion criteria. Author response: We do very much agree with the reviewers comment on participants involvement in other exercise/treatments, which may affect the clinical process. We do however ask participant to adhere to the allocated rehabilitation intervention and not to seek other health-care services specific to their THA or TKA during the course of the study (p. 8, line 205-209) and to contact their general practitioner or study principle as needed. Furthermore, one of the exclusion criteria of the trial is: “(…) receiving in-home rehabilitation by home care”. We believe this is the most pragmatic way to avoid study contamination and improve adherence to the study protocol. Lastly, as patient recruitment is finished, no alterations can be applied to the experimental procedures of the trial Action taken: No action taken. Reviewer 3, comment 3: The other thing not mentioned in the study is the discharge criteria. Whether or not the same discharge criteria are used for each participant and the details of these criteria contribute to the clarification of the clinical process. Therefore, I recommend including this in the method. Author response: We do agree with the reviewer’s comment. In Denmark the discharge criteria follow a fast-track pathway, which is standardised across the country. Discharge criteria are: obtainment of basic mobility (independent in transfer and walking with appropriate walking assistant) and acceptable pain levels. Action taken: Sentence added on page 5, line 95-97: “Before discharge from the hospital, patients with hip- or knee arthroplasties have obtained independent basic mobility (transfer and walking) with appropriate walking assistant and experiencing acceptable pain levels.” Reviewer 3, comment 4: Since THA and TKA are two different joints and surgeries, rehabilitation procedures and interventions are also different. In the study, these two different interventions were not clearly explained. The type, intensity and dosage of planned interventions or exercises should be detailed. Author response: The different rehabilitation procedures and interventions are described in detail in extended data, including the type, intensity and dosage. Action taken: No action taken." } ] } ]	1	https://f1000research.com/articles/10-146
https://f1000research.com/articles/10-956/v1	22 Sep 21	{ "type": "Systematic Review", "title": "Mesenchymal stem cell therapy efficacy in COVID-19 patients: A systematic review and meta-analysis", "authors": [ "Andrianto Andrianto", "Desak Ketut Sekar Cempaka Putri", "Makhyan Jibril Al Farabi", "Teuku Yusrizal", "Hanestya Oky Hermawan", "Teuku Yusrizal", "Hanestya Oky Hermawan" ], "abstract": "Objective: To evaluate mesenchymal stem cell (MSC) administration safety and efficacy in COVID-19 patients. Methods: We conducted a literature search on PubMed/MEDLINE, medRxiv, EBSCOhost/CINAHL, ProQuest, and Scopus with keywords adjusted to each search engine’s specifications on February 12, 2021. Interventional studies that reviewed MSC efficacy (mortality, hospitalization duration, need for mechanical ventilators, and inflammation markers) and/or safety (adverse events) in COVID-19 patients who were 18 years old or more were included in this study. Study eligibility, data extraction, and study quality assessment were conducted independently by each author. Results: A total of five studies of moderate to high quality with a total of 193 patients were included. One of the three randomized studies included did not apply blinding to either participants or medical professionals. Pooled OR (Odd Ratio) for mortality risk, adverse events incidence, and use of mechanical ventilators for patients on MSC therapy were 0.13 [95% CI: 0.02, 0.68], 0.91 [95% CI: 0.45, 1.86], and 0.42 [95% CI: 0.12, 1.47], respectively. Pooled mean difference for hospitalization duration in the MSC group versus the control was -3.54 [CI 95%: -4.68, -2.40] with 7% heterogeneity. All studies agreed that there was an increase of pro-inflammatory cytokines and a decrease of anti-inflammatory markers that were statistically different in the MSC group. Conclusion: Mesenchymal stem cell administration to COVID-19 patients is safe and effective in reducing mortality and hospitalization duration. Furthermore, a decrease of pro-inflammatory cytokines and an increase in anti-inflammatory cytokines were observed.", "keywords": [ "COVID-19", "efficacy", "safety", "mesenchymal stem cell." ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is a new disease that resembles pneumonia. As of August 2021, more than 200 million people worldwide have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the virus that causes COVID-19, and more than four million people have died.1 In Indonesia alone, COVID-19 has reached almost four million cases (as of August 2021) with more than 120,000 deaths.2\n\nBesides fever, cough, and shortness of breath that are typical of pneumonia, COVID-19 has various clinical manifestations ranging from sore throat, fatigue, dizziness, myalgia, chest pain, rhinitis, neurological manifestations, conjunctivitis, anorexia, diarrhea, skin manifestations such as rashes and urticaria, and some are even asymptomatic.3–5 Therefore, COVID-19 is also known as “the great imitator”. It is interesting that COVID-19 is less severe in children than in adults, while the elderly and people with comorbidities such as hypertension, obesity, diabetes mellitus, cardiovascular disease, malignancies, and other chronic diseases, tend to have more severe symptoms and have a higher mortality rate.3,6,7\n\nEven though it has a mortality rate that is not too high (case fatality rate of 2-3%), deaths from COVID-19 should not be underestimated.3 Various types of treatment for COVID-19 have been observed in trials such as various antivirals, dexamethasone, anti-inflammatory drugs, and even convalescent plasma therapy. However, most of these treatments have not proved effective and only the drug, remdesivir, has been shown to be effective and approved by the FDA (US Food and Drug Administration). Therefore, currently the majority of COVID-19 patients are treated symptomatically.8 Although the majority of patients have mild symptoms and recover without the use of mechanical ventilation, severe respiratory distress may occur in some patients, which can result in mortality.9 Therefore, various studies are still being conducted to find other potential treatments in COVID-19 management, one of which is stem cell therapy.\n\nMesenchymal stem cells (MSCs) are a type of multipotent stem cells in adults that can be found in various autologous and allogeneic sources, which have high proliferative abilities and can differentiate into various lineages. Several studies have shown that MSCs have immunomodulatory abilities that might help modulate proliferation, activation, and function of a wide variety of immune cells, both innate and adaptive cells. MSCs were previously used in graft vs host disease management and several immunological diseases caused by viruses such as HIV, chronic hepatitis B, and acute lung injury caused by influenza virus.9\n\nExcessive inflammatory reaction is found in COVID-19 patients due to the production of inflammatory factors including cytokines, chemokines, and reactive immune cells that cause cytokine storms. MSCs administration is thought to reduce immune reactions in COVID-19 patients, therefore preventing cytokine storms from occurring in the immune system and triggering endogenous repair. After intravenous injection is given, the trapped population of mesenchymal stem cells in the lungs functions to reduce inflammation by releasing anti-inflammatory mediators, improve lung microenvironment by releasing antimicrobial peptides, protect alveolar epithelial cells, prevent pulmonary fibrosis, and improve pulmonary dysfunction and pneumonia due to COVID-19.10,11\n\nThere are many advantages that MSCs have compared to other therapies, 1) mesenchymal stem cells can be extracted from various tissues such as bone marrow, adipose tissue, umbilical cord, dental pulp, menstrual blood, buccal fat tissue etc; 2) these stem cells are multipotent; 3) mesenchymal stem cells can be stored for repetitive use; and 4) there have been no studies showing adverse events of allogeneic stem cells.10\n\nMany studies have been conducted globally on the efficacy of MSCs against COVID-19. A case report from China by Liang et al.,12 reported a 65-year-old woman infected with SARS-CoV-2 who showed improvement in vital signs, blood and immune profiles, and CT scan results after MSC administration from umbilical cord. A randomized controlled trial conducted in the United States by Lanzoni et al.,13 reported that COVID-19 patients who were given stem cell therapy experienced significant symptom improvement compared to the control group and no adverse event was reported. However, one study reported a high incidence of adverse events.14 These controversial findings prompted the authors to conduct a systematic review and meta-analysis of MSC safety and efficacy in COVID-19 patients.\n\n\nMethods\n\nWe included interventional studies with/without randomization and blinding. Each study must’ve reported evidence of ethical clearance by the local research ethics committee. Only articles in English with a full manuscript available were included. The type of intervention was MSC administration, without any restrictions on cell seeding/harvesting method and MSC dose. There were no restrictions on the patient's social status, ethnicity, race, or nationality. The expected comparisons were administration of a standard therapy regimen according to local health protocols, placebo, or MSC vehicle.\n\nWe excluded studies that included patients with: 1) age under 18 years (pediatric patients); 2) multiorgan failure at the start of study; or 3) inherited/acquired immunity disorder. Each study reported at least one of these variables: mortality rates, adverse events, need for ventilators, treatment duration, time for clinical improvement, and changes in inflammatory markers. Case reports or case series studies were also excluded from this systematic review. Mortality was defined as deaths that occurred during hospitalization. Adverse events were evaluated within six hours of MSC administration, which included urticaria, palpitations, and pulmonary edema. Cardiac arrest within 24 hours after MSC administration was also considered as an adverse event.\n\nA systematic literature search of (Pubmed/MEDLINE, EBSCO/CINAHL, ProQuest, and Scopus from 1 January 2020 to 20 February 2021 was performed using the search strategy outlined in Table 1.15 Additional records were retrieved from Google Scholar. Duplicate articles were removed after the initial search. Two authors independently screened the title and abstract of the articles. Articles that passed the screening were assessed in full text based on the eligibility criteria. Disagreements were resolved by discussion with the senior author. This study was conducted following the Preferred Reporting Item for Systematic Reviews and Meta-Analysis (PRISMA) statement.16\n\nWe assessed the suitability of each study that appeared in the search engines by title and abstract. All appropriate articles were input in our database and duplicated using Zotero 5.0 (RRID:SCR_013784) citation manager application. All authors conducted an independent assessment of the eligibility of all studies. At this stage, the article eligibility assessment was conducted based on the full text of the assessed article. Any discrepancies that occurred were resolved by discussion.\n\nAll authors extracted the data independently. We developed a data extraction sheet that was referred to by the Cochrane Consumers and Communication Review Group.15,17 Some of the data we extracted included study design, age, number of participants, comorbidities, type of intervention, and all of the previously mentioned outcomes. Numerical representations were preferred over graphs to avoid misinterpretation/estimation. However, graphic presentation in each article was not a reason to exclude an article.\n\nThis meta-analysis compared MSC safety and efficacy in COVID-19 patients against primary (mortality) and secondary (adverse events, treatment duration, need for mechanical ventilation) clinical outcomes. For treatment duration, the input data are numerical, therefore we used the mean difference from each study to be processed in this meta-analysis. We used a 2 × 2 format to assess mortality, side effects, and need for mechanical ventilation outcomes, therefore OR (Odd Ratio) were obtained from each study. We compiled all the outcomes from each study by considering their weight, using the RevMan 5.0 (RRID:SCR_003581) application. Studies with high heterogeneity were analyzed using the DerSimonian and Laird random-effects model and Mantel-Haenszel fixed effect model was used if heterogeneity was low.\n\nQuality assessment was conducted by two independent reviewers (A and B). If there were differences in bias assessment results, a senior reviewer (C) was involved in making the final decision. Bias risk assessment in this study used the Risk Of Bias In Non-randomized Studies - of Interventions (ROBINS-I) tool and the revised Cochrane risk-of-bias (RoB) tool for randomized trials.18 The RoB instrument was used to assess the quality of interventional randomized studies with/without blinding.18 The ROBINS-I instrument was used to assess bias risk in interventional studies without randomization, according to Cochrane recommendations. The ROBINS-I tool used low, moderate, and serious as terminologies for risk stratification of the assessed studies.18\n\n\nResults\n\nThere were 137 articles that appeared in the initial search of the entire electronic database. Checking for duplicated articles was conducted using Zotero 5.0 (RRID:SCR_013784) citation manager application, where 133 articles were found free from duplication. We screened articles based on title and abstract suitability, and only seven articles matched. Two articles were excluded because they did not use controls (comparators) and did not report outcomes that matched the outcomes set in this meta-analysis. The PRISMA flow diagram detailing the literature search can be seen in Figure 1.\n\nFive studies were included from 137 results of the literature search.\n\nWe included five interventional studies with a total of 193 participants. Two out of the five studies did not apply randomization. One of the three randomized studies did not apply blinding to either participants or medical professionals. MSCs were taken from human umbilical cord, except for Leng et al., who did not specify the MSC origin used.19 Shi et al., administered MSC therapy at a dose of 2 × 106 cells/kg,20 while Lanzoni et al., used a fixed dose of 100 ± 20 × 106 cells.21 The other three studies did not mention the MSC dose administered explicitly.\n\nAll studies excluded pediatric patients. The mean age of participants across the studies was over 45 years. Meng et al., and Leng et al., excluded patients with comorbidities.14,19 The other three studies included comorbid patients, with a distribution/proportion that did not differ significantly between groups. The study characteristics and outcomes reported for each study can be seen in Tables 2 and 3.\n\n* Dosage was not reported.\n\n** Patient distribution was not significantly different between groups.\n\n* Outcome did not occur between groups.\n\nStudy quality assessment based on risk of bias using the RoB and ROBINS-I tools showed that the included studies were of moderate to good quality (Figure 2 and Table 4). A study by Leng et al., had moderate quality because there was no difference in the analysis of group characteristics.19 This might lead to confounding bias. Furthermore, a study by Shu et al., did not apply blinding, thus triggering performance bias, detection bias, and attrition bias.21\n\nResults showed that a study by Shu et al., did not apply blinding of the subject and personnel, thus triggering performance bias, detection bias, and attrition bias.\n\n*Mod: moderate. A study by Leng et al., 202018 had moderate quality because there was no difference in the analysis of group characteristics. This might lead to confounding bias.\n\nFour out of five studies reported mortality within 28 days after MSC therapy. Lanzoni et al., reported mortality incidence in one in 11 patients treated with MSC, and seven in 12 patients who were given standard therapy.21 This led to a statistically significant difference in patient mortality rates between groups, with an OR of 0.07 [CI 95%: 0.01, 0.75]. Leng et al., and Shu et al., did not report any mortality in the intervention group.18,21 Meanwhile, Shi et al., did not report any mortality in two groups.19\n\nCompilation of the study findings was conducted using a forest plot. The pooled OR for mortality risk for patients with MSC therapy was 0.13 [95% CI: 0.02, 0.68]. This value was obtained by the Mantel-Haenszel fixed effect model method because the heterogeneity was 0% and p value for heterogeneity was 0.76 (Figure 3).\n\nAdverse event is a parameter that could represent the safety of a product. The adverse events referred to were urticaria, palpitations, or pulmonary edema (within six hours) and/or cardiac arrest (within 24 hours) after MSC administration or placebo or MSC vehicle (without MSC). All studies reported this parameter in their articles. None of these studies reported a significant difference in adverse event incidence between groups. Meng et al., and Shu et al., found no adverse events in either group.14,21\n\nWith the Mantel-Haenszel fixed effect model method, it was found that the pooled OR for the MSC group that experienced adverse events was 0.91 [CI 95%: 0.45, 1.86], with a heterogeneity value of 51% (Figure 4).\n\nThe need for a mechanical ventilator could represent the respiratory distress severity of a patient. In this systematic review, three out of five studies reported the need for a mechanical ventilator post-MSC therapy during the treatment period. None of the three studies reported a significant difference in the number of mechanical ventilators needed between groups. Only Shi et al., reported a higher percentage of mechanical ventilator use in the MSC group.19\n\nIn this study, pooled OR for the MSC group that required a mechanical ventilator during treatment was 0.42 [95% CI: 0.12, 1.47]. This figure was obtained by Mantel-Haenszel fixed effect model, with a heterogeneity of 0% (Figure 5).\n\nThere were only two studies reporting hospitalization duration. Meng et al. reported that the mean hospitalization duration in patients on MSC therapy was 20.5 ± 2.0207 days, and 23.25 ± 2.0361 days for patients without MSC administration.14 Shu et al., also agreed that the mean hospitalization duration in patients on MSC therapy was shorter than patients who received standard therapy, with a mean difference of 4.00 [−5.44, −2.56] days.21 The results of meta-analysis using the Mantel-Haenszel fixed effect model method found that the pooled mean difference was −3.54 [CI 95%: −4.68, −2.40] with 7% heterogeneity (Figure 6).\n\nLeng et al., found a significantly greater increase in the IL-10 ratio and decrease in TNF-α in the intervention group compared to the control group within 10 days post-MSC administration.18 Meng et al., reported a significant reduction in IL-6 levels in patients receiving MSC therapy.14 Changes in IL-6 levels were not found to be significant in patients on standard therapy. Lanzoni et al., found a significant decrease in various inflammatory markers at six days post-MSC administration.20 Inflammation markers that were found to be significantly decreased were TNF-α, IL-2, IL-6, and IL-7. There was no significant decrease in these inflammatory markers in the control group. Shu et al., reported significantly lower levels of CRP and IL-6 in the MSC group compared to the control group at days three and seven post-MSC administration.21 On the same day, oxygenation index and lymphocyte count were found to be significantly higher in the MSC group.21\n\nThis systematic review and meta-analysis included five interventional studies. We assessed the heterogeneity of each outcome with the parameter I2 value in percent (%). Heterogeneity was noted to be quite low in all outcomes, except for the side effect outcome, which had moderate heterogeneity (51%). Moderate heterogeneity of side effects may result from different definitions or types of side effect reported in each study. Using a funnel plot, we noted that there was no significant risk of publication bias in all outcomes (Figure 7). The studies are evenly distributed in the right and left areas of the triangle forming a symmetrical funnel plot.\n\n\nDiscussion\n\nThis meta-analysis attempted to compare MSC administration safety and efficacy in COVID-19 patients, which were assessed by mortality, adverse events, treatment duration, need for mechanical ventilators, and inflammation markers. To answer the formulated clinical questions, we used five interventional studies, two of which did not apply randomization. For the most part, the studies included in this meta-analysis reported that MSC was effective and safe to be given to COVID-19 patients.\n\nWe found that MSC administration was successful in reducing hospitalization duration and significantly reduced mortality risk. The decrease in hospitalization duration was certainly related to disease resolution. At hematological level, we also found a significantly greater increase in IL-10 ratio and decrease in TNF-α in MSC treated patients within 10 days post-MSC administration.19 IL-10 is an anti-inflammatory mediator that counteracts TNF-α action. Increased IL-10 and decreased TNF-α levels indicate cytokine storm resolution. Significant decrease in TNF-α, IL-2, IL-6, and IL-7 levels in MSC treated patients was also reported in studies that were included in this systematic review.21–23\n\nCOVID-19 pathogenesis could not be separated from immunity dysregulation, which manifests as a cytokine storm. COVID-19 triggers immunity dysregulation by triggering pro-inflammatory cytokine production cascade activation, mainly IFN-I.24 Other cytokines such as IL-1, IL-2, IL-4, IL-7, IL-10, IL-12, IL-13, IL-17, macrophage colony-stimulating factor (MCSF), granulocyte colony-stimulating factor (G-CSF), MCP-1, MIP-1α, interferon gamma-induced protein-10 (IP-10), IFN -γ, TNF-α, and hepatocyte growth factor (HGF) have also been shown to be increased in COVID-19 patients, resulting in cytokine storms and a worsening of the patient's condition.25 This systematic review indicates that MSC administration plays a role in improving COVID-19 patient’s condition through improvement of immunological status by decreasing pro-inflammatory cytokines.\n\nFindings regarding MSC’s role in restoring immunological control in COVID-19 patients are supported by several studies that have proven beneficial effects. MSCs were shown to reduce TNF-α, IL-1 and IL-6 through hepatocyte growth factor (HGF), prostaglandin-E2 (PGE2), lipoxin A4-stimulated gene 6-protein (LXA4), TNF (TSG-6) release and suppression of inflammatory T-cell proliferation by indoleamine 2,3-dioxygenase expression, transition of Th1 and Th17 responses to Th2, and monocyte and mature myeloid dendritic cell inhibition.26\n\nTreatment with MSCs is a therapeutic strategy to stop the uncontrolled inflammatory cascade and, at the same time, reduce post-COVID-19 pulmonary fibrosis and abnormal lung function, decrease ground-glass opacification (GGO) and infiltrate zones in patients post-MSC administration. This indicates that MSC therapy does not only improve patient’s immunological status, but also plays a role in structural improvement and prevention of lung damage in COVID-19 patients.27,28 These findings are supported by an in vivo study, which found that MSCs interfere with pulmonary fibrosis activation pathways resulting in pulmonary protective effects against damage/injury.29\n\nEvidence that MSCs play a role in the immunomodulation process and lung structural repair supports the results that we found in this meta-analysis and systematic review, where MSCs effectively reduce mortality risk and reduce hospitalization duration. MSCs modulate oxidative stress, suppress systemic inflammation, and reduce lung damage progression.30\n\nIn addition, this meta-analysis also found that MSCs are safe to administer to COVID-19 patients. We noted no significant difference in adverse events incidence between groups. MSCs are a heterogeneous population of non-hematopoietic multipotent stromal cells with a specific cell surface expression pattern, low alloreactivity (expression of major histocompatibility complex (MHC)-I, MHC-II, and low co-stimulation molecules), and have the ability to differentiate tissues from favorable mesodermal lineage.31 Through these mechanisms, the body's resistance to MSC administration will be minimal. This confirms the findings of this meta-analysis that there are no adverse events in patients after MSC administration, and MSCs are safe to administer to COVID-19 patients.\n\nFrom our knowledge, there has been no meta-analysis or systematic review addressing the clinical questions explored in this article. Apart from the clinical questions that were successfully answered through this meta-analysis, there are several limitations that need to be underlined. First, the exclusion criteria were not applied for the MSC’s type of source and seeding methods. Second, all comorbid patients were included. Patients with degenerative or malignant diseases were perceived to have different immunological responses, which might affect MSC therapy efficacy. Third, some studies have low quality or a fairly high bias risk, especially those that did not apply blinding to their studies. In addition, the number of participants in each study and overall is still considered insufficient.\n\n\nConclusion\n\nGiving MSCs to COVID-19 patients is safe and effective in reducing mortality and hospitalization duration. Clinical improvement in patients occurred through cytokine storm resolution, which was characterized by decreased pro-inflammatory cytokines and increased anti-inflammatory suppression in 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\nFigshare: PRISMA checklist for ‘Mesenchymal stem cell therapy efficacy in COVID-19 patients: A systematic review and meta-analysis’, https://doi.org/10.6084/m9.figshare.16602203.v1.16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgment\n\nThis work was supported by Indonesian Endowment Fund for Education (LPDP) Republic of Indonesia.\n\n\nReferences\n\nDong E, Du H, Gardner L: An interactive web-based dashboard to track COVID-19 in real time. The Lancet Infectious Diseases. 2020 May; 20(5): 533–534. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMegasari NLA, Utsumi T, Yamani LN, et al.: Seroepidemiological study of SARS-CoV-2 infection in East Java, Indonesia. PLoS ONE. 2021 May; 16(5): e0251234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSinghal T: A Review of Coronavirus Disease-2019 (COVID-19). Indian J Pediatr. 2020; 87(4): 281–286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi V, Hsiao JL, Shi VY: COVID-19 skin manifestations: the new great imitator? Dermatol Online J. 2020 Nov; 15: 26(11). PubMed Abstract\n\nda Rosa MR, Francelino Silva Junior LC, Santos Santana FM, et al.: Clinical manifestations of COVID-19 in the general population: systematic review. Wien Klin Wochenschr. 2020 Nov 26; 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuki K, Fujiogi M, Koutsogiannaki S: COVID-19 pathophysiology: A review. Clin Immunol. 2020 Jun; 215: 108427. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou Y, Yang Q, Chi J, et al.: Comorbidities and the risk of severe or fatal outcomes associated with coronavirus disease 2019: A systematic review and meta-analysis. Int J Infect Dis. 2020 Oct 1; 99: 47–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeigel JH, Tomashek KM, Dodd LE, et al.: Remdesivir for the treatment of Covid-19. N Eng J Med. 2020 Oct; 383(19): 1813–1826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoudhery MS, Harris DT: Stem cell therapy for COVID-19: Possibilities and challenges. Cell Biol Int. 2020; 44(11): 2182–2191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGolchin A, Seyedjafari E, Ardeshirylajimi A: Mesenchymal Stem Cell Therapy for COVID-19: Present or Future. Stem Cell Rev Rep. 2020 Apr; 13: 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajarshi K, Chatterjee A, Ray S: Combating COVID-19 with mesenchymal stem cell therapy. Biotechnol Rep. 2020 Jun 1; 26: e00467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiang B, Chen J, Li T, et al.: Clinical remission of a critically ill COVID-19 patient treated by human umbilical cord mesenchymal stem cells. Medicine (Baltimore). 2020 Jul 31; 99(31). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanzoni G, Linetsky E, Correa D, et al.: Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind, phase 1/2a, randomized controlled trial. Stem Cells Transl Med. 2021 May; 10(5): 660–673. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeng F, Xu R, Wang S, et al.: Human umbilical cord-derived mesenchymal stem cell therapy in patients with COVID-19: a phase 1 clinical trial. Signal Transduct Target Ther. 2020 Aug 27; 5(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLefebvre C, Manheimer E, Glanville J: Searching for Studies. In: Cochrane Handbook for Systematic Reviews of Interventions. John Wiley & Sons, Ltd; 2008. p. 95–150.\n\nAndrianto A, Sari DKSC, Farabi MJA, et al.: Mesenchymal stem cell therapy efficacy in COVID-19 patients: A systematic review and meta-analysis. Figshare. 2021; Publisher Full Text\n\nRyan R, Hill S: Supporting implementation of Cochrane methods in complex communication reviews: resources developed and lessons learned for editorial practice and policy. Health Res Policy Syst. 2019 Mar 28; 17(1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins J, Green S: Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0. The Cochrane Collaboration. ; 2011 [cited 2020 May 7]. Reference Source\n\nLeng Z, Zhu R, Hou W, et al.: Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia. Aging Dis. 2020 Apr; 11(2): 216–228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi L, Huang H, Lu X, et al.: Treatment with human umbilical cord-derived mesenchymal stem cells for COVID-19 patients with lung damage: a randomised, double-blind, placebo-controlled phase 2 trial. medRxiv. 2020 Oct 21; 2020.10.15.20213553. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShu L, Niu C, Li R, et al.: Treatment of severe COVID-19 with human umbilical cord mesenchymal stem cells. Stem Cell Res Ther. 2020 Aug 18; 11(1): 361. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLanzoni G, Linetsky E, Correa D, et al.: Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind, phase 1/2a, randomized controlled trial. Stem Cells Transl Med. 2021 Jan; 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun P, Qie S, Liu Z, et al.: Clinical characteristics of hospitalized patients with SARS-CoV-2 infection: A single arm meta-analysis. J Med Virol. 2020; 92(6): 612–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo Y-R, Cao Q-D, Hong Z-S, et al.: The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak – an update on the status. Mil Med Res. 2020 Mar; 13: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTahaghoghi-Hajghorbani S, Zafari P, Masoumi E, et al.: The role of dysregulated immune responses in COVID-19 pathogenesis. Virus Res. 2020 Dec; 290: 198197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonguió-Tortajada M, Bayes-Genis A, Rosell A, et al.: Are mesenchymal stem cells and derived extracellular vesicles valuable to halt the COVID-19 inflammatory cascade? Current evidence and future perspectives. Thorax. 2021 Feb 1; 76(2): 196–200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThille AW, Esteban A, Fernández-Segoviano P, et al.: Chronology of histological lesions in acute respiratory distress syndrome with diffuse alveolar damage: a prospective cohort study of clinical autopsies. Lancet Respir Med. 2013 Jul 1; 1(5): 395–401. PubMed Abstract \| Publisher Full Text\n\nZhang Y, Ding J, Ren S, et al.: Intravenous infusion of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells as a potential treatment for patients with COVID-19 pneumonia. Stem Cell Res Ther. 2020 May 27; 11(1): 207. Publisher Full Text\n\nMcIntyre LA, Moher D, Fergusson DA, et al.: Efficacy of Mesenchymal Stromal Cell Therapy for Acute Lung Injury in Preclinical Animal Models: A Systematic Review. PLoS One. 2016; 11(1): e0147170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatthay MA, Calfee CS, Zhuo H, et al.: Treatment with allogeneic mesenchymal stromal cells for moderate to severe acute respiratory distress syndrome (START study): a randomised phase 2a safety trial. Lancet Resp Med. 2019 Feb 1; 7(2): 154–162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDominici M, Le Blanc K, Mueller I, et al.: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8(4): 315–317. PubMed Abstract \| Publisher Full Text" }	[ { "id": "97905", "date": "21 Jan 2022", "name": "Ali Golchin", "expertise": [ "Reviewer Expertise Stem cell research", "Cell-based therapy" ], "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 is a comprehensive and good systematic study. The authors have tried to consider all protocols and guidelines of systematic reviews and meta-analyses. Their conclusion is based on their results and real; however, it is possible that from Feb 2021 published new clinical reports about MSC therapy in COVID-19 patients. Moreover, it was expected in discussion and conclusion different issues of Mesenchymal stem cell therapy such as cost-utility, source limitations, personalized medicine aspects, etc. These issues make the most challenging aspects of stem cell therapy in different diseases, especially COVID-19 patients.\nReferences\nBasiri A, Mansouri F, Azari A, Ranjbarvan P et al (2021) Stem cell therapy potency in personalizing severe COVID-19 treatment. Stem Cell Rev Rep 17:193–213. https://doi.org/10.1007/s12015-020-10110-w\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-956
https://f1000research.com/articles/10-955/v1	22 Sep 21	{ "type": "Research Article", "title": "Modelling age-friendly environment for social connectedness: a cross-sectional study", "authors": [ "Lein Shi Ying", "Lai Ming Ming", "Lau Siok Hwa", "Lein Shi Ying", "Lau Siok Hwa" ], "abstract": "Background: The increase in aged populations in Malaysia has brought unprecedented challenges to families, policy makers, scholars, and business organisations. This paper adapted the WHO 2007 framework of features of age-friendly cities to examine age-friendly environment constructs and their linkages with social connectedness from the perspective of Malaysian middle-aged and older adults caring for themselves. Methods: A face-to-face cross-sectional survey was conducted on 402 middle-aged and older adults caring for themselves across selected states in west Malaysia, selected via purposive sampling. Firstly, features of age-friendly cities were explored through exploratory factor analysis involving 82 respondents. Subsequently, structural equation modelling was performed, involving 320 respondents. Results: The results indicated that the constructs of an age-friendly environment were built environment, community support and health services, civic participation, and employment as well as communication and information. The structural model provided evidence that implementing age-friendly initiatives relating to built environment, community support and health services, civic participation and employment as well as communication and information enables the ageing population to improve their connectedness with society. These findings supported the ecological theories, agreeing that age-friendly environments help middle-aged and older adults caring for themselves to increase their adaptability and reduce perceived pressure from the environment. This result was in line with the current literature in which an age-friendly environment is a form of support and an enabling environment to cultivate positive social relationships and connectivity. Conclusions: Creating an age-friendly environment that supports active and healthy living for middle-aged and older adults caring for themselves allows them to continue to share their experiences, knowledge, and wisdom. This is helpful and beneficial for societal well-being and economic development as well as for the future generations.", "keywords": [ "age-friendly", "social connectedness", "aging", "Malaysia", "environment" ], "content": "Introduction\n\nMalaysia is expected to become an aged nation by 2030 where 15.3%, approximately 5.8 million of its population, would comprise of individuals aged 60 and above according to the Department of Statistics, Malaysia.1 Malaysia is categorised as one of the nations that is ageing at the fastest pace because Malaysia would transform into an aged nation within 20 years, as individuals aged 60 and above only comprised of 7.6% of its population in year 2010.2 The main problems faced by ageing adults in Malaysia are illness, deteriorating physical functioning, existing building and facilities not being age-friendly designed, as well as less social interactions and feeling less connected to others.3\n\nAccording to The World Factbook, the majority of Malaysians (77.7%) live in urban areas. Urbanisation and population ageing have become issues for healthcare-related personnel, researchers, and town planners in developing healthy and liveable environments for the elderly.4 The threats found in urban areas are harmful to social well-being such as environmental pollution, tobacco and alcohol abuse, malnutrition, lack of physical activities, inaccessible transportation, poor infrastructure, and inadequate healthcare services.5 Furthermore, new buildings and most of the old buildings in Malaysia were built without meeting the statutory guidelines for disabled persons.6 The development of an age-friendly environment is essential because the current environment is built without taking into considerations the needs of people of all ages.7\n\nHaving the opportunity to deepen existing relationships and social connectedness with others is important for those who are ageing.8 Relationships become gradually more important when an individual ages; through an age-friendly environment initiative, one can improve one’s social connections and receive adequate social support, which is beneficial for maintaining health and well-being.9 In order to improve the well-being of the ageing population, an age-friendly environment should be developed to support social connectedness, participation, and integration among the elderly.10\n\nIn acknowledging the growth of the ageing population worldwide, the World Health Organisation (WHO) introduced two policy frameworks, namely, active ageing11 and global age-friendly cities.12 WHO (2007, p. 1)12 defined an age-friendly city as one that “encourages active ageing by optimizing opportunities for health, participation, and security in order to enhance quality of life” of elderly individuals. An age-friendly community helps the elderly “to maintain social connectedness while deepening existing relationships” (Emlet & Moceri, 2012, p. 2).10\n\nEmpirical studies of the age-friendly concept are few.13 Despite Malaysia being predicted to become an aged nation by 2030, studies conducted in relation to the ageing of the population are relatively limited compared to developed countries. There is an urgency to restructure policies for the ageing population that includes local factors that address the forthcoming health and social needs.14 The ageing of the population in Malaysia has created unprecedented challenges to policy makers, scholars, and business organisations as Malaysia will become an aged nation by 2030.\n\nIn an initiative to make environments better for the elderly and to accommodate their needs, the WHO Global Network of Age-friendly Cities and Communities was formed to facilitate mutual communication, discussions, and learning among countries, cities, and communities globally. Many developed countries, cities, and communities joined the network and adopted the global age-friendly cities guide.12 To date, the network consists of 847 cities and communities across 41 countries, which include more than 230 million people, and the network is expanding. Taiping city in Perak, Malaysia was chosen for the two-year age-friendly city pilot project in August 2019 and is on track to achieve age-friendly city status.\n\nIt should be pointed out that Malaysia has not adopted the WHO (2007) global age-friendly cities guide.12 Hence it is important to identify the constructs of age-friendly environments and subsequently examine the relationships between age-friendly environments and social connectedness from the perspectives of middle aged and older adults caring for themselves in Malaysia. This study empirically modelled an age-friendly environment and social connectedness model through structural equation modelling (SEM). SEM provides higher accuracy in predicting the research model as validity of the model is statistically evidenced through the assessment of goodness-of-fit indices.15\n\nThis study aims to empirically identify the significant constructs of age-friendly environments by adapting the WHO (2007) global age-friendly cities12 guide as well as examining the relationship between age-friendly environments and social connectedness by bridging the literature gap as suggested by Menec et al. (2011)16 and Emlet and Moceri (2012).10 This is among the first few attempts to model an age-friendly environment by identifying its significant constructs and subsequently linking to social connectedness from the perspective of middle-age and older adults caring for themselves in a developing country. The proposed research model is important and adds empirical evidence on the readiness and preparation of Malaysia for becoming an aged nation by 2030.\n\nAn age-friendly environment offers a comprehensive “age-integrated as well as age-targeted” initiative that helps the ageing adults to improve their social connectedness and self-worth12 (WHO, 2007, p. 72). The concept of social connectivity is an indispensable advantage of age-friendly environments given that an age-friendly environment forms a mutual connection between the ageing adults and their environment.16 The literature suggests that local authorities and practitioners should focus on neighbourhood design and establish community-based interventions to improve social connectedness and social participation among the elderly.17,18\n\nLawton’s ecological theory of ageing (Nahemow & Lawton, 1973)19 and Bronfenbrenner’s ecological theory (1977)20 are widely used to address the adaptation between environment and humans, whereby there should be a balance between both.19,20\n\nThe environment can bring certain pressures or hassle to daily living as an individual ages, which would create inconveniences for them.21 According to the ecological theory of Lawton (1974),21 there are two main causes for the decrease in capacity as one ages, namely perceived pressure from the environment and weakening of bio-physical functioning.\n\nBronfenbrenner (1977)20 explained that the ecological environment can be visualised as a set of nested environmental structures, which are arranged at each inclusion of another and interact bi-directionally. The structures are the microsystem, mesosystem, exosystem and macrosystem. Microsystem refers to interactions between an individual and their immediate environment such as family, home, friends, and workplace. Mesosystem refers to the interconnectivity of an individual with the interaction of multiple immediate environments. According to the Bronfenbrenner’s ecological theory (1977),20 individuals interact with the different environment structures in daily living. The ecological frameworks focus on the interconnectivity between humans and varied social and physical environments.22 Both the ecological frameworks emphasise the vitality of the social aspect of an environment.20,23\n\nLui et al. (2009)24 and Menec et al. (2011)16 indicated that elderly people would like to remain active and be involved in community activities in their silver days. An age-friendly environment supports and enhances social connectivity among the ageing population.\n\nIn the application of ecological frameworks to conceptualise an age-friendly environment, the environment should be dynamic to accommodate the changes in physical and cognitive functioning as well as social capacity as people grow older.10 Furthermore, social inclusion is a significant component in addressing age-friendly environments and could be improved by removing the obstacles brought by elements of the physical and social environments.25\n\nFeldman and Oberlink (2003)26 are amongst the pioneers of conceptualisation of age-friendly environments. They conducted focus group interviews involving adults aged 35 and above and community leaders from the United States. The study found four indicators of age-friendly environments, namely identifying basic necessities, promoting social and civic engagement, maximising independence for frail and disabled elderly and optimising physical and mental functioning and well-being. The study concluded that financial security, health and healthcare, social connections, housing and supportive services, transportation and safety were important factors for the elderly to age-in-place.\n\nSubsequently, WHO (2007)12 conducted focus group interviews to examine age-friendly city features from a wider perspective involving 1,485 elderly people aged 60 and above, 250 caregivers, and 515 service providers across 33 cities across continents worldwide. They found eight features that made a city age-friendly, which were outdoor spaces and buildings, transportation, housing, social participation, respect and social inclusion, civic participation and employment, community support and health services, as well as communication and information. On the other hand, Alley et al. (2007)27 studied elder-friendly communities using qualitative methods. They found that the significant criteria of elder-friendly communities were accessible and inexpensive transportation, safe residences, healthcare, safety, and being involved in the community.\n\nRichard et al. (2009)17 examined the associations between neighbourhood features and social participation among 282 urban elderly people in Canada through a survey. The results found that social participation had positive implications for their health. Hence, they suggested that the urban and local community authorities build a supportive neighbourhood that promoted social interaction and participation among the elderly.\n\nMenec et al. (2011)16 conceptualised the age-friendly community based on the WHO’s 2007 age-friendly city model12 and ecological theory. Their study proposed seven important domains, namely physical environment, housing, social environment, opportunities for participation, informal and formal community support and health services, transportation and communication and information. They also suggested that the concept of social connectivity served as a basic support to age-friendly communities. Subsequently, Menec et al. (2013)13 explored the characteristics of age-friendliness in Canada through a survey of 1,373 individuals. A larger percentage of the respondents scored highly on social age-friendly domains, such as social environment, participation opportunities, health-care services, and communication and information, in comparison with physical domains. Hence, they found that age-friendly domains in the social environment were easier to implement than in the physical environment.\n\nFrom the Asian perspective, Kadoya (2013)28 explored the barriers faced by the elderly when interacting with society through their participation in local community programmes in Japan and found that the ability to be mobile affected their social interaction. Hence, he concluded that social inclusion was an important factor for age-friendly cities. Lai et al. (2016)29 studied the WHO (2007) age-friendly12 environment features and linked them with active ageing. The study surveyed 211 informal caregivers and 402 adults who cared for themselves aged 45 and above in Malaysia through purposive sampling. They found that outdoor spaces and buildings, transportation, and housing as well as community support and health services were significant age-friendly environment features. The findings implied that the development of physical environment features such as a comprehensive public transportation system, elderly-friendly housing, outdoor spaces and buildings as well as medical and healthcare services should be carried out before social environment development.\n\nFranke et al. (2013)18 conducted a qualitative study to investigate the main areas that promoted physical activity among the elderly who were ageing actively in Canada. These areas were resourcefulness, social connections, and built environment. The study suggested that new policies addressing social connectedness and accessibility to social and physical environments were required to enhance and preserve independence and mobility among the elderly.\n\nScharlach and Lehning (2013)25 reviewed and described the various age-friendly efforts implemented in the United States, which helped to enhance social inclusion among the elderly from the perspective of physical and social environments. An environment that enabled the elderly to be mobile and with an accessible transportation system would help to promote social inclusion. In general, social inclusion could be improved by removing the hurdles found in the physical and social environments.\n\nRuza et al. (2015)30 compared the perspectives of age-friendliness among professionals and local community residents via focus group interviews. Both the professionals and local groups agreed that community and health services had the greatest significance in establishing age-friendliness of a city. Meanwhile, communication and information as well as civic participation and employment were found to have the least significance.\n\nRogers and Mitzner (2017)31 conducted a study to predict the personality, needs, and wants of the future elderly of 2050 by taking into account their independence, health, well-being, and social connectedness. There was a similarity between the future and current elderly; they wished to prolong their independence. Social connectedness and social activities were essential for the general well-being of older adults. Therefore, being socially connected with family, friends, and neighbours helped ageing adults to improve their life satisfaction.\n\nTo bridge the research gap and supported by ecological theories, this study empirically examines the constructs of age-friendly environments and proposed positive relationships between an age-friendly environment and social connectedness from a developing country perspective. Figure 1 illustrates the proposed research model of age-friendly environments and social connectedness; and the two hypotheses are as follows:\n\nH1: Outdoor spaces and buildings, transportation, housing, social participation, respect and social inclusion, civic participation and employment, community support and health services, as well as communication and information will be confirmed as constructs of an age-friendly environment.\n\nH2: An age-friendly environment is positively related to social connectedness.\n\nThis study examines eight features of age-friendly environments and social connectedness. These constructs are reflective models. As a result, the eight features form a latent variable of age-friendly environments. The latent construct of age-friendly environments is identified by a set of indicators. In short, the eight constructs of age-friendly environments are the indicators of age-friendly environments. Hence, an age-friendly environment is a formative model in which the valid constructs are confirmed through second-order confirmatory factor analysis (CFA).\n\n\nMethods\n\nThis research received approval (number EA1842021) from Research Ethics Committee of Multimedia University. Both informed and written consent were obtained from all participants prior to initiating the survey. When the respondents verbally agreed to participate, the questionnaire was given to them. The respondents who agreed to participate were required to tick on the column “If agree, please proceed to the next page” in the questionnaire.\n\nThis study employed a cross-sectional survey method to examine the research model of age-friendly environments and social connectedness. In line with the prior studies by Feldman and Oberlink (2003),26 Emlet and Moceri (2012),10 and Menec et al. (2013),13 middle-aged adults were used as targeted respondents. Middle-aged and older adults have witnessed death in their family and of their peers; thus, they may be compelled to improve their quality of life while they still have time.32 Perceptions of age-friendly environments from middle-aged adults are helpful as they portray the future needs of the elderly, which is beneficial for policy planning and enables them to voice their opinion for the future community in which they would age.10\n\nSelf-care refers to the ability of “people looking after themselves” (WHO, 2002, p. 37).11 By adapting the definition by Levinson (1986),33 middle-aged adults were individuals aged 45 to 65 years old, while older adults were individuals aged 65 and above.1 This study identified targeted respondents as middle-aged adults and older adults undertaking self-care, who were individuals aged 45 and above and have the ability to take care of themselves.\n\nPurposive sampling was used to collect data from respondents based on highly populated Malaysian states with high urbanisation levels and old-age dependency ratios. Hence the data was collected from Penang, Perak, Selangor, Wilayah Persekutuan Kuala Lumpur, Negeri Sembilan, Melaka, and Johor states in Malaysia. Since targeted respondents are middle-aged and old-age adults caring for themselves, places where many ageing adults are found or places which have high level of ageing adults’ activities were identified. The main research places of this study were the “Pusat Aktiviti Warga Emas (PAWE)”, or senior activity centers, in Pulau Pinang, Perak, Selangor, Wilayah Persekutuan Kuala Lumpur, Negeri Sembilan, Melaka, and Johor states. These senior activity centers provide a place where the elderly can gather and interact with one another. They are supported and governed by the Department of Social Welfare in Malaysia.\n\nThe study collected data from 402 self-care middle-aged and older adults, which fulfilled the minimum sample size as suggested by GPower 3 statistical software. The criteria were set by employing a priori power analysis with medium effect size, alpha at 0.05, power at 0.8, as well as eight constructs.34\n\nThe constructs of an age-friendly environment were adapted from the “checklist of essential features of age-friendly cities” (WHO, 2007b), which had not been empirically tested. Hence, the constructs of an age-friendly environment were firstly explored through exploratory factor analysis. Gpower 3 statistical software suggested the sample size N = 52 is required for regression analysis through a priori power analysis by considering a large effect size, alpha of 0.05, power of 0.8, and eight constructs (Faul et al., 2009).34\n\nIn performing the exploratory factor analysis, the study first selected 82 odd samples which comprised approximately 20% of the total sample size of 402. This sample, N = 82 fulfilled the minimum sample size required to analyse eight constructs based on a priori power analysis of large effect size, significance level at 0.05 and power of 0.8. Hence, the remaining sample of 320 was used to create the research model using structural equation modelling.\n\nThe survey had three sections, namely demographic variables, age-friendly environment, and social connectedness. Demographic variables examined respondents’ age, gender, nationality, race, and educational background. The age-friendly measurement items were developed by adapting the indicators of activity-friendly communities,35 checklist of essential features of age-friendly cities,36 and information, communication, and technology (ICT) scale by Cotten, Anderson, and McCullough (2013).37 The measurement items were examined using a five-point Likert scale ranging from 1 “very unimportant” to 5 “very important” to evaluate the level of importance of age-friendly environment features as perceived by the respondents. In examining social connectedness among respondents, the study adapted the social connectedness scale by Lee and Robbin (1995).38 This is a self-rated scale of emotional distance or connectedness between an individual and others in a society. In this study, the scale was evaluated through a five-point Likert scale that ranged from 1 “strongly disagree” to 5 “strongly agree”. Higher scores indicate greater social connectedness as well as freedom from social isolation. The original items were phrased negatively; therefore, the items were re-coded for analysis.\n\nThe questionnaire54 was developed in English and translated into Malay and Chinese using the direct translation method because this is an easy and simple procedure. The translation process began with direct translation of the questionnaire from English to the Malay and Chinese by language experts. The Malay and Chinese versions were reviewed and verified by a group of reviewers and researchers involved in this study who were familiar with English. The direct translation method can be improved through review by independent judges.39 It is worth noting that standard procedures for translating measurement items had yet to be established.39 There is “no empirical evidence in favour of one specific method” in translating measurement items (Acquadro et al., 2008, p. 509).40\n\nThe pilot study was conducted in Melaka, employing purposive sampling. The sampling location areas included local community areas such as parks, religious centres, and informal gatherings of middle-aged and older adults in residential areas. The pilot study was conducted in December 2013 among 30 middle-aged and older adults aged 45 and above who cared for themselves. Cronbach’s alpha reliability test was conducted to examine the reliability of the measurement items. The results showed that all variables were valid, indicated by a Cronbach’s alpha coefficient of more than 0.7. No major changes were made to the survey following the pilot study.\n\nThe actual data collection began from January 2014 until September 2014 and 402 responses were collected from middle-aged and older adults caring for themselves in the seven Malaysian states. Approval was sought from the Department of Social Welfare to allow the collection of data at senior activity centers in the selected states of Malaysia. It should be pointed out that the study only managed to collect 272 responses through face-to-face surveys administered by a graduate research assistant in the senior activity centers. These 272 responses did not achieve the minimum sample size required. Therefore, data was supplemented with data gathered from local community areas; for example, informal gatherings of middle-aged and older adults at religious places such as mosques, temples, or churches as well as gatherings of informal caregiver support groups of the Alzheimers Disease Foundation Malaysia in Petaling Jaya and Johor Bahru cities in Malaysia.\n\nThe Cronbach’s alpha test of reliability was conducted to examine the reliability of measurement items. Next, the 37 measures of age-friendly environments were explored through exploratory factor analysis (EFA) performed on 82 responses. EFA was conducted through principal axis factoring and promax rotation. Two-step structural equation modelling (SEM) was employed to examine the research model, which began with the specification of CFA and proceeded with structural model development. This study employed SPSS version 22 and Analysis of Moment Structures (AMOS) version 21.\n\nThe maximum likelihood method of estimation was utilised as it is the most commonly used method of estimation.41 This estimation method measures all the variables concurrently at a point in time and provides the most accurate estimation with the least variance when data is normally distributed.42 A common alternative to examine the normality of data is through monitoring the value of skewness and kurtosis and can be determined by the sample size. For a sample size greater than 300, “an absolute skew value larger than 2 or an absolute kurtosis larger than 7” are indicative that the data is not normally distributed (Kim, 2013, p. 53).43\n\nCFA allows the assessment of model goodness of fit and model validity. Model goodness of fit is indicated by insignificant p-value, normed chi-square value (CMIN/df) lower than 3, expected cross validation index (ECVI) near zero, standardised root mean residual (SRMR) lower than 0.08, root mean square error of approximation (RMSEA) lower than 0.07 with insignificant p-value, followed by goodness-of-fit index (GFI), adjusted GFI (AGFI), normed fit index (NFI), comparative fit index (CFI), and Tucker Lewis index (TLI) above 0.9.44-46\n\nConstruct validity of the specified model was examined through convergent validity and discriminant validity. The three criteria of convergent validity are standardised factor loadings, average variance extracted (AVE), and reliability.44 A measurement model achieved convergent validity when standardised factor loadings exceed 0.5 and preferably 0.7, AVE more than 0.5, as well as composite reliability (CR) larger than 0.7.44 Discriminant validity was determined using the method proposed by Fornell and Larcker (1981).47 Discriminant validity is achieved when AVE is larger than the value of squared correlation between constructs. When the constructs satisfy the model goodness of fit and validity assessment, a structural model for hypothesis testing can be specified. In this study, a structural model was specified by linking age-friendly environments and social connectedness.\n\n\nResults\n\nA total of 402 middle-aged and older Malaysian adults caring for themselves participated in the study. The sample had a greater proportion of female participants than male; 274 (68.2%) female and 128 (31.8%) male.55 The sample was evenly divided between 212 (52.7%) middle-aged adults and 190 (47.3%) older adults. The sample consists of a large number of Chinese respondents (240, 59.7%) and married respondents (260, 64.7%). The majority of the respondents (278, 69.2%) were not highly educated and their highest level of education was either primary level, secondary school level, or had attained Sijil Pelajaran Malaysia (SPM). An individual who has completed their secondary school education is awarded with Sijil Pelajaran Malaysia (SPM).48 Results of demographic profiles are presented in Table 1.\n\nCronbach’s alpha test was performed to examine the reliability of the variables. An item from the housing construct was removed to improve the coefficient. Variables were reliable given that the value of Cronbach’s alpha coefficient for each variable was larger than 0.7, as shown in Table 2.\n\n\n\na) Outdoor spaces and buildings\n\n\n\nb) Transportation\n\n\n\nc) Housing\n\n\n\nd) Social participation\n\n\n\ne) Respect and social inclusion\n\n\n\nf) Civic participation and employment\n\n\n\ng) Community support and health services\n\n\n\nh) Communication and information\n\nSubsequently, EFA through the principal axis factoring method and promax method of rotation was performed on 82 responses. A clean factor structure was achieved with four constructs, namely communication and information, built environment, community support and health services, and civic participation and employment. The result of the Kaiser-Meyer-Olkin (KMO) test was 0.84 and Bartlett’s test of sphericity was significant with p = 0.00. Pattern matrix analysis showed that factor loadings all exceeded 0.5, indicating that the analysis of factor structure was valid. The final factor structure suggested that the four factors be retained as each had eigenvalues higher than one. The results of EFA are shown in Table 3.\n\n\n\n1. Communication and information\n\n\n\na) Increases the quantity of my communication with others\n\n\n\nb) Contributes to my ability to stay in touch with people I know\n\n\n\nc) Makes me feel less isolated\n\n\n\nd) Makes it easier to meet new people\n\n\n\ne) Makes it easier for me to reach people\n\n\n\nf) Helps me feel more connected to friends and family\n\n\n\n2. Built environment\n\n\n\na) Drop off and pick up areas are available and conveniently located\n\n\n\nb) Presence of bicycle lanes and walking trails\n\n\n\nc) Public transportation is accessible by everyone including people with disabilities\n\n\n\nd) Outdoor safety is promoted by good street lighting during the night\n\n\n\ne) Taxis have discounts for elderly people\n\n\n\nf) Public transportation is available for elderly people to reach key destinations\n\n\n\ng) Public toilets are conveniently accessible\n\n\n\nh) Presence of traffic signals with adequate time for crossing for elderly people\n\n\n\n3. Community support and health services\n\n\n\na) Affordable physiotherapy services are offered to restore elderly health\n\n\n\nb) Health services are conveniently reachable\n\n\n\nc) Affordable medical care services are available for elderly people\n\n\n\nd) Adequate range of services are offered in the community to promote elderly well-being\n\n\n\n4. Civic participation and employment\n\n\n\na) Flexible paid opportunities for elderly people to work\n\n\n\nb) Elderly people have flexible voluntary options\n\n\n\nc) Workplaces are adapted to meet the needs of elderly people with disabilities\n\nThe EFA results showed that three features of age-friendly environment, namely outdoor spaces and buildings, transportation, and housing were grouped and explained as one single factor, which the study renamed as built environment. It was possible to re-classify these three features as a single factor given that they are the “key features of physical environment” (WHO, 2007, p. 9).12 This study named the combination of the three features as built environment because built environment comprehensively addresses the features of outdoor spaces and buildings, transportation as well as housing.27\n\nAt the same time, two features, namely respect and social inclusion as well as social participation, were removed as these items had cross-loading with other variables. WHO (2007, p. 9) identified respect and social inclusion, social participation, and civic participation and employment as “social environment and of culture that affect participation and mental wellbeing”.12 Social participation as well as civic participation and employment were re-grouped and explained as opportunities for participation because they were related to participation of the ageing adults in social activities.16 Civic participation and employment was found to be more practical in the context of Malaysia in addressing social participation among the ageing adults.\n\nSubsequently, first-order CFA of age-friendly environment and social connectedness features was carried out with 27 items. To examine the correlation among the constructs, they were allowed to covary. Overall first-order CFA of age-friendly environment and social connectedness features allowed the evaluation of convergent validity and discriminant validity.\n\nThe initial model did not achieve satisfactory model fit. Therefore, model respecification was performed by examining modification indices as standardised factor loadings had exceeded 0.5. Items with high modification indices were removed or allowed to co-vary in the model. This is because the items that highly explain a construct should be retained, which helps to improve the accuracy of a specified model.49 A satisfactory first order CFA of age-friendly environment and social connectedness features was achieved with a model fit of CMIN/df = 1.19, GFI = 0.96, AGFI = 0.94, RMSEA = 0.02, SRMR = 0.03, NFI = 0.98, TLI = 0.99, CFI = 0.99 and ECVI = 0.62. The results of CFA of age-friendly environment and social connectedness features as well as convergent validity are presented in Table 4.\n\n\n\n1. Communication and information\n\n\n\na) Makes it easier for me to reach people\n\n\n\nb) Contributes to my ability to stay in touch with people I know\n\n\n\nc) Makes me feel less isolated\n\n\n\n2. Built environment\n\n\n\na) Drop off and pick up areas are available and conveniently located\n\n\n\nb) Public transportation is accessible by everyone including people with disabilities\n\n\n\nc) Public toilets are conveniently accessible\n\n\n\n3. Community support and health services\n\n\n\na) Affordable physiotherapy services are offered to restore elderly health\n\n\n\nb) Affordable medical care services are available for elderly people\n\n\n\nc) Health services are conveniently reachable\n\n\n\n4. Civic participation and employment\n\n\n\na) Elderly people have flexible voluntary options\n\n\n\nb) Flexible paid opportunities for elderly people to work\n\n\n\nc) Workplaces are adapted to meet the needs of elderly people with disabilities\n\n\n\n5. Social connectedness\n\n\n\na) Disconnected from the world around me\n\n\n\nb) No sense of togetherness\n\n\n\nc) Don’t feel related to anyone\n\n\n\nd) Losing all sense of connectedness with society\n\nThe results showed that convergent validity was achieved with all the items, with satisfactory standardised factor loadings exceeding 0.7. The AVE indicated that all latent variables had good validity with all values greater than 0.5. For the reliability assessment, all the constructs were acceptable with composite reliability (CR) and Cronbach’s alpha coefficient exceeding 0.7.\n\nTable 5 shows the results of discriminant validity using the method of Fornell and Larcker (1981).47 The constructs achieved discriminant validity, as the bold values are larger compared to the other values.\n\n\n\n1. Communication and information\n\n\n\n2. Built environment\n\n\n\n3. Community support and health services\n\n\n\n4. Civic participation and employment\n\n\n\n5. Social connectedness\n\nSecond-order CFA indicated satisfactory standardised factor loading with three items exceeding 0.5 and one item near 0.5. The model was statistically fit with CMIN/df = 1.37, p-value = 0.05, GFI = 0.97, AGFI = 0.95, RMSEA = 0.03, SRMR = 0.03, NFI = 0.98, TLI = 0.99, CFI = 0.99 and ECVI = 0.39. The second-order model indicated that among the four, built environment had the highest standardised factor loading of 0.86 and the largest causal effect towards an age-friendly environment. Community support and health services followed next with a standardised factor loading of 0.80. Next was civic participation and employment with a standardised factor loading of 0.76. Communication and information, with a standardised factor loading of 0.49, had the smallest causal effect towards an age-friendly environment.\n\nAlthough the cut-off point for standardised factor loading was 0.5, communication and information, with a standardised factor loading of 0.49, was not removed from the model. This was because the model achieved satisfactory model fit, indicating no further model re-specification procedure was required. Moreover, a standardised factor loading of 0.49 is very close to 0.5. It is worth noting that in the CFA presented by Paúl et al. (2012)50 and Lee and Scott (2004),51 indicators were not removed when factor loading was less than 0.5.\n\nGiven these results, H1 can be rejected. Instead of eight, only four constructs, namely built environment, community support and health services, civic participation and employment, and communication and information were confirmed as constructs of an age-friendly environment. These four constructs are in line with the characteristics of age-friendly environments described by Feldman and Oberlink (2003)26 and Alley et al. (2007).27 An age-friendly environment included supportive and accessible social services as well as safe neighbourhoods, which was supported by the findings of Alley et al. (2007).27 The results of second-order CFA are presented in Figure 2.\n\nChiSq, chi-square; df, degrees of freedom; GFI, goodness-of-fit index; AGFI, adjusted GFI; RMSEA, root mean square error of approximation; NFI, normed fit index, TLI, Tucker Lewis index; CFI, comparative fit index.\n\nNext, a measurement model was specified by allowing age-friendly environment and social connectedness features to co-vary. The initial model achieved a fairly satisfactory fit as p-value was at 0.05. Model re-specification was carried out by removal of the item “I have no sense of togetherness with my peers” from social connectedness. Removing an item from the social connectedness variable was theoretically justified as the remaining three items still had a similar underlying sense of “connectedness” (Lee & Robbins, 1995, p. 236).38 Hence the final model achieved satisfactory fit with p-value = 0.08, CMIN/df = 1.22, GFI = 0.96, AGFI = 0.94, RMSEA = 0.03, SRMR = 0.05, NFI = 0.98, TLI = 0.99, CFI = 0.99.\n\nThe structural model was specified by linking age-friendly environments to social connectedness. The structural model was valid as all the items had standardised factor loadings of at least 0.5. It met satisfactory goodness of fit for all the indices with an insignificant p-value at 0.08, CMIN/df = 1.22, GFI = 0.96, AGFI = 0.94, RMSEA = 0.03, SRMR = 0.05, NFI = 0.98, TLI = 0.99 and CFI = 0.99. The model showed that age-friendly environments were found to be positively associated with social connectedness, with a standardised coefficient of 0.18.\n\nApproximately 3% of the variation in social connectedness was explained by the variation in age-friendly environment features. Hence, H2 was supported, indicating that age-friendly environments are positively related to social connectedness. This finding is in line with the findings of Menec et al. (2011)16 and Emlet and Moceri (2012).10 The positive relationship indicates that age-friendly environments help to promote and support social connectedness among middle-aged and older adults caring for themselves.16 Middle-aged and older adults caring for themselves who were leading an active and socially connected lifestyle were able to strengthen their emotional health, improve their general well-being, and delay their ageing process.31 The structural model of age-friendly environments and social connectedness is illustrated in Figure 3.\n\nChiSq, chi-square; df, degrees of freedom; GFI, goodness-of-fit index; AGFI, adjusted GFI; RMSEA, root mean square error of approximation; NFI, normed fit index, TLI, Tucker Lewis index; CFI, comparative fit index.\n\n\nDiscussion\n\nIt was worth noting that not all the eight features of WHO’s (2007)12 age-friendly cities were perceived to be significant in the Malaysia context. Built environment was found to be the most important construct of an age-friendly environment. Built environment increased protection and security for middle-aged and older adults caring for themselves when they move around and interact with the environment and community.16 As an environment becomes friendly, they can participate in social activities, exercise in the park, receive adequate healthcare, go shopping, and visit family and friends.10,16 A senior-friendly public transportation system was also important for their mobility.25,28 Having access to community healthcare services and recreational facilities could improve their social and physical well-being, which indirectly becomes an alternative to health promotion.18\n\nCommunity support and health services were found to be strongly associated with age-friendly environments. These findings are consistent with Ruza et al. (2015)30 and Lai et al. (2016).31 Age-friendly community support and health services enable middle-aged and older adults caring for themselves to receive necessary healthcare at the lowest cost to maintain a strong sound body and mind.9,12 Being healthy increases the capacity of middle-aged and older adults caring for themselves to interact with and live in their existing environment.21 Therefore, healthcare and medical services found within local communities are beneficial as these adults do not solely dependent on the services provided by the state or federal government.\n\nCivic participation and employment referred to voluntary options and paid opportunities in workplaces that take into consideration the disabilities of the ageing adults. The results indicated that middle-aged and older adults caring for themselves in Malaysia preferred to contribute and participate in society through paid or voluntary opportunities after their retirement. This result echoed the findings of Feldman and Oberlink (2003)26 who indicated that financial security as well as social and civic engagement were important age-friendly features.\n\nFourth, the communication and information construct was found to have a weak association with age-friendly environments, which was supported by Ruza et al. (2015).30 The result shows that communication and information obtained through the Internet assist with and ease communication among middle-aged and older adults caring for themselves, helping them to stay in touch with people, which in turn makes them feel less isolated. A possible explanation is that middle-aged and older adults caring for themselves are afraid of losing up-to-date information, news and being isolated from society.12\n\nOn the other hand, social participation and respect and social inclusion were not significant constructs of an age-friendly environment. A possible reason may be that poor public transport systems and service quality in Malaysia are disabling rather than enabling and may hinder some social participation. The public transportation system in Malaysia is not well connected or friendly to older people. Poor services and inaccessible public transportation in Malaysia discouraged people from using it.52\n\nThe results have implications for the Malaysian government and its related agencies to develop public policies for the welfare of its ageing population and older adults. The government must improve the living environment of the ageing population and focus on the physical environment. Furthermore, the results found that middle-aged and older adults caring for themselves were keen to participate in the labour force. Therefore, the authorities could encourage the private sectors to hire ageing workers by subsidising their training expenses or their salary. The Inland Revenue Board of Malaysia could develop schemes for income tax rebates or tax reliefs for companies that recruit older employees aged 60 years and above.\n\nThe results imply opportunities for a sustainable economy. An age-friendly workplace reduces the barriers to working opportunities for older employees.12 Business organisations can renovate their workplace or design an ergonomic workplace to accommodate the physical considerations of ageing adults. The findings support the development of an age-friendly business industry within the age-friendly environment. An age-friendly business industry is a solution to economic growth and sustainability as there are opportunities for new businesses,16 such as building senior-friendly housing with safety features, recreation facilities, and healthcare centres in the community. Another age-friendly business opportunity is to help middle-agers plan financially for their retirement, such as offering long-term care insurance and private retirement schemes.53\n\n\nConclusions\n\nThe overall findings indicate a simpler age-friendly environment in the Malaysian context, which evidenced only four age-friendly features instead of the eight recommended by WHO (2007).12 Built environment was found to be the most important construct, implying that governments of developing countries should give priority to developing features of the built environment, such as a supportive external environment and an accessible public transportation system. In Malaysia, many of the built environment features and an age-friendly transportation system have yet to be comprehensively formed.29 The poor public transportation system may affect the development of social environment features such as opportunities for civic engagement and social participation.28,30\n\nA non-probability sampling method was employed and most of the respondents were active adults caring for themselves, which may limit the generalisation of the findings. Therefore, attention should be paid when generalising the results to other developing countries or different settings. Future studies can examine the moderative effect of demographic variables such as gender and culture. An age-friendly environment index would enable the authorities to further identify areas of improvement for the benefit of the ageing population.\n\nThe results concluded that an age-friendly environment helps middle-aged and older adults caring for themselves to feel connected to the world and society as well as to feel related to anyone. Age-friendly environments help elderly people to stay active and participate in their community through accessible transportation systems and barrier-free environments.26 Social connectedness can be promoted by eliminating the physical barriers that arise from the built environment.25,29 Feeling connected with society, family, and friends can become a form of emotional resource.29 Age-friendly environments enable elderly people to improve their social connectivity, indirectly promoting independence as well as physical and social well-being.\n\n\nData availability\n\nFigshare: Elderly v2. https://doi.org/10.6084/m9.figshare.14870118.v4.55\n\nThis project contains the following underlying data:\n\n- Elderly pilot study.csv (raw data from pilot study)\n\n- Elderly.csv (raw data from main study)\n\n- Data dictionary.csv\n\nFigshare: Questionnaire (age-friendly). https://doi.org/10.6084/m9.figshare.16530777.v1.54\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 grateful for the financial support received from the Ministry of Higher Education, Malaysia. This work was inspired by Madam Tai Ching Kin’s personal health story. She is the mother of corresponding author, Lai Ming Ming and has granted permission to be named in this manuscript. We would like to thank Madam Tai and all participants in this survey.\n\n\nReferences\n\nDepartment of Statistics, Malaysia: Population projections (Revised) Malaysia 2010-2040. 2016; Putrajaya, Malaysia: Department of Statistics, Malaysia.\n\nMahmud A, Mazalan MF, Abdul Razak AR: Ageing phenomenon: Malaysia towards 2030. In: PopInfo Malaysia . 2017. Reference Source\n\nMohd Hashim N: Age friendly – cities and communities. Presented at the 12th ASEAN and Japan High Level Officials Meeting on Caring Socities, Japan. 2014, October.\n\nQuinn A: Healthy aging in cities. J Urban Health: Bulletin of the New York Academy of Medicine. 2008; 85(2): 151–153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organisation (WHO): Why urban health matters. 2010. 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Publisher Full Text\n\nLai MM, Lein SY, Lau SH, et al.: Modeling age-friendly environment, active aging, and social connectedness in an emerging Asian economy. J Aging Res. 2016; 2016: 2052380. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuza J, Kim JI, Leung I, et al.: Sustainable, age-friendly cities: An evaluation framework and case study application on Palo Alto, California. Sustainable Cities Society. 2015; 14: 390–396. Publisher Full Text\n\nRogers WA, Mitzner TL: Envisioning the future for older adults: Autonomy, health, well-being, and social connectedness with technology support. Futures. 2017; 87(2017): 133–139. Publisher Full Text\n\nJaques E: Death and the mid-life crisis. Int J Psychoanal. 1965; 46(4): 502–514. PubMed Abstract\n\nLevinson DJ: A conception of adult development. Am Psychol. 1986; 41(1): 3–13. 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J Counseling Psychol. 1995; 42(2): 232–241. Publisher Full Text\n\nManeesriwongul W, Dixon JK: Instrument translation process: A methods review. J Adv Nursing. 2004; 48(2): 175–186. PubMed Abstract \| Publisher Full Text\n\nAcquadro C, Conway K, Hareendran A, et al.: Literature review of methods to translate health-related quality of life questionnaires for use in multinational clinical trials. Value Health. 2008; 11(3): 509–521. PubMed Abstract \| Publisher Full Text\n\nSchermelleh-Engel K, Moosbrugger H, Müller H: Evaluating the fit of structural equation models: Tests of significance and descriptive goodness-of-fit measures. Methods Psychological Res Online. 2003; 8(2): 23–74.\n\nUllman JB: Structural equation modeling: Reviewing the basics and moving forward. J Pers Assess. 2006; 87(1): 35–50. PubMed Abstract \| Publisher Full Text\n\nKim HY: Statistical notes for clinical researchers: Assessing normal distribution (2) using skewness and kurtosis. Restor Dent Endod. 2013; 38(1): 52–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHair JF, Black WC, Babin BJ, et al.: Multivariate data analysis. 7th ed. New Jersey, USA: Pearson Prentice Hall; 2010.\n\nAwang ZH: Structural equation modelling using AMOS graphic. Selangor, Malaysia: UiTM Press2012.\n\nSchreiber JB, Nora A, Stage FK, et al.: Reporting structural equation modeling and confirmatory factor analysis results: A review. J Educational Res. 2006; 99(6): 323–338. Publisher Full Text\n\nFornell C, Larcker DF: Evaluating structural equation models with unobservable variables and measurement error. J Marketing Res. 1981; 18(1): 39–50. Publisher Full Text\n\nClark N: Education in Malaysia. World Education News & Reviews. 2014, December 2. Reference Source\n\nHayduk LA, Littvay L: Should researchers use single indicators, best indicators, or multiple indicators in structural equation models? BMC Medical Res Methodol. 2012; 12(1): 159–175. Publisher Full Text\n\nPául C, Ribeiro C, Teixeira L: Active ageing: An empirical approach to the WHO model. Current Gerontology Geriatrics Res. 2012; 2012: 1–10. Publisher Full Text\n\nLee JH, Scott D: Measuring birding specialization: A confirmatory factor analysis. Leisure Sci. 2004; 26(3): 245–260. Publisher Full Text\n\nZakaria Z, Hussin ZH, Batau MFA, et al.: Service quality of Malaysian public transports: A case study in Malaysia. Cross-Cultural Communication. 2010; 6(2): 84–92.\n\nKim JK: Challenges and opportunities in the era of population aging. Samsung Economic Research Institute (SERI) Quarterly. October, 2011; 4(4): 15–23.\n\nLai LSY, Lai M-M, Lau SH: Questionnaire (age friendly). figshare. Dataset. 2021; Publisher Full Text\n\nLai LSY, Lai M-M, Lau SH: Elderly v2. figshare. Dataset. 2021. Publisher Full Text\n\n\nFootnotes\n\n1 Individuals aged 45 to 50 are classified in the stage “entry life structure of middle adulthood” while individuals aged 60 to 65 are classified in the stage “late adult transition” 33 (Levinson, 1986, p. 7)." }	[ { "id": "95040", "date": "04 Oct 2021", "name": "Amy Yeo Chu May", "expertise": [ "Reviewer Expertise Generally on social media related areas but specifically more on social and emotional competences as well as general corporate governance" ], "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 investigates primarily on age-friendly environment and social connectedness in the context of a developing country. The authors have been able to demonstrate a well-designed research methodology which encumbrances the essential areas such as gap identification, research objectives, literature review right up to the end results. Well done for this!\nThe authors may want to consider tweaking the following areas for further improvement:\nLiterature review: Some literature concerning the old-age related studies have been examined in the authors’ country, as cited (Lai et al., 2016). The authors also have carefully examined some studies taken place in other part of the globe which is laudable! However, current literature (2019-2021) on old-age related studies at ‘national level’ seem to be less used in this study. I suggest the authors lay down the relevant literatures (national/international) before leading to the hypothesis I. The breakdown of each construct/variable (eg: outdoor sparse and building, transportation etc.) and the aged-friendly environment should be supported by relevant prior studies with succinct justifications.\nResearch Methods: This section was well articulated in terms of the appropriate research design, data collection, sampling, design of questionnaire and statistical tools that are suited to the current study.\nData analysis and results:\nSeveral statistical tools such as reliability and validity test as well as AVE were performed on the raw sample of 402. However, why was EFA only performed on 82 responses? It would be advisable for the authors to rationalise it for the benefit of potential readers who may not have the statistical knowledge. In other words, more interpretations are needed to make the statistical results more comprehensible.\nConclusion and implications: The authors concluded by highlighting only four age-friendly features are applicable in the context of Malaysia instead of eight recommended by WHO (2007). With this, the government of the country would have to consider putting up more built environment features and age-friendly transportation system. What about the other two features? Are they not as important as built environment and transportation? Basing on the outcome of this study, apart from the government intervention and support in ensuring an age-friendly environment and social connectedness, how about private sector involvement/participation so as to complement and supplement the government’s effort? Hence, to add value to the findings, I suggest the authors include the practical and theoretical implications arising from the results of the study.\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? Partly", "responses": [] }, { "id": "97564", "date": "04 Nov 2021", "name": "Peik Foong Yeap", "expertise": [ "Reviewer Expertise Organizational inclusiveness and behavior", "strategic management", "teaching and learning" ], "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 adapted the WHO 2007 framework of features of age-friendly cities to examine age-friendly environment constructs and their linkages with social connectedness from the perspective of Malaysian middle-aged and older adults caring for themselves. Structural equation modelling was performed with 320 samples. The structural model indicated four features i.e. built environment, community support and health services, civic participation and employment as well as communication and information enable the ageing population to improve their connectedness with society in Malaysia. The result of the study was in line with the present literature in which an age-friendly environment supports and cultivates positive social relationships and connectivity. Various implications for the Malaysian government and its related agencies to develop public policies for the welfare of its ageing population and older adults are discussed.\nThis study is interesting and timely to discuss the age-friendly environment for social connectedness among ageing population in Malaysia. However, is there any reason/s why Malaysia has not adopted the WHO (2007) global age-friendly cities guide as mentioned in the Introduction (para. 7, line 1)?\n\nBronfenbrenner’s ecological theory (1977) discusses the structures of microsystem, mesosystem, exosystem and macrosystem. There is a need to further explain exosystem and macrosystem, and how these four systems are interrelated in the literature review section.\nWhere was Alley et al.'s (2007) study of the elder-friendly communities using qualitative methods being carried out? Similar to Ruza et al. (2015) and Rogers and Mitzner (2017) studies respectively. More clarifications are needed.\nThe study collected data from 402 respondents from middle-aged and older adults caring for themselves in the seven Malaysian states. Among these data, 272 responses were collected at senior activity centers in the selected seven states of Malaysia. Meanwhile, 130 responses were data gathered from local community areas. The authors need to explain further the control mechanism that have been used to avoid duplication of data collection in the \"Data collection\" section.\nIt would be good to add more current literature and findings to enhance the robustness of the study.\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": "101635", "date": "22 Dec 2021", "name": "Mark Ward", "expertise": [ "Reviewer Expertise Ageing" ], "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 detailed and comprehensive piece of work which adds to the literature on age-friendly environments. I do however have some suggestions and queries that the authors might consider addressing. The most important of these relate to sampling and the choice of sub-samples for the factor analysis and SEM analyses.\nWhen describing Bronfenbrenner's ecological theory, it is important to also include the fifth system, the chronosystem, particularly when describing the social environment of older adults. After all, age(time) is a crucial factor to consider. Time is also important when considering things like the built environment as urban areas developed at different times may have more or less age friendly features. This is alluded to in paragraph 5 page 4 \"In the application of ecological frameworks...\".\nAs previous reviews have mentioned, there is a extensive more recent literature that could be referred to. For example, see DOI: 10.1080/13607863.2019.1652246 and work referred to within there.\nThe introduction and literature review are quite repetitive and this is not helped by the listing of previous literature. Rather than arranging these sections by study, why not arrange it by topic and reference whatever number of studies within each section?\nLast paragraph page 5. I am not familiar with the term 'reflective model'. What does it mean? The same paragraph. This is a needlessly verbose way of saying something very simple. That is, you have 8 domains derived from 37 (I think) indicators.\nThe following sentence does not belong in the study design section \"Middle-aged and older adults have witnessed death in their family and of their peers; thus, they may be compelled to improve their quality of life while they still have time\".\n\nI don't understand why the factor analysis included only 82 of the 402 participants? Similarly, why only include 320 in the SEM model? Could the full sample not have been used for both? In any event, please explain how/why these numbers were arrived at, beyond the fact that they are the minimum sample size according to your power calculation.\nI am not convinced that the sampling procedure was actually purposive. The purposive part appears to be the 272 recruited at senior activity centres. Sampling from local community areas strikes me more as convenience sampling. If truly purposive sampling, then what were the inclusion & exclusion criteria? What is simply age?\nMuch is made in some places of the distinction between middle-aged and older-aged but I can not see where this is considered analytically. While the study might not be powered to do so, did the authors consider stratifying the SEM model by these two age groups?\nIs is reasonable to suspect that evaluations of \"the level of importance of age-friendly environment features\" are partly informed by where people actually live. Therefore, I wonder what if results varied at all according to the seven locations from within which participants were recruited. Again, there might not be adequate power to assess this fully but it should at least be possible to adjust the standard errors for clustering in the SEM model.\nWhen referring to the SEM 'research model', do you mean 'measurement model'?\nPage 8: \"Bartlett’s test of sphericity was significant with p = 0.00\". A p value can never be truly zero. Please report this as <0.001.\nFinally, \"Approximately 3% of the variation in social connectedness was explained by the variation in age-friendly environment features. Hence, H2 was supported, indicating that age-friendly environments are positively related to social connectedness\". This is a very small effect size so the effect the age-friendly domains included here have on social connectedness are not over-stated. Do the authors have any thoughts on what other important factors there might be.\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?\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": [] } ]	1	https://f1000research.com/articles/10-955
https://f1000research.com/articles/10-526/v1	01 Jul 21	{ "type": "Case Report", "title": "Case Report: Pseudomonas can take a toll on a patient", "authors": [ "David K. Buchbinder", "Jasjit Singh", "Tuan Dao", "Aaron Sassoon", "Antonio Arrieta", "Jasjit Singh", "Tuan Dao", "Aaron Sassoon", "Antonio Arrieta" ], "abstract": "Pseudomonas aeruginosa (P. aeruginosa) is an aerobic Gram-negative bacterium that is implicated in the development of severe systemic infections among pediatric patients. It is identified in hospitalized chronically ill pediatric patients in association with genitourinary, respiratory tract, and skin or soft tissue infections as well as severe and life-threating infection including sepsis. A variety of immunologic mechanisms play a vital role in the host defense mechanisms against invasive infections with P. aeruginosa. Rarely, specific inborn errors of immune function are implicated in deficiencies that predispose to invasive infections with P. aeruginosa. Innate immune function including germ-line encoded pattern recognition receptors such as toll-like receptors (TLRs) and their downstream signaling is vital in the host defense against P. aeruginosa through the generation of antimicrobial peptides, cytokines/chemokines, and shaping of adaptive immune responses. Herein, we describe a previously healthy two-year-old female with an invasive skin, soft tissue, and central nervous system infection secondary to P. aeruginosa. The invasive nature of this infection prompted a careful evaluation for an inborn error of immunity. Decreased cytokine response to agonists of TLRs was documented. Targeted sequencing of interleukin-1 receptor-associated kinase (IRAK)-4 documented a homozygous deletion of exons 8-13 consistent with IRAK-4 deficiency. This report provides a vital educative message in the existing scientific literature by underscoring the importance of considering inborn errors of immunity in all patients with severe P. aeruginosa infections. Functional assessments of immune function often in combination with sequencing can accurately assign a diagnosis in a timely fashion allowing for definitive treatment and the use of necessary supportive care.", "keywords": [ "innate immunity", "toll like receptors", "interleukin-1 receptor-associated kinase-4", "case report" ], "content": "Introduction\n\nPseudomonas aeruginosa (P. aeruginosa) is an opportunistic aerobic Gram-negative bacterial pathogen associated with a variety of genitourinary (UTI), pulmonary as well as skin and soft tissue infections (SSTI) in hospitalized pediatric patients often in association with significant morbidity.1 Rarely, P. aeruginosa can be associated with severe and life-threatening infections among children without previously recognized associated risk factors.2 In these cases, it is vital to consider the possibility of an underlying inborn error of immunity. Invasive P. aeruginosa infections have been described in the setting of inborn errors of immunity including antibody deficiencies (agammaglobulinemia) Bruton (BTK), combined immunodeficiency disorders (severe combined immunodeficiency, ataxia telangiectasia), defects of phagocytes (chronic granulomatous disease, leukocyte adhesion deficiency), defects in actin-polymerization (Wiskott-Aldrich syndrome, MKL1-deficiency), chronic neutropenia and innate immunity including defects in canonical NFKB-signaling (e.g., NEMO/NFKBIA) as well as those that impair the downstream signaling of toll-like receptors (TLRs), such as defects in interleukin-1 receptor-associated kinase (IRAK)-4 and myeloid differentiation factor 88 (MyD88).2,3 We describe the presence of an invasive soft tissue and central nervous system infection with P. aeruginosa in a previously healthy two-year-old female which prompted evaluation for an inborn error of immunity.\n\n\nCase report\n\nA previously healthy two-year-old Hispanic female was evaluated for left hip and knee pain associated with a fever and a refusal to ambulate. She had been given cephalexin for presumed insect bites on her back and legs. Her past medical history was non-contributory. She was fully immunized. Her family history was negative for consanguinity and inborn errors of immunity. She was afebrile and her physical examination demonstrated a 3 cm circular erythematous lesion on mid left back which was non-indurated. Her left knee was minimally swollen, warm to touch, and non-erythematous. Her hips and knees remained in flexion with demonstrated resistance to knee extension. A nodular 1.5 cm mass in her popliteal fossa was also documented with tenderness upon palpation and she was unable to walk. Her laboratory investigation demonstrated a white blood cell count of 17.2 × 103/uL (normal range: 3.9-13.7), Hgb 10.4 g/dL (normal range:10.2-15.4), platelets 319 × 103 (normal range: 150-450), 64% neutrophils (normal range: 25-72), 26% lymphocytes (normal range: 24-71), 9% monocytes (normal range: 0-14), 0.6% eosinophils (normal range: 2-10), 0.3% basophils (normal range: 0-2), erythrocyte sedimentation rate (ESR) 126 mm/hr (normal range: 0-20), and C-reactive protein (CRP) 68.8 mg/L (normal range: 0-10). A blood culture was negative. Magnetic resonance (MR) imaging of her lower extremity demonstrated myositis and fasciitis involving the soft tissues of the distal left thigh, but no abscess (Figure 1A-C). Likely pathogens included methicillin sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus, and group A Streptococcus.\n\nAxial fat-saturated T1-weighted postcontrast MRI of the bilateral lower extremities (B) demonstrates associated enhancement of the vastus medialis, vastus intermedius, and soft tissues of the popliteal fossa. Findings are compatible with myositis with overlying cellulitis.\n\nEmpiric antibiotic therapy began with vancomycin (60 mg/kg/day intravenously divided every 6 hours for 3 days) and ceftriaxone (75 mg/kg/day intravenously every 24 hours for 3 days) which was then transitioned to cefazolin (100 mg/kg/day intravenously divided every 8 hours for 7 days), clindamycin (30 mg/kg/day intravenously divided every 8 hours for 7 days), amoxicillin-clavulanate (50 mg/kg/day orally divided every 12 hours for 4 days), and then to linezolid (30 mg/kg/day intravenously divided every 8 hours for 6 days) due to a lack of clinical and laboratory improvement. A lesion on her back was biopsied and demonstrated acute suppurative panniculitis and suppurative necrosis (Figure 2). Cultures were obtained with growth of P. aeruginosa and a 3-week course of cefepime (150 mg/kg/day intravenously divided every 8 hours) for SSTI was completed. Clinical and laboratory improvement occurred as demonstrated by an ESR 57 mm/hr (normal range: 0-20) and CRP <5 mg/L (normal range: 0-10). The patient was discharged while awaiting results of a workup for an inborn error of immunity.\n\nSkin punch biopsy demonstrates acute suppurative panniculitis with suppurative necrosis (20×, Hematoxylin and Eosin).\n\nShe then returned approximately 3 weeks later with malaise, an inability to stand upright, irritability, and pain on palpation of her back as well as a refusal to ambulate. On re-admission she was afebrile, and her laboratory investigation demonstrated an ESR 89 mm/hr (normal range: 0-20), and CRP <5 mg/L (normal range: 0-10). MR imaging of her spine demonstrated enhancement of T9-T11 with an epidural abscess (Figure 3A-B). A culture was obtained by computed tomography (CT)-guided needle aspiration and empiric therapy with meropenem (120 mg/kg/day intravenously divided every 8 hours for 2 days) was begun. Growth of P. aeruginosa was documented and a 6-week course of cefepime (150 mg/kg/day intravenously divided every 8 hours) was completed. Clinical improvement occurred; however, her laboratory investigation demonstrated an ESR 80 mm/hr (normal range: 0-20) and CRP <5 mg/L (normal range: 0-10) at the completion of therapy.\n\nAxial (A) and sagittal (B) fat-saturated volumetric interpolated breath-hold examination (VIBE) postcontrast magnetic resonance imagery (MRI) images of the thoracic spine demonstrate multilevel thoracic osteomyelitis and discitis with associated epidural and paravertebral abscesses.\n\nSecondary to the invasive nature of her P. aeruginosa infection, evaluation for an inborn error of immunity was completed. A normal neutrophil oxidative burst was noted. Additional laboratory assessments included a serum IgG 1331 mg/dL (normal range: 407-1009), IgA 85 mg/dL (normal range: 22-220), an elevated IgM 362 mg/dL (normal range: 43-163), and a IgE 3.8 kU/L (normal range: <97). Antibody responses documented a non-protective Haemophilus influenza type b antibody titer (0.30 mcg/L), a tetanus antibody titer which was protective (0.21 IU/mL), and pneumococcal titers which were protective (> 1.3 mcg/L) for nearly all serotypes covered by Prevnar 13. Lymphocyte immunophenotyping demonstrated a CD3+ count of 1340/ uL (normal range: 1484-5327), CD4+ count of 633 / uL (normal range: 733-3181), CD8+ count of 628/ uL (normal range: 370-2555), CD19+ count of 493/ uL (normal range: 370-2306), CD16/56+ count of 109/uL (normal range: 43-526). Decreased cytokine response to TLR agonists were documented (Figure 4). Targeted sequencing of IRAK-4 and MyD88 was performed. A homozygous deletion of exons 8-13 was documented in IRAK-4 consistent with a diagnosis of IRAK-4 deficiency.\n\nDecreased cytokine response to toll-like receptor (TLR) agonists: A) TNF alpha, B) IL-1b, and C) IL-6 was measured following peripheral blood mononuclear cell (PBMC) stimulation with PAM3CSK4 (TLR2 and TLR1), zymosan (TLR2 and TLR6), Poly (I:C) (TLR3), flagellin (TLR 5), CLO97 (TLR7 and TLR8), and LPS (TLR4).\n\nFollowing the diagnosis of IRAK-4 deficiency she was start prophylaxis with intravenous immunoglobulin (0.5 g/kg/dose intravenously every 4 weeks) as well as amoxicillin (250 mg orally each day). From her diagnosis at 2 years of age until 4 years of age she continued to experience infrequent infectious complications including a urinary tract infection (Escherichia coli), left knee swelling in association with a abscess (methicillin-susceptible Staphylococcus aureus), and a single admission for fever, cough, and post-tussive emesis. She is now 6 years of age and doing well without any recent infectious complications. She remains compliant with her prophylaxis therapy with intravenous immunoglobulin therapy and amoxicillin which she has tolerated without complications.\n\n\nDiscussion\n\nDetection of lipopolysaccharide and flagellin by TLRs results in the elaboration of pro-inflammatory cytokines.3 TLRs possess an intracellular domain known as the Toll–IL-1R domain (TIR). Upon activation of TLRs, the recruitment of TIR-containing cytosolic adaptors such as MyD88 occurs. The adaptor MyD88 then recruits cytosolic kinases, including the IRAK complex. The IRAK complex includes two kinases including IRAK-4 and two non-catalytic subunits. This results in the activation of downstream effectors including nuclear factor κB (NF-κB) and mitogen-activated protein kinases which support the synthesis of pro-inflammatory cytokines and chemokines, such as IL-1β, -6, -8, and -12 and tumor necrosis factor alpha.\n\nIRAK-4 deficiency is an autosomal recessive disorder which requires that affected patients have homozygous or compound heterozygous mutations in the IRAK-4.4,5 IRAK-4 deficient patients typically have normal basic immunological evaluations.4,5 Importantly, inflammatory responses are markedly blunted as demonstrated by the severe and life-threatening invasive bacterial infection with P. aeruginosa in our patient accompanied by an absence of CRP elevation. In these patients, CRP concentrations can be strikingly misleading as IRAK-4 deficient patients demonstrate impairment in the ability to increase CRP concentrations and to mount fever responses.\n\nAmong IRAK-4 deficient patients with invasive bacterial infection, S. pneumoniae is the most frequently (~50% of episodes) implicated organism.5 S. aureus and P. aeruginosa are less frequently (~20% of episodes) implicated organisms.5 Patients with IRAK-4 deficiency may also experience a variety of minor non-invasive bacterial infections such as upper respiratory tract infections (otitis, sinusitis, pharyngitis) as well as SSTI (furunculosis, folliculitis cellulitis).5 Once again, the most frequently implicated organisms in these non-invasive bacterial infections among IRAK-4 deficient patients are S. pneumoniae, S. aureus, and P. aeruginosa.\n\nCareful institution of aggressive supportive care measures is necessary for IRAK-4 deficient patients. Vaccines should include conjugated and nonconjugated vaccines for S. pneumoniae and N. meningitidis. Haemophilus influenzae type b conjugated vaccine should also be provided. Lifelong antibiotic prophylaxis with cotrimoxazole in combination with penicillin should be administered. Prophylaxis with intravenous or subcutaneous IgG should also be provided. Empiric parenteral antibiotic therapy against S. pneumoniae, S. aureus, and P. aeruginosa is critical whenever an infection is suspected or if the patient develops a fever. Inflammatory markers such as CRP should be considered unreliable. Importantly, patients may die from invasive bacterial infection despite prophylaxis even in the absence of fever or laboratory evidence of inflammation.\n\nAlthough the occurrence of invasive P. aeruginosa infections in IRAK-4 deficiency is not novel, there is a vital and important educative message that bears repeating. Astute clinical judgment is necessary in the evaluation of patients with a potential inborn error of immunity. Clinical findings such as severe infections with P. aeruginosa should support the consideration of inborn errors of immunity including IRAK-4 deficiency.\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 parent of the patient.", "appendix": "References\n\nKlockgether J, Tümmler B: Recent advances in understanding Pseudomonas aeruginosa as a pathogen. f1000Res. 2017 Jul 28; 6: 1261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsgari S, McLaren PJ, Peake J, et al.: Exome Sequencing Reveals Primary Immunodeficiencies in Children with Community-Acquired Pseudomonas aeruginosa Sepsis. Front Immunol. 2016 Sep 20; 7: 357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasanova JL, Abel L, Quintana-Murci L: Human TLRs and IL-1Rs in Host Defense: Natural Insights from Evolutionary, Epidemiological, and Clinical Genetics. Annu Rev Immunol. 2011; 29: 447–491. PubMed Abstract \| Publisher Full Text\n\nPicard C, Puel A, Bonnet M, et al.: Pyogenic bacterial infections in humans with IRAK-4 deficiency. Science. 2003 Mar 28; 299(5615): 2076–2079. PubMed Abstract \| Publisher Full Text\n\nPicard C, von Bernuth H, Ghandil P, et al.: Clinical features and outcome of patients with IRAK-4 and MyD88 deficiency. Medicine (Baltimore). 2010 Nov; 89(6): 403–25. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88936", "date": "05 Jul 2021", "name": "David Hagin", "expertise": [ "Reviewer Expertise Inborn Errors of Immunity/Primary Immunodeficiency 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\nI had a pleasure reading this case report. The authors nicely describe a rare case of IRAK-4 deficiency in a female patient with invasive pseudomonal infection.\nThe case report highlights several important issues. First, and most important, unusual and/or severe presentation should trigger further evaluation for possible underlying inborn errors of immunity (IEI). In this case, invasive infection and failure to respond to antibiotics raised this concern.\nNext, the case report highlights additional important laboratory findings, such as normal CRP levels in the presence of severe infection. In that regard, this case report shows that ‘normal’ CRP should not always be considered a reassuring sign, but rather the opposite, and in some cases could suggest a failure to mount a required inflammatory response.\nFinally, the case report describes a very elegant stepwise work-up approach, starting with identifying ‘clinical clues’ (such as normal CRP), followed by ordering the correct diagnostic assay, and finally performing targeted sequencing of a single candidate gene (sadly, not that common anymore).\nThere are several minor points I would like to suggest:\nDecreased cytokine response to TLR agonists: Was that performed by a commercial lab? If so, I think I would mention it so readers would know that it can be ordered. Also, which cells were used (fibroblasts/PBMCs)?\n\nWere the authors able to test for LPS-induced CD62L shedding in neutrophils?\n\nI don’t know if the patient has siblings, but perhaps the Discussion should include a recommendation to screen other family members for the same pathogenic variants.\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": [ { "c_id": "7044", "date": "21 Sep 2021", "name": "David K. Buchbinder", "role": "Author Response", "response": "Reviewer #1: Comment #1: Decreased cytokine response to TLR agonists: Was that performed by a commercial lab? If so, I think I would mention it so readers would know that it can be ordered. Also, which cells were used (fibroblasts/PBMCs)? Response #1: Thank you for this comment. The cytokine response to TLR agonists was performed by a commercial lab. We agree that this is important to point out for readers. Moreover, peripheral blood mononuclear cells were utilized for testing. This was also pointed out to help other readers/clinicians that may be facing similar clinical situations. Comment #2: Were the authors able to test for LPS-induced CD62L shedding in neutrophils? Response #2: We did not test for LPS-induced CD62L shedding in neutrophils. As suggested by Reviewer #2, additional discussion regarding the role of the CD62L shedding assay as a potential screening test has been added. Comment #3: I don’t know if the patient has siblings, but perhaps the Discussion should include a recommendation to screen other family members for the same pathogenic variants. Response #3: The patient does not have any siblings, but we agree that this is an important point to make from a clinical perspective. Additional discussion regarding the importance of screening other family members for the same pathogenic variants has been added." } ] }, { "id": "88939", "date": "29 Jul 2021", "name": "Shanmuganathan Chandrakasan", "expertise": [ "Reviewer Expertise Immunobiology of Immune dysregulation disorders", "HSCT and gene therapy for immune defects" ], "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\nVery well-written case report of a child with IRAK4 deficiency. I have no major comments, few minor comments include:\nBriefly discuss the role of CD62L shedding assay as another screening test.1\n\nExplain briefly why there is a need for Ig replacement in a disorder that is mainly myeloid lineage being affected- it might be of interest to the readers\n\nA line on long term prognosis might be helpful- the risk of invasive infection tend to improve with age\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": [ { "c_id": "7045", "date": "21 Sep 2021", "name": "David K. Buchbinder", "role": "Author Response", "response": "Reviewer #2: Comment #1: Briefly discuss the role of CD62L shedding assay as another screening test. Response #1: Thank you for this excellent and important suggestion. Additional discussion regarding the role of the CD62L shedding assay as a potential screening test has been added. The suggested reference has also been added. von Bernuth H, Ku CL, Rodriguez-Gallego C, Zhang S, et al.: A fast procedure for the detection of defects in Toll-like receptor signaling.Pediatrics. 2006; 118 (6): 2498-503. Comment #2: Explain briefly why there is a need for Ig replacement in a disorder that is mainly myeloid lineage being affected- it might be of interest to the readers Response #2: Thank you for this suggestion. Additional discussion focusing on explaining why immunoglobulin prophylaxis has a role in IRAK4 deficiency has been added. Comment #3: A line on long term prognosis might be helpful- the risk of invasive infection tend to improve with age Response #3: We apologize for this omission and agree that it is important to comment on the long-term prognosis of patients with IRAK4 deficiency." } ] } ]	1	https://f1000research.com/articles/10-526
https://f1000research.com/articles/10-953/v1	21 Sep 21	{ "type": "Software Tool Article", "title": "SureTypeSCR: R package for rapid quality control and genotyping of SNP arrays from single cells", "authors": [ "Ivan Vogel", "Lishan Cai", "Lea Jerman-Plesec", "Eva R. Hoffmann", "Ivan Vogel", "Lishan Cai", "Lea Jerman-Plesec" ], "abstract": "Genotyping of single cells using single nucleotide polymorphism arrays is a cost-effective technology that provides good coverage and precision, but requires whole genome amplification (WGA) due to the low amount of genetic material. Since WGA introduces noise, we recently developed SureTypeSC, an algorithm to minimize genotyping errors. Here, we present SureTypeSCR, an R package that integrates a state-of-the-art algorithm (SureTypeSC) for noise reduction in single cell genotyping and unites all common parts of genotyping workflow in a single tool. SureTypeSCR is built on top of the tidyverse ecosystem, which facilitates common operations over the data and allows users to create and experiment with the genotyping pipeline. Furthermore, the workflow of SureTypeSCR can also be used for standard genotyping of bulk DNA for batch processing in a single pipeline. SureTypeSCR is avaliable from: https://github.com/Meiomap/SureTypeSCR", "keywords": [ "single cell genotyping", "SNP array", "quality control", "machine learning", "tidyverse", "R package" ], "content": "Introduction\n\nSingle cell genotyping allows genomic discovery when material is limited such as in preimplantation genetic test of embryos for aneuploidy or monogenic disease. Furthermore, analysis of single cells also facilitates the discovery of heterogeneity of de novo mutations and copy number aberrations across a population.1-3 Whereas genotyping using single-nucleotide polymorphism (SNP) array technology benefits from high precision and good coverage of SNPs and is a cost effective way of reconstructing haplotypes when analyzing bulk DNA from a population of cells, single cell genotyping requires whole-genome amplification (WGA) prior to analysis.\n\nWGA is a necessary step in the workflow due to insufficient amount of DNA in single cells (8 pg) for SNP array analysis, which requires 100 ng or above.4 However, WGA introduces two categories of errors: (1) allele drop out (ADO) and (2) allele drop in (ADI). ADO occurs when WGA fails to amplify one of the alleles such as a heterozygous genotype (AB) is mistakenly genotyped as AA or BB. ADO is common and affects up to 30% of typed SNPs.5 ADI is less frequent than ADO and occurs when an AA or BB genotype is erroneously typed as AB. We previously showed that this occurs when the fluorescence signals of both alleles are suboptimal and an artefact of the normalization procedure.6 Multiple tools have been developed for analyzing the noise due to WGA in the sequencing data, whereas there are few experimental approaches for removing noise in SNP array data. They include increasing the genotyping scores based on the standard algorithms developed for bulk DNA7 or use parental support information to exclude erroneous variants.8 We previously developed a machine learning algorithm (SureTypeSC) that is trained on 28 million SNPs from 104 single cells that improves both recall and precision of the single cell data.6\n\nCurrently, analysis of SNP arrays is a multi-step process. The principle of SNP array genotyping by Illumina is measuring allelic ratio represented by red and green channel intensities for each allele (generically known as A and B). The intensities are stored in IDAT files and are then normalized using six-degree affine transformation and in GTC format.9 Illumina's GenomeStudio software is the standard tool for analyzing and quality checking of the genotypes and is compatible with both IDAT and GTC. However, including GenomeStudio in a pipeline with large sample batches can be impractical as the data loading process needs to be curated manually. Tools other than GenomeStudio designed for automated data conversion from IDAT to GTC include AutoCall (for Windows) and IAAP Genotyping CLI (for multiple platforms), both developed by Illumina. IDAT is a proprietary binary format and to our knowledge there is only one tool supporting its parsing - an R package illuminaio.10 Automated feature extraction from the GTC file can be done by Illumina's library IlluminaBeadArray that stores the features in numpy array,11 a data structure that allows convenient programmatic processing. There are tools that directly convert the GTC format to commonly used variant calling format (VCF) either issued by Illumina or available in the bioinformatics community (gtc2vcf).\n\nSureTypeSC, a Python library developed for precise single cell genotyping, requires optimization of certain parameters as well as manual curation of the GTC files in order to extract the genotype features by a 3rd party software (e.g. Illumina GenomeStudio). As this approach is experimental and requires programming knowledge of Python, we encapsulated the functionality of SureTypeSC together with automated feature extraction from the raw GTC data into an R package called SureTypeSCR. SureTypeSCR follows modern data science principles by using packages from tidyverse12 and allows rapid evaluation, visualization and presentation of SNP array data from single cells.\n\n\nMethods\n\nThe minimal set of input data for loading the Illumina SNP array data consists of a manifest file, cluster file, sample sheet and a set of GTC files, where each GTC file corresponds to one sample analyzed on the SNP array (Table 1). Both manifest file and cluster file are issued by Illumina per SNP array type. While manifest file describes SNP markers used on the array, cluster file contains information about genotype clusters per SNP marker gathered from population studies and used for scoring in the GenomeStudio software.13\n\nThe core of the package is implemented in a Python library and SureTypeSCR communicates with this library using reticulate. SureTypeSCR further uses Illumina's Python library IlluminaBeadArray to load the GTC files and then utilizes functions from the tidyverse ecosystem (packages dplyr and magrittr) to implement functions for assessing data quality. The data classification process then assigns a quality score to each analyzed single cell genotype (Figure 1).\n\nSureTypeSCR utilizes Illumina's IlluminaBeadArray library to load the metadata (Table 1) and raw genotype files. In case the data is in IDAT format, SureTypeSCR utilizes Illumina’s IAAP CLI software to convert it into GTC format. SureTypeSCR then implements various functions to check the quality of the data, perform intensity transformation, run dimension reduction algorithm and visualize the results. Subsequently, classification is performed using machine learning algorithm previously trained on large batch of single cell data with known ground truth.6 The algorithm is currently embodied in RF-GDA, which is part of the SureTypeSC Python library. An optional step is context dependent validation that can be implemented within SureTypeSCR in case parental or ploidy information are available.\n\nR (>4.0) and Python (>3.6) are required for installing and running the SureTypeSCR package. The software is installed using devtools. To ensure maximal reproducibility across different platforms, a virtual Python environment is created and all necessary Python dependencies are installed in this environment using reticulate. Subsequently, SureTypeSCR is built, installed and linked to the Python virtual environment The package was tested on three major platforms (Linux/Win/Mac). Data processing times depend on the number of samples in the batch and is estimated at 20s per sample on a single CPU with 4 GB RAM.\n\n\nUse cases\n\nTo demonstrate functionality of SureTypeSCR, we selected 23 single sperm samples from two families to explore the data and perform genotype classification (GEO database; accession GSE19247). The samples were amplified with multiple displacement amplification and processed on the Illumina Human CytoSNP array.8\n\nWe start the analysis with initializing the package, data and metadata (see Table 1 and code below). The R data package containing the sperm data can be downloaded from GitHub using devtools. Function data(.) then initializes data frame metadata, which stores the family information and other metadata that can be used in the analysis and samplesheet containing path to the downloaded samplesheet with the data. Manifest and cluster file are part of the SuretypeSCR installation. Function scbasic(.) loads the data into an R data frame. We then filter out SNPs, termed intensity only SNPs, that are used to detect copy number variant but do not provide genotyping information (filter(.) and str_detect(.)).\n\n\n\nCalculating call rates per individual and genotype reveals a high degree of heterozygosity (Figure 2A, AB rates), suggestive of significant ADI, since sperm are haploid cells and there were no aneuploidies reported in these samples:8\n\n(A) Call rates per individual and genotype calculated as proportions of called genotypes and all genotypes and no calls. No calls represent SNPs with itentisites below a baseline defined in the paper describing the original data.8 (B) PCA analysis accross all SNPs and genotypes from 23 samples that called in every sample. (C) MA plot on normalized intensities across 23 samples, the X axis corresponds to logarithmic average (a) and the Y axis is logarithmic difference (m). (D) MA plot across 23 samples after filtration with SureTypeSC. (E) Effect of used threshold on average heterozygosity (solid line) and average call rate (dashed line) across 23 sperm samples for both, SureTypeSC (grey line) and Illumina GenCall (yellow line). The ribbons represent the standard error of mean.\n\nThe principal component analysis (PCA) is performed using function plot_pca(.) that returns a ggplot object:\n\nAs shown in the code example above, users can customize which features (columns of the data frame) to use for the PCA analysis with the features parameter. There is an option to customize and add metadata to the ggplot object (currently, family information is supported) and a choice whether the PCA should be run per chromosome (by_chrom parameter) or on the whole data frame. While the per chromosome analysis can reveal aneuploid chromosomes, the latter is useful for validating kinship of the samples. This is demonstrated in Figure 2B, where the 23 sperm samples are separated into two clusters corresponding to two families defined in the metadata.\n\nTransformation of the intensities into a logarithmic scale minimizes the variability between the SNPs and samples and allows the patterns of the genotyping clusters to be detected.6 In order to evaluate the single cell genotypes using our classification algorithm, we calculate the logarithmic difference and logarithmic average of the intensities (m and a, respectively, Figure 2C). The following code performs the data transformation by adding four additional columns to the original data frame, two for the raw intensities and two for the normalized intensities for the X and Y channels. The user can then control the plotting by adjusting the fraction of points to be visualized, whether a smoothing spline should be applied to the transformed data and whether to use normalized intensities for plotting (parameters n, smooth and normalized in plot_ma(.)).\n\n\n\nNote that, by default, plot_ma(.) visualizes the plots per sample and we use stat_bin_2d(.) in Figure 2C to illustrate the point density and error distribution across the whole dataset. The MA plot in Figure 2C reveals an erroneous heterozygous cluster where m is close to zero and a is low that we previously showed is due to ADI.6 We subsequently perform sample genotype classification with SureTypeSC using:\n\nThe first layer of the classification algorithm (Random Forest) is loaded from the file. Then, the classification model is created per individual sample (group_by(.) and nest(.)) using Gaussian Discriminant Analysis to infer model parameters.6 The Gaussian Discriminant Analysis is conducted per individual sample rather than the combined dataset in order to avoid bias in the scoring function due to potential outliers in the data. The first two parameters of suretype_model(.) are formal and the last parameter defines the classifier (clf) to be used in the first layer (see the reference manual for a detailed description of all available parameters). After unnesting the df_model, the dataframe contains an additional column that contains the SureTypeSC classification score (rfgda_score). We can then apply a threshold (set_threshold(.)) and use MA plot again to observe how SureTypeSC has affected the quality of the data:\n\n\n\nFigure 2D shows the results from the entire dataset (using stat_bin_2d(.)). Unlike Figure 2C, which contains the data prior to SureTypeSC, the heterozygous cluster (m close to 0 and low a) caused by ADI is effectively removed and the data are concentrated along m = 4 and m = −4 representing homozygous AA and homozygous BB genotypes, respectively.\n\nFinally, we determined the call rate and % of heterozygous SNPs in the data as a function of the used threshold in both SureTypeSC and Illumina's GenCall (rfgda_score and score columns in the data frame, respectively):\n\n\n\nFigure 2E confirms that SureTypeSC is more specific towards noise whilst retaining higher call rates as the threshold increases compared to GenCall. This is consistent with our validation study published previously.6\n\n\nConclusions\n\nAlthough data from single cell genotyping using SNP arrays have been subjected to meta-analysis in the last decade to reconstruct haplotype maps,14,15 automated analysis has remained challenging. SureTypeSCR is an R package that aims to facilitate single cell SNP array analysis by encapsulating typical parts of the workflow into a common interface by following modern data science principles represented by the tidyverse ecosystem. The algorithm used for genotype classification is state-of-the-art in the single cell SNP array domain and is designed as a plug-in system for the SureTypeSCR package.6 We show typical use on real world data (Figure 2) with code snippets that demonstrate the functionality of the package. SureTypeSCR offers a single cell genotyping method with good precision in an easy-to-use R package, thus making it suitable for research and potentially clinical applications.\n\n\nData availability\n\nNCBI GEO: Preclinical Validation of a Microarray Method for Full Molecular Karyotyping of Blastomeres in a 24-hour Protocol, Accession number GSE19247: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE19247.\n\n\nSoftware availability\n\n\n\n• Software and source code available: https://github.com/Meiomap/SureTypeSCR.\n\n• Archived source code at time of publication: https://doi.org/10.5281/zenodo.4963845.16\n\n• License: GNU-GPL-3.", "appendix": "References\n\nMallory XF, Edrisi M, Navin N, et al.: Methods for copy number aberration detection from single-cell DNA-sequencing data. Genome Biology. August 2020; 21(1): 208. 1474-760X. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKermani BG: Artificial intelligence and global normalization methods for genotyping.December 2008. U.S. Patent No. 7, 035, 740. Washington, DC: U.S. Patent and Trademark Office. Reference Source\n\nSmith ML, Baggerly KA, Bengtsson H, et al.: illuminaio: An open source IDAT parsing tool for Illumina microarrays. F1000Res. December 2013; 2: 264. 2046-1402. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nVan Der Walt S, Colbert SC, Varoquaux G: The NumPy array: a structure for efficient numerical computation. arXiv:1102.1523 [cs]. February 2011. arXiv: 1102.1523.Publisher Full Text Reference Source\n\nWickham H, Averick M, Bryan J, et al.: Welcome to the Tidyverse. J Open Source Software. November 2019; 4(43): 1686. 2475-9066. Publisher Full Text Reference Source\n\nIllumina Inc.: Infinium Genotyping Data Analysis. 2014. Technical Note: Genotyping. Reference Source\n\nGruhn JR, Zielinska AP, Shukla V, et al.: Chromosome errors in human eggs shape natural fertility over reproductive life span. Science. American Association for the Advancement of Science Section: Report; September 2019; 365(6460): 1466–1469. 0036-8075, 1095-9203. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nOttolini CS, Newnham L, Capalbo A, et al.: Genome-wide recombination and chromosome segregation in human oocytes and embryos reveal selection for maternal recombination rates. Nat Genet. July 2015; 47(7): 727–735. 1061-4036. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVogel I, Cai L: Meiomap/SureTypeSCR: SureTypeSCR_v0.99.0(VersionRpackage_Zenodo). Zenodo. 2021, June 16. Publisher Full Text" }	[ { "id": "94990", "date": "22 Oct 2021", "name": "Joris Robert Vermeesch", "expertise": [ "Reviewer Expertise Genetics", "genomics", "cytogenetics", "embryo", "prenatal", "structural variation" ], "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 presents an R package that can contribute to the quality control and genotyping of SNP arrays generated from amplified single-cell DNA. The package builds on a previous R package, SureTypeSC. The package will be useful for laboratories using SNP arrays on amplified single-cell DNA. Overall, the descriptions are very detailed and clear, ready to be used.\n\nFigure 2(A) of the paper, why were \"NC\" values all the same for every sample?\n\nThe software requires extra data files, such as HumanCytoSNP-12v2_H.bpm and HumanCytoSNP-12v2_H.egt. It would be helpful to leave the links for downloading those files.\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": [] }, { "id": "245583", "date": "12 Mar 2024", "name": "Jason Torres", "expertise": [ "Reviewer Expertise Complex trait genetics", "genetic epidemiology", "molecular epigenomics" ], "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 this article ,the authors describe a newly developed R package - \"SureTypeSCR\" - that implements a previously-developed machine learning-based procedure (\"SureTypeSC\") for performing quality control of single-cell genotyping data. This package aims to streamline the quality control procedure (i.e. data formatting, normalisation and visualisation) within a single programming environment. This approach offers the greatest benefit to the analysis of single-cell genotyping data following whole genome amplification (WGA), as WGA introduces genotyping errors that require careful filtering.\nThe article provides a clear explanation of the rationale for the SureTypeSCR method, and its application to real-world data of 23 sperm samples from Johnson et al. 2010. My comments are relatively minor, but do require considered responses:\n\n1) The authors mention in the introduction that including \"GenomeStudio in a pipeline with large sample batches can be impractical as the data loading process needs to be curated manually.\" They further state that they \"encapsulated the functionality of SureTypeSC together with automated feature extraction from the raw GTC data into an R package\". It is not clear to me if this automated feature extraction addresses the impractical manual curation problem introduced previously. If so, it would be helpful to make the point explicitly.\n\n2) In the \"Metadata\" section, for completeness, it would be helpful to explain the content of the \"sample sheet\" input file, as all other input file types are address. It may also be helpful to provide example text of the fields present in each data type within Table 1.\n\n3) I was not able to successfully install SureTypeSCR within my R environment (4.3.1). During the installation of the python virtual environment, I was notified that the pip install sklearn command failed as sklearn was deprecated and instead required installation of scikit-learn. I then referred to the GitHub page to manually install SureTypeSCR using the command: R CMD INSTALL SureTypeSCR_0.99.0.tar.gz However, when I tried to download the tar.gz file from the \"current release\" page, I encountered \"404 - page not found' error. Therefore, I couldn't install and verify the functionality of SureTypeSCR.\n\n4) Similar to the other reviewer, it wasn't clear to me why all sperm samples had the exact same \"NC\" proportion in Figure 2A. Also, it would be informative to know how many SNPs were used in the analyses required to generate the PCA and MA plots.\n\nAlso, as a minor comment. The last sentence of the abstract states that SureTypeSCR can also be \"used for standard genotyping of bulk DNA for batch processing in a single pipeline\". However, from what I can discern, this was not explicitly demonstrated in this manuscript. It may therefore be helpful to include additional examples with real-world bulk DNA data. This could be perhaps included as an additional section within the GitHub tutorial.\n\nI'd greatly appreciate the authors' responses to these comments.\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? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "245600", "date": "14 Sep 2024", "name": "Kun Lu Lu", "expertise": [ "Reviewer Expertise Plant Genomics" ], "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 developed an R package called SureTypeSCR for SNP arrays analysis and single cell genotyping. Although this is an important and original work for single cell genotyping, there are still some suggestions for the manuscript as it stands.\n1.Besides calculating call rate, the number of SNPs and genotyping precision or accuracy per individual also should be calculated.\n2.In the step “data initialization and QC”, the IDAT or GTC files cannot be found in R codes.\n3.In Figure 2A, the genotype reveals a high degree of heterozygosity (AB rates), however, how many heterozygous SNPs were caused by ADI error? In addition, how to detect ADO error due to ADO is common and affects up to 30% of typed SNPs.\n4.In Figure 2E, the authors used different threshold to determine the call rate and percentage of heterozygous SNPs. What does the threshold mean? What is the relationship between the threshold and heterozygous SNPs?\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": [] } ]	1	https://f1000research.com/articles/10-953
https://f1000research.com/articles/10-446/v1	04 Jun 21	{ "type": "Research Article", "title": "Health professionals’ attitudes toward religiosity and spirituality: a NERSH Data Pool based on 23 surveys from six continents", "authors": [ "Alex Kørup", "Jens Søndergaard", "Nada A Alyousefi", "Giancarlo Lucchetti", "Klaus Baumann", "Eunmi Lee", "Azimatul Karimah", "Parameshwaran Ramakrishnan", "Eckhard Frick", "Arndt Büssing", "Esther Schouten", "Wyatt Butcher", "René Hefti", "Inga Wermuth", "Rocio de Diego-Cordero", "Maria Cecilia Menegatti-Chequini", "Niels Christian Hvidt", "Jens Søndergaard", "Nada A Alyousefi", "Giancarlo Lucchetti", "Klaus Baumann", "Eunmi Lee", "Azimatul Karimah", "Parameshwaran Ramakrishnan", "Eckhard Frick", "Arndt Büssing", "Esther Schouten", "Wyatt Butcher", "René Hefti", "Inga Wermuth", "Rocio de Diego-Cordero", "Maria Cecilia Menegatti-Chequini", "Niels Christian Hvidt" ], "abstract": "Background In order to facilitate better international and cross-cultural comparisons of health professionals (HPs) attitudes towards Religiosity and/or Spirituality (R/S) we updated the NERSH Data Pool.\nMethods We performed both a network search, a citation search and systematic literature searches to find new surveys.\nResults We found six new surveys (N=1,068), and the complete data pool ended up comprising 7,323 observations, including 4,070 females and 3,253 males. Most physicians (83%, N=3,700) believed that R/S had “some” influence on their patients’ health (CI95%) (81.8%–84.2%). Similarly, nurses (94%, N=1,020) shared such a belief (92.5%–95.5%). Across all samples 649 (16%; 14.9%–17.1%) physicians reported to have undergone formal R/S-training, compared with nurses where this was 264 (23%; 20.6%–25.4%).\nConclusions Preliminary analysis indicates that HPs believe R/S to be important for patient health but lack formal R/S-training. Findings are discussed. We find the data pool suitable as a base for future cross-cultural comparisons using individual participant data meta-analysis.", "keywords": [ "Health professionals", "Religion", "Spirituality", "Data pool", "International collaboration" ], "content": "Introduction\n\nIn 2015, our international research collaborative Network of Research in Spirituality and Health (NERSH.org) decided to build a large global data pool of health professionals’ (HPs) religiosity and/or spirituality (R/S) based on two pre-selected questionnaires.1 The establishment and evolvement of the NERSH collaboration including the NERSH questionnaire have been described elsewhere,1 as are two prior releases of the data pool.2,3\n\nThe aim of this article is to 1) report the results of a newly updated network, citation and literature search leading to the 3rd version of the NERSH Data Pool, 2) describe characteristics of the data pool, and descriptive statistics of observations’ demographics, 3) report selected details about the physicians and nurses in the data pool regarding their attitudes towards influence of R/S on patient health, and their degree of training in handling R/S in medicine, and finally 4) to share ideas for future cross-cultural projects.\n\n\nBackground\n\nOne important part of R/S in healthcare concerns the R/S of the health professionals delivering care, and their attitudes toward R/S in clinical practice. Quantitative measurement and cross-cultural comparisons of human values and attitudes are notoriously cumbersome and debated,4 and probably especially so when the topic of interest lies at the nexus of religiosity, spirituality and health.5,6\n\nMigration and population growth continually change the landscapes of cultures and faiths of the world’s countries7 creating new demands of healthcare systems that historically were developed to function within a single belief system (i.e. Christian Samaritanism etc.). This creates a need for a cross-cultural understanding and adds yet another challenging factor to this research field, requiring the highest levels of data quality and data integrity for us to limit information bias and optimize statistical measurements.\n\nComparing research findings have hitherto been difficult because of limited comparability of study designs and study outcome, as described by Garssen et al. in their meta-analysis of R/S and mental health where they had to exclude 100 out of 181 eligible studies based on either methodological issues or incompatible study design or outcome measures. These differences are known throughout this research field and have made data pooling less feasible. Opportunities are missed and statistical analyses of greater precision are left unexplored.\n\nCurrent differences build upon a history where only two decades ago, research within R/S in healthcare was almost solely based on populations of developed countries, and predominantly adhering to Christian worldviews. Although still dominated by North American and European research,6 today this research field has roots in countries from all parts of the globe spanning six continents with all major faiths and spiritual orientations. The expansion of this field into other cultures and worldviews have created a more mosaic and complex picture of R/S in medicine world-wide.\n\nDespite national and cultural differences, we believe a common denominator of the human existence exists through which we experience and use R/S or secularism, and we find it important to take steps toward distilling those common characteristics within medical care that relate to HPs’ R/S. We believe a small step in that direction is to openly and respectfully share our data and exchange our ideas, as is the purpose of the NERSH Data Pool.\n\nIt is our experience that research communities in general have become better at exchanging experiences and sharing research data. The increased interest has been seen in the amount of publications mentioning ‘data sharing’ in their abstract over the last four decades from 46 in 1980 to 5,960 in 2019;8 and this is with good reason, as sharing and pooling research data have been linked to higher research quality,9 and have been recognized to forge fruitful research collaborations.9,10\n\nUnderlining the arguments for sharing and pooling research data, the current Covid-19 pandemic has shown us, on an historic scale, just how interconnected countries, and cultures, have become, bringing forth the imperative of healthcare systems to not only support cross-cultural care and understanding, but also prioritize international cooperation including sharing and pooling of data.\n\n\nMethods\n\nEligible surveys were found using a combination of a network search (the NERSH collaboration), and both a citation search and a systematic literature review.\n\nThe network search utilized the global collaboration of researchers in NERSH. Past, on-going or planned surveys using either the Religion and Spirituality in Medicine, Perspectives of Physicians (RSMPP) questionnaire11 or the NERSH Questionnaire are the topic of frequent correspondence between collaborators, the NERSH Questionnaire being basically RSMPP with several additions including support for the DUREL index. Also, several research groups have joined the collaboration with a priority to share their survey data once collected and published locally. Local restrictions may apply so that survey data are not released to be included into the data pool until certain criteria are met, why included data are not necessarily added in chronological order.\n\nCitation and literature searches were performed by the first author in the period of January to February 2020. For the citation search we looked up citations in Web of Science referencing eight articles on the original RSMPP-survey published by Curlin in the period 2005 to 2008.11-18 All citing articles were reviewed on abstract level, and if the data source was the RSMPP, or the data source was unclear based on the title or abstract, the entire article was screened. In order to ensure we found all surveys based on the RSMPP, and to also find potential surveys based on the more recent NERSH Questionnaire, our search strategy also included a literature search in Google Scholar, Web of Science, Embase, Medline and PsychInfo using the search strings in Table 1. Survey data already included in the data pool were ignored. All searches were limited to English productions, and as the previous version of the data pool was based on the same search strategy in 2016, we only assessed publications from the year 2016 and forth.\n\nResults limited to publication year 2016 and later.\n\nWe only included data on health professionals based on either of the two questionnaires: Religion and Spirituality in Medicine, Perspectives of Physicians (RSMPP)11 or NERSH Questionnaire,1 the latter being a further development of the first. Customized versions were accepted if they were mainly the RSMPP or NERSH Questionnaire. Observations that were missing information on gender or only contained empty answers were excluded. Also, we set a minimum age of 18 years.\n\nThe data pool versions were created consecutively upon earlier releases. Thus, only survey samples not already included in the 2nd version were added to the 3rd version. All importations were based on raw original data samples sent to us by the local researchers. Data were sent to us in various data formats (Stata, RRID:SCR_012763; IBM SPSS, RRID:SCR_002865; or Microsoft Excel, RRID:SCR_016137), and all were converted into Stata datasets (.dta) before import. For an open-access alternative to the statistical software the R Project for Statistical Computing (RRID:SCR_001905) can perform equivalent analyses. In case interpretation of the raw data was not straightforward, or vital information could not be extracted from published articles, the relevant researchers were contacted by e-mail or phone until the issue was resolved.\n\nThe data pool was created to comprise a total of 98 variables, of which 76 stem from the RSMPP, two are part of the DUREL-index not included (in complete form) in the RSMPP, and the remaining 20 variables are calculated variables aiding study categorization and usage of the included scales.\n\nA codebook was created documenting available original and calculated variables and scales.19\n\nAll observations in the data pool have been anonymized. The data pool is physically located in a secure server environment in Odense, Region of Southern Denmark, and containing only anonymized data upholds the latest security requirements of the General Data Protection Regulation (GDPR) of the European Union. The project was approved by the University of Southern Denmark Research & Innovation Organization (reg.nr: 10.312).\n\n\nResults\n\nFor the network search, knowledge about past or on-going research projects within the NERSH group were assessed. We knew from an earlier query that Baumann and Lee were in possession of survey data from 138 German chaplains using the NERSH Questionnaire from 2012 to 2014, and that their data had now been approved to be included into the NERSH Data Pool.20 Also, a Swiss data sample of 104 general practitioners from a survey by Hefti et al. in 2015 was now available.21 The Swiss survey was published in German language and would thus not have been found by the citation or literature searches.\n\nThe eight citation searches performed found a total of 763 hits including many duplicates. References were screened by the first author who identified three eligible surveys that were based on parts of the original RSMPP. A survey by Cordero et al. from Seville, Spain, examined 75 graduate students (nurses, podiatrists and physiotherapists) using the RSMPP and the DUREL index in 2017,22 and the same research group also examined Portuguese nursing students in 2016 using the same questionnaires.23 In addition, Menegatti-Chequini et al. in performed two surveys of psychiatrists in 2013–2014, a local facility sample in São Paulo (N = 84), and a nation-wide sample among members of the Brazilian Psychiatric Association (N=508) using a questionnaire based on the RSMPP.24,25 Details of the citation search hits are found in Table 2.\n\nThe literature search resulted in a total of 4,929 hits. Restricting the search to articles published 2016 or later reduced the count to 1,133 hits. 20 articles were retrieved in full length but did not prove eligible. In summary, the literature search did not find any eligible surveys not already found by the network and citation searches.\n\nIn total, six new surveys were eligible to import into the NERSH Data Pool. The researchers were contacted and invited to submit their original data, and all agreed with written confirmation. The surveys varied from surveys sampled across an entire nation to surveys done at a single facility or hospital. The study with Spanish nursing students (Cordero, 2019) was sampled among graduate students from The Faculty of Nursing, Podiatry and Physiotherapy in Seville.22 By the same research group, the Portuguese nursing students (Cordero, 2018) were 3rd- and 4th-year students from the School of Health of University of Algarve and School of Health of Polytechnic Institute of Santar.23 The data on German hospital chaplains was collected in a nation-wide survey by Lee et al.,20 while the Swiss sample of General practitioners were a random sample from the region of Bale and Aarau by Hefti et al.21 From Menegatti-Chequini et al. we received two samples, the first based on a survey sent to members of the Brazilian Psychiatry Association (ABP) and an additional sample from a single psychiatric department Instituto de Psiquiatria, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (IPqHC-FMUSP). Duplicates from the ABP and IPqHC-FMUSP samples were removed.\n\nAfter applying exclusion criteria, a total of 1,068 unique observations were added to the NERSH Data Pool. The entire 3rd version of the data pool ended up comprising 7,323 observations, including 4,070 females and 3,253 males. Mean age (SD) was 41.4 (12.5). Response rates ranged from 18% (116 responses out of 642 questionnaires sent in New Zealand with no possibility for follow-up on non-responders) to 95% (Brazil) and 99% (Indonesia), the latter two secured due to tight follow-up including personal meetings and encouragements to complete the forms. Crude response rate was 54.7% for all currently included studies.\n\nA total of 4,872 physicians, 1,319 nurses, and 286 midwifes were included. Other HP occupations were included in smaller numbers (Table 2). Medical specialties represented are listed in Table 3 grouped by study. The largest group of specialties are gynecology/obstetrics with 1,788 participants mainly from the German sample from perinatal care professionals, followed by 1,591 working in psychiatry, 953 from Internal Medicine, 842 from general practice, 447 from surgical specialties, 236 within pediatric medicine, and 143 from paraclinical specialties (i.e. laboratory sciences and supportive branches of medicine not directly involved patient care).\n\nStudents not included.\n\nAlmost all health professionals supplied information about their religious affiliation (N = 7,158; 97.7%). If the responder did not want to supply this information the answer was treated as missing. All questionnaires had a “No affiliation” option, and some also included the options “None”, “Atheism” and/or “Agnosticism”. These options were all grouped together based on their common denominator of not being affiliated with a religion. Answers of religious affiliation were categorized in groups of the major faiths: Buddhism, Hinduism, Judaism, Mormonism, Islam and Christianity. Christian denomination (i.e. Orthodoxy, Catholicism or Protestantism) was registered for samples providing this information. Answers that did not fit any of these groups were placed in the “Other” group.\n\nReligious affiliations from predominantly Islamic cultures were almost entirely Muslim (Saudi Arabia 100% and Indonesia 86%), whereas Hinduism was predominant in the Indian sample, N = 195; 71%.\n\nLooking at the entire data pool the largest group was Christian denominations (N = 4,189; 59%), represented in all samples apart from the sample from Saudi Arabia. Second largest was the group of responders that declared themselves not affiliated with a religion, atheist or agnostic (N = 1,529; 21%). Muslims comprised the third largest group in the data pool (N = 504; 7%) (Table 4).\n\nResponders were asked about the potential influence of R/S on patient health in general. Across all samples 3,700 physicians answered this question, of which 1,767 (48%, CI95% 46.4%–49.6%) replied that R/S has “Much” or “Very much” influence on patient health (Figure 1). The number of physicians believing that R/S has at least “Some” influence on patient health was 3,078 (83%, 81.8%–84.2%). In comparison, for 1,020 nurses these proportions were 658 (65%, 62.1%–67.9%) and 955 (94%, 92.5%–95.5%) respectively. Due to the large between-group heterogeneity, statistical significance test of differences was not feasible.\n\nPhysicians, N = 3,700. Nurses, N = 1,020.\n\nAcross all samples 649 (%, CI95%) (16%, 14.9%–17.1%) physicians reported to have undergone any formal training regarding R/S in medicine with the lowest proportion was 4% (Lucchetti, 2016) and the highest 28% (Ramakrishnan, India, 2014). For nurses this amounted to 264 (23%, 20.6%–25.4%) ranging from 18% (Schouten-Wermuth, 2016) to 35% (Hvidt-Frick, 2014) (Figure 2).\n\nPhysicians, N = 3,987. Nurses, N = 1,158.\n\n\nDiscussion\n\nOur discussion here will focus first on the reported statistics and general composition of the data pool, and second, we will discuss the objectives behind building the NERSH Data Pool.\n\nUsing both network, citation and systematic literature searches we found six new survey samples based on the RSMPP or NERSH Questionnaire, which were added to the preexisting second version of the data pool, now released as the NERSH Data Pool 3.0. Here we have presented the demographics of the complete data pool and select variables regarding attitudes toward influence of R/S on patient health, and whether responders had any formal training in R/S in medicine.\n\nThe included samples were collected in very distinct locations, some were collected from national organizations, and others were collected at single facilities. The cultures and religious landscapes of the represented countries differ, and participants' attitudes are likely affected thereof. Some samples include only HPs of a specific profession (i.e. only physicians, nurses or chaplains), where others included a variety of health professions.\n\nThe heterogeneity between studies was expected as part of the design and is the reason why it did not make sense to test between-sample differences of descriptive variables. As expected, when looking at attitudes toward potential influence of R/S on patient health, we see large variations between studies, and based on the 95% confidence intervals nurses more often reported this influence on patient health than the physicians did. This is in line with earlier research on differences in nurses' and physicians' attitudes toward R/S,26,27 although we must underline that the crude descriptive findings reported here are at study-level and not controlled for within-study confounders. This was beyond the scope of this descriptive analysis of the data pool.\n\nAlso, the reported R/S training of the physicians and nurses in the data pool varied largely. Again, we read two important points out of this. 1) Physician and nurses are largely under-educated when it comes to handling R/S in the clinical setting (i.e. 16% and 23% respectively had some education, leaving approximately four out of five physicians and nurses as having no formal education in this matter). We have no information about the degree or length of educations that were reported. 2) Large differences between sample may signify how the focus on R/S education local is influenced by local culture.\n\nWithin the research field “R/S and health” several traditional meta-analyses have been published based on both cross-sectional, mixed or longitudinal studies showing varying results although mainly showing a positive correlation.6 A recent meta-analysis by Garssen et al. based on longitudinal studies28 also reported a positive association between religiosity and mental health, albeit religiosity was only able to explain 0.6% of the variation in mental health. The limited effect found, and the study conclusion, were later questioned, in part, due to the chosen definition of mental health.29 A meta-analysis by Hackney et al. based on 34 cross-sectional studies on the relationship between religiosity and mental health found religiosity to account for only 1% of the variation in mental health (r = 0.1).30 The latter meta-analysis also demonstrated that simply by adjusting the definitions of religiosity and psychological adjustment they were able to get a result that either supported a positive, negative or no relationship at all.30 Both studies were thoroughly executed and are simply brought to attention here in order to exemplify two important challenges our research field must overcome. The first is the need to continually strive for the improvement of our scientific methods and tools. Below we will argue for a wider usage of meta-analyses based on individual participant data (IPDMA) as one way of mitigating some of the limitations of traditional meta-analysis. Second, we fear that continually separate efforts will be fruitless if researchers fail to develop common conceptual definitions of key concepts within R/S research, and instead keep proposing own definitions and instruments whose face validity are often limited to a local population. We cannot claim to offer a solution to this latter problem, but we argue below for the advantages we ourselves experience through our international and cross-cultural collaboration network.\n\nMAs and IPDMAs\n\nTraditional meta-analyses (MAs) are the recommended approach when comparing results of several studies. MAs examining HPs’ attitudes in clinical practice are still scarce, and IPDMAs even more so.\n\nIn this study we have reported how we have built a pool of individual HP survey responses that enables us to perform IPDMA. Already a single recent IPDMA study compared the religiosity of physicians from seven countries, and their self-reported influence of own religiosity on their clinical practice. Religiosity and influence of religious beliefs were most pronounced in India, Indonesia, and a European faith-based hospital, and half (50%) the physicians examined reported to be influenced that their work as a physician was influenced by their own religiosity.31 Using individual participant data, the authors were able to conduct a sensitivity analysis of potential confounders at sample level, and thus demonstrated the potential of this data pool.\n\nKoenig et al. recently expressed detailed concerns about the biases introduced when using meta-analyses within R/S research.29 They describe R/S and mental health research as a social science where meta-analyses should only be used to describe heterogeneity and not as much searching for consistency and generalizability of study findings across populations. Koenig et al. highlights several common critique points of MAs including when studies measure different variables, incomplete or unstandardized results, inability to account for inter-study variation, heterogeneity due to broad inclusion criteria without the possibility to limit the analysis to sub-groups within the samples, and susceptibility to the ecological fallacy. All these critique points are mitigated by the improved IPDMA-design, which we enable when collecting individual participant data.32\n\nMeta-analyses are considered one of the gold standards behind evidence-based health care, and the number of published meta-analyses have increased markedly over the last decades although average study quality has been questioned.33 One of the caveats is that when collections of samples are very heterogeneous, comparisons of variables using a traditional meta-analysis design are likely to lead to biased results if within-sample confounders are not controlled for at the individual-level (i.e. ecological fallacy).34 This fallacy is suspected to bias interpretations of meta-analyses where individual participant data were not available to the researchers, who thus had to rely on simple aggregated effect measures of the included studies (i.e. at study-level). Sometimes effect measures even differ between the included studies, hence they must be converted to a common effect measure by meta-analysis researchers. This introduces yet another step in the meta-analysis and thus a risk for bias/error.\n\nKoenig et al. also problematized the risks introduced when study results are reduced to a single value.29 For the social and health sciences this is however a key concept that has driven scientific research to where it is today. This is not saying that this reductionism does not include biases and caveats that researchers must understand and respect in their interpretation of their results. Especially within psychometrics (i.e. regarding R/S and mental health) measurements may lack the validity and reliability compared to measurements from more objective sciences. Still limited by these known biases, psychometrics have not only made quantifiable scientific comparisons possible within mental health research, but have also generated a wealth of crucial clinical instruments positively affecting lives and disease courses of patients, some examples are scales for measuring severity of mental illnesses during treatment like Hamilton Depression Rating Scale,35 Global Assessment of Functioning,36 IQ tests like WAIS-VI37 and more. Measuring aspects of R/S is notoriously difficult, mostly due the personal and subjective dimensions that are natural parts of R/S experiences. Challenges caused by imprecise measurements, and/or attempts to compare results from studies using different or custom instruments (i.e. comparing apples and oranges) does not implicitly negate those instruments, but rather demonstrate a lack of collaboration amongst researchers of this field.\n\nWe thus believe, that the use of an instrument, or scientific method like meta-analyses, that over time has demonstrated its ability to advance health care and/or enrich the research thereof, should not be discontinued because of its imprecisions before another instrument or scientific method with improved characteristics are suggested to take its place.\n\nAssimilating research designs and measurements\n\nIt has been argued that MAs are so complex that mistakes are inevitable.33 We truly acknowledge the difficulty in conducting MAs, and most likely not a single MA is 100% perfect. Still, this is not an argument against the use of this method, because the argument of embedded error is applicable to practically most research methods including both quantitative and qualitative methods. Planning and deciding on research designs, deciding how to collect and filter the data, how strictly to enforce an interview guide, the handling of missing answers/observations, the weighing of pros and cons of different statistical strategies for analysis, choosing which statistics to report and how to interpret them. These are just some examples where research projects are susceptible to subjective decisions by human researchers that are prone to make mistakes and bad judgements. Rather than giving up on these methods we believe this calls for our continuing focus on our own human biases, and also for the need to systematize research methods using internationally recommended gold standards (like PRISMA) that precisely aims to document and limit these errors.38 Also, instead of limiting research on R/S and health to theological and qualitative methods, we should welcome diverse and mixed research strategies, all adding valuable perspectives on a theoretical common consensus among researchers. This way we will continually equip ourselves with the latest and best instruments, with which we will attempt to prove or disprove the current hypotheses about how R/S is related to patient health and health care in general. Only this way can we make reason for decision makers to level the importance of R/S in health care with other central health topics crucial for patient health and well-being.\n\nLike any other scientific research design, IPDMA has weaknesses, the largest being heterogeneity which are to a large degree caused by differences in sampling (location, culture, profession and sampling method), and differences in subjective judgements in the local samples.\n\nStill, pooling survey data in the NERSH Data Pool will enable us to perform meta-analyses using the individual data of survey participants. In summery IPDMA carries both statistical and clinical advantages over regular traditional MA.32,39 Some advantages include: 1) the ability to utilize data from yet unpublished studies and or outcome measures thus reducing publication bias; 2) standardized statistical analysis across studies; 3) ability to perform analysis on sub-groups participants (i.e. certain religious affiliations, occupations or medical specialties); 4) ensure consistent inclusion and exclusion criteria; 5) standardized handling of missing values across samples; and 6) overcoming the ecological fallacy of traditional meta-analysis by enabling analysis at the individual level.34\n\n\nConclusions\n\nThe above results and discussion highlight several important prerequisites for this research field, that we argue in favor of the following. 1) The psychometric constructs within R/S should be measured only with validated and broadly accessible instruments. 2) Ideally, researchers should stick to the same validated instruments. Even an average measure with known limitations, and used by everyone, is worth much more to this research field than a more precise measure used only by the few. 3) International and cross-cultural collaborations should be developed in order to bring researchers together. Our own experience from the NERSH-collaboration networking across national borders, and not least cultures, states and promotes a whole-hearted respect of each other’s worldviews, while cultivating an assimilated professional work ethic that demands the highest scientific standards. 4) We highly recommend sharing research data, in order to utilize statistical analysis of greater power at the individual level (i.e. enabling IDPMA). We see the advantages of this within the niche of HPs R/S through the NERSH Data Pool, but data pooling like this could lift the entire research field of “R/S and health” and “Spiritual care” into a new era of scientific research.\n\nThe NERSH Data Pool of health professionals’ attitudes towards R/S in medicine is our attempt to help lift the quality of meta-analyses within this field. We have no knowledge about a similar data pool, and we look forward to test and retest hypotheses about R/S in medicine using its qualities.\n\n\nLimitations\n\nLarge between-samples heterogeneous was expected due to differences in sampling and culture of the background populations. Due to the reduced external validity, any attempt to compare local survey results must be done with caution and should control for within-sample confounders.\n\nWe have not been able to control for cohort effects because none of the samples have performed a follow-up survey.\n\nThe 2012 survey of Brazilian physicians40 was based on interviews rather than self-administered questionnaires, which may have led respondents to give less extreme answers in fear of stigmatization. Contrary, it may be argued that face-to-face interviews limit acquiescence bias where responders tire out in written questionnaires and give the same answer to multiple subsequent questions.\n\n\nPerspectives\n\nResearching and developing spiritual care at a national level is as important as ever, but it is not until we undertake the challenge of understanding international and cross-cultural differences that we can hope to truly develop our own culturally framed healthcare system. Also, we support that both researchers and health care stakeholders take candid and openhearted interest in healthcare systems from other cultures in order to allow a united and global growth in healthcare.\n\nIn the near future, we will use the described data pool to test and retest hypotheses about R/S in medicine using IPDMA designs. Some planned analyses are: 1) the association of HPs’ attitudes and self-reported behavior regarding R/S in the clinical encounter; 2) R/S characteristics and attitudes of physicians from different medical specialties; and 3) Attitudes of HPs considering controversial ethical situations in healthcare.\n\nWe believe this work will better our understanding of how HPs value work in clinical practice, and aiding the development of R/S curricula that will help HPs learn how to incorporate spiritual care into their treatment of patients from any culture, and despite potentially differing world-views.\n\n\nInvitation to collaborate\n\nPlease contact last author (NCH) if you are interested in joining the NERSH collaboration.\n\n\nData availability\n\nOpen Science Framework: Extended data for ‘Health professionals’ attitudes toward religiosity and spirituality: a NERSH Data Pool based on 23 surveys from six continents’. http://doi.org/10.17605/OSF.IO/J79PT.19\n\nThis project contains the following extended data:\n\n• NERSH Data Pool 3.0 Codebook. Excel file describing the structure, variables and scales included in the NERSH Data Pool 3.0. The file also includes an overview of the 23 samples included.\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\nHvidt NC, Kørup AK, Curlin FA, et al.: The NERSH International Collaboration on Values, Spirituality and Religion in Medicine: Development of Questionnaire, Description of Data Pool, and Overview of Pool Publications. Religions. 2016; 7(8): 107. Publisher Full Text\n\nKørup AK, Nielsen CT, Søndergaard J, et al.: The International NERSH Data Pool—A Methodological Description of a Data Pool of Religious and Spiritual Values of Health Professionals from Six Continents. Religions. 2017; 8(2): 24. Publisher Full Text\n\nKørup AK, Søndergaard J, Alyousefi NA, et al.: The International NERSH Data Pool of Health Professionals’ Attitudes Toward Religiosity and Spirituality in 12 Countries. J Relig Health. 2020. PubMed Abstract \| Publisher Full Text\n\nHall DE, Koenig HG, Meador KG: Conceptualizing “Religion”: How Language Shapes and Constrains Knowledge in the Study of Religion and Health. Perspect Biol Med. 2004; 47(3): 386–401. PubMed Abstract \| Publisher Full Text\n\nla Cour P, Hvidt NC: Research on meaning-making and health in secular society: secular, spiritual and religious existential orientations. Brazilian J Soc Sci Med. (1982) 2010; 71(7): 1292–9. PubMed Abstract \| Publisher Full Text\n\nKoenig H, Koenig HG, King D, et al.: Handbook of religion and health. Oup Usa; 2012.\n\nPew Research Center: The Changing Global Religious Landscape.2017.\n\nHulsen T: Sharing Is Caring—Data Sharing Initiatives in Healthcare. Int J Environ Res Public Health. 2020; 17(9): 3046. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInstitute of Medicine (IOM): Sharing clinical research data: Workshop summary.2013.\n\nPopkin G: Data sharing and how it can benefit your scientific career. Nature. 2019; 569(7756): 445–7. PubMed Abstract \| Publisher Full Text\n\nCurlin FA, Lantos JD, Roach CJ, et al.: Religious characteristics of U.S. physicians: a national survey. J Gen Intern Med. 2005; 20(7): 629–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Chin MH, Sellergren SA, et al.: The Association of Physicians' Religious Characteristics with their Attitudes and Self-reported Behaviors regarding Religion and Spirituality in the Clinical Encounter. Med Care. 2006; 44(5): 446–53. PubMed Abstract \| Publisher Full Text\n\nCurlin FA, Dugdale LS, Lantos JD, et al.: Do religious physicians disproportionately care for the underserved? Ann Fam Med. 2007; 5(4): 353–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Lawrence RE, Chin MH, et al.: Religion, Conscience, and Controversial Clinical Practices. N Engl J Med. 2007; 356(6): 593–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Lawrence RE, Odell S, et al.: Religion, spirituality, and medicine: psychiatrists' and other physicians' differing observations, interpretations, and clinical approaches. Am J Psychiatry. 2007; 164(12): 1825–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Odell SV, Lawrence RE, et al.: The relationship between psychiatry and religion among U.S. physicians. Psychiatr Serv. 2007; 58(9): 1193–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Sellergren SA, Lantos JD, et al.: Physicians' Observations and Interpretations of the Influence of Religion and Spirituality on Health. Arch Intern Med. 2007; 167(7): 649–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurlin FA, Nwodim C, Vance JL, et al.: To die, to sleep: US physicians' religious and other objections to physician-assisted suicide, terminal sedation, and withdrawal of life support. Am J Hosp Palliat Care. 2008; 25(2): 112–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKørup AK: NERSH Data Pool 3.0 Codebook. Open Science Framework (osr.io). 2021. Publisher Full Text\n\nLee E, Zahn A, Baumann K: How do Psychiatric Staffs Approach Religiosity/Spirituality in Clinical Practice? Differing Perceptions among Psychiatric Staff Members and Clinical Chaplains. Religions. 2015; 6(3): 930–47. Publisher Full Text\n\nHefti R, Wartenweiler T, Merz O: Der Einfluss von Religiosität und Spiritualität auf die Haltung von Schweizer Ärzten gegenüber ethisch umstrittenen medizinischen Fragen. Praxis. 2018; 107(7): 373–8. Publisher Full Text\n\nde Diego Cordero R, Lucchetti G, Fernández-Vazquez A, et al.: Opinions, Knowledge and Attitudes Concerning “Spirituality, Religiosity and Health” Among Health Graduates in a Spanish University. J Relig Health. 2019. PubMed Abstract \| Publisher Full Text\n\nCordero RDD, Romero BB, De Matos FA, et al.: Opinions and attitudes on the relationship between spirituality, religiosity and health: A comparison between nursing students from Brazil and Portugal. J Clin Nurs. 2018; 27(13-14): 2804–13. PubMed Abstract \| Publisher Full Text\n\nMenegatti-Chequini MC, Gonçalves JPB, Leão FC, et al.: A preliminary survey on the religious profile of Brazilian psychiatrists and their approach to patients' religiosity in clinical practice. BJPsych Open. 2016; 2(6): 346–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMenegatti-Chequini MC, Maraldi EO, Peres MF, et al.: How psychiatrists think about religious and spiritual beliefs in clinical practice: findings from a university hospital in Sao Paulo, Brazil. Braz J Psychiatry. 2019; 41(1): 58–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalboni MJ, Sullivan A, Enzinger AC, et al.: Nurse and Physician Barriers to Spiritual Care Provision at the End of Life. J Pain Symptom Manage. 2014; 48(3): 400–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKristeller JL, Zumbrun CS, Schilling RF: 'I would if I could': how oncologists and oncology nurses address spiritual distress in cancer patients. Psychooncology. 1999; 8(5): 451–8. PubMed Abstract \| Publisher Full Text\n\nGarssen B, Visser A, Pool G: Does Spirituality or Religion Positively Affect Mental Health? Meta-analysis of Longitudinal Studies. Int J Psychol Relig. 2020: 1–17. Publisher Full Text\n\nKoenig HG, Hill TD, Pirutinsky S, et al.: Commentary on “Does Spirituality or Religion Positively Affect Mental Health?”. Int J Psychol Relig. 2020: 1–18. Publisher Full Text\n\nHackney CH, Sanders GS: Religiosity and Mental Health: A Meta-Analysis of Recent Studies. J Sci Study Religion. 2003; 42(1): 43–55. Publisher Full Text\n\nKorup AK, Sondergaard J, Lucchetti G, et al.: Religious values of physicians affect their clinical practice: A meta-analysis of individual participant data from 7 countries. Medicine (Baltimore). 2019; 98(38): e17265. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiley RD, Lambert PC, Abo-Zaid G: Meta-analysis of individual participant data: rationale, conduct, and reporting. BMJ. 2010; 340. PubMed Abstract \| Publisher Full Text\n\nBailar JC: The Promise and Problems of Meta-Analysis. N Engl J Med. 1997; 337(8): 559–61. PubMed Abstract \| Publisher Full Text\n\nReade MC, Delaney A, Bailey MJ, et al.: Bench-to-bedside review: Avoiding pitfalls in critical care meta-analysis – funnel plots, risk estimates, types of heterogeneity, baseline risk and the ecologic fallacy. Crit Care. 2008; 12(4): 220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams JBW: A Structured Interview Guide for the Hamilton Depression Rating Scale. Arch Gen Psychiatry. 1988; 45(8): 742. PubMed Abstract \| Publisher Full Text\n\nHall RCW: Global Assessment of Functioning. Psychosomatics. 1995; 36(3): 267–75. Publisher Full Text\n\nDrozdick LW, Raiford SE, Wahlstrom D, et al.: The Wechsler Adult Intelligence Scale—Fourth Edition and the Wechsler Memory Scale—Fourth Edition. Contemporary intellectual assessment: Theories, tests, and issues. 4th ed.New York, NY, US: The Guilford Press; 2018; 486–511.\n\nMoher D, Liberati A, Tetzlaff J, et al.: 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\nDebray TPA, Moons KGM, van Valkenhoef G, et al.: Get real in individual participant data (IPD) meta-analysis: a review of the methodology. Res Synth Methods. 2015; 6(4): 293–309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLucchetti G, Ramakrishnan P, Karimah A, et al.: Spirituality, Religiosity, and Health: a Comparison of Physicians' Attitudes in Brazil, India, and Indonesia. Int J Behav Med. 2016; 23(1): 63–70. PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 In this paper we will use R/S as a broad concept embracing both religiosity and spirituality. Religiosity being defined as the beliefs, practices and experiences pertaining to an organized religion or belief system; where spirituality is defined by a spectrum from evolved humanistic values through striving for an inner truth to contact with the transcendent. When we use them in unison here it is because this makes the most sensitive measure, and because we lack more specific definitions that are globally accepted. This is also the notation used in the original RSMPP questionnaire and the updated NERSH Questionnaire." }	[ { "id": "89010", "date": "02 Aug 2021", "name": "Anja Visser", "expertise": [ "Reviewer Expertise spiritual care", "spirituality", "coping", "oncology" ], "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 invitation to review your paper, discussing the expansion of the NERSH database, its (potential) function and the attitudes toward and training in spiritual care of physicians and nurses in the database.\nTo me, the most valuable point of this paper lies in the discussion of the potential of meta-analyses based in individual participant data (IPDMA). Discussion about the use of meta-analysis in psychology in general and in the psychology of religion in particular is becoming stronger and I think the authors offer an important avenue for mitigation of these issues with the suggestion to make more use of IPDMA and to build a database like the NERSH database to enable these types of studies. I would, therefore, recommend that the authors place more emphasis on this issue in their paper. For example, by moving this section of the discussion to the introduction section. This would also resolve the lack of clarity about the purpose of the paper that the introduction now suffers from.\n\nAt the same time, there is a weakness in the argumentation for the NERSH database as a 'good example' for IPDMA research, which is shown very clearly in the demonstration of the use of the database to examine the attitudes toward and training in spiritual care of physicians and nurses. As the authors correctly emphasize, IPDMA allows for a reduction of bias introduced in meta-analyses because of the heterogeneity between studies. However, even in the reasonably large dataset of NERSH, no statistical tests could be applied because of between-study heterogeneity (p. 8). A more thorough discussion of this point is needed in the paper, I think, if the authors really want to make a convincing argument for the use of databases such as NERSH: What is needed to reduce this heterogeneity, what might be disadvantages of a homogenizing approach and how much information is necessary to enable sufficient statistical power? The authors briefly discuss some of these issues on p13, but I recommend expanding this.\nSome more minor suggestions I have concerns of a lack of clarity about method.\nOn p4, section 'building the NERSH Data Pool' it is stated that only survey samples that weren't already included were selected. However, I wondered if this meant that any repeated-measures samples would be excluded? Though I understood toward the end of the paper that such samples were non-existent, I suggest changing the word 'samples' to 'datasets', to avoid this misunderstanding.\n\nI also wasn't fully clear on the search strategy. Though I could follow the text, I became confused when looking at the upper half of Table 1; Have you looked for studies that cited those studies by Curlin et al.? If so, why did you choose these studies as for the citation search?\n\nDo I understand it correctly that Table 2 contains all of the datasets currently in the NERSH database and that the ones listed on p7 are the new ones that were added to the third version? If so, please make this more clear in the text.\n\nWhy have you chosen to only examine the data of physicians and nurses?\n\nHave I understood correctly that the exclusion criteria mentioned on p8 are missing information on gender, only containing empty answers and not being 18years of age or older? Perhaps this could be made more clear in the methods section?\nFinally, there are some misspellings and awkward sentences throughout the text that another careful reading should be able to get out.\n\nOverall, I really appreciate the efforts of the authors to advance research methodology in the field of religion, spirituality, and health, and I think this paper helps the field make an important step in that direction..\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? Partly", "responses": [ { "c_id": "7118", "date": "21 Sep 2021", "name": "Alex Kørup", "role": "Author Response", "response": "Response to reviewer Anja Visser Thank you for reviewing our manuscript. Below I attend to all of your comments. The changes will be included in the 2nd version of the article uploaded along this response. This reply and the updated version of the article has been approved by all authors. Comment #1 ”… Discussion about the use of meta-analysis in psychology in general and in the psychology of religion in particular is becoming stronger and I think the authors offer an important avenue for mitigation of these issues with the suggestion to make more use of IPDMA and to build a database like the NERSH database to enable these types of studies. I would, therefore, recommend that the authors place more emphasis on this issue in their paper. For example, by moving this section of the discussion to the introduction section. This would also resolve the lack of clarity about the purpose of the paper that the introduction now suffers from.” Response #1 Thank you for pointing this out. We have moved the suggested section and strengthened the introduction section which are now more in line with the main purpose of the paper: To suggest, and present an example of, how data pooling can enable future IPDMA studies. Comment #2 “… As the authors correctly emphasize, IPDMA allows for a reduction of bias introduced in meta-analyses because of the heterogeneity between studies. However, even in the reasonably large dataset of NERSH, no statistical tests could be applied because of between-study heterogeneity (p. 8). A more thorough discussion of this point is needed in the paper, I think, if the authors really want to make a convincing argument for the use of databases such as NERSH: What is needed to reduce this heterogeneity, what might be disadvantages of a homogenizing approach and how much information is necessary to enable sufficient statistical power? The authors briefly discuss some of these issues on p13, but I recommend expanding this.” Answer #2 Yes, heterogeneity is a significant challenge in the data pool. Doing international and cross-cultural research we expected a significant degree of heterogeneity, and because we collected already existing datasets, we were not able to reduce the heterogeneity already present between the raw samples. Still, we know from another project/article (currently undergoing peer-review elsewhere) that we can reduce the between-study heterogeneity by grouping the physicians by medical specialty. Further stratification of the health professionals in the data pool would likely further reduce the heterogeneity making comparisons more feasible. A disadvantage of this approach would be the risk of diluting a potential effect by creating small groups. The current article is however not a meta-analysis, and we have chosen to report the complete content of the data pool here. Another and more long-term strategy to reduce heterogeneity would be to reduce methodological biases of sampling of future datasets. This could be done for instance by ensuring thorough cross-cultural validation of local questionnaires including qualitative feedback from health professions who completed the questionnaire, ensuring that responders are presented with explaining definitions of central terms and concepts referenced in the questionnaire, and unifying the planning and execution of the local surveys internationally. We have added a short discussion of this to the Limitations section of the article. Regarding an estimate of sample size needed to receive sufficient statistical power, for the current study this would be purely theoretical and of little practical relevance. It could however make sense to calculate such estimate before conducting future hypothesis-testing studies considering the expected variance of the selected measurements of those specific studies. Comment #3 “On p4, section 'building the NERSH Data Pool' it is stated that only survey samples that weren't already included were selected. However, I wondered if this meant that any repeated-measures samples would be excluded? Though I understood toward the end of the paper that such samples were non-existent, I suggest changing the word 'samples' to 'datasets', to avoid this misunderstanding.” Answer #3 No samples were repeated so there were no repeated-measures samples excluded. If we had access to any repeated-measures samples they would be of great value to us and would have been included in the data pool. In the section ‘Building the NERSH Data Pool’ we made two clarifications using ‘dataset’ rather than samples. Comment #4 “I also wasn't fully clear on the search strategy. Though I could follow the text, I became confused when looking at the upper half of Table 1; Have you looked for studies that cited those studies by Curlin et al.? If so, why did you choose these studies as for the citation search?” Answer #4 The eight articles by Curlin used in the citation search were selected by Hvidt and Kørup in 2016 for the first systematic search. It was important for us to use the exact same approach in this study. Articles were chosen because they all rest on the RSMPP and were the first articles published by Curlin in the years 2005 to 2008. Looking for publications using the RSMPP we judged it unlikely that at least one of these articles would not be referenced. Today we have executed this search strategy twice (in 2016 and 2020), both times followed by a rigorous literature search as also described in the article. Both times the literature search was not able to find further samples that we had not already found using the network and citation searches. I have added this elaboration to the article as well to clarify this part of the study design. Comment #5 “Do I understand it correctly that Table 2 contains all of the datasets currently in the NERSH database and that the ones listed on p7 are the new ones that were added to the third version? If so, please make this more clear in the text.” Answer #5 Yes, it was a bit misleading in the text. We have rephrased it to “After inclusion of the found datasets the complete data pool comprised 4,872 physicians, 1,319 nurses, 286 midwifes, and other HP occupations in smaller numbers (See Table 2 for details)”. Comment #6 “Why have you chosen to only examine the data of physicians and nurses?” Answer #6 We present these statistics to give a small view into the characteristics of the health professionals in data pool. Physicians and nurses comprise 85% of the observations in the data pool why they were suited for this purpose without grouping different occupations together. On the other hand, we did not want to perform any actual statistical comparison between the groups due to the heterogeneity between samples (Such analysis would require a study of its own). We decided to report these statistics to give readers a hint of the scope and potential of the data pool in relation to future studies and collaborations. Comment #7 “Have I understood correctly that the exclusion criteria mentioned on p8 are missing information on gender, only containing empty answers and not being 18years of age or older? Perhaps this could be made more clear in the methods section?” Answer #7 Yes, you are correct. This information is already available in the Methods section under the title ‘Inclusion and exclusion criteria’. I have added clarifications in the text. Comment #8 “Finally, there are some misspellings and awkward sentences throughout the text that another careful reading should be able to get out. “ Answer #8 We have attended to several minor misspellings/grammatical issues through-out the manuscript. Comment #9 “Overall, I really appreciate the efforts of the authors to advance research methodology in the field of religion, spirituality, and health, and I think this paper helps the field make an important step in that direction..” Answer #9 Thank you." } ] } ]	1	https://f1000research.com/articles/10-446
https://f1000research.com/articles/10-948/v1	21 Sep 21	{ "type": "Research Article", "title": "Encoding Retina Image to Words using Ensemble of Vision Transformers for Diabetic Retinopathy Grading", "authors": [ "Nouar AlDahoul", "Hezerul Abdul Karim", "Myles Joshua Toledo Tan", "Mhd Adel Momo", "Jamie Ledesma Fermin", "Hezerul Abdul Karim", "Myles Joshua Toledo Tan", "Mhd Adel Momo", "Jamie Ledesma Fermin" ], "abstract": "Diabetes is one of the top ten causes of death among adults worldwide. People with diabetes are prone to suffer from eye disease such as diabetic retinopathy (DR). DR damages the blood vessels in the retina and can result in vision loss. DR grading is an essential step to take to help in the early diagnosis and in the effective treatment thereof, and also to slow down its progression to vision impairment. Existing automatic solutions are mostly based on traditional image processing and machine learning techniques. Hence, there is a big gap when it comes to more generic detection and grading of DR. Various deep learning models such as convolutional neural networks (CNNs) have been previously utilized for this purpose. To enhance DR grading, this paper proposes a novel solution based on an ensemble of state-of-the-art deep learning models called vision transformers. A challenging public DR dataset proposed in a 2015 Kaggle challenge was used for training and evaluation of the proposed method. This dataset includes highly imbalanced data with five levels of severity: No DR, Mild, Moderate, Severe, and Proliferative DR. The experiments conducted showed that the proposed solution outperforms existing methods in terms of precision (47%), recall (45%), F1 score (42%), and Quadratic Weighted Kappa (QWK) (60.2%). Finally, it was able to run with low inference time (1.12 seconds). For this reason, the proposed solution can help examiners grade DR more accurately than manual means.", "keywords": [ "Diabetic Retinopathy Grading", "Ensemble Learning", "Imbalanced Data", "Vision Transformer", "Self-attention Mechanism" ], "content": "Introduction\n\nDiabetes mellitus (DM) is a group of metabolic disorders that are characterized by high levels of blood glucose and are caused by either the deficient secretion of the hormone insulin, its inaction, or both. Chronically high levels of glucose in the blood that come with DM may bring about long-term damage to several different organs, such as the eyes.1,2 DM is a pandemic of great concern3-6 as approximately 463 million adults were living with DM in 2019. This number is expected to rise to about 700 million by the year 2045.4\n\nHigh levels of glucose in the blood damage the capillaries of the retina (diabetic retinopathy [DR]) or the optic nerve (glaucoma), cloud the lens (cataract), or cause fluid to build up in the macula (diabetic macular edema), thereby causing diabetic eye disease.6-11 DR is the leading cause of blindness among adults in the working age12 and has brought about several personal and socioeconomic consequences,13 and a greater risk of developing other complications of DM and of dying.14 According to a meta-analysis that reviewed 35 studies worldwide from 1980 to 2008, 34.6% of all patients with DM globally have DR of some form, while 10.2% of all patients with DM have vision-threatening DR.15\n\nA study found that screening for DR and the early treatment thereof could lower the risk of vision loss by about 56%,16 proving that blindness due to DR is highly preventable. Moreover, the World Health Organization (WHO) Universal Eye Health: A Global Action Plan 2014–2019 advocated for efforts to reduce the prevalence of preventable visual impairments and blindness including those that arise as complications of DM.\n\nMany tests can be used for the screening of DR. While sensitivity and specificity are certainly important, the data about performance of tests for DR are different. Researchers employ different outcomes to measure sensitivity, e.g., the ability of a screening test to detect any form of retinopathy, and the ability to detect vision-threatening DR. Additionally, some tests may detect diabetic macular edema better than the different grades of DR according to World Health Organization. Diabetic retinopathy screening: a short guide. Copenhagen: WHO Regional Office for Europe. The examiner’s skill is also a source of variation in the test results. A systematic review found that the sensitivity of direct ophthalmoscopy (DO) varies greatly when performed by general practitioners (25%–66%) and by ophthalmologists (43%–79%).17\n\nDR grading is an essential step in the early diagnosis and effective treatment of the disease. Manual grading is based on high-resolution retinal images examined by a clinician. However, the process is time-consuming and is prone to misdiagnosis. This paper aims to address the matter by developing a fast and accurate automated DR grading system. Here, a novel solution that is based on an ensemble of vision transformers was proposed to enhance grading. Moreover, a public DR dataset proposed in a 2015 Kaggle challenge was used for the training and evaluation.\n\nTraditional machine learning (ML) methods have been used to detect DR. Typically, these ML methods require hand-tuned features extracted from small datasets to aid in classification. These traditional methods may involve ensemble learning18; the calculation of the mean, standard deviation, and edge strength19; and the segmentation of hard macular exudates.20,21 However, these methods require tedious and time-consuming feature engineering steps that are sensitive to the chosen set of features. Work that employs traditional ML methods to detect DR usually yield favorable results using one dataset but fail to obtain a similar success when another dataset is used.18,19 This is a common limitation of hand-crafted features.\n\nDeep neural networks, such as CNNs, with much larger datasets have also been used for classification tasks in the diagnosis and grading of DR. These methods involve CNNs developed from scratch to grade the disease using images of the retinal fundus22; transfer learning based on Inception-v3 neural network to perform multiple binary classification (moderate versus worse DR, and severe or worse DR)23; segmentation prior to detection by pixel classification24 or patch classification.25 A deep learning (DL)-based framework that uses advanced image processing and a boosting algorithm for grading of DR was also proposed by.26 This is one of only a handful of works that have effectively employed transfer learning to train large neural networks for this purpose. Recently, ResNet, a deep CNN, was proposed to address the problem brought about by imbalanced datasets in DR grading.27 Additionally, a bagging ensemble of three CNNs: a shallow CNN, VGG16, and InceptionV3, was used to classify images as DR, glaucoma, myopia and normal.28\n\nPreviously, a transformer was also proposed by Vaswani et al.29 for natural language processing tasks especially for machine translation. Inspired by the successes of the transformers in NLP, transformers were transferred to computer vision tasks e.g. image classification.\n\n\nMethods\n\nIn this section, the DR detection dataset is explored. Additionally, the vision transformer, a DL model that was used on these data, is discussed in detail.\n\nThe DR detection dataset is highly imbalanced and consists of high-resolution images with five levels of severity including No_DR, Mild, Moderate, Severe, and Proliferative_DR. It has significantly more samples for the negative (No_DR) category than for the four positive categories. Table 1 shows the class distribution of the training and testing sets. Figure 1, on the other hand, shows a few samples from each class. The images come with different conditions and were labeled with subject IDs. The left and right fields were provided for every subject. The images were captured by different cameras, thus affecting the visual appearance of the images of left and right eyes.\n\nThe images have various sizes but were resized uniformly.\n\nThe samples of the training set were rescaled between 01, cropped to remove their black borders, and augmented by randomly flipping the samples horizontally and vertically, and by randomly rotating the samples by 360°. The samples of the test set were only cropped and rescaled. Figure 2 shows a few augmented samples from the training set.\n\nA vision transformer is a state-of-the-art DL model that is used for image classification and was inspired by Dosovitskiy et al.30 Figure 3 shows the architecture of the vision transformer. In this paper, a retinal image that has a sequence of patches encoded as a set of words was applied to the transformer encoder as shown in Figure 3. The original image’s patchesN=H×W/P2were extracted with a fixed patch size PP where P=16, W is the image width, H is the image height, and N is the number of patches. The extracted patches were flattened and each patch xp belonged to ℝP2.C, where C is the number of channels.\n\nAs a result, the 2D image was converted into a sequence of patches x∈ℝN×P2.C. Each patch in the sequence x was mapped to a latent vector with hidden size D=768. A learnable class embedding z00=xclass was prepended for the embedded patches, whose state at the output of the transformer’s encoder zL0 serves as the representation y of the image. After that, a classifier was attached to the image representation y. Additionally, a position embedding Epos was added to the patch embeddings to capture the order of patches that were fed into the transformer encoder. Figure 4 illustrates the architecture of transformer’s encoder with L blocks, each block containing alternating layers of multi-head self-attention (MSA)29 and multi-layer perceptron (MLP) blocks. The layer normalization (LN)31 was applied before every block, while residual connections were applied after every block.30\n\nEnsemble learning is a ML ensemble meta-algorithm. Bagging (Bootstrap Aggregating) is a type of ensemble learning that uses “majority voting” to combine the output of different base models to produce one optimal predictive model and improve the stability and accuracy.32\n\nThe advantage of ensemble bagging several transformers is that aggregation of several transformers, each trained on a subset of the dataset, outperforms a single transformer trained over the entire set. In other words, it leads to less overfit by removing variance in high-variance low-bias datasets. To increase the speed of training, the training can be done in parallel by running each transformer on its own data prior to result aggregation, as shown in Figure 5.\n\nThe images available in this dataset were resized to H=256,W=256, the latent vector hidden size was set to D=768, the number of layers of the transformer to L=12, the MLPsize to 3072, the MSAheads to 12, and the default value of the patch size to P=16. Thus, the sequence’s number N was 256.\n\nIn the experiments conducted, 20% from each class in the training set were selected for validation. All transformers were fine-tuned using the weights of the transformer pre-trained on ImageNet-21K.33\n\nFor optimization, the ADAM algorithm34 was utilized with a batch size of 8. Furthermore, the mean squared error loss function was used. The training process for each transformer consists of two stages:\n\n1) All layers in the transformer backbone were frozen and the regression head that was initialized randomly was unfrozen. Then, the regression head was trained for five epochs.\n\n2) The entire model (transformer backbone + regression head) which was trained for 40 epochs was unfrozen.\n\nData augmentation, early stopping, dropout, and learning rate schedules were used to prevent overfitting and loss divergence. Figure 6 shows the attention map of a few samples extracted from the transformer.\n\nThe classification heads of all transformers were removed and replaced by a regression head with one node instead of logits. The regression output of a transformer was interpreted as shown in Table 2 to be converted into a category.\n\nAn ensemble of ten transformers with similar architectures and hyperparameters was used. The samples were divided randomly into ten sets and each transformer was trained on each one. After interpreting the regression output from each transformer, the predicted classes from ten transformers were aggregated with “majority voting” to predict the final class.\n\nTraining, validation, and testing were carried out using the TensorFlow framework on an NVIDIA Tesla T4 GPU.\n\n\nResults and discussion\n\nIn this section, the results of the proposed ensemble of transformers are discussed. The performance metrics, such as precision, recall, and F1 score were calculated. Additionally, the quadratic weighted kappa (QWK) metric was utilized in this dataset because these data needed specialists to label the images manually since the small differences among the classes can only be recognized by specialist physicians. QWK which lies in the range −1+1 measures the agreement between two ratings and is calculated between the scores assigned by human raters (doctors) and predicted scores (models) as shown in Table 3. The dataset has five ratings: 0, 1, 2, 3, 4.\n\nQWK was calculated as follows:\n\n1) The confusion matrix O between predicted and actual ratings was calculated.\n\n2) A histogram vector was computed for each rating in the predictions and in the actual.\n\n3) The E N×N matrix which represents the outer product between the two histogram vectors was calculated.\n\n4) The W (N×N) weight matrix was constructed representing the difference between the ratings as shown in Table 4.35\n\nWhere 1 ≤ i ≤ 5, 1 ≤ j ≤ 5\n\n5) QWK was defined as follows35:\n\nwhere N is the number of classes.\n\nTable 5 shows the performance metrics of ten transformers with each one trained on a subset of data. It is obvious that there is a big difference among the performances of these individual transformers. Transformer_1 was able to yield a Kappa of 55.1%. On the other hand, transformer_10 yielded a Kappa of 30.9%. Ensembles of various numbers of transformers including all ten transformers, four transformers (1,3,8,9), and other configurations were also evaluated. The best model was an ensemble of two transformers (1,3) which yielded a Kappa of 60.2%.\n\nThis Kappa is at the boundary between moderate and substantial agreement. The previous results confirm that the performance of the ensemble of transformers (1,3) trained with fewer training images outperformed the ensemble of ten transformers trained with five times the number of images. Table 6 compares the performance of the ensemble of transformers with the ensemble of ResNet50 CNNs. The ResNet50 CNN was transferred from ImageNet 1K. The top layers were replaced by a support vector machine that was tuned with this dataset. The proposed ensemble of transformers outperformed the ensemble of ResNet50 CNNs significantly by >18% Kappa.\n\nThe confusion matrix of each configuration including ensembles of transformers with ten, four, and two transformers, and the ensemble of two ResNet50\n\nCNNs were shown in Figure 7. The confusion matrix (c) which represents the best Kappa of 60.2% shows that the model was able to recognize the categories of severe and proliferative DR from one side, and NO DR and mild DR from the second side.\n\n\nConclusion\n\nThis study is a new attempt to demonstrate the capability of the ensemble bagging of vision transformers applied on retinal image classification for the grading of DR into five levels of severity. The experiments conducted showed that even when the dataset was challenging, the proposed method was able to yield promising performance measures in terms of precision (47%), recall (45%), F1 score (42%), and QWK (60.2%). Furthermore, the inference time was low at 1.12 seconds. Hence, we intend to enhance the performance by utilizing a collection of various DR datasets. This can increase the size and variety of training data to train the proposed model from scratch instead of starting from the weights of the ImageNet 21K-based model. By doing so, we can ultimately enhance performance.\n\n\nAuthor contributions\n\nConceptualization by N.A., M.A.M.; Data Curation by N.A.; Formal Analysis by N.A., H.A.K., M.A.M.; Funding Acquisition by H.A.K.; Investigation by N.A., J.L.F; Methodology by N.A., H.A.K., M.A.M.; Project Administration by H.A.K.; Software by N.A., M.A.M.; Validation by N.A., M.J.T.T.; Visualization by N.A.; Writing – Original Draft Preparation by N.A., M.A.M, J.L.F.; Writing – Review & Editing by N.A., H.A.K., M.J.T.T., M.A.M, J.L.F.\n\n\nEthics and consent\n\nAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\n\nThe Retinal images are public third part dataset provided by EyePACS, a free platform for retinopathy screening.\n\n\nCompeting interests\n\nNone of the authors declare any competing interests.\n\n\nGrant information\n\nThis research project was funded by Multimedia University, Malaysia.\n\n\nData availability\n\nThe dataset used in this work is accessible to the public on the Kaggle website. It was created in 2015 for the Kaggle Diabetic Retinopathy Detection competition. This competition is sponsored by the California Healthcare Foundation. Retinal images were provided by EyePACS, a free platform for retinopathy screening.", "appendix": "References\n\nAmerican Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care. Jan. 2010; 33(Suppl 1): S62–S69. PubMed Abstract \| Publisher Full Text\n\nFowler MJ: Microvascular and Macrovascular Complications of Diabetes. Clin Diab. Apr. 2008; 26(2): 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou B, et al.: Worldwide trends in diabetes since 1980: a pooled analysis of 751 population-based studies with 4.4 million participants. Lancet. Apr. 2016; 387(10027): 1513–1530. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Diabetes Federation: IDF Diabetes Atlas. 9th ed. Brussels, Belgium: International Diabetes Federation; 2019, pp. 32–61.\n\nNarayan KMV: The Diabetes Pandemic: Looking for the Silver Lining. Clinical Diabetes. Apr. 2005; 23(2): 51–52. Publisher Full Text\n\nCheloni R, Gandolfi SA, Signorelli C, et al.: Global prevalence of diabetic retinopathy: protocol for a systematic review and meta-analysis. BMJ Open. Mar. 2019; 9(3): e022188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu L, Fernandez-Loaiza P, Sauma J, et al.: Classification of diabetic retinopathy and diabetic macular edema. World J Diabetes. Dec. 2013; 4(6): 290–294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang W, Lo ACY: Diabetic Retinopathy: Pathophysiology and Treatments. Int J Mol Sci. Jun. 2018; 19(6): 1816. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong BJ, Aiello LP, Pasquale LR: Presence and Risk Factors for Glaucoma in Patients with Diabetes. Curr Diab Rep. Dec. 2016; 16(12): 124–124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPollreisz A, Schmidt-Erfurth U: Diabetic cataract-pathogenesis, epidemiology and treatment. J Ophthalmol. 2010; 2010: 608751–608751. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDas A, McGuire PG, Rangasamy S: Diabetic Macular Edema: Pathophysiology and Novel Therapeutic Targets. Ophthalmology. 2015; 122(7): 1375–1394. PubMed Abstract \| Publisher Full Text\n\nCheung N, Mitchell P, Wong TY: Diabetic retinopathy. Lancet. Jul. 2010; 376(9735): 124–136. Publisher Full Text\n\nRees G, et al.: Association Between Diabetes-Related Eye Complications and Symptoms of Anxiety and Depression. JAMA Ophthalmol. Sep. 2016; 134(9): 1007–1014. PubMed Abstract \| Publisher Full Text\n\nKramer CK, Rodrigues TC, Canani LH, et al.: Diabetic Retinopathy Predicts All-Cause Mortality and Cardiovascular Events in Both Type 1 and 2 Diabetes. Diabetes Care. May 2011; 34(5): 1238. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYau JWY, et al.: Global Prevalence and Major Risk Factors of Diabetic Retinopathy. Diabetes Care. Mar. 2012; 35(3): 556–564. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRohan TE, Frost CD, Wald NJ: Prevention of blindness by screening for diabetic retinopathy: a quantitative assessment. BMJ. Nov. 1989; 299(6709): 1198–1201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHutchinson A, et al.: Effectiveness of screening and monitoring tests for diabetic retinopathy--a systematic review. Diabet Med. Jul. 2000; 17(7): 495–506. PubMed Abstract \| Publisher Full Text\n\nBhatia K, Arora S, Tomar R: Diagnosis of diabetic retinopathy using machine learning classification algorithm. 2016 2nd Int Conf Next Generation Computing Technologies (NGCT). 2016, pp. 347–351. Publisher Full Text\n\nAsha PR, Karpagavalli S: Diabetic Retinal Exudates Detection Using Extreme Learning Machine. Emerging ICT for Bridging the Future - Proceedings of the 49th Annual Convention of the Computer Society of India CSI Volume 2, Cham. 2015; pp. 573–578. Publisher Full Text\n\nSopharak A, Uyyanonvara B: Automatic exudates detection from diabetic retinopathy retinal image using fuzzy c-means and morphological methods. Proceedings of the 3rd IASTED International Conference of Advances in Computer Science and Technology. 2007; pp. 359–364. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsareh A, Mirmehdi M, Thomas B, et al.: Automatic recognition of exudative maculopathy using fuzzy C-means clustering and neural networks. Proc Medical Image Understanding Analysis Conference. 2001, vol. 3, pp. 49–52.\n\nPratt H, Coenen F, Broadbent DM, et al.: Convolutional Neural Networks for Diabetic Retinopathy. Procedia Computer Sci. Jan. 2016; 90: 200–205. Publisher Full Text\n\nGulshan V, et al.: Development and Validation of a Deep Learning Algorithm for Detection of Diabetic Retinopathy in Retinal Fundus Photographs. JAMA. Dec. 2016; 316(22): 2402–2410. PubMed Abstract \| Publisher Full Text\n\nPrentašić P, Lončarić S: Detection of exudates in fundus photographs using deep neural networks and anatomical landmark detection fusion. Comput Methods Programs Biomed. Dec. 2016; 137: 281–292. PubMed Abstract \| Publisher Full Text\n\nvan Grinsven MJJP, van Ginneken B, Hoyng CB, et al.: Fast Convolutional Neural Network Training Using Selective Data Sampling: Application to Hemorrhage Detection in Color Fundus Images. IEEE Transactions Medical Imaging. May 2016; 35(5): 1273–1284. Publisher Full Text\n\nWang Y: A Deep Learning Based Pipeline for Image Grading of Diabetic Retinopathy. Master of Science: Virginia Polytechnic Institute and State University. 2018.\n\nSallam MS, Asnawi AL, Olanrewaju RF: Diabetic Retinopathy Grading Using ResNet Convolutional Neural Network. 2020 IEEE Conference on Big Data and Analytics (ICBDA). 2020, pp. 73–78. Publisher Full Text\n\nSmaida M, Yaroshchak S: Bagging of convolutional neural networks for diagnostic of eye diseases. CEUR Workshop Proceedings. 2020; 2604, 715–729.\n\nVaswani A, et al.: Attention Is All You Need. arXiv:1706.03762 [cs]. Dec. 2017. Accessed: May 30, 2021. Reference Source\n\nDosovitskiy A, Lucas B, Alexander K, et al.: An image is worth 16x16 words: Transformers for image recognition at scale. ICLR. 2021.\n\nBa JL, Kiros JR, Hinton GE: Layer Normalization. arXiv:1607.06450 [cs, stat]. Jul. 2016. Accessed: Jun. 30, 2021. Reference Source\n\nFan W, Zhang K: Bagging In: Encyclopedia of Database Systems. LIU L, ÖZSU MT, Eds. Boston, MA: Springer US; 2009, pp. 206–210. Publisher Full Text\n\nRidnik T, Ben-Baruch E, Noy A, et al.: ImageNet-21K Pretraining for the Masses. arXiv:2104.10972 [cs]. Jun. 2021. Accessed: Jun. 30, 2021. Reference Source\n\nKingma DP, Ba J: Adam: A Method for Stochastic Optimization. arXiv:1412.6980 [cs]. Jan. 2017. Accessed: Jun. 30, 2021. Reference Source\n\nTymchenko B, Marchenko P, Spodarets D: Deep Learning Approach to Diabetic Retinopathy Detection. arXiv:2003.02261 [cs, stat]. Mar. 2020. Accessed: Jun. 30, 2021. Reference Source" }	[ { "id": "94946", "date": "25 Nov 2021", "name": "Shruti Jain", "expertise": [ "Reviewer Expertise Image and Signal Processing", "Soft Computing", "Internet-of-Things", "Pattern Recognition", "Bio-inspired Computing and Computer-Aided Design of FPGA and VLSI circuits" ], "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\nAdd a section highlighting the main contributions of your methodology, with detailed reference to, and comparison with, existing work.\n\nI find it difficult to understand, from the abstract, the proposed methodology by which you seek to solve the problem of your paper. For example, the authors could clarify the following: “To enhance DR grading, this paper proposes a novel solution based on an ensemble of state-of-the-art deep learning models called vision transformers”. What are vision transformers? Are authors proposing this or what is new in it? https://viso.ai/deep-learning/vision-transformer-vit/\nLikewise the authors write \"A challenging public DR dataset proposed in a 2015 Kaggle challenge was used for training and evaluation of the proposed method.\" Yet there are many datasets for DR grading, so why only Kaggle? The author can validate their model with other datasets.\n\nAdd some latest papers and cite them.\n\nRead the whole manuscript for typos and grammatical mistakes.\n\nRecheck, not all references that could be given are given. In 'Dataset Overview', in the sentence “the quadratic weighted kappa (QWK) metric was utilized in this dataset because of these data...” the reference is missing. Likewise there are places where authors can give references.\n\nFinally, your conclusion needs to be more tailored to your findings. The authors write \"Hence, we intend to enhance the performance by utilizing a collection of various DR datasets. This can increase the size and variety of training data to train the proposed model from scratch instead of starting from the weights of the ImageNet 21K-based model. By doing so, we can ultimately enhance performance”\nYet, why don’t authors have tried increasing the datasets utilized already? Furthermore, the ImageNet 21K-based model is mentioned significantly in the conclusion without having been elaborated upon in the main text of the article: why was it introduced, what is it, and what is it for and how does it improve performance? Likewise, there is no mention of ensemble transformers or vision transformers in the conclusion.\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/10-948
https://f1000research.com/articles/10-945/v1	21 Sep 21	{ "type": "Research Article", "title": "Dermatoglyphics and abdominal resistance in female children and adolescents: a cross-sectional study", "authors": [ "Renan Souza", "Adriano Alberti", "Gabriel Gastélum Cuadras", "Rodrigo Gomes de Souza Vale", "Eliton Marcio Zanoni", "Eloel Benetti Zavorski", "Josiane Aparecida De Jesus", "Ben Hur Soares", "Fabiane Pertille", "Gracielle Fin", "Bruna Becker da Silva", "Leoberto R. Grigollo", "Aline D. Schlindwein", "Viviane Freiberger", "Leticia Ventura", "Luana Quadros", "Ramón Alfonso González Rivas", "Oscar Núñez Enríquez", "Samuel Alfredo Islas Guerra", "Felipe Valenzuela Jurado", "Arturo Iván Chávez Erives", "Arturo Martínez Treviz", "David Arnoldo García Fernández", "Graziela Marques Leão", "Clarissa Martinelli Comim", "Rudy José Nodari Junior", "Renan Souza", "Gabriel Gastélum Cuadras", "Rodrigo Gomes de Souza Vale", "Eliton Marcio Zanoni", "Eloel Benetti Zavorski", "Josiane Aparecida De Jesus", "Ben Hur Soares", "Fabiane Pertille", "Gracielle Fin", "Bruna Becker da Silva", "Leoberto R. Grigollo", "Aline D. Schlindwein", "Viviane Freiberger", "Leticia Ventura", "Luana Quadros", "Ramón Alfonso González Rivas", "Oscar Núñez Enríquez", "Samuel Alfredo Islas Guerra", "Felipe Valenzuela Jurado", "Arturo Iván Chávez Erives", "Arturo Martínez Treviz", "David Arnoldo García Fernández", "Graziela Marques Leão", "Clarissa Martinelli Comim", "Rudy José Nodari Junior" ], "abstract": "Background: Dermatoglyphics is considered, in the scientific milieu, to be an epigenetic marker. The objective of this study was to analyze the presence of dermatoglyphic marks characteristic of neuromotor capacity and abdominal resistance in children and adolescents. Methods: This is a cross-sectional study. The sample consisted of 1,002 individuals, female children and adolescents between the ages of 10 and 16, from public and private schools in the city of Joaçaba, Santa Catarina, Brazil. The protocol selected for analyzing the fingerprints was dermatoglyphics, proposed by Cummins and Midlo using a Dermatoglyphic Reader. The Brazilian Sports Project Manual - PROESP 2015 was used to collect data on muscle strength motor tests. Results: The results showed the presence of a dermatoglyphic mark characteristic of abdominal motor capacity and muscle strength in females. A higher frequency of arches was identified in MET4 and whorls in MET5 and MDT4 in the Risk Zone group. In the Healthy Zone group, ulnar loop was found to be more frequent in MET4, MET5, and MDT4 fingers. Conclusions: The results demonstrated a predictive marker for abdominal motor capacity and strength in females through dermatoglyphics.", "keywords": [ "Dermatoglyphic", "Mark", "Strength", "Female." ], "content": "Introduction\n\nIn childhood and adolescence, in addition to physiological implications related to biological maturation, the body is influenced by environmental and behavioral factors acting positively or negatively (Guedes, 2011). Childhood comprises the period between two and 10 years of age and adolescence from 10 to 20 years of age (Gallahue et al., 2013).\n\nIn terms of the development and improvement of motor skills, there is an interaction of factors inherited before birth and environmental factors (Barros et al., 2017). The factors inherited before birth are directly linked to the individual's genetics, or to fetal development, which is the period of development over the nine months of gestation, and the parents' lifestyle will be the major influencer in this process (Nodari et al., 2014b). The environment and maternal lifestyle are influential in the development of fingerprints, so dermatoglyphics is considered, in the scientific milieu, to be an epigenetic marker (Garufo et al. (2017).\n\nStudies on dermatoglyphics have investigated factors related to the prognosis and diagnosis of diseases in fields such as anthropology, legal medicine, and health, in which fetal developmental markers are compared and related to different physical fitness variables (Santos et al., 2007). This is another tool that may identify or even help health professionals in guiding patients towards a better quality of life. Therefore, the objective of this study was to analyze the presence of dermatoglyphic marks characteristic of neuromotor strength capacity and abdominal resistance in children and adolescents.\n\n\nMethods\n\nThis was a cross-sectional study. The participants of the current study were female children and adolescents between the ages of 10 and 16, from public and private schools in the city of Joaçaba, Santa Catarina, Brazil. The sample consisted of 1,002 individuals, belonging to the database of the Laboratory of Exercise Physiology and Evaluation Measures, from the University of the West of Santa Catarina (Unoesc), Joaçaba campus. Only female were studied, as women have different dermatoglyphic showing fewer lines compared to men (Nodari & Fin, 2016).\n\nThe data is available in (Souza & Alberti, 2021). The database has 3,074 individuals investigated in the school census in 2013 and 2014, according to the National Institute of Educational Studies and Research Anísio Teixeira (INEP). The sample constituted only of individuals who met the inclusion and exclusion criteria.\n\nAll participants who had dermatoglyphic and strength data collected and stored in the database were included in the study. Participants who failed to perform abdominal strength and resistance tests, as well as those with anomalous or invalid fingerprints, which are scarred fingerprints or excessive sweating, were excluded. For the participants who participated in the research in both years, 2013 and 2014, the data collected from 2013 and 2014 was the data taken into account.\n\nFor the collection, processing, and analysis of the fingerprints, the computerized process of dermatoglyphic reading validated by Nodari et al. (2014b) was used. The Dermatoglyphic Reader from Salus Dermatoglyphic Luzerna-SC/Brasil. Consists of an optical scanner that collects and interprets the image and constructs, in binary code, a dermatoglyphic drawing, which is processed by the reader’s specific software for the treatment and reconstruction of real and binarized images in black and white, as validated by Nodari Júnior et al. in 2008. This study can also be performed with the traditional dermatoglyphic method of paper ink and magnifying glass.\n\nThe Brazilian Sports Project Manual - PROESP 2015 was used to collect data on muscle strength motor tests. The instrument is composed of a set of tests, set of criteria and evaluation standards. It is used to evaluate patterns of body growth, nutritional status, physical fitness and sports performance (Gaya et al., 2015). In the abdominal resistance test, classification was done using established cutoff points (Table 1) and later stratified, by age and sex, into two categorical scales: health risk zone (values below the cutoff point) or healthy zone of fitness (values above the cutoff point) (Gaya et al., 2015).\n\nGaya et al. (2015).\n\nAt the time of fingerprinting collection, the individual under study held the phalange (ulna side) on the scanner of the Dermatoglyphic Reader, performing a bearing, in its longitudinal axis, to the lateral (radio) side (Nodari, 2009). The collection of the fingerprints began with the little finger AKA ‘pinky’ of the left hand, followed by the left ring finger, left middle finger, left index finger and left thumb, in sequence to the right thumb, right index finger, right middle finger, right ring finger, ending in the right little finger (Nodari, 2009).\n\nOn the other hand, the evaluation of the motor capacity (muscle strength) was performed using the abdominal resistance test, according to the protocol (Gaya et al., 2015). The test consists of performing the highest number of abdominal exercise within the given time of one minute. The participant must be lying supine on a mat, knees bent at a 45 degree angle and arms crossed over the thorax. The participant should flex the trunk until the elbows touch the thighs and then return to the initial position. The largest number of complete repetitions should be recorded within an established time (Gaya et al., 2015).\n\nTo analyze genetic potential and fetal development through fingerprints, the chosen protocol was dermatoglyphic protocol proposed by Cummins & Midlo (1961) On the collection of the fingerprints obtained using the Dermatoglyphic Reader®, the evaluator marks the core and delta points and consequently, the software qualitatively identifies the image and quantifies lines, generating a computerized worksheet resulting from the processed data (Nodari, 2009).\n\nThe data (Souza & Alberti, 2021) was processed using Statistical Package for the Social Science (SPSS), version 20.0, allowing the calculation of descriptive and analytical statistics. In the comparison between the two groups and their quantitative variables, the Kolmogorov-Smirnov test was used to observe the distribution of normality. After the test was applied, a non-normal distribution of the data was observed. The non-parametric test, called Mann-Whitney (two independent samples in the case of the abdominal strength test - Risk Zone and Healthy Zone) was used as the inference for comparisons among the following numerical variables: sum of the number of lines in left hand finger 1 - thumb (MESQL1), sum of the number of lines in left hand finger 2 - index (MESQL2), sum of the number of lines in left hand finger 3 - middle finger (MESQL3), sum of the number of lines in left hand finger 4 – ring finger (MESQL4) and sum of the number of lines in left hand finger 5 - little finger (MESQL5); sum of the total number of left hand lines (SQTLE); sum of the number of lines in right hand finger 1 – thumb (MDSQL1), sum of the number of lines in right hand finger 2 - index (MDSQL2), sum of the number of lines in right hand finger 3 - middle finger (MDSQL3), sum of the number of lines in right hand finger 4 - ring finger (MDSQL4) and sum of the number of lines in right hand finger 5 - little finger (MDSQL5); sum of the total number of right hand lines (SQTLD); sum of the total number of lines in both hands (SQTL), with a significance level of p <0.05.\n\nFor the comparison of categorical variables: arch, radial loop, ulnar loop, whorl, on the following fingers: left hand drawing finger 1 (MET1), finger 2 (MET2), finger 3 (MET3), finger 4 (MET4) and finger 5 (MET5), and right hand drawing finger 1 (MDT1), finger 2 (MDT2), finger 3 (MDT3), finger 4 (MDT4) and finger 5 (MDT5) the Chi-square test was used.\n\nWhen a significant difference between the patterns expressed by the two groups was observed based on the Chi-square test, the recommendation made by Pereira (2001) is to perform an analysis of adjusted residuals. In this case, the data were compared with each other, observing the standard value of 1.96, that is, all the results found above the standard demonstrate the presence of a significant difference between the groups and which of the pictures in the fingerprints is more frequent.\n\nThe data used in this research is part of the research project: the kineanthropometric profile, motor development and socioeconomic profile of children and adolescents, led by Prof. Dr. Rudy José Nodari Júnior. The research was approved with under protocol number 449.924 of the Research Committee on Human Beings of Unoesc/HUST (CEP), in accordance with the ethical standards of norms and regulatory directives of research involving human beings, in accordance with Resolution 466, 2012, of the National Health Council and the Declaration of Helsinki. All participants and their parents or guardians were informed about the study procedures, possible risks, and benefits. Free and informed written documented consent was obtained from the parents or custody holders.\n\n\nResults\n\nDermatoglyphics, as a scientific study of fetal development markers, makes it possible to carry out investigations based on the potential of each individual, according to the approach and results found in this study through dermatoglyphic marks. A total of 1,002 individuals were analyzed in this study. Table 2 shows the sociodemographic characteristics, age, height and weight of the individuals investigated.\n\nWhen analyzing the abdominal resistance test scores in relation to the quantitative variables, no differences were found when comparing the Risk Zone and Healthy Zone groups (Table 3).\n\nHowever, when analyzing the qualitative variables (types of patterns) in females, differences were observed between the Risk Zone and Healthy Zone groups in the fingers MET4, MET5, and MDT4 (Table 4).\n\n*p <0.05.\n\nAfter the analysis of adjusted residuals, a higher frequency of arches was identified in MET4 and whorls in MET5 and MDT4 in the Risk Zone group. In the Healthy Zone group, ulnar loop was found to be more frequent in MET4, MET5, and MDT4 fingers. The obtained results may point to the presence of a dermatoglyphic marker for different levels of abdominal strength/resistance, related to the physical fitness for health, in the study population (Table 5).\n\n\nDiscussion\n\nGenetic studies may provide an additional method of predicting genetically predisposed performance characteristics of pre-full-length adults in untrained children by considering the profile of combinations of genetic variants associated with a particular trait. Dermatoglyphics follows this premise by being able to refer people according to their performance characteristics through dermatoglyphic marks (Guth & Roth, 2013). Research is being directed to verify dermatoglyphic marks including traces, patterns, and lines in fingerprints that are related to certain pathologies.\n\nAn example of the evolution of research in dermatoglyphics related to health is the study of Nodari et al. (2014a), with a sample consisting of 400 individuals, of which 200 were women with a positive diagnosis of breast cancer (clinical and mammography) and 200 women who represented a control group and were healthy in relation to cancer. This study showed a significant difference in all fingers of the left hand between the control group and the group with breast cancer; the radial loop tag was more frequent in the control group, which could comprise a marker of protection against breast cancer. This is in addition to other studies conducted with dermatoglyphics that show a link with diabetes and middle age (Kahn et al., 2009) and obesity (Alberti et al., 2019; Alberti et al., 2021) in diseases such as cancer (Chintamani et al., 2007). All identified a strong dermatoglyphic relation with the pathology.\n\nFor the present study, we sought to identify the level of abdominal strength and resistance in the test performed, so that we could predict health levels, physical conditioning and relate those to the dermatoglyphic patterns found. The results showed significant differences in the qualitative dermatoglyphic characteristics (types of dermatoglyphic drawing) in females of the Risk Zone and Healthy Zone groups.\n\nIt is observed in the study by Vialle et al. (2014) that a low level of physical fitness can lead to health problems. When diseases such as lower back pain occur, it can lead to frequent visits to the doctor, which in turn leads to an increase in medical leave from work. Kumar et al. (2010) state that this is due to a lack of strengthening in primary abdominal muscle groups and low levels of flexibility.\n\nThe higher frequency of ulnar loop observed on the MET4, MET5 and MDT4 fingers in the Healthy Zone group found in the present study. It cannot be stated, but it can serve as a potential dermatoglyphic marker for healthy individuals. In addition, studies have demonstrated a relationship between the ulnar loops and high performance and speed among track athletes (Linhares et al., 2009; Nodari & Fin, 2016). It is also possible to analyze that the greater presence of (LU) characteristic of activities or exercises of short duration and high intensity, coincides with the one found in the present research for the group Healthy Zone that had the results within the established standards.\n\nThe greater presence of the arch and whorl patterns in the Risk Zone group of the present study may point to a new predictive marker of individuals with abdominal strength/resistance. The need for such motor conditioning is a major factor in the fight against diseases related to physical inactivity, such as obesity (Ronque et al., 2007). Abdominal fat can be a major health risk factor, since it is observed that when it is deposited on a large scale as intravisceral fat, this can lead to greater chances of terminal diseases, such as cancer (Dutra et al., 2016).\n\nThe present research aimed to verify kineanthropometric and dermatoglyphic variables in children and adolescents at the stage of puberty when great and constant changes take place. The knowledge of the greater number of variables and their tendencies as a function of growth and development facilitates a better understanding of the moment when the young person is present and provides more effective work with this audience (Kumar et al., 2016). Moreover, it may be possible for both, the quieter and the more adequate insertion in the competitions, as well as the orientation of talents consistent with the level of development of each individual which can lead to fewer prognostic errors Dermatoglyphics can help in this process as a hallmark of fetal development.\n\nAs a limitation, the present study did not perform comparisons with different ethnic groups, because in the present study it was not possible to analyze this due to the ethnic limitation of the sample. Different ethnic groups may have different dermatoglyphic marks.\n\n\nConclusion\n\nThe results showed the presence of dermatoglyphic marks that may be characteristic of motor capacity and abdominal muscle strength in females. But it can be said, but dermatoglyphia can be a marker that can help. Thus, dermatoglyphics is an effective method that allows the observation of the neuromotor potentials, such as muscular strength, predisposed in an individual from the genetic and fetal development marks present in their fingerprints.\n\n\nData availability\n\nZenodo: Dermatoglyphic and abdominal resistance of children and adolescents of female sex https://doi.org/10.5281/zenodo.5048427 (Souza & Alberti, 2021) This project contains the following underlying data:\n\n- Dermatoglyphic and abdominal resistance of children and adolescents of female sex.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAlberti A, Kupek E, Comim CM, et al.: Dermatoglyphical impressions are different between children and adolescents with normal weight, overweight and obesity: a cross-sectional study [version 1; peer review: 2 approved, 1 approved with reservations, 1 not approved]. F1000Res. 2019; 8: 964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlberti A, Ruiz Reyes MA, De Jesus JA, et al.: Identification of obesity in children and teenagers. Minerva Pediatr. 2021. PubMed Abstract \| Publisher Full Text\n\nBarros JDSV, de Lima MVM, Sampaio AN, et al.: Análise das Capacidades Motoras nos Estágios Maturacionais de Adolescentes do o Sexo Feminino. J Hum Growth Dev. 2017; 27(2): 206–212. Publisher Full Text\n\nChintamani, Khandelwal R, Mittal A, et al.: Qualitative and quantitative dermatoglyphic traits in patients with breast cancer: a prospective clinical study. BMC Cancer. 2007; 7: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCummins H, Midlo C: Finger prints, palmas, and soles: An introduction to dermatoglyphics. New York: Dover Publication. 1961. Reference Source\n\nDutra GF, Kaufmann CC, Pretto ADB, et al.: Sedentarismo e práticas alimentares inadequadas na infância: um estudo de coorte. Ciência e Saúde Coletiva. 2016; 21(4): 1051–1059. Publisher Full Text\n\nGallahue DL, Ozmun JC, Goodway JD: Compreendendo o Desenvolvimento Motor: Bebês, Crianças, Adolescentes e Adultos (7th ed). Porto Alegre: Artmed. 2013. Reference Source\n\nGarufo L, Messina G, Nodari R: La Dermatoglifia, analisi genetica delle capacità motorie in sportivi di differenti discipline. (Doctoral thesis, Università Degli Studi Di Palermo, Italia). 2017. Publisher Full Text\n\nGaya A, Lemos A, Gaya A, et al.: Projeto Esporte Brasil: Manual de testes e avaliação. Porto Alegre: UFRGS. 2015. Reference Source\n\nGuedes DP: Crescimento e desenvolvimento aplicado à Educação Física e ao Esporte. Rev bras educ fís esporte. 2011; 127–140. Publisher Full Text\n\nGuth LM, Roth SM: Genetic influence on athletic performance. Curr Opin Pediatr. 2013; 25(6): 653–658. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nINEP: National Institute of Studies and Educational Research Anísio Teixeira. [date unknown]. Educational indicators. [accessed 2018 July 5]. Reference Source\n\nKahn HS, Graff M, Stein AD, et al.: A fingerprint marker from early gestation associated with diabetes in middle age: the Dutch Hunger Winter Families Study. Int J Epidemiol. 2009; 38(1): 101–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar S, Sharma VP, Shukla R, et al.: Comparative efficacy of two multimodal treatments on male and female sub-groups with low back pain (part II). J Back Musculoskelet Rehabil. 2010; 23(1): 1–9. PubMed Abstract \| Publisher Full Text\n\nKumar S, Stecher G, Tamura K: MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33(7): 1870–1874. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinhares RV, Matta MO, Lima JRP, et al.: Efeitos da maturação sexual na composição corporal, nos dermatóglifos, no somatótipo e nas qualidades físicas básicas de adolescentes. Arq Bras Endocrinol Metab. 2009; 53(1). Publisher Full Text\n\nNodari RJ Jr: Protótipo de escaneamento informatizado: possibilidade em diagnóstico em saúde por meio das impressões digitais. Revista de Salud Publica. 2009.\n\nNodari RJ Jr, Fin G: Dermatoglifia: Impressões digitais como marca genética e de desenvolvimento Fetal. Joaçaba: Ed. Unoesc. 2016. Reference Source\n\nNodari RJ Jr, Fin G, Fortunatti D: Perfil dermatoglífico de portadoras de câncer de mama do Meio-Oeste de Santa Catarina. 2014a, in press.\n\nNodari RJ Jr, Heberle A, Emygdio RF, et al.: Dermatoglyphics: Correlation between software and traditional method in kineanthropometric application. Revista Andaluza de medicina del deporte. 2014b; 7(2): 60–65. Publisher Full Text\n\nPereira JCR: Análise de dados qualitativos: estratégias metodológicas para as ciências da saúde, humanas e sociais. São Paulo: Edusp. 2001.\n\nRonque ER, Cyrino ES, Dórea V, et al.: Diagnóstico da aptidão física em escolares de alto nível socioeconômico: avaliação referenciada por critérios de saúde. Rev Bras Med Esporte. 2007; 13(2): 71–76. Publisher Full Text\n\nSantos LC, Dantas PMS, Filho JF: Características Genotípicas E Fenotípicas Em Atletas Velocistas. Motricidade. 2007; 4: 50–57. Reference Source\n\nSouza R, Alberti A: Dermatoglyphic and abdominal resistance of children and adolescents of female sex. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.5048427\n\nVialle EN, Vialle LRG, Mariúba ESO, et al.: Results of a domiciliary rehabilitation program for chronic low back pain patients. Coluna/Columna. 2014; 13(4): 287–290. Publisher Full Text" }	[ { "id": "94862", "date": "05 Oct 2021", "name": "Lavanya Prathap", "expertise": [ "Reviewer Expertise Dermatoglyphics", "Exercise", "Epigenetics", "Cancer", "Chronic diseases." ], "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 appreciate the author for working on this project Dermatoglyphics and the abdominal neuromuscular efficiency. The author attempted to compare the quantitative and qualitative dermatoglyphic variables namely fingerprints and the patterns respectively with the abdominal neuromuscular strength. The author has used the appropriate methods and statistical tools to analyze the data and has come out with appropriate results.\nThe author could have detailed with little more explanations with respect to grouping, parameters used (both dermatoglyphics and abdominal resistance exercises/tools/tests) to make the study more reproducible and applicable.\n\nIntroduction: The scope and purpose of the study can be added with elaboration.\nMethods:\nGrouping can be made clear\n\nMore Clarity in describing the dermatoglyphic and tests for abdominal resistance -variables used.\n\nMore Clarity in inclusion and exclusion criteria\nResults and Discussion:\nThe first paragraph of the discussion states breast cancer and its association with dermatoglyphics and its role in epigenetics. The quoted literature123 can add further value to the point and evidence for the readers of this manuscript to get introduced to that concept.\n\nResult summary to be added as the first paragraph of discussion section followed by correlation with previous studies.\n\nFuture scope and recommendations of the study to be added before the conclusion.\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": [] }, { "id": "94863", "date": "11 Oct 2021", "name": "Kewal Krishan", "expertise": [ "Reviewer Expertise Biological anthropology", "Forensic anthropology", "Anthropometry", "Dermatoglyphics" ], "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\nPlease do not use “dermatoglyphic mark”, ‘mark’ doesn’t fit here. You can mention “dermatoglyphic marker”.\n\nSome of the sentences are not clear such as “The protocol selected for analyzing the fingerprints was dermatoglyphics, proposed by Cummins and Midlo using a Dermatoglyphic Reader”. This manuscript would benefit with the help of an English language editor. There are many sentences in the text which are similar and alter the meaning if not correctly written.\n\nPlease write the full form of MET4, MET5, and MDT4 in the abstract.\n\nPlease explain with scientific evidence why neuromotor strength capacity and abdominal resistance may be related to the dermatoglyphic characteristics?\n\nThe introduction needs extensive revision as the relevant review of literature and significance of the study are missing.\n\nThe sentences such as “Only female were studied, as women have different dermatoglyphic showing fewer lines compared to men (Nodari & Fin, 2016)” are meaningless. There are many grammatical and technical mistakes, I couldn’t understand what the authors want to convey here.\n\nPlease give details, when the data were collected? Who collected the fingerprints etc.\n\nAccording to the authors, only females were studied but table 1 shows males also.\n\nWhat are the kineanthropometric variables in the study? Couldn’t understand?\n\nThe conclusion is vague and the authors have not given details of the accuracy and reliability of the study in medical and diagnostic terms.\n\nIn addition to all these flaws, I failed to understand how the study is contributing to the advancement of medical science. My question is “can we identify the abdominal strength and resistance by studying the fingerprint characteristics?\n\nThe article needs extensive revision on all of these points.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-945
https://f1000research.com/articles/10-527/v1	01 Jul 21	{ "type": "Research Article", "title": "Delicate balance: the relationship between internal astigmatism and lens astigmatism", "authors": [ "Meng Liu", "Cheng Dai", "Guimei Zhou", "Xiaodong Lv", "Jingjia Li", "Binzhong Li", "Meng Liu", "Cheng Dai", "Guimei Zhou", "Xiaodong Lv", "Jingjia Li" ], "abstract": "Background: Due to lack of equipment for directly measuring crystal morphology, there has been little research on crystal astigmatism. The purpose of this study was to accurately explore the correlation between internal astigmatism and lens astigmatism in patients with ametropia. Methods: This is a cross-sectional study conducted in the Affiliated Hospital of North Sichuan Medical College, China, in September 2020. Diopter values (refractive astigmatism, RA) of patients with ametropia was recorded, and the corneal and lens biological parameters were measured by CASIA2 (corneal/anterior segment optical correlation tomography analyzer). Biometric parameters, including the total corneal astigmatism (TCA), anterior and posterior curvature radius of the lens (ACL, PCL), internal astigmatism (IA), anterior and posterior astigmatism of the lens (AAL, PAL) were measured. Grouping and comparisons were made according to gender and age. Results: In total, 151 participants (293 eyes) were included in the analysis. There were significant (P<0.05) differences in the IA (Z=-2.194, P=0.028) according to gender, but not in the other parameters. By age group, there were statistically significant differences in the TCA (H=10.609, P=0.005), IA (F=3.722, P=0.025), and PAL (H=8.254, P=0.016), but not in the others. The IA was positively correlated with the age (r=0.155, P=0.008), RA (r=0. 534, P<0.001), AAL (r=0.308, P<0.001), and was negatively correlated with the TCA (r=-0.244, P<0.001). The regression equation between the IA and AAL was: Y (IA) =-0.626 +0.447X (AAL). Conclusions: There is a delicate balance between internal astigmatism and intraocular astigmatism. There were no significant differences in the other parameters except IA for different gender groups. For different ages, there are symbol differences in the TCA, IA and PAL, but not in the other parameters. In the analysis of IA with lens astigmatism, it was found that internal astigmatism was mainly related to AAL, but not to PAL.", "keywords": [ "Astigmatism", "Internal astigmatism", "Lens astigmatism", "Corneal astigmatism", "Refractive error" ], "content": "Introduction\n\nAstigmatism is the most common refractive error in the world, which may come from congenital or acquired factors1. Astigmatism can change from birth, and its development is influenced by many factors such as heredity, extraocular muscle tension, visual feedback and eyelid pressure2. According to World Health Organisation (WHO) statistics in 2018, the prevalence rates of astigmatism in children and adults are 14.9% and 40.4%, respectively3. Similarly, studies have found that 47% of cataract patients suffer from astigmatism ≥1 degree4. Uncorrected ametropia is the second leading cause of blindness in the world, and astigmatism is a major factor that damages human vision5. At present, we have a clear understanding of the detection of refractive astigmatism and corneal astigmatism, but due to the lack of methods to observe crystal morphology, there are few studies on lens astigmatism. The CASIA2 (Tomey Corp., Nagoya, Japan) system is a new type of anterior segment scanner. This novel device can provide accurate measurement of anterior segment parameters, and has good repeatability and reproducibility6,7. Based on the advantages of the CASIA2 system, this paper combines corneal and lens parameters and optometry data to analyze the correlation between internal astigmatism and lens astigmatism.\n\n\nMethods\n\nAll the subjects were ametropia patients who came to the Affiliated Hospital of North Sichuan Medical College naturally. The experimenter (Meng Liu) explained this study and examined it with the permission of the patients. Since this research does not infringe on any rights and interests of patients, it can be conducted with the authorization of the Ethics Committee of North Sichuan Medical College. In order to reduce bias, we carefully screened the subjects. All patients had simple ametropia. Patients with organic diseases, such as those affecting the ocular surface and fundus, and patients who had undergone operations affecting refraction, the ocular surface or the fundus in the past were excluded.\n\nWritten informed consent was provided by the patient or their family/guardian if under the age of 18 years. As this study is an extended retrospective study of relevant prospective approved studies8 [approval number 2020ER(A) 068] and does not harm the interests of patients, it received an exemption by the Ethics Committee of North Sichuan Medical College.\n\nBiometric parameters of the lens were measured by the same experimenter (ML). An ARK-510A autorefractor (NIDEK, Co., Ltd, Gamagori, Japan) and DK-700 optometry system (Topcon, Japan) were used to perform standardized optometry, and the CASIA2 system (Tomey Corp., Nagoya, Japan) was applied to obtain other measurements.\n\nOutcome parameters included the following: (1) Sex and age; (2) RA: The refractive astigmatism obtained by optometry; (3) TCA, ACL and PCL directly recorded by the CASIA2 system; (4) IA calculated by RA minus TCA; (5) AAL, PAL: Convert radius of curvature to curvature by formula9 (Ø=n′-nr, Ø = curvature; n’=Exit square refractive index; n=Incident square refractive index; r=the radius of curvature).The default refractive index of CASIA2 crystal is 1.4085, and the refractive index of aqueous humor is 1.336.The radius of the anterior and posterior curvature of the lens can be obtained directly from CASIA2.\n\nThe sample size was estimated by Stata 16.0 software. The test efficiency β= 0.1, α=0.05, bilateral test, and the correlation coefficient is 0.3. The calculated sample size is estimated to be 112. SPSS 25.0 statistical software was used for analysis. All data were first tested by the Kolmogorov-Smirnov (K-S) goodness-of-fit normality test before comparisons. Data with a normal distribution were compared using a paired sample t test or ANOVA analysis. If the distribution was not normal, a nonparametric test was used for comparison. In the correlation analysis(Internal astigmatism with Age, Refractive astigmatism, Total corneal astigmatism, Anterior astigmatism of the lens, Posterior astigmatism of the lens), data with a normal distribution are presented as the mean±standard deviation (X±S), a Pearson correlation analysis was applied, and a scatter plot was used to describe the correlation. If the data did not conform to a normal distribution, the median±quartile spacing (M±Q) was used, and a Spearman correlation analysis was carried out. Multivariate linear regression analysis was performed for parameters with multiple correlations. P < 0.05 was considered to indicate statistical significance.\n\n\nResults\n\nThis was a cross-sectional study of 151 patients (293 eyes, 77 males and 74 females), including 93 patients with 179 eyes aged 4–12 years, 17 patients with 32 eyes aged 13–18 years and 41 patients with 82 eyes aged 19–40 years26.\n\nTable 1 shows the comparison results of all parameters grouped by gender. It can be seen that there is no statistical difference in other parameters except IA (Z=-2. 194, P=0. 028).\n\n* indicates that the comparison is statistically significant. RA: refractive astigmatism; TCA: total corneal astigmatism; IA: internal astigmatism; AAL: anterior astigmatism of the lens; PAL: posterior astigmatism of the lens.\n\nComparison of all parameters by age group is shown in Table 2. It can be seen that there are statistically significant differences in TCA (H=10. 609, P=0. 005), IA (F=3. 722, P=0. 025), and PAL (H=8. 254, P=0. 016), and others have no statistically significant differences.\n\n* indicates that the comparison is statistically significant. RA: refractive astigmatism; TCA: total corneal astigmatism; IA: internal astigmatism; AAL: anterior astigmatism of the lens; PAL: posterior astigmatism of the lens.\n\nAfter K-S test, the IA and the AAL showed normal distribution, so Pearson correlation analysis was used to analyze the correlation between them. Other parameters had non-normal distribution, so Spearman correlation analysis was used, and the results are shown in Table 3.\n\n* indicates that the comparison is statistically significant. RA: refractive astigmatism; TCA: total corneal astigmatism; IA: internal astigmatism; AAL: anterior astigmatism of the lens; PAL: posterior astigmatism of the lens.\n\nThe IA was positively correlated with the age (r=0. 155, P=0. 008), RA (r=0. 534, P < 0.001), and was negatively correlated with the TCA (r=-0. 244, P < 0.001). It can be seen that in the correlation analysis between IA and lens astigmatism, IA was positively correlated with AAL (r=0. 308, P < 0.001), but not with other lens astigmatism parameters. Therefore, for the regression analysis of IA and AAL, the regression equation is Y (IA) =-0.626 +0.447 X (AAL) (F=30. 461, P < 0.001, R2=0. 095) (Figure 1).\n\n\nDiscussion\n\nThe types of astigmatism in human eyes include refractive astigmatism, corneal astigmatism, internal astigmatism and lens astigmatism. Internal astigmatism compensates for corneal astigmatism from birth, but the efficiency of its decreases with age10. The active compensation between corneal astigmatism and internal astigmatism in childhood helps maintain refractive stability, which is mainly due to the high convergence of the wavefront incident on the lens due to corneal refraction11,12. However, due to the lack of precise equipment for observing anterior segment, the relationship between internal astigmatism and lens is still controversial. For instance, one study found that internal astigmatism gradually increases with age, and it mainly comes from lens13. Nevertheless, some studies believe that the prevalence rate of astigmatism increases with age, and the refractive and corneal astigmatism shift to ATR (against-the-rule). But the continuous corneal changes seem to be the cause of the age trend of refractive astigmatism, and the severity of lens opacity plays a small role in the change of internal astigmatism14. Therefore, based on the advantages of the CASIA2 system, this paper comprehensively analyzed the correlation between internal astigmatism and lens astigmatism by combining corneal and lens parameters. Inevitably, our research also has certain limitations. Because of the complexity and instability of astigmatism, this study only analyzed the power of astigmatism, but did not involve the axial direction. However, through this article, we have made clear the quantitative relationship between internal astigmatism and lens astigmatism, and lens astigmatism mainly comes from the anterior surface of the lens. This can provide reference and ideas for more accurate research on astigmatism.\n\nAccording to gender, there were statistical differences in internal astigmatism, which were lower in boys than girls, but not in other parameters. Li et al.15 found that corneal astigmatism and internal astigmatism seemed to be higher in girls than in boys. Similarly, Liu et al.10 also found that girls had greater internal astigmatism than boys. This may be due to the fact that girls' physical development is earlier than boys', and the difference caused by the growth rate of the axial length. Gender is highly correlated with the growth of the axial length, and the growth of the axial length has also been proved to be related to internal astigmatism16.\n\nThen, we found that corneal astigmatism, internal astigmatism and posterior astigmatism of lens were different according to age. Firstly, corneal astigmatism has been changing since birth. Naeser et al.17 proved that corneal astigmatism is not stable until the age of 50. Under normal circumstances, corneal astigmatism changes regularly by 0.25 D every 10 years. Secondly, the compensation effect of internal astigmatism on reducing corneal astigmatism is very significant among preschool children, and then this compensation effect gradually weakens with age18,19. Finally, we found that there were differences in the posterior astigmatism of lens, but there was not in the anterior. Birkenfeld et al.20 have also found that with the increase of age, the astigmatism of the lens changes significantly, but the difference is that they have significant changes in the anterior lens. We all know that the curvature of the anterior lens changes more than posterior in the process of accommodation21, but this is not completely equivalent to the greater astigmatism of the anterior surface with the change of age, which is the direction for further research.\n\nIt can be seen that internal astigmatism is highly correlated with the anterior astigmatism of the lens, but not with the posterior and the internal astigmatism increases with the increase of anterior astigmatism of the lens. Although the refractive index of lens is gradient, its astigmatism is close to anterior surface astigmatism20. However, this does not mean an absolute correlation between internal astigmatism and the anterior astigmatism of the lens, because the state of the lens is unstable. For example, Pérez et al.22 found that in the relaxed state, spherical terms account for the majority of anterior lens surface irregularity (47%) and posterior lens astigmatism (70%); however, in the accommodation lens, astigmatism is the main irregularity of anterior lens surface (90%). The optical characteristics of the lens depend on its shape and refractive index distribution23, which can affect its astigmatism to a great extent, thus further causing internal astigmatism to change. It seems that corneal and internal astigmatism cancel each other out24. The unity of changes among corneal astigmatism, lens astigmatism and refractive astigmatism, do not occur individually25.\n\nTo sum up, we found that there are gender and age differences in some astigmatism parameters and the relationship between internal astigmatism and lens astigmatism is clarified. Internal astigmatism increases with the increase of lens astigmatism, and the source of lens astigmatism is mainly the anterior. However, further research is required to determine whether lens astigmatism can be equated with internal astigmatism, and the correlation between internal astigmatism and anterior astigmatism of the lens is related to the mechanism of accommodation.\n\n\nData availability\n\nOpen Science Framework: Delicate balance: relationship between internal astigmatism and lens astigmatism. https://doi.org/10.17605/OSF.IO/7X9FD26.\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 Professor Changjun Lan (Department of Ophthalmology, Affiliated Hospital of North Sichuan Medical College) for his expert technical assistance.\n\n\nReferences\n\nWoltsche N, Werkl P, Posch-Pertl L, et al.: [Astigmatism]. Ophthalmologe. 2019; 116(3): 293–304. PubMed Abstract \| Publisher Full Text\n\nRead SA, Collins MJ, Carney LG: A review of astigmatism and its possible genesis. Clin Exp Optom. 2007; 90(1): 5–19. PubMed Abstract \| Publisher Full Text\n\nHashemi H, Fotouhi A, Yekta A, et al.: Global and regional estimates of prevalence of refractive errors: Systematic review and meta-analysis. J Curr Ophthalmol. 2018; 30(1): 3–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson DF, Dhariwal M, Bouchet C, et al.: Global prevalence and economic and humanistic burden of astigmatism in cataract patients: a systematic literature review. Clin Ophthalmol. 2018; 12: 439–452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarb EN, Wildsoet CF: Origins of Refractive Errors: Environmental and Genetic Factors. Annu Rev Vis Sci. 2019; 5: 47–72. PubMed Abstract \| Publisher Full Text\n\nShoji T, Kato N, Ishikawa S, et al.: In vivo crystalline lens measurements with novel swept-source optical coherent tomography: an investi gation on variability of measurement. BMJ Open Ophthalmol. 2017; 1(1): e000058. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKimura S, Morizane Y, Shiode Y, et al.: Assessment of tilt and decentration of crystalline lens and intraocular lens relative to the corneal topographic axis using anterior segment optical coherence tomography. PLoS One. 2017; 12(9): e0184066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDai C, Liu M, Li B: Relationship between lens morphology and axial length in adolescent. Journal of the Third Military Medical University, China. 2021; 43(6).\n\nBinzhong L: Applied Optics. Science Press, Beijing, China, 2017. Reference Source\n\nLiu Y, Cheng Y, Zhang Y, et al.: Evaluating internal and ocular residual astigmatism in Chinese myopic children. Jpn J Ophthalmol. 2017; 61(6): 494–504. PubMed Abstract \| Publisher Full Text\n\nHarvey EM, Miller JM, Twelker JD, et al.: Longitudinal change and stability of refractive, keratometric, and internal astigmatism in childhood. Invest Ophthalmol Vis Sci. 2014; 56(1): 190–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu T, Thibos LN: Compensation of corneal oblique astigmatism by internal optics: a theoretical analysis. Ophthalmic Physiol Opt. 2017; 37(3): 305–316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLimaiem R, Baba A, Bouraoui R, et al.: [Internal astigmatism with other ocular lesions]. J Fr Ophtalmol. 2012; 35(4): 277–83. PubMed Abstract \| Publisher Full Text\n\nLiu YC, Chou P, Wojciechowski R, et al.: Power vector analysis of refractive, corneal, and internal astigmatism in an elderly Chinese population: the Shihpai Eye Study. Invest Ophthalmol Vis Sci. 2011; 52(13): 9651–7. PubMed Abstract \| Publisher Full Text\n\nLi H, Li SM, Liu L R, et al.: Astigmatism and its components in 12-year-old Chinese children: the Anyang Childhood Eye Study. Br J Ophthalmol. 2019; 103(6): 768–774. PubMed Abstract \| Publisher Full Text\n\nWu L, Weng C, Xia F, et al.: Internal Astigmatism and Its Role in the Growth of Axial Length in School-Age Children. J Ophthalmol. 2018; 2018: 1686045. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaeser K, Savini G, Bregnhøj JF: Age-related changes in with-the-rule and oblique corneal astigmatism. Acta Ophthalmol. 2018; 96(6): 600–606. PubMed Abstract \| Publisher Full Text\n\nWang Z, Huang D, Chen X, et al.: Preschool Children Exhibit Evident Compensatory Role of Internal Astigmatism in Distribution of Astig matism: The Nanjing Eye Study. Invest Ophthalmol Vis Sci. 2019; 60(1): 73–81. PubMed Abstract \| Publisher Full Text\n\nMathur A, Atchison DA, Tabernero J: Effect of age on components of peripheral ocular aberrations. Optom Vis Sci. 2012; 89(7): E967–76. PubMed Abstract \| Publisher Full Text\n\nBirkenfeld J, De Castro A, Marcos S: Astigmatism of the Ex Vivo Human Lens: Surface and Gradient Refractive Index Age-Dependent Contributions. Invest Ophthalmol Vis Sci. 2015; 56(9): 5067–73. PubMed Abstract \| Publisher Full Text\n\nSchachar RA, Mani M, Schachar I H: Image registration reveals central lens thickness minimally increases during accommodation. Clin Ophthalmol. 2017; 11: 1625–1636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPérez-Merino P, Velasco-Ocana M, Martinez-Enriquez E, et al.: OCT-based crystalline lens topography in accommodating eyes. Biomed Opt Express. 2015; 6(12): 5039–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBirkenfeld J, De Castro A, Ortiz S, et al.: Contribution of the gradient refractive index and shape to the crystalline lens spherical aberration and astigmatism. Vision Res. 2013; 86: 27–34. PubMed Abstract \| Publisher Full Text\n\nRozema JJ, Hershko S, Tassignon MJ, et al.: The components of adult astigmatism and their age-related changes. Ophthalmic Physiol Opt. 2019; 39(3): 183–193. PubMed Abstract \| Publisher Full Text\n\nNamba H, Kawasaki R, Sugano A, et al.: Age-Related Changes in Ocular Aberrations and the Yamagata Study (Funagata). Cornea. 2017; 36 Suppl 1: S34–S40. PubMed Abstract \| Publisher Full Text\n\nLiu M, Dai C, Zhou G, et al.: Delicate balance: relationship between internal astigmatism and lens astigmatism. 2021. http://www.doi.org/10.17605/OSF.IO/7X9FD" }	[ { "id": "92837", "date": "07 Sep 2021", "name": "Samira Heydarian", "expertise": [ "Reviewer Expertise Optometry" ], "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\nHere are some comments that require clarification as given below.\nAbstract:\nIt is better to use lenticular astigmatism instead of crystal astigmatism.\n\nPlease clarify what the difference is between internal astigmatism and intraocular astigmatism.\n\nThe conclusion is not appropriate and it is just the repetition of the results.\nIntroduction:\n“Similarly, studies have found that 47% of cataract patients suffer from astigmatism≥1 degree”. What do you mean by \"degree\"? It seems that it should be changed to \"diopter\".\nMethod: Subjects:\nHow were the subjects screened to reduce bias? Please clarify more.\n\nPlease add the inclusion criteria. For instance, is there any age limit for the study?\nProcedures:\nWhat are the other measurements that the authors measured using CASIA and what is the standardized optometry?\n\nDue to the effect of accommodation on the lens and even on corneal curvature, cyclorefraction and comparing changes before and after cycloplegia seems useful.\nOutcome parameters:\nAs a general rule, all non-standard abbreviations/acronyms should be written out in full on first use (in both the abstract and the paper itself) and followed by the abbreviated form in parentheses. Please consider this point throughout the manuscript.\n\nDo the authors consider the curvature of the anterior and posterior surface of the lens as AAL and PAL? Astigmatism is the difference between the main curvatures, not just the curvature.\nStatistical analysis:\nPlease cite the reference that is used for the calculation of the sample size.\nResults:\nPlease add the mean age of the participants.\n\nHow did they compare astigmatism without considering the effect of the axis? Vector analysis seems necessary for an appropriate comparison.\nDiscussion\nThe discussion is weak and needs to be improved.\n\nThe effect of the posterior surface of the cornea and retinal astigmatism was not considered and discussed 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? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7130", "date": "20 Sep 2021", "name": "Binzhong Li", "role": "Author Response", "response": "Dear Professor Samira Heydarian, Thank you very much for your many meaningful comments on this article, so as to make changes according to your suggestions. The changes are as follows: 1. Abstract: 1.1 It is better to use lenticular astigmatism instead of crystal astigmatism. Author response: Thank you for your suggestion, it has been modified. 1.2 Please clarify what the difference is between internal astigmatism and intraocular astigmatism. Author response: Thank you for your suggestion. As explained in the method and first paragraph of the discussion section, internal astigmatism in this paper is the difference between human total astigmatism (refractive astigmatism) and corneal total astigmatism, which is the overall internal astigmatism including intraocular astigmatism. 1.3 The conclusion is not appropriate and it is just the repetition of the results. Author response: It has been modified. 2. Introduction: 2.1 “Similarly, studies have found that 47% of cataract patients suffer from astigmatism≥1 degree”. What do you mean by \"degree\"? It seems that it should be changed to \"diopter\". Author response: It has been modified. 3. Methods 3.1 How were the subjects screened to reduce bias? Please clarify more. Author response: Thank you for your suggestion, it has been added. 3.2 Please add the inclusion criteria. For instance, is there any age limit for the study? Author response: It has been added. 3.3 What are the other measurements that the authors measured using CASIA and what is the standardized optometry? Author response: It has been modified. That is, the optometry process to obtain the best corrected vision and diopter. 3.4 Due to the effect of accommodation on the lens and even on corneal curvature, cyclorefraction and comparing changes before and after cycloplegia seems useful. Author response: Yes, your point of view is worth thinking about. As you said, the changes related to adjustment before and after mydriasis are very important, which is worth our next research direction. 3.5 As a general rule, all non-standard abbreviations/acronyms should be written out in full on first use (in both the abstract and the paper itself) and followed by the abbreviated form in parentheses. Please consider this point throughout the manuscript. Author response: It has been added in the abstract. 3.6 Do the authors consider the curvature of the anterior and posterior surface of the lens as AAL and PAL? Astigmatism is the difference between the main curvatures, not just the curvature. Author response: We are discussing the main curvature, which has been made clear. Thanks again. 3.7 Statistical analysis: Please cite the reference that is used for the calculation of the sample size. Author response: It has been added. 4. Results: 4.1 Please add the mean age of the participants. Author response: It has been added. 4.2 How did they compare astigmatism without considering the effect of the axis? Vector analysis seems necessary for an appropriate comparison. Author response: It has been added. 5. Discussion 5.1 The discussion is weak and needs to be improved. Author response: According to your suggestion, the discussion section has been modified. 5.2 The effect of the posterior surface of the cornea and retinal astigmatism was not considered and discussed throughout the manuscript. Author response: Total corneal astigmatism of CASIA2 is a general index combined with anterior and posterior corneal surfaces. Due to the lack of equipment to detect retinal astigmatism, we did not discuss retinal astigmatism, but under the condition of clear refractive medium it seems that physiological retinal astigmatism is very small, which deserves further study and is also a new direction for us to learn. Thank you for your suggestion. Best wishes, Binzhong Li (on behalf of all authors)" } ] }, { "id": "91892", "date": "01 Nov 2021", "name": "Hu Liu", "expertise": [], "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\nAlthough this manuscript raised an interesting topic, i.e. the relationship between lens astigmatism and internal astigmatism, it had multiple mistakes in the methods and failed to provide convincing results and logical discussion to prove this relationship. Here are some examples:\nThe authors described that the internal astigmatism was calculated by refractive astigmatism minus total corneal astigmatism; however, internal astigmatism should be the vectorial difference between refractive astigmatism and anterior corneal astigmatism. The wrong method for calculating internal astigmatism makes all the following results unreliable.\n\nAll the subjects were ametropia, which might influence the relationship between the lens astigmatism and internal astigmatism, just like age and gender. Its influence should be clarified further.\n\nWere the subjects under cycloplegia or not when they were taking the examinations? If they were not under cycloplegia, it is necessary to consider whether the accommodation will affect the degree of lens astigmatism and the reliability of the results.\n\nI noticed that all the correlation analyses were taken without adjusting for confounding factors, which quite impacts the quality of the results. Without adjustment, however, could the authors say “internal astigmatism was mainly related to AAL, but not to PAL”? Although I admit that internal astigmatism mainly comes from a lens, I cannot agree with the conclusion made in the manuscript because of the powerless results.\n\n“Because of the complexity and instability of astigmatism, this study only analyzed the power of astigmatism, but did not involve the axial direction.” However, the axis might play an important role.\n\nIn addition, there are some grammar mistakes.\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? 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/10-527
https://f1000research.com/articles/10-470/v1	14 Jun 21	{ "type": "Research Article", "title": "Development, validation and reliability of a questionnaire for assessment of physician's knowledge, attitude and practices (KAPs) regarding foodborne diseases in the Kingdom of Saudi Arabia", "authors": [ "Mohammed AL-Mohaithef" ], "abstract": "Background: The burden of foodborne illness is considered to be high across the world. Based on the Knowledge-Attitude-Practice (KAP) model, physician’s awareness is essential for conducting individualized treatments, thus reducing the burden of foodborne illness. However, there have been no validated questionnaires specific to the awareness of physicians with foodborne diseases. This study aims to develop and validate a KAP questionnaire for physicians to assess their awareness about the diagnosis and management of foodborne illness.\n\nMethods The questionnaire was developed in three phases: a comprehensive literature review, face and content validity, followed by a reliability test by internal consistency. A cross-sectional study was designed in Abha, Saudi Arabia. Physicians (n=125) were opportunistically recruited from both public and private primary healthcare centers. The questionnaire’s content and validity were confirmed by experts in their corresponding fields. After signing the informed consent, the study participants received the questionnaire to evaluate their KAPs on foodborne diseases. Results: A total of 160 physicians from both public and private primary health care centers were approached to enrol 125 study participants into the survey (response rate 78.13%). Of the 31 items designed for assessing the KAP of physicians on foodborne illnesses, three items were excluded after Cronbach’s α analysis. In total, 29 items were included in the final set of the questionnaire. Results of different validity and reliability analyses suggest the questionnaire has a high face and content validity as well as good reliability in internal consistency and stability. Conclusions: This study introduces a newly developed questionnaire with good reliability and validity values that can assess physician’s awareness of foodborne disease. The awareness questionnaire, as a study instrument, had a favourable acceptance among physicians. It is a sound method for evaluating and measuring levels of foodborne disease-related awareness among physicians in Abha, Saudi Arabia.", "keywords": [ "Content validity", "foodborne illness", "knowledge", "attitudes", "and practices (KAPs)", "physician", "reliability", "Saudi Arabia" ], "content": "Introduction\n\nFoodborne illness is a major, but preventable, public health problem across the world.1 The pattern of foodborne illness has changed dramatically due to the lifestyle and behavioral changes of the population. The frequency of foodborne illness outbreaks is growing because of newly discovered pathogens.2 The WHO has estimated that, globally, 1 in 10 people fall ill yearly and 420,000 die as a result of food contamination.3 However, the most recent data on the burden of foodborne diseases in the Kingdom of Saudi Arabia have not yet been published.\n\nThe major concern in the Kingdom of Saudi Arabia is the hajj season, where millions of pilgrims come from different parts of the world. The government is very particular about the quality of food items in the restaurant. However occasionally there have been reports on foodborne disease outbreak due to Salmonella, Bacillus cereus and Staphylococcus aureus.4\n\nThere has been a constant increase in the number of food poisoning cases in the past few years in the Kingdom of Saudi Arabia, especially during the summer and the Hajj season.5 According to the World Health Organization (WHO), populations in the developing countries including the Middle East, are more likely at risk of foodborne illness due to the restricted strategies in the disease surveillance and prevention and control programs. After the African and South-East Asian regions, it is estimated that the highest burden of foodborne diseases was reported in the Middle East and North African (MENA) region.6 Several studies, including literature reviews and primary studies related to foodborne illnesses in the Kingdom of Saudi Arabia have been conducted to assess the burden.7–12\n\nMost of the countries across the globe have some kind of mechanism to report foodborne illness. In the last ten years, countries such as Canada, England, Japan and the United States generated an annual report on the cases of foodborne diseases. It is apparent that the cases of staphylococcus intoxication have decreased in most of the countries except in Latin America, due to high consumption of unpasteurized milk and cream-filled desserts. However, the cases of salmonella increased all over the world and new agents such as Escherichia coli 0157:H7 were reported in many countries.13,14\n\nCases of Vibrio vulnificus septicaemias have been increasingly reported during the last few years in hospitals in these regions, especially in the Gulf of Mexico states. The patients are more likely to die if the treatment is not given at the appropriate time. The death associated with foodborne diseases is multifactorial and it is also based on the toxicity of the agents.15\n\nHepatitis E is one of the major public health issues in the entire world. It is mainly transmitted through contaminated food. The sporadic cases of hepatitis E are widely reported in many industrialized nations in Europe, Asia and North America. The burden due to hepatitis E can be reduced through implementation of an effective disease surveillance program and health education.16\n\nThe public awareness of food safety has been increasing in the last few years. Though the national statistics of several countries showed a steady decrease in foodborne illness; still the threat to the population continues due to new emerging pathogens. In order to tackle the new emerging pathogens, the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have introduced a risk-based concept such as Acceptable Level of Protection (ALOP) and Food Safety Objectives (FSOs). These two organisations started using a risk-based approach primarily for controlling the public health hazards due to food insecurity. They have adopted and utilized risk analysis as their main framework to ensure food safety. Microbiological risk assessment has been carried out by the government authority as a part of evaluation of foodborne microbiological hazards to public health. The government has started to make a decision on microbiological safety based on this Microbiological risk assessment.17\n\nGeneral physicians play an important role in the detection and treatment of foodborne illness as they are the first point of contact and a trusted primary source of information for the general public.16 Primary health centers are the first point of contact of the healthcare delivery system, where people in all countries visit for accessing the healthcare services.17\n\nPhysicians play a significant role in communicating public health messages to the patients, as the information given is trusted by the patient. Therefore, the physicians must provide information regarding foodborne diseases, at least to the vulnerable population. However, some physicians may not value the counselling session provided for the patients about the foodborne diseases due to the lack of knowledge on food safety, lack of time and lack of perceived benefit.18\n\nEven though there are common guidelines for the diagnosis and treatment of foodborne illness, these vary across physicians. The diagnosis and treatment procedures also vary between physicians working in public and private primary healthcare centers. The literature indicates that misunderstandings occur between the healthcare providers and the patients due to the absence of cultural knowledge and awareness, as well as through the lack of understanding and flexibility.3\n\nFurthermore, a one-dimensional biomedical perspective is frequently found among the healthcare service providers rather than a holistic perspective for providing patient care.5,7,8 The self-administered questionnaire is a tool for assessing the insight and experiences of individuals who work alongside the healthcare organization. It is also considered to be another way for evaluating the acceptability and awareness about the multiplicity in healthcare institutions.9\n\nValidated and reliable instruments are lacking for the measurement of the knowledge, attitudes, and practices (KAPs) regarding foodborne illness among physicians in the Kingdom of Saudi Arabia, as well as in all the Gulf Cooperation Council (GCC) countries. The aim of this study was to construct a valid and reliable instrument for assessing the KAPs of physicians in the Kingdom of Saudi Arabia toward foodborne illness.\n\n\nMethods\n\nThis is a cross-sectional study conducted in the selected public and private primary health care centers in the city of Abha located in the south western region of Asir in the Kingdom of Saudi Arabia. By using the Raosoft sample size calculator19; the sample size has been calculated with a 5% margin of error, and confidence interval of 95 %, a response distribution of 50% and a total population size of 180 (n = 180). The final sample size was n = 125.\n\nMultistage cluster sampling was used in order to recruit the physicians from the selected public and private primary health care centers. A formal permission was obtained from the ministry of health for conducting this particular study on primary health care centers. Permission from the private primary health care centers was obtained separately in order to collect the data for this study.\n\nPhysicians in the selected public and private primary health care centers in the city of Abha were approached. Individuals who agreed to participate and had given written consent were included in this study.\n\n\nQuestionnaire development\n\nThere were various phases involved in developing the KAP tool for confirming the validity and reliability. The items in the instrument were designed by a group of experts from the fields of nutrition, microbiology and public health during the first phase. The pilot study, in addition to face, content, and construct validity (primarily designed for validating the structure of the questionnaire) was carried out to determine the validity and reliability of the questionnaire. Factor analysis and the test-retest reliability were performed to ensure the internal consistency and the stability of the questionnaire. Same study participants were asked to complete the questionnaire for the second time within the interval of seven days, to measure the test-retest reliability.\n\n\nEvaluating item clarity\n\nThe questionnaire was distributed to 125 physicians from both the public and private healthcare sectors to collect information about foodborne illness. After collecting the completed questionnaires, each response was examined carefully looking at the consistency of the answers indicating the questions were widely comprehensible. The questionnaire was then modified to enhance its clarity.\n\n\nEvaluating face validity\n\nTo appraise the face validity of the questionnaire, ten experts from the Department of Public Health, College of Health Sciences, Saudi Electronic University were selected based on their research experience. They were provided with the questionnaire and the specific objectives of this research. The experts were asked to provide their opinions about the questionnaire related to these research objectives. The feedback from the experts regarding the questionnaire was accepted and incorporated to endorse the face validity.\n\n\nEvaluating content validity\n\nTo improve content validity, the experts were randomly selected from the authors listed in the publication derived from the Pubmed using key word “foodborne illnesses” and were invited to evaluate the content of the questionnaire. The specialist team included public health professionals, microbiologists, and nutritionists. Their recommendations and perspectives were deliberated and integrated into the questionnaire, thereby helping to establish the content validity of the questionnaire.\n\nThe final version of the questionnaire contains information about the KAPs related to foodborne illness and it is available as an extended data.20 Knowledge, in this paper, refers to the accurate technical knowledge among the physicians about foodborne illness, whereas attitude represents the perceptions of physicians regarding foodborne illness. Practices denote the existing management and treatment modalities that the physicians follow when handling food poisoning cases. The questionnaire comprises of four sections. Section one consists of eight questions on collecting various socio-demographic information from the physicians; section two consists of seven questions for determining the knowledge level among the physicians about foodborne diseases; section three consists of seven questions describing the perceptions of physicians about foodborne illness; and section four consists of ten questions for evaluating the current practices of physicians related to the diagnosis and management of foodborne diseases.\n\n\nData analysis\n\nThe data analysis was carried out using SPSS (Version 16.0, SPSS Inc. Chicago, IL, USA). The raw data of the food Poisioning study has been deposited as underlying data (v5.5).21 The internal consistency of the questionnaires was determined using Cronbach's α at a satisfactory level that varied between 0.70 and 0.95.22,23 Pearson's correlation coefficient was used to calculate the test-retest reliability. Cross tabulation and the kappa measure of agreement (k) were used to measure the test-retest agreement of the KAP questionnaire. A two-sided p-value <0.05 was considered statistically significant. As per Cade et al. large correlation coefficients were defined as 0.5 or greater, which indicated higher reliability.24,25 The strength of agreement was calculated using kappa, and according to Masson et al., the kappa measure of agreement (k) can be classified as follows: poor (<0.20), fair (0.21–0.40), moderate (0.41–0.60), good (0.61–0.80), and very good (0.81–1.00).26\n\n\nEthical considerations\n\nEthical permission was obtained from the research ethics committee from the College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia (REC#2017-03-08). The study was conducted in Abha, Asir Region, where King Khalid University is a designated institutional ethical committee for providing approval for all the research that are confined to the Asir Region. Written consent was obtained from all study participants. Anonymized data were used for analysis and interpretation. The questionnaire and the consent form were securely stored in locked file cabinets and access was given to the specific researchers involved in data analysis.\n\n\nResults\n\nA total of 125 from 160 physicians who participated in this study were selected from the government and private primary healthcare centres in the Abha city, with a response rate of 78.13%. Table 1 shows the demographic information of the study participants.\n\nMajority of the physicians (41.6%) were in age group of 35-44 years, 68.8% were male and 35.2% practice family medicine. Majority of physicians (54.4%) reported practising experience of 6-15 years.\n\nTable 2 provides scale statistics for the 31 items. The mean value was 65.33 with a variance of 95.18 and a standard deviation of 9.39. The reliability values were estimated using Cronbach's α. The Cronbach's α, which represents the raw or unstandardized value of alpha, was 0.723. However, Cronbach's alpha, which is based on standardized items, was 0.704; thus, the inference showed that the stronger items are inter-related. From the 31 items that were included in the analysis for reliability, three questions were not in the acceptable range of Cronbach's alpha. These were excluded in the main study. These results prove that the tests are expected to be reliable.\n\nTable 3 shows the results from the test-retest analysis using the Pearson correlation coefficient (r). Correlation coefficients were very high for the majority of questions (> 0.7 for 29/31 questions) in the questionnaire. The highest correlation was observed for gender and the lowest correlation was observed for the target microorganisms in canned goods. According to the results from the bivariate correlations, the highest and lowest agreements from test to retest were observed for the same variables as for the Pearson correlation coefficient. Twenty questions from the questionnaire showed a very good strength of agreement, and the remaining nine questions had a good strength of agreement.\n\n\nDiscussion\n\nA study was conducted among 125 physicians from selected governmental and non-governmental primary healthcare centres in Abha city to evaluate the KAPs among physicians toward foodborne illness. The content and face validity assessments were completed by specialists in related fields, inclusive of microbiologists and public health practitioners. Item clarity was tested by conducting a pilot study among these 125 physicians. The same evaluations of the face and content validity were performed in a study conducted in Malaysia to validate the questionnaire on KAPs related to lifestyle.27 The internal reliability between the questions was calculated by using Cronbach's α. Our KAP questionnaire on foodborne illness showed adequate internal uniformity (α = 0.723). Numerous previously published studies on Cronbach's α have projected a satisfactory α value that varied between 0.70 to 0.95 for this test.28,29 The results of the present study, α = 0.723, are supported by the values mentioned in recently published research articles.30 A study conducted in Iran for assessing the KAPs of disaster preparedness reported an internal consistency of α = 0.785, which is somewhat greater than the current study.30 Another study conducted in Malaysia for assessing the KAPs related to dengue fever prevention, also demonstrated internal consistency with Cronbach's α = 0.798, which is higher than the results of the present study.22A similar value of Cronbach's α was obtained in a study aimed at measuring the KAPs for the prevention of dengue fever among the male population in the Maldives.23 A study among Iraqi patients about the KAPs related to immunization, reported an internal consistency with Cronbach's α score of 0.812, which is also higher than that observed in our study.31\n\nA study conducted in Spain for assessing the development and validation of the questionnaire related to academic stress in secondary education found that the internal consistency Cronbach's α value is 0.77, which is slightly higher than the current study.32 Similarly, a study in Malaysia of a KAP questionnaire among the childcare providers reported a Cronbach's α coefficient ranging between 0.89 to 0.90, which is much higher than the present study.33\n\nLikewise, a study conducted in Iran for evaluating the KAPs related to obesity among adults and adolescents revealed a Cronbach's α score of 0.72, which is similar to the current study.34 A similar study conducted among Iranian adults regarding the KAPs related to oral health reported a Cronbach's α of 0.82, which is higher than the value reported in this study.35 To determine the KAPs regarding vitamin D among adults in Tehran, Amiri P. et al. presented an internal consistency value of 0.60, which is less than recorded in our study.36\n\nThe current study results showed that the correlation coefficient was high for many questions in the KAPs questionnaire about foodborne illness. The results of a study by Rachel et al.37 on the test-retest reliability of a new questionnaire on diet and eating behaviors, found that the correlation coefficient was high for most questions, which supports the results of this study.\n\nThe present study indicated that 29 of 32 items had a good level of reliability. Similarly, a study conducted by Luísa. et al.38 in Brazil on the reliability of a questionnaire for the assessment of food safety knowledge, perceptions, and practices found significance in stability and reliability of the questions. Comparable results were observed in the studies that were conducted previously about the knowledge, perceptions and behaviors related to food safety, which also support the results of the current study.39,40\n\nThere are certain limitations in this study. Although the study had representative samples of physicians from both public and private primary health care centers; the sample was drawn only from one particular region (Asir region) in the Kingdom of Saudi Arabia. Self-reported questionnaire was the primary source for collecting the information from the physicians, therefore, the quality of information delivered by the study participants is arguable.\n\n\nConclusion\n\nA questionnaire was framed and developed to evaluate the KAPs among physicians toward foodborne illness. It was shown to have adequate validity and reliability. This questionnaire can be classed as a noteworthy instrument for assessing the KAPs related to foodborne illness among physicians across the country. The study highlights the importance of a standardized questionnaire to assess KAPs among physician towards foodborne illness to initiate an appropriate interventional program for physicians to reduce the burden of foodborne illnesses.\n\n\nData availability\n\nHarvard Dataverse, V1: Data-Food Poisoning. http://doi.org/10.7910/DVN/QP1KVI21.21\n\nThe project contains the following underlying data:\n\n[Data-Food Poisioning] (Raw, unaveraged qPCR data).\n\nHarvard Dataverse, V1: “Data-Food Poisioning”.\n\nhttps://doi.org/10.7910/DVN/G67UW3.20\n\nThis project contains the following extended data:\n\n• Annexure 1: Questionnaire on Socio-demographic Variables\n\n• Annexure 2: Questions regarding the knowledge, attitude and practices about food borne illness\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 express our gratitude to the Deanship of Scientific Research, Saudi Electronic University, Riyadh, Kingdom of Saudi Arabia.\n\n\nReferences\n\nWorld Health Organization: Food safety and food-borne illness. Fact Sheet No 237. 2007. Reference Source\n\nTauxe RV: Emerging foodborne pathogens. Int J Food Microbiol. 2002; 78: 31–44. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: WHO's a first ever global estimate of foodborne diseases.2015. Reference Source\n\nTodd ECD: Foodborne disease and food control in the Gulf States. Food Control. 2017; 73: 341–366. Publisher Full Text\n\nAl-Mazrou YY: Food poisoning in Saudi Arabia: Potential for prevention? Saudi Med J. 2004; 25: 11–14. PubMed Abstract\n\nTodd ECD: Foodborne Disease in the Middle East. In: Badran A, Murad S, Baydoun E, et al.. (eds) Water, Energy & Food Sustainability in the Middle East. Springer; 2017: 389–440. Publisher Full Text\n\nBharathirajan R, Gopinathan R, Prakash M: Microbial Management of Household Cold Storage Exploratory Study in Jeddah, Saudi Arabia. Int. J. Curr. Microbiol. Appl. Sci. 2012; 1: 50–55.\n\nAl-Dughaym AM, Altabari GF: Safety and quality of some chicken meat products in Al-Ahsa markets-Saudi Arabia. Saudi J Biol Sci. 2009; 17: 37–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomily AM, Sayyed SB, Habib HA, et al.: Salmonella isolates' serotypes and susceptibility to commonly used drugs at a tertiary care hospital in Riyadh, Saudi Arabia. J Infect Dev Ctries. 2012; 6: 478–482. PubMed Abstract \| Publisher Full Text\n\nMinistry of Health: Annual Food Poisoning Report 1422 Riyadh. Food Poisoning Department. 2003: 12.\n\nHayajneh F, Mahmoud F, Althomali AMH, et al.: Prevalence and characterization of hydatidosis in animals slaughtered at Al Taif abattoir, Kingdom of Saudi Arabia. Open J. of Animal Sci. 2014; 4: 38–41. Publisher Full Text\n\nWorsley A: Perceived reliability of sources of health information. Health Educ Res. 1989; 367–76(1989): 4. Publisher Full Text\n\nTodd ECD: Worldwide Surveillance of Foodborne Disease: the Need to Improve. J Food Prot. 1996; 59(1): 82–92. PubMed Abstract \| Publisher Full Text\n\nTebbutt GM, Wilson D, Holtby I: A study of 279 general outbreaks of gastrointestinal infection in the north-East region of England. Int J Environ Res Public Health. 2009; 6(2): 547–557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTodd ECD: Preliminary Estimates of Costs of Foodborne Disease in the United States. J Food Prot. 1989; 52(8): 595–601. PubMed Abstract \| Publisher Full Text\n\nTodd ECD: Microbiological safety standards and public health goals to reduce foodborne disease. Meat Science. 2004; 66(1): 33–43. PubMed Abstract \| Publisher Full Text\n\nYugo DM, Meng XJ: Hepatitis E virus: foodborne, waterborne and zoonotic transmission. Int J Environ Res Public Health. 2013; 10(10): 4507–4533. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharon Hurd CP, Hoefer D, Hadler JL, et al.: Assessment of Physician Knowledge and Practices Concerning Shiga Toxin–Producing Escherichia coli Infection and Enteric Illness. Foodborne Diseases Active Surveillance Network (FoodNet). STEC Knowledge among Surveyed Physicians. Clin Infect Dis. 2012; 54: 446–452. PubMed Abstract \| Publisher Full Text\n\nSeymour JW, Polsky DE, Brown EJ, et al.: The Role of Community Health Centers in Reducing Racial Disparities in Spatial Access to Primary Care. J Prim Care Community Health. 2017: 147–152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArendt S, Rajagopal L, Strohbehn C, et al.: Reporting of foodborne illness by U.S. consumers and healthcare professionals. Int J Environ Res Public Health. 2013; 10(8): 3684–3714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaosoft, Sample size calculator: 2004. Reference Source\n\nHarvard Dataverse, V1: Data-Food Poisioning.Publisher Full Text\n\nHarvard Dataverse, V1: Data-Food Poisioning.Publisher Full Text\n\nAbdullah MN, Wan Azib WNH, Mohd Harun MF, et al.: Reliability and Construct Validity of Knowledge, Attitude and Practice on Dengue Fever Prevention Questionnaire. Am Int J Contemp Res. 2013; 3: 69–75.\n\nAhmed N: Knowledge, attitude and practice of dengue fever prevention among the people in Male, Maldives. J Health Res. 2007; 22: 33–37.\n\nField AP: Discovering statistics using SPSS: (and sex and drugs and rock 'n' roll) 2009; 6th Edition. Los Angeles. 2009.\n\nCade J, Thompson R, Burley V, et al.: Development, validation and utilization of food-frequency questionnaires – a review. Public Health Nutr. 2002; 5: 567–587. PubMed Abstract \| Publisher Full Text\n\nMasson LF, McNeill G, Tomany JO, et al.: Statistical approaches for assessing the relative validity of a food-frequency questionnaire: use of correlation coefficients and the kappa statistic. Public Health Nutr. 2003; 6: 313–321. PubMed Abstract \| Publisher Full Text\n\nHiew CC, Chin Y, Chan YM, et al.: Development and Validation of Knowledge, Attitude and3 Practice on Healthy Lifestyle Questionnaire (KAP-HLQ) for Malaysian Adolescents. J Nutr Health Sci. 2015; 2: 1–11.\n\nCohen R, Swerdlik ME: Psychological testing and assessment. 19th Edition. Boston: McGraw-Hill Higher Education; 2010.\n\nDeVellis R: Scale development: theory and applications 2003. 2nd ed. Thousand Okas: Theory and application; 2003.\n\nTavan H, Waleyeh M, Azadi A, et al.: Questionnaire to Measure Nurses' Disaster Preparedness. J Clin Diag Res. 2016; 10: 6–9.\n\nAwadh AI, Hassali MA, Al-lela OQ, et al.: Immunization knowledge and practice among Malaysian parents: a questionnaire development and pilot-testing. BMC public health. 2014; 14: 1100–1107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcía-Ros R, Pérez-González F, Tomás J: Development and Validation of the Questionnaire of Academic Stress in Secondary Education: Structure, Reliability and Nomological Validity. Int J Environ Res Public Health. 2018; 15(9): 2023. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZakria NM, Tengku Ismail TA, Wan Mansor WNA, et al.: Validation of Infant and Young Child Feeding Questionnaire for the Assessment of Knowledge, Attitudes and Practices among Child Care Providers: The IYCF-CCPQ. Int J Environ Res Public Health. 2019; 16(12): 2147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGolshiri P, Parastoo Y, Sarrafzadegan N, et al.: Developing and validating questionnaires to assess knowledge, attitude, and performance toward obesity among Iranian adults and adolescents: TABASSOM study. ARYA Atherosclerosis J. 2012; 7: 119–124.\n\nRasouli-Ghahroudi AA, Rokn AR, Afshin K, et al.Designing and standardizing a questionnaire for evaluating knowledge, attitude, and practice of Iranian adults with cardiovascular diseases about oral health. ARYA Atheroscler J. 2013; 9: 350–356. PubMed Abstract \| Free Full Text\n\nAmiri P, Golaleh A, Sadrosadat H, et al.: Psychometric Properties of a Developed Questionnaire to Assess Knowledge, Attitude and Practice Regarding Vitamin D. Nutrients. 2017; 9: 471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMyr RK, Bere E, Øverby NC: Test-retest reliability of a new questionnaire on the diet and eating behaviour of one year old children. BMC Res Notes. 2015; 8: 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHelena Maia LL, William W, Bento VA: Reliability of a questionnaire to assess food safety knowledge, perceptions, and practices among outpatients with human immunodeficiency virus. Cad. Saúde Pública. 2007; 23: 971–976. PubMed Abstract \| Publisher Full Text\n\nMedeiros LC, Hillers VN, Chen G, et al.: Design and development of food safety and attitude scales for consumer food safety education. J Am Diet Assoc. 2004; 104: 1671–1677. PubMed Abstract \| Publisher Full Text\n\nHaapala I, Probart C: Food safety knowledge, perceptions and behaviors among middle school students. J Nutr Educ Behav. 2004; 36: 71–76. PubMed Abstract \| Publisher Full Text" }	[ { "id": "87510", "date": "29 Jun 2021", "name": "Nazik Eltayeb Musa Mustafa", "expertise": [ "Reviewer Expertise Food safety", "bacteriology", "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\nGeneral comments:\nThis is a significant study concerned with the development of tools used to investigate knowledge, attitude, and practices of physicians engaged in the diagnosis and treatment of food-borne diseases.\n\nThe study tackles important issues related to food safety KAP and it participates actively in providing knowledge about physician food safety KAP. However, food safety descriptive findings related to knowledge attitude and practices could be included in the manuscript to maximize the benefit of this research for instance the descriptive data could be extended beyond the demographic data to Include (concisely) the major findings of physician knowledge attitude and practices instead of presenting them only in the forms of Internal consistency and test-retest reliability.\n\nNevertheless, the study gives a deep inside look at the construction of the questionnaire in terms of reliability and validity and it gives substantial weight to general questionnaire construction techniques.\n\nSpecific comments:\nAbstract:\nThe phrase ‘Physicians (n=125) were opportunistically recruited’ does not clearly reflect the multistage cluster sampling technique applied for selected public and private primary health care centers mentioned in the method section\nIntroduction:\nIn the 2nd paragraph, line 2 the sentence ‘The government is very particular about the quality of food items in the restaurant; may need modification to reflect more generalized food control activities i.e., food establishments instead of the phrase ‘the restaurant’.\n\nIn the same paragraph bacterial names Bacillus cereus and Staphylococcus aureus should be in italics (there is no need to italicize the name Salmonella).\n\nThe bacterial names Escherichia coli (paragraph 4 line 5) should also be italicized.\n\nSame should be done for Vibrio vulnificus (paragraph 5 line 1).\n\nParagraph 6 needs revision to specify the Saudi government, a slight reordering of the sentences in this paragraph will increase its readability.\n\nI suggest merging Paragraphs 7 and 8 to avoid redundancy in specifying the importance of the Physicians roles in food safety.\n\nMethods:\nA slight change in the abstract is needed to reflect the methods followed in selecting samples.\n\nIt appears to the reader that evaluating face validity and evaluating content validity for the questionnaire were done in the same way, hence no need to repeat the sentences, I suggest merging the evaluation of face and validity.\n\nIn the sentence ‘experts were randomly selected from the authors listed in the publication derived from the PubMed using the keyword “foodborne illnesses”'. It is not clear if the author includes Saudi Arabia among search keywords? More elaboration is needed here about the responders’ number and distribution in fields.\nThe questionnaire: In annexure 2:\nSome of the questions, for example, question 13 could have had been asked with less ambiguity for example question 13 (The target microorganism in canning is?) could be asked in a more straightforward way (i.e. The most encountered food poisoning microorganisms from canned food is?).\n\nSome of the questions regarding the knowledge, attitude, and practices about foodborne illness are barely related to food safety issues generally tackled by physicians, for example, question 19 is quite lengthy and hardly fulfills the required target.\n\nResults and discussion:\nThese are professionally written sections with clear tables and main finding paragraphs.\n\nI could not find the descriptive results of the findings related to knowledge, attitude, and practices of physicians towards food safety. It seems that it is the author's preference not to present descriptive data in this regard. Adding such data will benefit readers from the field with fewer statistics knowledge and increase the manuscript audience.\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? Partly", "responses": [ { "c_id": "7086", "date": "02 Sep 2021", "name": "Mohammed AL-Mohaithef", "role": "Author Response", "response": "We have incorporated the reviewer comments and uploaded the new version of the manuscript." } ] } ]	1	https://f1000research.com/articles/10-470
https://f1000research.com/articles/10-482/v1	16 Jun 21	{ "type": "Systematic Review", "title": "High flow nasal oxygen for acute type two respiratory failure: a systematic review", "authors": [ "Asem Abdulaziz Alnajada", "Bronagh Blackwood", "Abdulmajeed Mobrad", "Adeel Akhtar", "Ivan Pavlov", "Murali Shyamsundar", "Asem Abdulaziz Alnajada", "Bronagh Blackwood", "Abdulmajeed Mobrad", "Adeel Akhtar", "Ivan Pavlov" ], "abstract": "Background: Acute type two respiratory failure (AT2RF) is characterized by high carbon dioxide levels (PaCO2 >6kPa). Non-invasive ventilation (NIV), the current standard of care, has a high failure rate. High flow nasal therapy (HFNT) has potential additional benefits such as CO2 clearance, the ability to communicate and comfort. The primary aim of this systematic review is to determine whether HFNT in AT2RF improves 1) PaCO2, 2) clinical and patient-centred outcomes and 3) to assess potential harms. Methods: We searched EMBASE, MEDLINE and CENTRAL (January 1999-January 2021). Randomised controlled trials (RCTs) and cohort studies comparing HFNT with low flow nasal oxygen (LFO) or NIV were included. Two authors independently assessed studies for eligibility, data extraction and risk of bias. We used Cochrane risk of bias tool for RCTs and Ottawa-Newcastle scale for cohort studies. Results: From 727 publications reviewed, four RCTs and one cohort study (n=425) were included. In three trials of HFNT vs NIV, comparing PaCO2 (kPa) at last follow-up time point, there was a significant reduction at four hours (1 RCT; HFNT median 6.7, IQR 5.6 – 7.7 vs NIV median 7.6, IQR 6.3 – 9.3) and no significant difference at 24-hours or five days. Comparing HFNT with LFO, there was no significant difference at 30-minutes. There was no difference in intubation or mortality. Conclusions: This review identified a small number of studies with low to very low certainty of evidence. A reduction of PaCO2 at an early time point of four hours post-intervention was demonstrated in one small RCT. Significant limitations of the included studies were lack of adequately powered outcomes and clinically relevant time-points and small sample size. Accordingly, systematic review cannot recommend the use of HFNT as the initial management strategy for AT2RF and trials adequately powered to detect clinical and patient-relevant outcomes are urgently warranted.", "keywords": [ "High flow nasal oxygen", "high flow nasal therapy", "acute type 2 respiratory failure", "acute hypercapnic respiratory failure", "acute exacerbation of chronic obstructive pulmonary disease" ], "content": "Introduction\n\nAcute type two respiratory failure (AT2RF) is characterised by arterial hypercapnia (PaCO2 >6 kPa or >45 mmHg) and its treatment requires ventilator support in a significant proportion of cases.1 Chronic obstructive pulmonary disease (COPD) is the second-most widespread disease in the UK, with 1,201,685 cases reported in 2013. Acute exacerbations of COPD (AECOPD) account for 100,000 admissions annually in England. Of these, around 20% will present with or develop hypercapnia, an indicator of increased risk of death.2,3 Development of AT2RF in patients with COPD is associated with a significantly increased risk for requiring invasive ventilation and mortality rate,4,5 with mortality rates up to 15% in patients who require admission to the intensive care unit (ICU).\n\nThe treatment of AT2RF is aimed at the underlying pathological processes such as fluid overload, bronchospasm and infection along with controlled oxygen therapy, to decrease the work of breathing. Patients often require ventilator support that may be non-invasive ventilation (NIV) or invasive mechanical ventilation (IMV). Current guidelines recommend the use of NIV.1 Current evidence has established the role of NIV in improving arterial oxygenation, hypercapnia, acidosis, mortality and intubation rates.6 However, the NIV failure rate ranges from 15 to 25%, with some evidence stating a failure rate as high as 60%.7–10 The factors leading to NIV failure include non-compliance due to claustrophobia, delirium, sputum retention, reduced communication and skin compromise such as skin necrosis in the nasal bridge.1,10,11\n\nHigh flow nasal oxygen or insufflation (described as high flow nasal therapy (HFNT) in this manuscript) is novel respiratory support that integrates humidified air with a high flow rate of up to 60 L/minute. Reported benefits from HFNT include consistent fractional inspired oxygen delivery, dead space washout, reduced work of breath, comfort and tolerability, ability to communicate, mucous clearance and NIV-like effects, which makes it a more tolerable method for patients.12–15 In type I ARF, HFNT has been demonstrated to lead to improved oxygenation, lower rates of endotracheal intubations and lower mortality.16\n\nIn the last 10 years, evidence has emerged for its increasing use and a role for these modalities in clinical practice for the treatment of AT2RF.15,17 Several observational studies have suggested potential benefits of HFNT for AT2RF as demonstrated by improved gas exchange and acidosis,18,19 and reductions in the respiratory rate and work of breathing.20–22 Individual studies have shown that HFNT improves blood gas levels in AT2RF patients21–23 and is associated with improved comfort.23\n\nAdequate respiratory support through controlled oxygen, reduced work of breathing and CO2 clearance is essential to prevent intubation and invasive ventilation. NIV, despite its frequent use, has limitations and a high failure rate. HFNT might overcome the limitations of NIV and could be used in AT2RF patients as an initial intervention or in patients who do not tolerate NIV. Despite the increase in current literature suggesting benefits from the use of HFNT in AT2RF, current evidence is limited. Other systematic reviews are exploring the use of HFNT for the management of AT2RF post-extubation and after initial stabilization of the patient using NIV.24,25 However, there is no systematic review that focuses on the use of HFNT as an initial management strategy for AT2RF.\n\nThe primary objective of this systematic review was to determine whether the use of HFNT for patients with AT2RF improves PaCO2 in comparison to LFO or NIV. Secondary objectives were to examine whether HFNT in patients with AT2RF improves other clinical or patient-centred outcomes and to assess any potential harms.\n\n\nMethods\n\nThe systematic review was registered in the PROSPERO database (CRD42019148748, 05/09/2019) and published a priori. We conducted this systematic review according to the PRISMA guidelines (see Reporting guidelines).26,27\n\nRandomized controlled trials, uncontrolled trials and cohort studies were included if they compared the use of HFNT with a flow rate >20 L/minutes versus LFO or NIV. We included studies of adult (≥18 years old) patients with AT2RF (>6 kPa or >45 mmHg) managed as inpatients in an acute care setting (emergency department, respiratory ward or critical care units). We excluded reports that described the use of HFNT in peri-operative settings, drug overdose, or ventilator weaning.\n\nThe primary outcome for this review was the change in PaCO2 post-intervention (measured at time points reported by authors). The secondary outcomes were: respiratory parameters including pH, the partial arterial pressure of oxygen (PaO2), dyspnoea score, tidal volume and minute volume; mucous clearance (before, during or after HFNT application); the level of consciousness; patient comfort; intubation rate; length of stay in hospital; mortality; post-discharge COPD exacerbation rate and readmission rate secondary to AECOPD.\n\nWe searched the electronic databases MEDLINE, EMBASE and the Cochrane Central Register of Controlled Trials from January 1999 to January 2021. The databases search was conducted on 15/01/2021. Language restrictions were not applied. In addition, we searched Google Scholar and references of all articles for any additional studies. With the assistance of a professional librarian, we developed a systematic search strategy using appropriate keywords and MeSH terms and these are detailed in the data availability section (see Extended data).27 The systematic review software management system Covidence was used to store citations, remove duplication and aid screening.\n\nTwo review authors (AAA and MS) independently screened the titles and abstracts of all citations. The full texts of all potentially eligible studies were independently reviewed for inclusion confirmation. Any disagreement was resolved through discussion within the review team.\n\nData were independently extracted from included studies using a standardized data extraction form by two reviewers (AAA and MS). The information extracted included type and setting of the study, recruitment information, participant characteristics (age and underlying conditions), inclusion criteria, nature of interventions, in each group (e.g. flow rate and method of delivery), time-points of measurement and outcomes. Any disagreement was resolved through discussion with BB. Data that were unavailable or insufficient from publications were requested from study authors.\n\nTwo reviewers (AAA and MS) independently assessed the quality of included studies using the Cochrane risk of bias tool for RCTs and the Newcastle-Ottawa scale for cohort studies.28,29 Each potential source of bias was marked as high, low or unclear. We assessed the quality of the evidence associated with HFNT for AT2RF using GRADE to determine the strength of the evidence into one of four grades: high, moderate, low or very low.30 The quality of evidence is reported in the Summary of Findings (SOF) tables (Tables 1,2,3).\n\n* Abbreviations: HFNT, High flow nasal therapy; IQR, Interquartile range; kPa, Kilopascal; MD, mean difference; NIV, Non-invasive ventilation; OR, Odd ratio; SD, Standard deviation.\n\n‡ N/R - Not reported.\n\n* Abbreviations: HFNT, High flow nasal therapy; IQR, Interquartile range; MD, mean difference; n/N, Number of patients NIV, Non-invasive ventilation; OR, Odd ratio; SD, Standard deviation.\n\n* Abbreviations: HFNT, High flow nasal therapy; LFO, kPa, Kilopascal; Low flow oxygen; MD, mean difference; SD, Standard deviation.\n\nMeasurement of effect\n\nRevMan software (Review Manager, version 5.3) was used for data analysis. Results are reported as odds ratios (ORs) with 95% confidence intervals (CIs) for binary variables and mean differences (MD) with 95% CIs for continuous variables. A meta-analysis was planned, but there were insufficient studies and results are presented narratively.\n\nSubgroup and sensitivity analysis\n\nThe planned subgroup analyses of patient conditions (COPD, neuromuscular disorders, and interstitial lung disease), and the planned sensitivity analysis excluding trials with a high risk of bias could not be undertaken due to the low number of trials.\n\n\nResults\n\nThe search identified 727 records. Following the removal of duplicates and non-eligible studies, 39 full-text studies were screened and 34 studies were excluded. Five studies with 425 participants were included in this review (Figure 1).22,23,31–33\n\nThe characteristics of the included studies are summarized in Table 4. Four studies were RCTs22,23,31–33 and one was a cohort study.22 Four studies compared HFNT with NIV22,31–33 and one RCT compared HFNT with LFO using simple nasal prongs.23 The disease state of interest was an acute-moderate hypercapnic respiratory failure (n = 88) in one study,22 and AECOPD (n = 337) in the remaining four studies.23,31–33\n\n* Abbreviations: ACOPD, Acute chronic obstructive pulmonary disease; COPD, Chronic obstructive pulmonary disease; HFNT, High flow nasal therapy; n/N: Number of patients; NIV: Non-invasive ventilation; RCT, Randomized controlled trial.\n\nThe risk of bias assessments for the four RCTs are described in Figure 2.23,31–33 Blinding of participants and personnel were not possible in the trials. One trial showed a high risk for selection bias due to unexplained randomization sequence and allocation concealment.32 The trials showed a high risk or unclear risk of detection bias due to no31,32 or unclear23 blinding of the outcomes assessor. One trial showed a high risk of attrition bias due to unreported incomplete data.31 The cohort study showed a low risk of bias in all domains and did not describe how the outcomes were assessed.22\n\nChanges in PaCO2 after the intervention was reported in all five studies (Table 5),22,23,31–33 four studies compared HFNT to NIV.22,31–33 Doshi et al.31 reported no significant difference in PaCO2 at one hour between HFNT and NIV but there was a significant reduction in PaCO2 at four hours (HFNT 6.7, 5.6 – 7.7 vs NIV 7.6, 6.3 – 9.3 (Median, interquartile range (IQR)). In the other studies comparing HFNT to NIV,22,32,33 there was no significant difference in PaCO2 at various time-points with a similar trend in PaCO2 (Figure 3). Pilcher et al.23 compared HFNT with LFO at various five-minute time intervals with no significant difference, but when adjusted for the baseline PaCO2, they reported a significant improvement in PaCO2 by HFNT when compared to LFO.\n\n* Abbreviations: RCT, Randomized controlled trial; HFNT, High flow nasal therapy; NIV, Non-invasive ventilation; LFO, Low flow oxygen; n/N, Number of patients; SD, Standard deviation; min, minutes.\n\npH level was reported in three studies.22,31,32 Doshi et al.31 reported no significant difference in pH between HFNT and NIV at one hour (HFNT 7.36, 7.34-7.42 vs NIV 7.31, 7.27-7.37, (Median, IQR)) or four hours (HFNT 7.38, 7.34-7.42 vs NIV 7.35, 7.33-7.37, (Median, IQR)). Cong et al.32 reported no significant difference in pH between HFNT and NIV at 12 hours (MD -0.10, 95% CI -0.13, 0.06) or five days (MD -0.05, 95% CI -0.08, -0.01). Lee et al.22 showed no significant difference in pH between HFNT and NIV at six hours (MD 0.02, 95% CI -0.16, 0.20) or 24 hours (MD 0.00, 95% CI -0.03, 0.03). The PaO2 level was reported in four trials.22,31–33 Doshi et al.31 reported no significant difference in PaO2 between HFNT and NIV at one hour (HFNT 13.2, 10.7-19.2 vs NIV 15.1, 8.8-22.9, (Median, IQR)) or four hours (HFNT 11.1, 5.3-13.2 vs NIV 11.7, 10.3-12.9, (Median, IQR)). Cong et al.32 reported no significant difference in PaO2 between HFNT and NIV at 12 hours (MD 0.00 kPa, 95% CI −0.70, 0.70) or five days (MD −0.10 kPa, 95% CI −0.72, 0.52). Lee et al.22 reported no significant difference in PaO2 between HFNT and NIV at six hours (MD 0.00 kPa, 95% CI −1.30, 1.30) or 24 hours (MD −0.10 kPa, 95% CI −1.28, 1.08).\n\nPatient comfort was reported in three RCTs.23,32,33 Patient comfort assessed using a self-designed survey in Cong et al.32 a 10-point numerical rating scale in Cortegiani et al.33 and the Likert scale in Pilcher et al.23 showed that HFNT was more comfortable than LFO but louder than LFO (Table 6).\n\n* Answers to questions were made on a 1-5.\n\n‡ Abbreviations: RCT, Randomized controlled trial; HFNT, High flow nasal therapy; n/N, Number of patients; SD, Standard deviation; NIV, Non-invasive ventilation; MD, mean difference; SNP, Simple nasal prongs; MD, mean difference.\n\nThe intubation rate was reported in three studies comparing HFNT with NIV.22,31,33 Doshi et al.31 demonstrated no significant difference in intubation rate at 72 hours (RCT; OR 0.33 95% CI 0.06, 1.81). Cortegiani et al.33 reported no significant difference in intubation rate at two hours (RCT; OR 0.32 95% CI 0.01, 8.02) or six hours (RCT; OR 0.97 95% CI 0.06, 16.14). Lee et al.22 reported no significant difference at 30 days (cohort; OR 0.89 95% CI 0.34, 2.30) (Table 7).\n\n* 10-point numerical rating scale: where 0 is no discomfort and 10 is maximum discomfort\n\n‡ Abbreviations: HFNT, High flow nasal therapy; n/N, Number of patients; NIV, Non-invasive ventilation; IQR, Interquartile range.\n\nThe mortality rate was reported in two studies22,33 and there was no difference between HFNT and NIV groups (Table 7).\n\nThe dyspnoea score, measured by Modified Borg score, a self-reported rating of perceived dyspnoea on a scale of one to 10, with 10 being the worst, was reported in two trials.31,33 The reduction in the dyspnoea score was similar between HFNT and NIV at different time points in both trials (Table 8).\n\n‡ Abbreviations: High flow nasal therapy; n/N, Number of patients; NIV, Non-invasive ventilation\n\nLength of stay in hospital was reported by three trials31–33 comparing HFNT and NIV with no difference between the two groups (Table 9).\n\n* Abbreviations: HFNT, High flow nasal therapy; n/N: Number of patients; NIV: Non-invasive ventilation; OR: Odd ratio\n\n* Borg Modified Score: a self-reported rating of perceived dyspnoea on a scale of one to 10, with 10 being the worst.\n\n‡ Abbreviations: n/N, Number of patients; IQR, Interquartile range; HFNT, High flow nasal therapy; NIV, Non-invasive ventilation\n\n* Abbreviations: n/N, Number of patients; IQR, Interquartile range; HFNT, High flow nasal therapy; NIV, Non-invasive ventilation; MD, mean difference; SD, standard deviation\n\n\nDiscussion\n\nWithin the AT2RF patient population where HFNT is used as the initial management strategy, this systematic review has identified very few studies: four comparing HFNT with NIV and one comparing HFNT with LFO. HFNT, compared with NIV, showed a significant difference in PaCO2 after four hours of treatment,31 although the difference was not demonstrated at 24 hours,22 five days,32 six hours33 and a similar lack of difference is seen when compared to LFO at 30 minutes.23 The reduction in PaCO2 between the two groups at four hours demonstrated in Doshi et al.31 is not adjusted for the baseline difference in PaCO2 between the two groups. The absolute reduction of PaCO2, when compared to the baseline, was 0.8 kPa for the HFNT group and 0.99 kPa in the NIV group, which suggest that the significant difference was secondary to baseline difference rather than true clinical superiority. Compared with NIV or LFO, HFNT showed no difference in pH and PaO2 and has similar intubation rates, mortality and hospital length of stay. HFNT, when compared to NIV, is associated with better comfort as presented by Cong et al.32 and Cortegiani et al.,33 although this was not replicated in Pilcher et al.23 This systematic review found that despite the potential benefit of improved patient comfort and increasing use of HFNT in the treatment of AT2RF, the current evidence is quite poor. The certainty of the evidence was primarily impacted by the small number of trials and sample sizes, selection bias and few RCTs. Lack of blinding is a potential source of bias but the nature of the intervention precludes blinding, while the objective nature of the outcome measures reduces the risk of bias. Hence, objective outcome measures were not downgraded for lack of blinding while subjective measures such as comfort score and dyspnoea score were downgraded.\n\nIn AT2RF, the production of CO2 is increased due to additional work of breathing, increased metabolism and failure to clear CO2. NIV failure occurs in a quarter of these patients needing further IMV. The extent of reduction in pH, associated with the elevated CO2, is significantly associated with NIV failure.34 Any medical optimization introduced early after the detection of AT2RF should be aimed at improving CO2 clearance and pH because the development of respiratory acidaemia post-admission is associated with a mortality of 33%.3 While current evidence has convincingly established the benefits of NIV for AT2RF, evidence for newer and better-tolerated technologies to reduce hypercapnia is urgently required due to the high intolerance rate leading to a late failure.10\n\nIn this systematic review focused on early intervention for AT2RF patients, there is no difference in various respiratory parameters between HFNT and NIV except for one study showing an improvement in PaCO2 at a single time-point. HFNT is associated with a reduction in PaCO2 and an increase in pH similar to NIV. While this could suggest that HFNT is non-inferior to NIV, HFNT cannot be recommended as an alternative management strategy to reduce PaCO2 due to the low quality of evidence, lack of standardization of time-points for PaCO2 measurement and the lack of adequately powered sample sizes. A similar response in CO2 to HFNT is reported in COPD patients with stable type 2 respiratory failure,35 post-acute NIV,36 post NIV failure,37–39 post-extubation40 and during breaks in NIV.20\n\nStudies have shown a reduction in intubation rate and mortality between NIV versus usual care36 and a reduction in the length of hospital stay, lower incidence of complications with a longer-term benefit of fewer readmissions to hospital in the following year between NIV and IMV41 with one study suggesting a mortality benefit.42 HFNT, if equivalent to NIV, should ultimately reduce important outcomes such as intubation, mortality and health resource use. Three studies found no difference in intubation rate22,31,33 and three studies found no difference in length of stay31–33 thus suggesting therapeutic equivalence but the studies were not powered for these outcomes. Doshi et al.,31 showed that HFNT when compared to NIV had a similar therapy failure rate of approximately 25%. Patients receiving HFNT had a trend towards a shorter ICU stay, likely driven by a lower intubation rate in the HFNT group (5.9%) when compared to the NIV group (16.1%), which did not achieve statistical significance in this study that was not powered for this outcome.\n\nA key balancing outcome is an increase in adverse outcomes that have been highlighted in studies comparing NIV to usual care that include a delay in escalation to IMV,43 increased mortality when compared to immediate IMV, and increased mortality when IMV is delayed.44 In this systematic review, Lee et al.22 and Cortegiani et al.33 taken together with lower intubation rate in the HFNT arm,31 suggests that HFNT is unlikely to be associated with harm through delayed initiation of IMV, but this hypothesis needs to be confirmed in a clinical trial.\n\nOne of the putative benefits of HFNT is patient comfort due to the lack of a tight-fitting mask, prevention of skin breakdown, better communication and mucous clearance.23 HFNT, when compared to NIV, was shown to be associated with improved comfort in Cong et al.32 and Cortegiani et al.33 In this review, Doshi et al.31 and Cortegiani et al.33 did not detect any difference in dyspnoea between HFNT and NIV. The lack of demonstrable benefit is likely secondary to the earlier time points in the studies investigating the role of HFNT in the initial management of AT2RF.\n\nHFNT is increasingly emerging as a therapeutic option for AT2RF, but various studies have combined it with other clinical scenarios such as post-extubation,39 NIV interruption,20 or physiological studies23 and even in studies that explored its efficacy in acute exacerbations, the place of intervention could lead to bias, for example after initial management in the emergency medicine department, thus introducing unintentional bias such as lead-time bias as well selection bias.32 The location of patients in a closely monitored environment, as opposed to a general ward,44 might mask any adverse outcomes due to deterioration through earlier intervention. Hence, it is essential to investigate its utility in the early management of AT2RF in the emergency medicine department.\n\nHigh flow nasal cannula can flush anatomical dead space, provide mild positive distending pressure, improve mucociliary clearance as well as be better tolerated.45 Depending on the type of respiratory failure, type 1 or 2, a specific nasal cannula design that alters flow pattern could have a differential effect. A small-bore nasal cannula as seen in high flow nasal insufflation might purge the anatomical dead space more efficiently, thereby providing minimal ventilator assistance.45,46\n\nThe strength of the systematic review is that it was conducted to a high standard following recommended methods for the conduct, quality assessment and reporting,47 using a comprehensive search strategy of all electronic databases. Despite this, the recommendations of the review are limited by the small number of trials, which highlights the need for further adequately powered trials.\n\nWe recommend that future research needs to address the following research gaps in the evidence base for the use of HFNT in AT2RF. Future trial designs should be randomized controlled trials, they should include sufficiently large patient numbers to ensure they are adequately powered for important clinical outcomes. Outcomes should be standardized with clear definitions including clinical outcomes, use validated scales and relevant time points.48 The role of nasal cannula diameter in the efficiency of CO2 clearance should be tested to determine whether the type of device used has an impact on therapy efficacy.49 Studies should also encompass a robust health economic analysis, include outcome analysis of patients who fail therapy and identify any features to predict the outcome of the therapy to allow patient selection.\n\nIn conclusion, this review found very few studies investigating the clinical efficacy of HFNT in AT2RF. A similar reduction in PaCO2 was seen between HFNT and NIV at various time-points,32,33 while a significantly higher PaCO2 clearance with HFNT, when compared to NIV, was demonstrated at an early time point in one study.31 Similarly, HFNT use was associated with better comfort in two studies,32,33 while a similar benefit was not shown in the other study.23 The evidence is also moderate in quality and the benefit demonstrated is limited to clinically irrelevant time points, with no studies powered to detect clinical outcomes. Therefore, a change in practice cannot be recommended until further high-quality clinical trials are conducted.\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\nQueen’s University Belfast institutional data repository (Pure system): High flow nasal therapy for acute type 2 respiratory failure: A systematic review. https://doi.org/10.17034/4080c4eb-38f0-4c02-91ee-37129ceb65a6.27\n\nThis project contains the following extended data:\n\n- Search strategy for Medline for research article High flow nasal therapy for acute type two respiratory failure. A systematic review.pdf\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReporting guidelines\n\nQueen’s University Belfast institutional data repository (Pure system): PRISMA checklist for “High flow nasal therapy for acute type 2 respiratory failure: A systematic review”. https://doi.org/10.17034/4080c4eb-38f0-4c02-91ee-37129ceb65a6.27\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 acknowledge support from the Deanship of Scientific Research at King Saud University and Queen’s University Belfast librarian, Richard Fallis, for his support in developing the search strategy.\n\n\nReferences\n\nDavidson A, Banham S, Elliott M, et al.: BTS/ICS guideline for the ventilatory management of acute hypercapnic respiratory failure in adults. Thorax. 2016; 71: ii1–ii35. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "87742", "date": "14 Jul 2021", "name": "Ben Messer", "expertise": [ "Reviewer Expertise Acute respiratory support" ], "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 thorough systematic review which has appropriate methodology and identifies the paucity of evidence available comparing HFNO to NIV. There are minimal differences detectable and no clinically important differences.\nThe conclusions drawn are appropriate and importantly, mention is made of the benefits of NIV which provide a rationale for further study including a trial of NIV vs HFNO.\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": [ { "c_id": "7114", "date": "20 Sep 2021", "name": "Asem Alnajada", "role": "Author Response", "response": "We would like to thank the reviewer for his time in reviewing this manuscript. The comments have been responded to individually as stated below. We feel that the manuscript has improved with his input and hope that he is satisfied by our response and additional changes. C1: This is a thorough systematic review which has appropriate methodology and identifies the paucity of evidence available comparing HFNO to NIV. There are minimal differences detectable and no clinically important differences. R1: Thank you for this comment, as you mentioned the paucity has been identified in HFNO vs NIV. This is an important point to mention as this is currently a signal that HFNO is non-inferior to NIV in managing mild to moderate acute type 2 respiratory failure but the evidence base is poor and important clinical outcomes need to be robustly investigated. C2: The conclusions drawn are appropriate and importantly, mention is made of the benefits of NIV which provide a rationale for further study including a trial of NIV vs HFNO. R2: We agree with the rational comment you had given. Further studies are required to thoroughly evaluate the clinical significance between the treatment groups. We once again thank the reviewer for the time and effort taken in reviewing the manuscript and providing the comments." } ] }, { "id": "89642", "date": "29 Jul 2021", "name": "Federico Longhini", "expertise": [ "Reviewer Expertise Respiratory failure", "HFNC", "NIV", "invasive 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\nI have reviewed the manuscript and there are some criticism requiring discussion:\nPlease note that at least two articles should be included in the review for HFNC vs NIV for the primary outcome (Sklar 2018 and Papachatzakis 2020).\n\nI suggest to check for other articles in the review by Pisani et al (PMID: 31591056), that also merits to be cited in the manuscript.\n\nPlease update findings and discussion according to the aforementioned points.\n\n\"While this could suggest that HFNT is non-inferior to NIV, HFNT cannot be recommended as an alternative management strategy to reduce PaCO2 due to the low quality of evidence, lack of standardization of time-points for PaCO2 measurement and the lack of adequately powered sample sizes.\" I would mitigate this message. In case of failure of NIV due to interface intolerance, with improving blood gases, I would attempt to shift the treatment to HFNC, in order to avoid intubation. noteworthy, several studies have demonstrated that delay in intubation for hypercapnic respiratory failure does not impact on patients' outcome and survival.\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": [ { "c_id": "7115", "date": "20 Sep 2021", "name": "Asem Alnajada", "role": "Author Response", "response": "We would like to thank the reviewer for his time in reviewing this manuscript. The comments have been responded to individually as stated below. We feel that the manuscript has improved with his input and hope that he is satisfied by our response and additional changes. C1: Please note that at least two articles should be included in the review for HFNC vs NIV for the primary outcome (Sklar 2018 and Papachatzakis 2020). R1: Thank you for this comment, the studies cited have their merits in exploring the role of HFNT for AHRF. However, the studies cited don’t fit our inclusion criteria which have been described in the protocol published in the PROSPERO database (CRD42019148748) and the methodology section of the systematic review, therefore, cannot be included in our SR. Specifically, the systematic review focusses on studies that have utilised HFNO as the initial management strategy for acute hypercapnic respiratory failure (AHRF). This systematic review is unique in that respect as evidence synthesis in this emergency clinical scenario is lacking with studies that have utilised at later stages of management such as post initial NIV use, interspersed with NIV and indeed studies not limited to AHRF are included in previous systematic reviews. Sklar et al have conducted a systematic review to investigate the impact of HFNT for patients with immunocompromise which don’t meet most of the inclusion criteria which we established as our review include only randomized controlled trials, uncontrolled trials and cohort studies focusing on HFNT as initial treatment when compared to LFO and/or NIV for AHRF. Papachatzakis et al was excluded due to various reasons including the inclusion of a mixed population and did not utilise HFNT as an initial treatment plan for the patient. C2: I suggest to check for other articles in the review by Pisani et al (PMID: 31591056), that also merits to be cited in the manuscript. R2: Thank you for the comment. We have cited the review by Pisani et al whose group have done a lot of work in this area. The various papers included in that review were also captured through our search and included in our review if they conformed to our protocol published in the PROSPERO database. C3: Please update findings and discussion according to the aforementioned points. R3: The citations suggested are outside the scope of the systematic review inclusion criteria and hence not included for outcome analysis. To give a broader picture of the field and the scope of HFNO, we have amended the background section to include the articles suggested above. C4: While this could suggest that HFNT is non-inferior to NIV, HFNT cannot be recommended as an alternative management strategy to reduce PaCO2 due to the low quality of evidence, lack of standardization of time-points for PaCO2 measurement and the lack of adequately powered sample sizes.\" I would mitigate this message. In case of failure of NIV due to interface intolerance, with improving blood gases, I would attempt to shift the treatment to HFNC, in order to avoid intubation. noteworthy, several studies have demonstrated that delay in intubation for hypercapnic respiratory failure does not impact on patients' outcome and survival. R4: Thank you for this comment. The current evidence base to suggest HFNO in patients’ failing NIV is limited to small studies with no definitive efficacy studies. The authors do agree that it is an area that requires immediate attention. The paragraph has been amended to reflect the reviewers’ comments. “While this could suggest that HFNT is non-inferior to NIV, HFNT cannot be recommended as an alternative management strategy to reduce PaCO2 due to the low quality of evidence, lack of standardization of time-points for PaCO2 measurement and the lack of adequately powered sample sizes. Similarly, in patients failing NIV due to compliance issues, HFNO may be a promising option to limit mechanical ventilation. This recommendation falls beyond the scope of this systematic review and is a clinical scenario that requires urgent attention\". We once again thank the reviewer for the time and effort taken in reviewing the manuscript and providing the comments." } ] } ]	1	https://f1000research.com/articles/10-482
https://f1000research.com/articles/10-584/v1	16 Jul 21	{ "type": "Case Report", "title": "Case Report: Diabetic nephropathy aggravates the progression and prognosis of COVID-19-associated acute limb ischemia", "authors": [ "Decsa M. Hertanto", "Henry Sutanto", "Soebagijo Adi", "Decsa M. Hertanto", "Henry Sutanto" ], "abstract": "Hypercoagulation is a hallmark of both the novel coronavirus disease (COVID-19) and type 2 diabetes mellitus (T2DM). It increases the risk for vascular thrombosis, including peripheral artery diseases. Among others, acute limb ischemia (ALI) is one of most common complications that requires immediate and prompt treatments to reduce morbidity and mortality. However, the complex interplay between COVID-19, T2DM and its complications (e.g., diabetic nephropathy), and ALI creates a great challenge in the management of the disease. Here, we present a case of a 59-year-old diabetic female with progressive pain in her left leg in the last five years, which was significantly intensified following COVID-19 diagnosis. Bluish coloration, numbness and functional impairments were observed during examinations with no palpable pulsation on left posterior tibial and dorsalis pedis arteries. The patient also had diabetic nephropathy (stage III), hypoalbuminemia, anemia and a urinary tract infection that complicated the management of the disease. Due to the excruciating pain and the worsening of the limb conditions, right leg revascularization and left leg amputation were performed at day 14 after admission. Following the surgeries, no more pain was observed and patient was discharged for further follow-up at the outpatient clinic.", "keywords": [ "acute limb ischemia", "COVID-19", "diabetes mellitus", "diabetic nephropathy", "limb amputation", "hypercoagulability", "vascular thrombosis", "case report" ], "content": "Introduction\n\nThe 2019 coronavirus disease (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome-associated coronavirus type 2 (SARS-CoV-2). It was first encountered in Wuhan city, Hubei province, China in late 2019 and since early 2020, it has been declared a pandemic by the World Health Organization (CDC, 2021; WHO, 2021). The viral disease infects healthy people through respiratory or micro droplets released by infected individuals during sneezing, speaking or coughing (WHO, 2021). Following its entry into the respiratory tract of the contracted individuals, the virus binds to the angiotensin converting enzyme type 2 (ACE-2) receptors in the alveolar cells type 2 to interact with the host’s cellular immunity (Hertanto, Sutanto, & Wungu, 2021). In the first months of the pandemic, the virus was believed to solely affect the respiratory system, causing severe pneumonia. However, it is now understood that SARS-CoV-2 affects various organs and subsequently worsens other comorbidities, including type 2 diabetes mellitus (T2DM) (Mokhtari et al., 2020). Interestingly, T2DM is also one of the biggest contributors to the worsening of COVID-19 severity, creating a vicious cycle. The COVID-19-associated mortality has been reported to be doubled in patients with T2DM as compared to those without (Aggarwal et al., 2020; Parveen et al., 2020).\n\nMeanwhile, peripheral artery disease is one of the most common complications in T2DM. Previous studies reported that diabetic patients had a two to four-fold higher risk for developing peripheral artery diseases (e.g., critical limb ischemia [CLI]) than those with no T2DM (Liao et al., 2013; Luscher et al., 2003). Of note, 500-1000 new cases of CLI per million people per year were estimated in the Western Europe and the United States (Davies, 2012; Spreen et al., 2016). In an acute phase, peripheral artery disease can rapidly progress into acute limb ischemia (ALI), and such progression is expected to happen within two weeks of significant reduction of limb perfusion. Thus, such a condition can increase morbidity and mortality. Additionally, uncontrolled blood glucose also facilitates the worsening of ALI, and could potentially end up with limb amputation (Fukuda et al., 2015; Kaur et al., 2020; Spreen et al., 2016; Waspadji, 2014).\n\nAltogether, the hypercoagulability caused by both COVID-19 and T2DM predisposes ALI and therefore increases mortality. Thus, such an emerging and challenging situation requires immediate and prompt treatments. However, research about this particular topic and fixed guidelines to manage such complex conditions are currently limited (Galanis et al., 2020; Kaur et al., 2020). Moreover, the presence of diabetic nephropathy could add more challenges in diagnosing and treating those patients. Here, we present a complex case of a patient with COVID-19, ALI, T2DM and diabetic nephropathy that was hospitalized and treated in a tertiary referral hospital in Indonesia.\n\n\nCase report\n\nA 59-year-old Asian housewife was referred to the emergency department (ED) of Dr. Soetomo General Hospital (SGH) with bluish coloration, numbness and progressive pain in her left leg. The patient had been hospitalized in another hospital for shortness of breath due to COVID-19 two days before the referral, but because of the limb coloration, she was referred to SGH. Similar pain was also felt in her right leg, which was reddish in color. The patient had complained about the pain for five years (initially sensed as frequent tingling), which worsened one week before her visit at the hospital. The pain was intermittent and was mostly felt while moving or walking, and worsened at night during sleep. Most of the time, the patient hanged her legs down to alleviate the pain. During initial assessment, fever, occasional coughing and intermittent dyspnea were observed. The patient also complained about pain during micturition five days before the hospitalization. There were no issues regarding appetite and defecation. The patient also had a four-year history of uncontrolled T2DM with a bad compliance to oral antidiabetic (glimepiride 2 mg 1 – 0 – 0) and untreated hypertension. No relevant family medical history was reported.\n\nThe physical examinations at the ED showed a lethargic condition with Glasgow Coma Scale (GCS) of E4M5V6. Her blood pressure was 150/90 mmHg with heart rate of 110 beats/minute (bpm). Her respiratory rate was 24 times/minute, and her temperature was 36.7°C with blood oxygen saturation (SpO2) of 97%. Her pain was assessed using the Visual Analogue Scale (VAS) and resulted in a score of 6 out of 10. Her weight, height and body mass index (BMI) were 70 kg, 155 cm and 29.1, respectively. Head and neck examinations showed anemic conjunctivae with no icterus or cyanosis. Her cardiopulmonary examination was within a normal range (i.e., both sides of thorax were symmetrical and no retraction), as well as the abdominal examination (i.e., fluffy abdomen, bowel sounds were normal, liver and spleen were not palpable). Interestingly, her extremity assessment revealed a bluish coloration in her left leg with cold sensation during palpation and no pulse was detected (Table 1). Detailed left leg examinations also discovered a necrotic region below her ankle, with a cold sensation up to one-third of her left lower extremity. Furthermore, the motoric and sensory functions of the left leg were poor. Meanwhile, her right leg was warm, had no necrosis, and exhibited normal sensory and motoric functions (Figure 1A). Ankle Brachial Index (ABI) was negative on the left and 0.9 on the right.\n\nA) The clinical presentation of the lower extremities at day 0 of hospitalization. B) The worsening of ALI at day 8. C) The progress of COVID-19-associated pneumonia before referral, at day 1 and day 4 of hospitalization. Left: bilateral infiltrates were observed; middle: bilateral pneumonia was slightly increased; right: an improvement was visible.\n\nThe laboratory examinations showed hemoglobin (Hb) of 10.8 g/dl, hematocrit of 35%, leucocyte count of 24460 /μl and platelet count of 627000/μl. The differential blood count of leucocytes highlighted neutrophil domination (88.1%) followed by lymphocytes (5%). Hemostatic parameters, such as prothrombin time (PT), activated partial thromboplastin time (aPTT), and D-dimer were 14.6, 30.8 and 9740 ng/ml, respectively. Her liver functions of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were 70 U/l and 193 U/l respectively. Blood albumin was 2.89 g/dl, fasting blood glucose was 128 mg/dl, HbA1c was 9%. Renal functions test revealed a serum creatinine of 1.5 mg/dl and blood urea nitrogen (BUN) of 61 mg/dl. Her C-reactive protein (CRP) was 22.5 mg/l, sodium 132 mg/dl, potassium 4.7 mg/dl, chloride 96 mg/dl. HbsAg, anti-HCV and HIV were all negative. COVID-19 polymerase chain reaction (PCR) test was positive. The urinalysis showed following results: nitrite positive, leucocytes positive, glucose 4+, A/C< 30, P/C<0,15, while her arterial blood gas analysis (BGA) showed pH 7.42, pCO2 38, pO2 146, HCO3 23.4 and SpO2 99%. Additionally, her chest X-ray showed bilateral pulmonary infiltrates (Figure 1C).\n\nTaking into account the patient’s complaints, history, physical, laboratory and radiological examinations, the patient was then diagnosed with COVID-19-associated pneumonia with left inferior extremity dead limb and bilateral inferior extremity ALI, as well as urinary tract infection, anemia, hypoalbuminemia, T2DM and chronic kidney disease (CKD) stage III. Subsequently, she was treated with oxygen via simple mask at 6 liters per minute (lpm), sodium chloride (NaCl) 0.9% infusion 1000 ml/24 hours, subcutaneous heparin injection (2×5000 unit), intravenous (iv) dexamethasone injection (6 mg 1×1), remdesivir 1×200 mg, ceftriaxone 2×1 g, metronidazole 3×500 mg, insulin aspart (novorapid) 14 – 16 – 16, insulin detemir (levemir) 0 – 0 – 10, albumin 20% transfusion 100 ml/4 hours, oral isoprinosine 3×500 mg, oral vitamin D 1×5000 IU and oral cilostazol 1×100 mg. Her diet was also maintained within 1900 kcal/day.\n\nAt day four, the dyspnea was improved but the leg started to feel burnt and the pain progressed. Blood pressure was 149/84 mmHg, heart rate 68 bpm, respiratory rate 20 times/minute, SpO2 98%, VAS score 5, COVID-19 PCR negative, leucocyte count was 27540 /μl with the proportion of neutrophil 87.4%, serum creatinine 1.4 mg/dl, blood albumin 2.8 mg/dl, D-dimer 6540 ng/ml, ferritin 1602 ng/ml, fibrinogen 642.6 mg/dl and procalcitonin (PCT) of 1.08. At day 5, the patient only complained about her progressive leg pain, while her daily examinations revealed blood pressure of 127/74 mmHg, heart rate 86 bpm, respiratory rate 20 times/minute, SpO2 99% and VAS score of 5. Meanwhile, the COVID-19 PCR was negative, leucocyte count was 31390/μl with neutrophil 86.6%, serum creatinine 1.3 mg/dl, blood albumin 3 mg/dl, D-dimer 5570 ng/ml, ferritin 1572 ng/ml, fibrinogen 542 mg/dl and PCT of 0.23. Additionally, an improvement of the bilateral pulmonary infiltrates on chest X-ray was also observed (Figure 1C).\n\nAt day 7, the patient complained that the pain in her leg had increased significantly. Her blood pressure was 130/80 mmHg, heart rate 98 bpm, respiratory rate 20 times/minute and SpO2 98%. The urine culture was positive for Escherichia coli sensitive to cefoperazone sulbactam. Because of the progressive extremity pain, the patient was referred to the cardiothoracic and vascular surgery (CTS) division and, consistent with the first diagnosis, she was diagnosed with left dead limb and bilateral ALI (Figure 1B), and was advised to perform computed tomography angiography (CTA) and undergo an above-knee amputation. At day 9, the right lower extremity CTA (Figure 2) discovered total occlusion due to 6 cm thrombus on right popliteal artery (from 1 cm above right femorotibial joint toward inferior). Right anterior tibial artery received contrast flow from collateral arteries and no contrast flow was seen in right posterior tibial, peroneal and dorsalis pedis arteries. Meanwhile, the left lower extremity CTA showed total occlusion due to 12.7 cm thrombus on left popliteal artery (from 6 cm above left femorotibial joint toward inferior). Moreover, no contrast flow was seen in left anterior tibial, posterior tibial, peroneal and dorsalis pedis arteries.\n\nThe CTA revealed an abdominal aortosclerosis with abnormalities on both limbs. Right limb: total occlusion due to 6 cm thrombus on right popliteal artery (from 1 cm above right femorotibial joint toward inferior), right anterior tibial artery received contrast flow from collateral arteries and no contrast flow was seen in right posterior tibial, peroneal and dorsalis pedis arteries. Left limb: total occlusion due to 12.7 cm thrombus on left popliteal artery (from 6 cm above left femorotibial joint toward inferior). No contrast flow was seen in left anterior tibial, posterior tibial, peroneal and dorsalis pedis arteries. (This figure has been edited in Microsoft PowerPoint 2016 to obscure patient’s data.)\n\nAt day 11, the patient was still waiting for surgery and the pain increased. Her blood pressure was 130/80 mmHg, heart rate 88 bpm, respiratory rate 20 times/minute, SpO2 98% and VAS equals 7. Laboratory parameters showed Hb 10.3, leucocyte count 22610/μl, platelet 509000/μL, D-dimer 3960 ng/ml and random blood glucose of 171. Subsequently, the patient received 8 lpm oxygen through a simple mask, diet type-B 1900 kcal/24 hours, iv fluid with NaCl 0.9% 1000 ml/24 hours, cefoperazone sulbactam 2×1 g iv, subcutaneous novorapid 14 – 16 – 16 unit, subcutaneous levemir 0 – 0 – 10 unit, subcutaneous heparin 2×5000 unit and oral cilostazol 1×100 mg. At day 13, her blood pressure was 127/70 mmHg, heart rate 90 bpm, respiratory rate 20 times/minute and SpO2 98%. Laboratory results showed Hb 9, leucocyte count 11800/μL, platelet 410000/μL, BUN 20 mg/dl, serum creatinine 1.3 mg/dl and random blood glucose of 160. At this point, the patient was assigned for antegrade right femoral artery thrombectomy and left above-knee amputation at the following day. At day 14, the surgeries (both amputation and thrombectomy) were performed (Figure 3) and 10 cm thrombus was retrieved in the right femoral artery. Lastly, as a follow-up, at day 17, no symptoms were observed, therefore the patient was discharged.\n\nTwo days post-surgery, the patient was safely discharged and followed up regularly for recurring symptoms at the outpatient clinic.\n\n\nDiscussion\n\nALI is a pathological condition in which the perfusion toward extremities suddenly and significantly drops. Such condition often threatens the viability of the extremity, with a high risk of limb loss if the perfusion is completely obstructed. In most cases, the disease rapidly progresses within two weeks of the initial complaint. Therefore, optimal history-taking and physical examinations are crucial in ALI diagnosis and treatment. Cold sensation in extremity (poikilothermy), pain, pallor, pulselessness, sensory disturbance (paresthesia) and motoric alteration (paralysis) are commonly found in the disease trajectory of ALI (Gerhard-Herman et al., 2017; Obara, Matsubara, & Kitagawa, 2018). Additionally, limb examinations, including the pulsation palpation and ABI assessment, are essential. Due to the complex pathophysiology of the disease and the interplay with other comorbidities, the risk factors of ALI (e.g., T2DM, hypertension, smoking, chronic pulmonary diseases and CKD) also need to be explored (Fukuda et al., 2015).\n\nThe American College of Cardiology (ACC) and American Heart Association (AHA) proposed that ALI can be divided into 3 categories: first grade, viable body parts with no immediate threat; second grade, threatened body parts (IIa: marginally threatened, indicating that body parts can be salvaged if immediate and prompt treatments were performed; IIb: immediately threatened, indicating that threatened body parts require immediate revascularization); and third grade, irreversible damage of body parts (Gerhard-Herman et al., 2017). In the case we presented above, we can classify the left lower extremity of the patient as third grade because no pulsation was observed in left posterior tibial and dorsalis pedis arteries. Moreover, the SpO2 was negative on the left lower limb with some necrotic regions, indicating the nonviability of the left lower limb. Meanwhile, the right lower extremity can be classified in the second grade because the pulsations and SpO2 persisted. Adjunctive (radiological) examinations, such as duplex ultrasound, CTA or magnetic resonance angiography (MRA) could also assist the diagnosis of ALI by locating the obstruction and guide revascularization procedure (Davies, 2012; Kinlay, 2016). Studies have shown that CTA is 89-99% sensitive and 83-97% specific for ALI (Fukuda et al., 2015).\n\nIn this case, we also saw that the rapid progression of ALI happened after the SARS-CoV-2 infection. COVID-19 often induces a hypercoagulable state and hyperinflammation which could progress into disseminated intravascular coagulation (DIC) (Asakura & Ogawa, 2021). Such a condition is commonly marked by the elevation of D-dimer and fibrinogen (Eljilany & Elzouki, 2020). On the other hand, SARS-CoV-2 infection can also damage the endothelial cells, causing endothelial dysfunction and initiate the expression of tissue factors, activate platelets, and increase von Willebrand factor and factor VIII (Bonaventura et al., 2021; Evans et al., 2020). Altogether, they facilitate the formation of thrombin and blood clot (thrombus). Thrombin triggers inflammation through its platelet modulation and stimulation of neutrophil, which further activates endothelial cells and monocytes, allowing the formation of micro thrombus. Such condition can increase the thromboembolic risk in designated locations.\n\nInterestingly, COVID-19 is able to instigate hyperglycemia, even without previous history of T2DM. Such hyperglycemic condition causes oxidative stress, which increases inflammation and coagulation. This increases the risk of coagulation and facilitates thrombosis as well (Ceriello, 2020). Due to the complex interactions, the prognosis of peripheral artery diseases in T2DM relies on the comorbidities, the presence of concomitant infections, neuropathy and the patient’s immune status. Uncontrolled T2DM could speed up the atherosclerosis and was shown to have a five to 10-fold higher risk of amputation (Li et al., 2007; Serrano Hernando & Martin Conejero, 2007).\n\nIn general, the management of first grade ALI comprises pharmacological treatments, while the second grade requires revascularization and presumably amputation if the limb cannot be salvaged. Common drugs for peripheral artery diseases, such as antiplatelets, statins, antihypertensives, anticoagulants, cilostazol and pentoxifylline could be administered in ALI, together with lifestyle modifications to control blood glucose level and smoking cessation.\n\nThe presented case highlighted the effectiveness of revascularization and limb amputation in treating end-stage ALI (grade IIb and III) in patients with complex diseases such as T2DM, diabetic nephropathy and COVID-19. This case also exemplified that anticoagulation might not be adequate to treat ALI in the presence of COVID-19 and T2DM, and that early revascularization should be considered in such situations. Nonetheless, the COVID-19 infectivity has to be taken into account to maintain the safety of the healthcare workers during the procedure. In the presented case, we had to wait until we obtained two-times negative COVID-19 PCR results before sending the patient for CTA and surgical interventions. This local policy could be called a ‘double-edged sword’, in that on the one hand it protected the healthcare workers from COVID-19, but on the other hand, it delayed the procedure for the patient and facilitated the progression of ALI. In the future, an improved understanding of COVID-19 infectivity and pathogenesis would hopefully reduce the time to interventions and diminish ALI-associated morbidity and mortality.\n\n\nConclusion\n\nHere, we presented a 59-year-old female with COVID-19, T2DM, third grade ALI and CKD stage III (diabetic nephropathy). In this patient, revascularization of the right lower extremity via thrombectomy and left lower limb amputation were performed based on findings in physical examinations, supported by CTA. At day 17, the patient was discharged and recovered from COVID-19, confirmed by two negative PCR tests. This case highlights the complexity of ALI in the presence of COVID-19 and comorbidities (i.e., T2DM and diabetic nephropathy), which requires intensive monitoring and prompt immediate treatments to reduce morbidity and mortality.\n\n\nConsent\n\nA written informed consent for the publication of her clinical details and clinical images was obtained from the patient.\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\nAggarwal G, Lippi G, Lavie CJ, et al.: Diabetes mellitus association with coronavirus disease 2019 (COVID-19) severity and mortality: A pooled analysis. J Diabetes. 2020; 12(11): 851–855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsakura H, Ogawa H: COVID-19-associated coagulopathy and disseminated intravascular coagulation. Int J Hematol. 2021; 113(1): 45–57. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nObara H, Matsubara K, Kitagawa Y: Acute Limb Ischemia. Ann Vasc Dis. 2018; 11(4): 443–448. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParveen R, Sehar N, Bajpai R, et al.: Association of diabetes and hypertension with disease severity in covid-19 patients: A systematic literature review and exploratory meta-analysis. Diabetes Res Clin Pract. 2020; 166: 108295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerrano Hernando FJ, Martin Conejero A: [Peripheral artery disease: pathophysiology, diagnosis and treatment]. Rev Esp Cardiol. 2007; 60(9): 969–982. PubMed Abstract \| Publisher Full Text\n\nSpreen MI, Gremmels H, Teraa M, et al.: Diabetes Is Associated With Decreased Limb Survival in Patients With Critical Limb Ischemia: Pooled Data From Two Randomized Controlled Trials. Diabetes Care. 2016; 39(11): 2058–2064. PubMed Abstract \| Publisher Full Text\n\nWaspadji S: Kaki Diabetes. In: Setiati S (Ed.), Buku Ajar Ilmu Penyakit Dalam VI ed. 2014." }	[ { "id": "91878", "date": "16 Aug 2021", "name": "Ketut Suastika", "expertise": [ "Reviewer Expertise Clinical and adult endocrinologist" ], "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 Case Report was reported detailly on clinical findings, supporting diagnosis examinations, and management of the patients. It was also written well. The case report has become a reminder that in the patient with T2DM and CPVID-19, morbidity or mortality does not come from lung complications only.\n\nTo the authors: please note the value of eGFR by formula calculation to know the degree of CKD stage.\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": [ { "c_id": "7139", "date": "17 Sep 2021", "name": "Henry Sutanto", "role": "Author Response", "response": "The Case Report was reported detailly on clinical findings, supporting diagnosis examinations, and management of the patients. It was also written well. The case report has become a reminder that in the patient with T2DM and CPVID-19, morbidity or mortality does not come from lung complications only. Report: We would like to thank the reviewer for the time and willingness to assess the quality of our manuscript. To the authors: please note the value of eGFR by formula calculation to know the degree of CKD stage. Report: In the new version of the manuscript, we have added the estimated Glomerular Filtration Rate (eGFR) during admission (38 ml/min/1.73 m2 or stage 3B) and before discharge (45 ml/min/1.73 m2 or stage 3A), which indicated an improvement of the diabetic nephropathy after treatments." } ] }, { "id": "92346", "date": "01 Sep 2021", "name": "Joyce Bernardi", "expertise": [ "Reviewer Expertise cardiac electrophysiology" ], "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 in detail the case of a 59-year-old female with T2DM, presenting with bilateral acute limb ischemia that aggravated after COVID-19 infection. A revascularization of the right lower limb through thrombectomy and amputation of the necrotic left lower limb have been performed. The manuscript highlights how the presence of COVID-19 infection and other comorbidities is critical in the evolution of ALI and how a timely intervention is important to reduce morbidity and mortality. The background of the case is well described, together with an extensively detailed description of the progression, diagnosis, and treatment. In the discussion the authors give a good overview of what is known about the interaction between COVID-19 and other comorbidities, such as T2DM and ALI, pointing out the importance of a prompt intervention.\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": [ { "c_id": "7140", "date": "10 Sep 2021", "name": "Henry Sutanto", "role": "Author Response", "response": "The authors describe in detail the case of a 59-year-old female with T2DM, presenting with bilateral acute limb ischemia that aggravated after COVID-19 infection. A revascularization of the right lower limb through thrombectomy and amputation of the necrotic left lower limb have been performed. The manuscript highlights how the presence of COVID-19 infection and other comorbidities is critical in the evolution of ALI and how a timely intervention is important to reduce morbidity and mortality. The background of the case is well described, together with an extensively detailed description of the progression, diagnosis, and treatment. In the discussion the authors give a good overview of what is known about the interaction between COVID-19 and other comorbidities, such as T2DM and ALI, pointing out the importance of a prompt intervention. Response: We would like to thank the reviewer for the time and willingness to assess the quality of our manuscript." } ] } ]	1	https://f1000research.com/articles/10-584
https://f1000research.com/articles/10-130/v1	19 Feb 21	{ "type": "Study Protocol", "title": "Salivary biomarkers associated with the progression of disease in people living with HIV: A scoping review", "authors": [ "Priyanka Prasad", "Viola D’Souza", "Prasanna Mithra", "Raghu Radhakrishnan", "Priyanka Prasad", "Viola D’Souza", "Prasanna Mithra" ], "abstract": "Background: Biomarkers are measurable indicators of normal biological processes, which provide an objective assessment of the physiologic state of living systems. Saliva contains several biomarkers that serve as a diagnostic tool in health and disease. Evaluation of a multitude of salivary components could potentially predict the clinical outcome. This is especially critical in a chronic, potentially life-threatening condition like human immunodeficiency virus (HIV) infection. Scrupulous evaluation of relevant biomarkers could facilitate the early detection of HIV, determine the stage of infection and monitor the disease progression. Currently, there is a paucity of validated biomarkers in saliva predicting the disease progression in people living with HIV. In this scoping review, we aim to provide an overview of the available evidence on salivary markers associated with the progression of disease in people living with HIV. Methods: The authors shall develop a tailored search strategy for each database using relevant keywords. We will search for eligible studies indexed in the following databases: MEDLINE, EMBASE, and the Cochrane Library, Cumulative Index to Nursing and Allied Health Literature (CINAHL) and gray literature. We will restrict the search to studies published in the English language. Following deduplication, all search results will be exported to the EPPI reviewer web, where two independent reviewers using a data extraction tool developed and pretested by the review authors will screen eligible studies. The result of this review will be reported using the Preferred Reporting Items for Systematic Review and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist and reporting guidelines. Discussion: The proposed scoping review protocol will enable the identification and assessment of salivary biomarkers, which can predict disease progression in patients with HIV infection. The synthesis of evidence from this review will assist in improving our current understanding of biomarkers used to evaluate the progression of HIV infection.", "keywords": [ "People living with HIV", "PLWHA", "HIV", "AIDS", "Biomarker", "Saliva" ], "content": "Introduction\n\nThe human immunodeficiency virus (HIV), belonging to the Retroviridae family, targets the body’s immune system1. Since it has high affinity for the receptors present on the surface of CD4+ T-lymphocytes and macrophages, it makes a person vulnerable to infection2. Infection of the target cell by HIV results in the production of progeny virions depleting the CD4+ lymphocytes and ensuing immunosuppression of the host. The pathophysiology of HIV involves a dynamic host-virus interaction, resulting in acquired immunodeficiency syndrome (AIDS) in severe cases3. The incubation period for the virus is around 5–10 years in adults4,5. This broad interval between HIV infection and the development of symptoms can be attributed to several hosts and virus-related factors such as the development of new viral strains, the immune status of the host, as well as environmental cofactors6.\n\nHIV viral load, CD4+ T-cell count in peripheral blood and quantitative measurements of soluble markers present in plasma, like neopterin, tumor necrosis factor-alpha (TNFα), interleukins (ILs), beta 2-microglobulin (B2M), soluble CD8, etc have been used as surrogate markers to assess the progression of HIV infection in patients. CD4+ T-cell count also evaluates the efficacy of the host’s immune response to antiretroviral therapy (ART)7–10. The onset of AIDS, which implies a progression of HIV infection, could be predicted accurately by monitoring the percentage of CD4+ T lymphocytes in the peripheral blood11. However, in patients on ART, the CD4+ T-cell counts are not reliable markers to recognize virologic failure in the individual12.\n\nAccording to the National Institutes of Health (NIH), “a biomarker is an objectively measured and evaluated indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”13. Essentially, a biomarker can represent any entity and can exist as antibodies, microbes, DNA, RNA, lipids, metabolites, or proteins14. These biomolecules provide crucial information that helps us understand the physiologic state of a biological system. Any alteration in their concentration, structure, function, or action within a biological system can be correlated with disease characteristics such as onset, progression, or even regression of the particular disease or a measure of the host response to foreign bodies15. According to a review by Kanekar et al. in 2010, the clinical utility of biomarkers to assess the disease progression for HIV infection is inconclusive16.\n\nSaliva is a complex biological fluid that can mirror the body's health17. It contains several biomolecules such as enzymes, hormones, antibodies, growth factors, antimicrobial constituents, etc. which can function as useful prognostic markers18. A good salivary biomarker to detect the progression of HIV (with high sensitivity and specificity) would help clinicians and oral pathologists to monitor the deterioration of clinical condition in people living with HIV/AIDS (PLWHA)15,19.\n\nThe literature reveals the dearth of evidence on validated biomarkers16. Much of the information on the topic is largely experimental, which has to be systematically compiled and objectively assessed. Therefore, this scoping review is aimed at synthesizing available evidence on salivary markers for disease progression in HIV infection.\n\n\nObjectives\n\nTo identify pertinent salivary biomarkers consistent with the progression of HIV infection in HIV positive individuals\n\nTo systematically review the existing literature on biomarkers in HIV to identify key concepts and gaps\n\nTo assess the current levels of evidence, the quality of evidence and provide a synthesis of the currently available salivary biomarkers in HIV infection\n\n\nMethods\n\nPeople diagnosed with HIV/AIDS (PLWHA) as per WHO clinical case definition is “an individual with HIV infection irrespective of the clinical stage (including severe or stage 4 clinical disease, also known as AIDS) confirmed by laboratory criteria according to country definitions and requirements”. We will include longitudinal studies that have measured outcomes of at least two different time points. Cross-sectional studies measuring clinical parameters at only one point will be excluded. Only studies that have reported an association between salivary biomarkers and change in the clinical measure will be included in our scoping review. Due to lack of sufficient resources, studies will be excluded if English language texts are not available.\n\nWe will not limit our inclusion based on age, gender, duration of HIV infection, ART status, or demography. Only studies that have reported measurable and quantifiable biological parameters associated with salivary biomarkers will be included. These parameters include, but are not limited to the presence of specific biomolecules, their biologic concentrations, specific gene-phenotype distribution in a population.\n\nIts association with disease progression, its potential to be generalizable to PLWHA irrespective of their age, gender, sensitivity, specificity, reliability, ease of measurement, safety and acceptance to the patient. Additionally, it should reflect a true change in the clinical condition and remain unaffected by symptomatic treatment. We will exclude those studies that fail to meet the criterion of biomarker parameters mentioned. Studies that have not specified the type of surrogate marker used or include the objective measures of a particular biomarker or related only to specific opportunistic infections in HIV will be excluded.\n\nThe methodological framework proposed by Arksey and O'Malley20 and the methodological enhancement developed by Levac et al.21 were referred to for this scoping review. The six-stage methodical framework for conducting a scoping review include: “(1) identifying the research question; (2) identifying relevant studies; (3) selecting studies; (4) charting the data; (5) collating, summarizing and reporting the results and (6) consulting with relevant stakeholders.”\n\nThe research question developed by the research team in consultation with key stakeholders will address the role of salivary biomarkers in assessing the progression of disease in PLWHA. For this review, a quality indicator is ‘an explicitly and measurable item which act as building blocks in the assessment of care’.\n\nSearch terms were finalized based on the feedback from the research team, subject experts, and extensive literature review. An experienced search scientist developed the search strategy and co-authors as per the Medline format and tailored to other databases and sources. The search strategy used in this scoping review included: (PLWHA OR PLHIV OR PLWH OR PLWA OR HIV OR (people living with HIV/AIDS) OR (people living with AIDS) OR (acquired AND (immunodeficiency OR immune‐deficiency OR immuno‐deficiency) AND syndrome) OR Immunocompromised OR immune-compromised OR Slim disease) AND ((HIV related oral lesions) OR (Periodontal disease) OR Periodontitis OR (periodontal infection) OR Xerostomia or (dry mouth) OR (salivary gland disease)) OR (Oral candidiasis) OR (hairy leukoplakia) OR (Kaposi sarcoma) OR (linear gingival erythema) OR (necrotizing ulcerative periodontitis) OR (aphthous ulcer) OR (wasting disease))) AND ((biological marker) OR biomarker OR saliva* OR biomolecule* OR (bacterial burden) OR marker)\n\nThe selected search terms will be searched in the title and/or abstract as well as subject headings keywords (eg, MeSH, EMTREE) as appropriate. We will include all articles from the beginning of the databases until October 2020. Only English language studies will be included. The search results from each database will be downloaded and imported onto Mendeley for the removal of duplicates. Following de-duplication, the remaining studies will be imported into the EPPI reviewer Web.\n\nWe will use the PICO (Population, Intervention, Control and Outcomes) strategy for formulating a foreground research question (Table 1). Primary studies indexed in the following databases will be searched for inclusion in our review: MEDLINE, EMBASE, the Cochrane Library, Cumulative Index to Nursing and Allied Health Literature (CINAHL). The reference lists of all included studies will be hand searched for potentially relevant studies.\n\nTo ensure that all information pertinent to the research question is adequately captured, our search will include several grey literature sources from relevant databases (e.g. Grey Literature Report, OpenGrey, Web of Science Conference Proceedings). We will further conduct a targeted search of grey literature in the websites of organizations working on HIV/AIDS research on the local, provincial, national, and international levels. Any studies, reports, and conference abstracts identified through these databases, which are of relevance to this review, will be included.\n\nWe will undertake a two-step screening process to include all potentially relevant articles in this review: (1) title and abstract screening; (2) full-text screening. In the first stage of screening, two review authors (VD and PP) will independently screen the title and abstract of all retrieved citations for inclusion against a set of minimum inclusion criteria. These criteria will be determined by testing on a sample of abstracts before beginning the abstract review to ensure that they are robust enough to capture all studies pertinent to the primary objective. Articles will be included for full-text screening if either one or both of the review authors deem them relevant to the research question.\n\nAll the studies included in the T&A stage will be subject to full-text screening. In this step, both the investigators (VD and PP) will independently screen the full-text articles to assess if they meet the inclusion/exclusion criteria. We will calculate Cohen's κ statistics at both the T&A review stage and the full article review stage to determine inter-rater agreement. Studies will be reviewed another time if there is any discordance regarding the study eligibility. If there are further disagreements, it will be resolved through discussion with a third investigator (RR) until a consensus is reached. A flow diagram will be used to represent the inclusion and exclusion of retrieved studies\n\nThe research team will develop a data collection instrument to extract information from the included studies and to confirm study relevance. Study characteristics including publication year, publication type (eg, original research), study design, country, study setting, a specific biomarker used, statistical analysis performed, the association between biomarker tested and disease progression, the effect of therapeutic agents on biomarker changes, economic aspects and acceptability of biomarker, etc will be extracted (see Table 2). The research team will review and pretest the form to make sure that the data extraction form captures all the required information from the included studies accurately.\n\nData from the included studies will be extracted independently by the two review authors (VD and PP) using the EPPI reviewer22. To ensure a high degree of accuracy of the data extraction, we will compare the independently abstracted data of each reviewer. Both the review authors to ensure consistency in the extracted data will discuss any discrepancies identified in the collected data. The data will be compiled by the EPPI reviewer22.\n\nThe quality tool developed by McGhee et al. in 2014 to assess the quality of surrogate biomarkers will be used to assess the overall methodological quality of the studies23.\n\nWe will synthesize the data narratively for each biomarker. All the outcomes stated in the studies will be reported. Additionally, we will present a summary of the range of outcomes where feasible.\n\nWe will assess the relationship between HIV infection and salivary biomarkers. We will report the effects of this relationship by variables reported in the studies, which were accounted for in the analysis. This review will further include a table of research implications, which will be extracted from each paper by research priorities. Additionally, we will report implications for clinical practice, where relevant. We will report the scoping review according to the PRISMA statement on reporting scoping reviews24.\n\nThe results of this scoping review will be disseminated through stakeholder meetings, conference presentations and peer-reviewed publications.\n\nThe search strategy and final plan for data extraction are complete. Formal screening of search results against eligibility criteria is ongoing.\n\n\nDiscussion\n\nThe proposed scoping review protocol will enable the identification and assessment of salivary biomarkers, which can predict disease progression in patients with HIV infection. Salivary components that mimic HIV infection progression can act as early predictors of the deteriorating clinical condition and serve as an alternative method to monitor the clinical condition. Moreover, its ease of extraction will correspond to greater compliance amongst patients when compared to other biofluids like blood.\n\nThe synthesis of evidence from this review will assist in improving our current understanding of biomarkers used to evaluate HIV disease progression. This paper will be the pilot in a series of studies aimed at identifying and validating a salivary biomarker for the potential development of a point of care device, which can assess HIV infection progression.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nGerman Advisory Committee Blood (Arbeitskreis Blut), Subgroup ‘Assessment of Pathogens Transmissible by Blood’: Human Immunodeficiency Virus (HIV). Transfus Med Hemother. 2016; 43(3): 203–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCloyd MW: Human Retroviruses. In: Medical Microbiology 4th edition. 1996. Reference Source\n\nMunshi SU, Panda H, Holla P, et al.: MicroRNA-150 is a potential biomarker of HIV/AIDS disease progression and therapy. PLoS One. 2014; 9(5): e95920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHira SK, Shroff HJ, Lanjewar DN, et al.: The natural history of human immunodeficiency virus infection among adults in Mumbai. Natl Med J India. 2003; 16(3): 126–31. PubMed Abstract\n\nBacchetti P, Moss AR: Incubation period of AIDS in San Francisco. Nature. 1989; 338(6212): 251–3. PubMed Abstract \| Publisher Full Text\n\nAl-Jabri AA: Mechanisms of Host Resistance Against HIV Infection and Progression to AIDS. Sultan Qaboos Univ Med J. 2007; 7(2): 82–96. PubMed Abstract \| Free Full Text\n\nShi M, Taylor JM, Fahey JL, et al.: Early levels of CD4, neopterin, and beta 2-microglobulin indicate future disease progression. J Clin Immunol. 1997; 17(1): 43–52. PubMed Abstract \| Publisher Full Text\n\nFahey JL, Taylor JM, Manna B, et al.: Prognostic significance of plasma markers of immune activation, HIV viral load and CD4 T-cell measurements. AIDS. 1998; 12(13): 1581–90. PubMed Abstract \| Publisher Full Text\n\nZangerle R, Steinhuber S, Sarcletti M, et al.: Serum HIV-1 RNA levels compared to soluble markers of immune activation to predict disease progression in HIV-1-infected individuals. Int Arch Allergy Immunol. 1998; 116(3): 228–39. PubMed Abstract \| Publisher Full Text\n\nKaslow RA, Duquesnoy R, VanRaden M, et al.: A1, Cw7, B8, DR3 HLA antigen combination associated with rapid decline of T-helper lymphocytes in HIV-1 infection. A report from the Multicenter AIDS Cohort Study. Lancet. 1990; 335(8695): 927–30. PubMed Abstract \| Publisher Full Text\n\nHulgan T, Shepherd BE, Raffanti SP, et al.: Absolute count and percentage of CD4+ lymphocytes are independent predictors of disease progression in HIV-infected persons initiating highly active antiretroviral therapy. J Infect Dis. 2007; 195(3): 425–31. PubMed Abstract \| Publisher Full Text\n\nMoore DM, Awor A, Downing R, et al.: CD4+ T-Cell Count Monitoring Does Not Accurately Identify HIV-Infected Adults With Virologic Failure Receiving Antiretroviral Therapy. J Acquir Immune Defic Syndr. 2008; 49(5): 477–84. PubMed Abstract \| Publisher Full Text\n\nBiomarkers Definitions Working Group: Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001; 69(3): 89–95. PubMed Abstract \| Publisher Full Text\n\nIlyin SE, Belkowski SM, Plata-Salamán CR: Biomarker discovery and validation: technologies and integrative approaches. Trends Biotechnol. 2004; 22(8): 411–6. PubMed Abstract \| Publisher Full Text\n\nYoshizawa JM, Schafer CA, Schafer JJ, et al.: Salivary Biomarkers: Toward Future Clinical and Diagnostic Utilities. Clin Microbiol Rev. 2013; 26(4): 781 LP-791. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanekar A: Biomarkers predicting progression of human immunodeficiency virus-related disease. J Clin Med Res. 2010; 2(2): 55–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang L, Xiao H, Wong DT: Salivary Biomarkers for Clinical Applications. Mol Diagn Ther. 2009; 13(4): 245–59. PubMed Abstract \| Publisher Full Text\n\nKinney JS, Ramseier CA, Giannobile WV: Oral fluid-based biomarkers of alveolar bone loss in periodontitis. Ann N Y Acad Sci. 2007; 1098: 230–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHegde MN, Attavar SH, Hegde ND: Saliva as a Biomarker in Detection of Human Immunodeficiency Virus - A Clinical Perspective. Clin J Surg. 2019; 2(1): 1–4. Reference Source\n\nArksey H, O’Malley L: Scoping studies: towards a methodological framework. Int J Soc Res Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nLevac D, Colquhoun H, O’Brien KK: Scoping studies: advancing the methodology. Implement Sci. 2010; 5(1): 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas J, Graziosi S, Brunton J, et al.: EPPI-Reviewer: advanced software for systematic reviews, maps and evidence synthesis. London: UCL Social Research Institute: EPPI-Centre Software. 2020.\n\nMcGhee DJM, Ritchie CW, Thompson PA, et al.: A systematic review of biomarkers for disease progression in Alzheimer’s disease. PLoS One. 2014; 9(2): e88854. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018; 169(7): 467–73. PubMed Abstract \| Publisher Full Text" }	[ { "id": "85113", "date": "18 May 2021", "name": "Sudhir Prabhu", "expertise": [ "Reviewer Expertise Community medicine", "vaccines", "non-communicable diseases", "maternal and child health", "HIV", "infectious diseases" ], "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 planned is clinically relevant and scientifically sound without any major errors.\nHowever the following minor technical issues might need to be addressed for the readers mainly for better clarity and understanding the purpose of the review:\n1. Objective 2 mentions \"to identify key concepts and gaps\" it is not clear whether it is with respect to existing biomarkers or only salivary biomarkers or both. Are the authors trying to convey through this review that due to limitations of biomarkers (lab/clinical), salivary biomarkers could fill that knowledge gap in the natural history of HIV?\n2. Outcome variables in HIV is normally assessed using Clinical, Immunological (CD4) and Virological (viral load) progression, when the authors mention outcomes in the PICO framework to be \"quantifiable\" does it mean only CD4 and viral load, both of them couldn't be seen in MeSH terms.\n3. Also progression of HIV which is mentioned as clinical staging, can be better mentioned as WHO staging and could be cited. Were there any other staging used for monitoring?\nAfter reviewing the article I would like to give the status as \"Approved\" for this article as the comments or queries raised don't alter the quality of the review. Even if the authors don't comply with the changes suggested here, the scientific and ethical validity of the systematic review planned by them will not change.\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? Yes", "responses": [ { "c_id": "6705", "date": "24 May 2021", "name": "Raghu Radhakrishnan", "role": "Author Response", "response": "Dear Reviewer The authors thank the reviewer for their kind comments. We are pleased to respond to the reviewer's comments. Comment: Objective 2 mentions \"to identify key concepts and gaps\" it is not clear whether it is for existing biomarkers or only salivary biomarkers or both. Are the authors trying to convey through this review that due to limitations of biomarkers (lab/clinical), salivary biomarkers could fill that knowledge gap in the natural history of HIV?. Response: We are systematically reviewing the existing literature only on salivary biomarkers in HIV. Comment: Outcome variables in HIV are normally assessed using Clinical, Immunological (CD4) and Virological (viral load) progression, when the authors mention outcomes in the PICO framework to be \"quantifiable\" does it mean only CD4 and viral load, both of them couldn't be seen in MeSH terms. Response: Often scientists do not include outcome measures in the search strategy, because many abstracts do not contain a description of these outcome measures. (Laboratory Animals 2012;46: 24–31. DOI: 10.1258/la.2011.01108) Comment: The progression of HIV, which is mentioned as clinical staging, can be better mentioned as WHO staging and cited. Was there any other staging used for monitoring? Response: Progression was with the immunological and clinical staging of HIV (https://www.who.int/hiv/pub/guidelines/HIVstaging150307.pdf)." } ] }, { "id": "87039", "date": "21 Jun 2021", "name": "Pascale Ondoa", "expertise": [ "Reviewer Expertise Laboratory science", "HIV immunology and virology", "Laboratory system strengthening", "diagnostics" ], "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\n30/06/2021 - Updated review (In response to the author’s comments): I acknowledged that I mistakenly reviewed the paper as a Systematic Review Study Protocol instead of a Scoping Review Study Protocol.\nI still think that the paper has many gaps and should not be accepted as is. The authors should revise their piece and include the requested information. Looking at the JBI manual for scoping review https://wiki.jbi.global/display/MANUAL/11.2.7+Data+extraction, https://guides.library.unisa.edu.au/ScopingReviews/Protocol the minimum information one expects to see in a protocol are not provided.\n21/06/2021 - Original review: This paper intends to introduce a systematic review of available data on markers for HIV disease progression in saliva.\nThe introduction is brief and does not correctly bring the rationale for identifying markers of disease progression. Viral Load is considered a reliable and early indicator of virological failure in patients receiving ART and is recommended by the WHO. Alternative markers might be useful, but we need to understand why. The discussion on the lesser value of CD4 to swiftly identify disease progression is an old one. Maybe the authors would like to develop assays that do not require blood collection and molecular testing? Then it should be more clearly outlined.\nIn addition to the poor background information, a systematic review protocol is supposed to provide a detailed plan of the methodology and analysis beforehand. Should the research question not be defined at this stage? The following are missing in the paper:\nThe search terms (key words) for the search. The time frame of coverage. The search strategy. The extraction of data. The outcome measures: how is a pertinent salivary marker defined? The strategy to handle biases\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? No", "responses": [ { "c_id": "6848", "date": "30 Jun 2021", "name": "Raghu Radhakrishnan", "role": "Author Response", "response": "Dear Reviewer Thank you for your comments. As a corresponding author, I am pleased to respond to your comments. Please find my responses as follows. Query - 1: This paper intends to introduce a systematic review of available data on markers for HIV disease progression in saliva. This paper intends to introduce a systematic review of available data on markers for HIV disease progression in saliva Response - 1:This paper is a scoping review and not a systematic review. This paper aims to ‘map the literature of available data on markers for HIV disease progression in saliva and to provide an opportunity to identify key concepts; gaps in the research; and types and sources of evidence to inform practice, policymaking, and research’ (Daudt et al.) Query - 2: The introduction is brief and does not correctly bring the rationale for identifying markers of disease progression. Viral Load is considered a reliable and early indicator of virological failure in patients receiving ART and is recommended by the WHO. Alternative markers might be useful, but we need to understand why. The discussion on the lesser value of CD4 to swiftly identify disease progression is an old one. Maybe the authors would like to develop assays that do not require blood collection and molecular testing? Then it should be more clearly outlined. Response - 2: We agree with the reviewer that “Viral Load is considered a reliable and early indicator of virological failure in patients receiving ART and is recommended by the WHO”. However, they might not be readily available in resource-limited settings (Ford et al; Ferreyra et al.) Query - 3: In addition to the poor background information, a systematic review protocol is supposed to provide a detailed methodology and analysis plan beforehand. Should the research question not be defined at this stage? The following are missing in the paper: Response - 3: This is a protocol for a scoping review and not a systematic review and the following information are quoted from the manuscript. The search terms (keywords) for the search, The time frame of coverage. I wish to inform the reviewer that this paper intends to introduce a systematic review of available data on markers for HIV disease progression in saliva. References: Daudt HM, van Mossel C, Scott SJ. Enhancing the scoping study methodology: a large, inter-professional team's experience with Arksey and O'Malley's framework. BMC Medical Research Methodology. 2013;13:48. DOI: 10.1186/1471-2288-13-48. Ford N, Meintjes G, Pozniak A, Bygrave H, Hill A, Peter T, et al. The future role of CD4 cell count for monitoring antiretroviral therapy. Lancet Infect Dis. Elsevier Ltd; 2015;15(2):241–7. Ferreyra C, Yun O, Eisenberg N, Alonso E, Khamadi AS, Mwau M, et al. Evaluation of Clinical and Immunological Markers for Predicting Virological Failure in an HIV/AIDS Treatment Cohort in Busia, Kenya. PLoS One. 2012;7(11)" }, { "c_id": "6864", "date": "02 Jul 2021", "name": "Raghu Radhakrishnan", "role": "Author Response", "response": "Dear Reviewer We acknowledge the reviewer’s comments and thank F1000 for inviting Dr. Pascale Ondoa to review our manuscript. The authors would like to humbly respond to the information that the reviewer has sought. For ease of understanding, we would like to draw information from our manuscript for each JBI criteria and presented it below for your kind consideration. (1) An introduction detailing: Definitions Per our manuscript: “According to the National Institutes of Health (NIH), “a biomarker is an objectively measured and evaluated indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”13. Essentially, a biomarker can represent any entity and can exist as antibodies, microbes, DNA, RNA, lipids, metabolites, or proteins” Overall review objective From the manuscript: “Objectives - To identify pertinent salivary biomarkers consistent with the progression of HIV infection in HIV positive individuals To systematically review the existing literature on biomarkers in HIV to identify key concepts and gaps To assess the current levels of evidence, the quality of evidence and provide a synthesis of the currently available salivary biomarkers in HIV infection” Aim: Per our manuscript: “aimed at synthesizing available evidence on salivary markers for disease progression in HIV infection”. Details of any preliminary searches undertaken Explanation of the need for review Per our manuscript: \"The literature reveals the dearth of evidence on validated biomarkers16. Much of the information on the topic is largely experimental, which has to be systematically compiled and objectively assessed.\" Eligibility criteria (with contextualization and rationalisation) From the manuscript: “People diagnosed with HIV/AIDS (PLWHA) as per WHO clinical case definition is “an individual with HIV infection irrespective of the clinical stage (including severe or stage 4 clinical disease, also known as AIDS) confirmed by laboratory criteria according to country definitions and requirements”. We will include longitudinal studies that have measured outcomes of at least two different time points. Cross-sectional studies measuring clinical parameters at only one point will be excluded. Only studies that have reported an association between salivary biomarkers and change in the clinical measure will be included in our scoping review. Due to a lack of sufficient resources, studies will be excluded if English language texts are not available. We will not limit our inclusion based on age, gender, duration of HIV infection, ART status, or demography. Only studies that have reported measurable and quantifiable biological parameters associated with salivary biomarkers will be included. These parameters include, but are not limited to the presence of specific biomolecules, their biologic concentrations, specific gene-phenotype distribution in a population. (2) Sample Search Strategy From the manuscript: “The search strategy used in this scoping review included: (PLWHA OR PLHIV OR PLWH OR PLWA OR HIV OR (people living with HIV/AIDS) OR (people living with AIDS) OR (acquired AND (immunodeficiency OR immune‐deficiency OR immuno‐deficiency) AND syndrome) OR Immunocompromised OR immune-compromised OR Slim disease) AND ((HIV related oral lesions) OR (Periodontal disease) OR Periodontitis OR (periodontal infection) OR Xerostomia or (dry mouth) OR (salivary gland disease)) OR (Oral candidiasis) OR (hairy leukoplakia) OR (Kaposi sarcoma) OR (linear gingival erythema) OR (necrotizing ulcerative periodontitis) OR (aphthous ulcer) OR (wasting disease))) AND ((biological marker) OR biomarker OR saliva* OR biomolecule* OR (bacterial burden) OR marker)” 3) Explanation of search approach, including: Which black and grey literature will be searched? Per manuscript: “To ensure that all information pertinent to the research question is adequately captured, our search will include several grey literature sources from relevant databases (e.g. Grey Literature Report, OpenGrey, Web of Science Conference Proceedings). We will further conduct a targeted search of grey literature on the websites of organizations working on HIV/AIDS research on the local, provincial, national, and international levels. Any studies, reports, and conference abstracts identified through these databases, which are of relevance to this review, will be included.” Justification for choices From the manuscript: “To ensure that all information pertinent to the research question is adequately captured” (4) Study selection process, including resolving disagreements between reviewers From the manuscript: “We will undertake a two-step screening process to include all potentially relevant articles in this review: (1) title and abstract screening; (2) full-text screening. In the first stage of screening, two review authors (VD and PP) will independently screen the title and abstract of all retrieved citations for inclusion against a set of minimum inclusion criteria. These criteria will be determined by testing on a sample of abstracts before beginning the abstract review to ensure that they are robust enough to capture all studies pertinent to the primary objective. Articles will be included for full-text screening if either one or both of the review authors deem them relevant to the research question. All the studies included in the T&A stage will be subject to full-text screening. In this step, both the investigators (VD and PP) will independently screen the full-text articles to assess if they meet the inclusion/exclusion criteria. We will calculate Cohen's κ statistics at both the T&A review stage and the full article review stage to determine inter-rater agreement. Studies will be reviewed another time if there is any discordance regarding the study eligibility. If there are further disagreements, they will be resolved through discussion with a third investigator (RR) until a consensus is reached. A flow diagram will be used to represent the inclusion and exclusion of retrieved studies” (5) A draft charting table/form for data extraction and accompanying explanation Data charting form: Table number 2. From the manuscript: “The research team will develop a data collection instrument to extract information from the included studies and to confirm study relevance. Study characteristics including publication year, publication type (eg, original research), study design, country, study setting, a specific biomarker used, statistical analysis performed, the association between biomarker tested and disease progression, the effect of therapeutic agents on biomarker changes, economic aspects and acceptability of biomarker, etc will be extracted (see Table 2). The research team will review and pretest the form to make sure that the data extraction form captures all the required information from the included studies accurately.” (6) How results and data will be presented (e.g. draft chart, figure or table) From the manuscript: “We will synthesize the data narratively for each biomarker. All the outcomes stated in the studies will be reported. Additionally, we will present a summary of the range of outcomes where feasible. We will assess the relationship between HIV infection and salivary biomarkers. We will report the effects of this relationship by variables reported in the studies, which were accounted for in the analysis. This review will further include a table of research implications, which will be extracted from each paper by research priorities. Additionally, we will report implications for clinical practice, where relevant. We will report the scoping review according to the PRISMA statement on reporting scoping reviews24.” (7) Data Extraction: We have grouped the information and presented it across the JBI charting form for your reference and ease in table 2 Author(s): From the manuscript: “Author”. Year of publication: From the manuscript: “Year of publication”. Origin/country of origin (where the source was published or conducted): From the manuscript: “country”. Aims/purpose: From the manuscript: “Aim/ objective of the study”. Population and sample size within the source of evidence (if applicable): From the manuscript: “population, sample size”. Methodology/methods: From the manuscript: “Study design, settings, age, duration of infection”. Intervention type, comparator, and details of these (e.g. duration of the intervention) (if applicable). Duration of the intervention (if applicable): From the manuscript: “biomarker used, biomarker classification. Outcomes and details of these (e.g. how measured) (if applicable) (method of biomarker obtained): From the manuscript: “Main findings of the study, the association between biomarker tested and disease progression, does the biomarker associated with increase mortality, Correlation, Effect of therapeutic agents on biomarker changes, The method used for statistical analysis, Acceptability of biomarker. Key findings that relate to the scoping review question/s.: From the manuscript: “Conclusion, Confounders adjusted, most relevant findings”. The authors once again wish to place on record our sincere gratitude and appreciation to the editorial board for providing us an opportunity to contribute to F1000. Thanking you. Kind regards Raghu" } ] }, { "id": "85305", "date": "23 Aug 2021", "name": "Purnima Madhivanan", "expertise": [ "Reviewer Expertise Systematic Reviews and Meta-analysis", "Infectious Diseases", "HIV/AIDS", "Diagnostics for developing world" ], "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 review was conducted by Namoonga Mantina and Abidemi Okechukwu under the guidance of Purnima Madhivanan.\n\nGeneral Review This scoping review protocol was reviewed using the PRISMA-SCR checklist and the JBI Manual for Evidence Synthesis.\nGeneral comments\nIn general, the methodology section of this protocol addresses most of the items in the PRISMA-SCR checklist.\n\nThis protocol as it is written, however it communicates conflicting statuses to the reader. In some sections, it reads as a protocol and in other instances, it reads like the final report of the scoping review. In the methodology section, the authors noted that the search terms were finalized, however, within the same section, the authors noted that they “will use the PICO…strategy for formulating a foreground research question”.\n\nSpecific comments\nTitle:\nAuthors did not specify in the article title that the paper was a scoping review protocol. Leaves the reader to figure it out at the very end of the manuscript.\nIntroduction:\nWhile the salivary biomarkers for disease progression in HIV infection appears to be an under researched area, the area of study aligns well with the authors’ decision to conduct a scoping review. However, the introduction fails to explain why the questions and objectives of this study support a scoping review rather than a systematic review.\n\nParagraph 2, first statement is missing a citation. Please include citations for the statement.\n\nParagraph 5: The authors state that there is a “dearth of evidence of validated biomarkers”. As a general statement (encompassing all conditions), this seems inaccurate. If the authors mean to specifically refer to HIV/the condition being evaluated, then this should be added for clarification.\n\nThe objectives of the study did not specify key components of the scoping review, which should be population or participants, concepts, and context. The authors might want to consider revising the objectives to specify each element. The PRISMA_SCR checklist can provide additional details.\n\nIn addition, Objective 2 is not specific to salivary biomarkers implying a much broader scope for review. This needs to be clarified along with clearly defining the limits of the scoping review. It seems this objective aims to explore all biomarkers related to HIV yet the stated premise of the review is to review salivary biomarkers. This objective seems beyond the scope of the review.\n\nThe terminology to identify people living with HIV has be to used appropriately. We no longer use HIV positive individuals as a term to address people living with HIV.\n\nThe case to evaluate saliva as a biomarker could be further explanation. From the information provided it seems that the only reason for choosing saliva is because CD4+ T-cell count aren’t reliable to detect virologic failure under ART. Has there been research to indicate that saliva would perform better under this condition and be worth investigating?\nMethodology:\nParagraph 1: Provide a reference for the clinical definition of PLWHA.\n\nGiven the statement written by the authors that there is a dearth of studies on salivary biomarkers for monitoring HIV progression (paraphrased), limiting the search to studies published in English language may leave out important studies and limit the breadth of the scoping review. This is contrary to the very basis for conducting this review. Furthermore, the review, if limited to English language only, will bias the findings and the scope of the review will be further reduced.\n\nThe third objective stated that “level of evidence” and “quality of evidence” will be assessed. How are the authors distinguishing these two? Furthermore, the methods section only details methodological quality. Furthermore, additional information on the tool being used would be helpful.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "7163", "date": "05 Jan 2022", "name": "Raghu Radhakrishnan", "role": "Author Response", "response": "Dear Reviewers, The authors thank the reviewer for their kind comments. We are pleased to respond to the reviewer's comments. Title: Authors did not specify in the article title that the paper was a scoping review protocol. Leaves the reader to figure it out at the very end of the manuscript. - Changes have been made in the manuscript and re uploaded Introduction: While the salivary biomarkers for disease progression in HIV infection appears to be an under researched area, the area of study aligns well with the authors’ decision to conduct a scoping review. However, the introduction fails to explain why the questions and objectives of this study support a scoping review rather than a systematic review. – A sentence in this regard is now added at the end of Introduction. Paragraph 2, first statement is missing a citation. Please include citations for the statement. - Changes have been made in the manuscript and re uploaded. Paragraph 5: The authors state that there is a “dearth of evidence of validated biomarkers”. As a general statement (encompassing all conditions), this seems inaccurate. If the authors mean to specifically refer to HIV/the condition being evaluated, then this should be added for clarification. - Changes have been made in the manuscript and re uploaded. The objectives of the study did not specify key components of the scoping review, which should be population or participants, concepts, and context. The authors might want to consider revising the objectives to specify each element. The PRISMA_SCR checklist can provide additional details. Objectives: To identify pertinent salivary biomarkers consistent with the progression of HIV infection in HIV positive individuals. To systematically review the existing literature on biomarkers in HIV to identify key concepts and gaps. To assess the current levels of evidence the quality of evidence and provide a synthesis of the currently available salivary biomarkers in HIV infection. P - HIV positive individuals E - Salivary biomarker O - progression of the disease In addition, Objective 2 is not specific to salivary biomarkers implying a much broader scope for review. This needs to be clarified along with clearly defining the limits of the scoping review. It seems this objective aims to explore all biomarkers related to HIV yet the stated premise of the review is to review salivary biomarkers. This objective seems beyond the scope of the review. - Changes have been made in the manuscript and re uploaded. The terminology to identify people living with HIV has be to used appropriately. We no longer use HIV positive individuals as a term to address people living with HIV – Changes have been made in the manuscript and re uploaded. The case to evaluate saliva as a biomarker could be further explanation. From the information provided it seems that the only reason for choosing saliva is because CD4+ T-cell count aren’t reliable to detect virologic failure under ART. Has there been research to indicate that saliva would perform better under this condition and be worth investigating? - We have added more explanation in the introduction section in this regard. Methodology: Paragraph 1: Provide a reference for the clinical definition of PLWHA. - Changes have been made in the manuscript and re uploaded. Given the statement written by the authors that there is a dearth of studies on salivary biomarkers for monitoring HIV progression (paraphrased), limiting the search to studies published in English language may leave out important studies and limit the breadth of the scoping review. This is contrary to the very basis for conducting this review. Furthermore, the review, if limited to English language only, will bias the findings and the scope of the review will be further reduced. - This is beyond the scope of the current review The third objective stated that “level of evidence” and “quality of evidence” will be assessed. How are the authors distinguishing these two? Furthermore, the methods section only details methodological quality. Furthermore, additional information on the tool being used would be helpful. – Changes have been made in the manuscript and re uploaded." } ] } ]	1	https://f1000research.com/articles/10-130
https://f1000research.com/articles/10-938/v1	17 Sep 21	{ "type": "Research Article", "title": "Pulmonary function parameters among marble industry workers in Lahore, Pakistan", "authors": [ "Imran Maqsood Butt", "Tajammal Mustafa", "Shahnaz Rauf", "Anjum Razzaq", "Javaria Anwer", "Imran Maqsood Butt", "Shahnaz Rauf", "Anjum Razzaq", "Javaria Anwer" ], "abstract": "Background: Occupational contact with dust particles is a well-known phenomenon, particularly in developing countries of the world. Crystalline silica present in marble dust is the main etiology of a rising prevalence of obstructive lung diseases in marble stone workers, who are in direct contact with marble dust in the surrounding environment during their regular work. The purpose of this study was to compare the pulmonary function parameters of workers in marble workshops and age matched healthy individuals in the Lahore District of Pakistan. Methods: The study included 164 male individuals, 82 individuals working in marble workshops and 82 healthy individuals from the same community. Data were collected through in-person interviews using a structured questionnaire after obtaining written consent. A Spiro Lab spirometry for pulmonary function tests was used to identify any change in the lung function parameters. FVC% (forced vital capacity), FEV 1 (forced expiratory volume in first second) and FEV1 / FVC ratio were evaluated. Results: Mean age in the exposed group (marble workers) and non-exposed group (healthy individuals) were 29.92 ± 6.19 and 30.58 ± 6.37 years, respectively. The mean years of work experience of the exposed group was 11.92 ± 5.67 years. A statistically insignificant difference was observed between marble exposed workers & healthy individuals from the demographic variables. Lung function parameters in marble workers exhibited a highly significant (P < 0.001) decrease in FVC%, FEV1 & FEV1 / FVC ratio when compared to healthy individuals. Seventy-one percent of marble workers had abnormal pulmonary parameters whereas 34% of workers had restrictive pulmonary impairment. Marble workers who had worked for more than 15 years had a highly significant risk of developing abnormal pulmonary function (P < 0.001). Conclusion: Continuous exposure to marble dust deteriorates the lung function of marble workers.", "keywords": [ "Pulmonary functions", "Spirometry", "Marble dust", "Marble workers" ], "content": "Introduction\n\nToxic elements, fumes, and respirable dust produced in marble workshops can cause a health-related risk to the workers in different units of marble workshops. Marble is a semi-translucent very fine to coarse grained crystalline rock. It mainly contains calcite, serpentine and dolomite and is mostly used in making monuments, headstones, and floors. Finely ground marble is used as a whitening agent in toothpastes, paint, and paper.1,2 Marble dust mainly contains free silica, which means “free of elements” as it is not combined with other elements or silicon dioxide (SiO2). Silica is a common ingredient of the Earth’s crust3 and can be found in alpha quartz, beta quartz, moganite, granite, slate, sandstone, and keatite, and it is toxic to the human respiratory system. Occupational exposure to dust containing crystalline silica occurs in the stone, granite, construction, mining, metal foundry, ceramic production and glass industries.4 Airborne dust (free silica) is produced in marble workshops during quarrying, grinding, mining, cutting, and polishing activities and is the main causative factor for many occupational lung diseases such as pneumoconiosis, chronic obstructive pulmonary disease (COPD), silicosis and asthma.5,6\n\nContinuous inhalation of respirable silica causes many diseases including silicosis characterized by inflammation and pulmonary fibrosis. Silica dust particles enter the alveoli, increase the production of inflammatory mediators in the peripheral airway, and cause emphysema. The particles are mainly deposited on airway surfaces where air flow changes direction. Silica particles having a size 0.2 to 2 micrometers get deposited on the walls of the airway and particles less than 0.2 micrometers enter the terminal respiratory epithelial surfaces and finally diffuse into alveolar gas.7,8 Deposited crystalline silica particles cause respiratory mucosal irritation, mucosal hypersecretion in the large airways, mucosal gland hypertrophy of trachea and bronchi, an increase in the number of goblet cells in small airways, and excessive mucus formation, which results in the formation of a mucous plug in the lumen, and fibrosis of small airways.7,8 Obstruction in the air flow in airways results in decreased FVC (forced vital capacity), FEV1 (forced expiratory volume in first second) and FEV1/FVC ratio airways.9,10 The results of recent research showed a significant association between pulmonary problems and inhalation of silica dust in Bangladesh.11 Previously published data from Australia demonstrated that stone workers working in factories that perform cutting and grinding activities produce higher contents of respirable silica, which is associated with a higher rate of severe silicosis.12 A previous cross-sectional study among Indian stone-crush workers reported a noteworthy decrease in FEV1, FVC and FEV1/FVC parameters.13 Several previous studies conducted in Austria, Nigeria, and Lebanon demonstrated that longer exposure to occupational dust (silica) as well as smoking leads to a gradual decrease in Pulmonary Function Tests (PFTs).14-16\n\nIn low and middle economic countries, silica associated lung illness remains a major health threat. In China more than half a million silicosis cases were documented between 1991 and 199517,18 and over ten thousand silicosis associated deaths were reported in South African miners.19 Despite better dust control safety measures and advancements in developed countries, there is a constant need to control dust due to a recent pneumoconiosis outbreak in Australia and the USA.20,21\n\nRisk of developing lung diseases among silica exposed workers was higher with prolonged exposure to respirable silica dust. The incidence of silica associated silicosis was 12% among the workers who worked for 30 years or more.22 Sharma et al. reported that silica is a key factor for other autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, Caplan syndrome, and Erasmus syndrome, which derives from silica exposure and cytoplasmic antibody related vasculitis.23\n\nMarble workers are at a greater risk because of illiteracy, low socioeconomic status, poor knowledge of personal protective measures (PPM), lack of safety rules and their enforcement, and exploitation by employers.24\n\nIn developing countries such as Pakistan, plenty of evolutionary projects such as roads, flyovers, underpasses, housing schemes, hospitals etc. are commissioned by the local government and federal agencies. These projects demand people working in the marble sector. Vast research has been done in the stone cutting sector globally but data on pulmonary function parameters for marble industry workers is scarce.25 Thousands of individuals work in small to medium sized marble workshops in a poorly ventilated hazardous environment. The marble workers working in marble workshops are among the most neglected and work in poorly organized areas. The present study measured the impact of silica dust exposure on pulmonary function tests of these workers. The objective of the current study was to compare pulmonary function parameters among the workers of Lahore based marble workshops and age matched healthy individuals.\n\n\nMethods\n\nThe study was carried out in the Ichra and Township areas of the Lahore District, which are heavily populated commercial areas having the majority of small to medium sized marble workshops. The study unit consisted of marble workers working in wet cutting and dry cutting (grinding) units of the marble workshops. The study group (exposed) comprised male marble workers, aged 18 to 40 years inclusive exposed to marble dust for more than one year, and working 30 hours or more per week. Adult male workers with a diagnosed respiratory system disease (asthma, tuberculosis, bronchiectasis), those with thoracic deformity, those who underwent chest surgery, those who had history of pneumothorax, hemoptysis, recent abdominal or eye surgery, known respiratory malignancy, unstable cardiovascular status (recent heart attack, aneurysm), and those who were unfit for spirometry were excluded.\n\nNormal healthy male individuals from the same community residing near each study site having similar socio demographic status but free from respiratory problems were selected as the control (non-exposed) group.\n\nThe sample size was calculated with the WHO sample size calculator using the formula:\n\nAssuming 90% power of study, 5% significance level, Z1-β = 90% power of study, population variance (σ2) = 0.5329, anticipated mean for study group I (μ1) = 2.77 (Isara et al.),15 anticipated mean for study group II (μ2) = 3.14 (Isara et al.),15 the calculated sample size (n) for one group was 82. The total required sample size for both groups was 164.\n\nA total of 164 subjects were selected, 82 marble workers (study exposed group) by simple random sampling (lottery method) and 82 healthy individuals (study control group) were selected by convenience sampling.\n\nThe study was approved by the Ethical Committee of the Institute of Public Health (IPH), Lahore, vide letter number 478, dated July 23, 2018. The data was collected from February to April 2019. The data collection techniques (clinical examination, questionnaire, and pulmonary function parameters) were similar for both study groups (marble dust exposed workers and healthy individuals). The study procedures were explained to every study subject and a written consent was obtained from each individual who agreed to participate in the study. All participating subjects were interviewed in-person using functional proforma developed by the American Thoracic Society (ATS)26 and the pulmonary function tests of each subject were addressed as outlined in the Medical Research Council Questionnaire27 (https://mrc.ukri.org/documents/pdf/questionnaire-on-respiratory-symptoms-1986/). Some items of ATS proforma, like some symptoms and family history, were not included in the questionnaire as these did not relate to our study objectives. The first section of the functional proforma captured information on gender, area of residence, date of birth, marital & educational status. While the second section of the questionnaire documented information regarding occupational history, nature of work, working hours, and total duration of work in years. The height in centimeters and weight in kilograms were measured for all study participants.28,29 Spirometry was performed by using a Spirolab spirometer (MIR SRL, Roma, Italy). Spirolab spirometry was used throughout the study period for both study groups for PFTs. Spirometry pre-requisites (any potential contraindication, strenuous exercise an hour before test, consumption of bronchodilators) were followed in accordance with the standard protocols and guidelines by ATS.30 The purpose of spirometry and the procedure were explained to each participant.30,31 The spirometry values obtained were reported with correction for body temperature at ambient pressure, saturated with water vapor (BTPS). Lung volumes (FVC and FEV1) were measured in a standing position from a sequence of a minimum of three readings, having adequate start. After probing the data from whole usable curves, the highest FVC & FEV1 were noted even though they didn’t belong to the same curve. The FEV1/FVC was also calculated from the same tracing.\n\nSpirometry designates the presence of pulmonary impairment, if any of the following recordings were observed32:\n\n• FEV < 80% - predicted normal\n\n• FEV1 < 80% - predicted normal\n\n• FEV1/FVC ratio < 0.7\n\nThe percentages of the predicted values were calculated by Bellamy et al.’s following formula32:\n\nFVC (Reading/Predicted Values) × 100% = %age of predicted value\n\nFEV1 Reading/(Predicted Values) × 100% = %age of predicted value\n\nFEV1/FVC Reading/Reading %\n\nThe airflow obstruction can be defined as follows according to the NICE COPD guideline33:\n\n• Mild airflow obstruction - FEV1 more than 80%\n\n• Moderate airflow obstruction - FEV1 between 50–80%\n\n• Severe airflow obstruction - FEV1 between 30–49%\n\n• Very Severe airflow obstruction - FEV1 <30% predicted\n\nObstructive Type Impairment: is characterized by a complete or partial narrowing of airways at any point, which results in an increased resistance and air flow.34 Narrowing of airways is the main reason of obstructive type impairment and is typically found in asthma and COPD (http://www.irishthoracicsociety.com). Restrictive Type Impairment: The lung capacity is lower than the predicted value for age, sex & size. In this type of impairment, the capacity of lung volume is lower because the lungs are firm & less compliant. There is a decrease in the level of lung parenchymal mobility or chest wall.34 Pneumoconiosis and pulmonary fibrosis are examples of restrictive types of impairment, which causes scarring (fibrosis) of the lungs. Combined Restrictive/Obstructive: In combined type of impairment, all the values of FVC, FEV1, & FEV1/FVC are decreased. It may be associated with two conditions i.e. asthma and another lung pathology or some pulmonary conditions (http://www.irishthoracicsociety.com).\n\nFigure 1 presents operational definitions of pulmonary impairment as recommended by spirometry flow chart for diagnosis (reference). The underlying study data (Table 1: Data Marble Feb 19 2021.sav) is available at Figshare.35\n\nThe data were analyzed in SPSS version 24 and organized in tabulated form according to the frequency distribution table. The continuous variables such as age, duration of dust exposure, pulmonary function values were summarized in mean, median, standard deviation (SD), and categorical variables like nature of work were depicted as frequencies and percentages. The SD was calculated to understand the variation in the study data. The student t-test was employed to compare mean pulmonary function indicators (FVC, FEV1, and FEV1/FVC ratio) between marble workshop workers and healthy community controls. The ANOVA was applied to compare pulmonary function indicators (FVC, FEV1, and FEV1/FVC ratio) by years of exposure; Tukey’s method was employed for post-hoc pairwise comparisons. ANCOVA was employed to control and test for confounding due to smoking on pulmonary function indicators. Chi-square test was used to test the difference in the proportion of smokers and pulmonary impairment between marble workshop workers and healthy community controls. The p-value of 0.05 or less was taken as statistically significant for all analyses.\n\n\nResults\n\nThe current study showed that the average age, height, and weight of the study exposed group (participants exposed to marble dust) were 29.92 ± 6.19 years, 172.84 ± 10.2 cm and 74.39 ± 13.77 kg respectively and for the control group (non-exposed group) were 30.58 ± 6.37 years, 170.45 ± 9.56 cm and 74.89 ± 12.54 kg respectively. There was no statistically significant difference in the sociodemographic characteristics among marble dust exposed workers and the control group (p > 0.05). About 89% of the exposed group had education levels up to matriculation or below, as compared to 13.4% of the non-exposed group (statistically significant with p < 0.001). In the study exposed group, 48% of the subjects were married compared to 39% of participants among the non-exposed group (p = 0.353). Among the exposed group, 19.5% were smokers compared to 4.9% of non-exposed (healthy individuals), and this was statistically significant (p = 0.007) (Table 1).\n\nAbout 71% of workers from the exposed group had statistically significant (p < 0.001) abnormal spirometry results whereas none of the healthy individuals (non-exposed) had abnormal spirometry results. The vast majority of lung impairments were of the restrictive type 40.25%; including mild (34.15%) and moderate (6.1%) restriction respectively followed by obstructive type 25.62% of impairment; which includes mild obstruction (10.98%), moderate obstruction (7.32%), moderate-severe obstruction (6.1%), and very severe obstruction (1.22%) respectively, while combined type of impairment was 4.88%.\n\nThe mean values ± SD of FVC%, FEV1% and FEV1/FVC ratio of non-exposed (healthy individuals) were 95.68 ± 10.66, 92.85 ± 9.78, and 80.73 ± 4.71 respectively and in the marble dust exposed group were 85.36 ± 14.80, 75.93 ± 15.65 and 74.34 ± 12.68, respectively (Table 2). The FVC%, FEV1%, and FEV1/FVC ratio for participants exposed to marble dust were significantly lower than healthy individuals (p < 0.001) (Figure 2).\n\n* Statistically significant at α ≤ 0.05.\n\nComparison of PFTs (mean ± SD) in study population.\n\np-value ≤ 0.05, p-value ≤ 0.01, *p-value ≤ 0.001 vs healthy group.\n\nComparison of pulmonary function parameters (mean ± SD) in the study population showed the average duration of work in marble workshop workers was 11.2 ± 5.677 years, and 51.2% had been working for more than 10 years (Table 3).\n\nAll three pulmonary function parameters (FVC%, FEV1% and FEV1/FVC ratio) among marble workers by years of exposure showed a statistically significant reduction with increase in duration of work (p < 0.001). The maximum reduction in pulmonary function indices was observed among marble workers who had been working for 16–20 years (Table 4).\n\n Statistically significant at α ≤ 0.05.\n\nTable 5 shows pairwise comparison of pulmonary function indices among marble workers with years of work in marble industry categories. Compared to participants with 1–5 years’ work exposure, the FVC% and FEV1% were significantly decreased among individuals with 6–10 years’ work exposure, individuals with 11–15 years’ work exposure, and individuals with 16–20 years’ work exposure (p < 0.001). Compared to participants with 1–5 years’ work exposure, FEV1/FVC ratio was significantly less among workers with 16–20 years’ work exposure. No other significant differences between groups were observed.\n\n* Statistically significant (p ≤ 0.05).\n\n\nDiscussion\n\nPFTs are the main tool to help identify pulmonary abnormalities. PFTs also provide information about airways, pulmonary bed integrity, and parenchymal size. Sharma et al. concluded in his study that airborne crystalline silica significantly reduced the FVC and FEV1 values in workers who were engaged in marble factories when they were exposed to marble dust as an air pollutant.23 These particles cause respiratory irritation mechanically and release of histamine mediators, which leads to obstruction in the airway.\n\nThe current study showed a significant reduction (p < 0.001) in mean values of PFT indices (FVC%, FEV1% and FEV1/FVC ratio) of marble dust exposed workers as compared to the non-exposed (healthy individuals) group. These results were also in agreement with data reported by Ophir et al. in his recent study, who found that ultrafine silica particles are strongly associated with and responsible for the deterioration of pulmonary function parameters among stone factory workers.36 In another study, Ullah et al. reported that exposure to high concentration silica particles by stone crushing workers significantly affects their lungs.25 Researchers also observed that carbon containing particles have a contrary effect on the dysfunction of pulmonary functions in addition to silica particles.\n\nAhmed et al. documented direct association of marble dust on lung functions in marble factory workers who had constant exposure to silica dust.37 The spirometry values (FVC%, FEV1) were statistically significant (p < 0.01) and had similar differences between the control group and marble workers to our study. Furthermore, many previous studies in India,38 Thailand,39 Iran,40 Egypt,41 Nigeria,15 and Libya16 reported similar findings and mean values of pulmonary function parameters were found to be remarkably deteriorated resulting in pulmonary dysfunction.\n\nThe inhaled dust particles get deposited in lungs, cause irritation, and cause an inflammatory reaction leading to fibrosis, faulty oxygen diffusion and abnormal lung functions.6 The change in the elasticity and viscosity of mucous influences its clearance and results in a luminal mucous plug, which is responsible for obstruction to the air flow resulting in decrease in FVC, FEV1 and FEV1/FVC ratio.8\n\nReduced values of FEV1 and FVC are indications of obstructive and restrictive types of lung changes respectively. The deviations of pulmonary function indicators (FVC, FEV1, FEV1/FVC ratio) are the major characteristic features of restrictive lung disease.31 In our study, 71% of marble workers had abnormal PFTs while 29% had normal PFT values. Among abnormal PFTs, 40.25% had the highest proportion of pulmonary impairment labelled as restrictive lung pathology, which was followed by 25.62% obstructive type, while 4.88% of cases demonstrated a mixed type of impairment. Our current study findings are quite similar to past studies where researchers reported that 20.1% and 24.3% of lung impairments were of restricted type and 1.5% and 5.8% were of obstructive type.42,43 Similar results (restriction 23.3% and obstruction 6.2%) were also reported in a previous study among stone crushing industrial workers.25 In contrast to our study, notably less restrictive (2.5%) and obstructive (6.7%) types of pulmonary impairment were reported in a recent study on stone crushing workers.11 The major causes of restrictive type impairment are chest wall limitations, neuromuscular disorder, fibrosis of lung parenchyma, and pleural disorder, whereas obstructive type of pulmonary impairment is commonly seen in chronic bronchitis, asthma and emphysema.11\n\nAge of the study subject could be considered an important causal factor for variation in lung functions and it accounts for a 1.41 fold higher incidence rate using a fixed ratio of variation of lung function parameters.44 Previously published data showed that the anthropometric parameters have a strong association with lung function parameters.45,46 The present study showed an equal distribution of age group among marble dust exposed workers and non-exposed healthy individuals, both groups were insignificant (p < 0.491). Similar studies on marble stone workers found no statistically significant (p > 0.05) differences in the ages of marble workers and control groups.47,48\n\nIn our study, the results of sociodemographic characteristics showed no statistical difference in age (p < 0.491), weight (p < 0.808) and height (p < 0.1274). Our findings for the mean age, height, and weight are similar to previously reported data by Vyas,48 Shaik et al.,38 and Dostbil et al.49 for case control studies among marble stone workers.\n\nIn the current study, only 20 (12.2%) of the total study participants were smokers, while the proportion of marble workers who smoked were 16 (19.50%) only, compared to four healthy individuals (4.90%). The smoking effect on all PFT parameters was statistically not significant (p > 0.05). Similar findings were reported in a previous study, which showed that smoking did not (p < 0.98) affect PFTs15 although other authors, Rathod et al.,50 Sheikh et al.,51 in India, Ullah et al., in Pakistan,25 and Jaber et al.,42 in Cairo reported a significant association (p < 0.05) of smoking on pulmonary function impairment, which contradicts our study. In our study the possible reason of these non-significant effects could be due to shorter smoking duration, the low intensity (only four packs per year) of smoking in the study population, a fewer number of study participants, and less inhalation during smoking.\n\nThe mean years of work experience of our participants (marble workers) was 11.20 ± 5.67 years, which was statistically highly significant (p < 0.001). The majority of the marble workers (23; 28.05%) were working for a period of more than 16 years and their PFT values markedly decreased due to the high duration of exposure (p < 0.001). The maximum reduction in pulmonary function indices were noted among the marble workers who had been working for 16–20 years. All three pulmonary functions parameters, FVC%, FEV1% and FEV1/FVC ratio, showed significant reduction with increase in duration of work (p < 0.001).\n\nOur study results are analogues to a previous study in which the study author reported a statistically highly significant (p < 0.001) reduction in PFT values.52 He claimed that the duration of exposure to silica is most important and documented predictor of decreased pulmonary functions in the workers exposed to silica dust.\n\nOur study results are parallel to an earlier study in which the researchers documented a statistically highly significant (p < 0.05) reduction in pulmonary function parameters with longer duration of work exposure in marble factories.50 The researchers also reported that the deterioration of PFTs is strongly associated with increased duration of exposure to silica.50\n\nSimilar types of results were also reported among quarry workers in Nigeria where the mean values of PFTs, the negative correlation between longer work exposure and FEV1 (p < 0.05), and lower lung function values were reported.52 In concurrence to our study, researchers (Shaik et al.38 Nandini et al.,53) also reported a significant reduction in FVC, FEV1 and FEV1/FVC ratio (p < 0.001) in quarry and granite workers having greater than 10 years of silica dust exposure.\n\nThere is a possibility of confounding due to age and smoking. Age matched participants from the community (control group) were selected, hence confounding due to age is unlikely. No differences in the pulmonary function test values between smokers and non-smokers were observed. Also, on ANCOVA analysis, smoking was not statistically significant, therefore, confounding due to smoking is also unlikely.\n\n\nConclusion\n\nPulmonary function parameters were found to be significantly decreased among marble workers who have marble dust exposure compared to healthy individuals from the same community. The current study disclosed that the continuous occupation of the marble industry for more than 15 years resulted in further reduction of workers’ pulmonary function parameters, which ultimately would have harmful effects on their health status.\n\n\nLimitations\n\nExposure to marble dust is not the only reason for pulmonary function deterioration. There could be other possible factors such as toxic materials in the work environment as well as undetectable concentrations of dust particles other than silica. Dust exposed particle sampling was not used to quantify its composition and concentration, and a comparison to silica dust was not made in both study groups. In addition to this, further respiratory procedures like chest radiographs should be carried out for comparison with previous literature in order to verify findings.\n\n\nData availability\n\nFigshare: Underlying data for ‘Pulmonary function parameters among marble industry workers in Lahore, Pakistan’, https://figshare.com/s/7a24edab6fc0d64c526c.35\n\nThis project contains the following underlying data:\n\n• Table 1: Data Marble Feb 19 2021.sav.\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 statement\n\nWritten informed consent was taken from each study participant, the informed consent form was in Urdu, the national language. Under the confidentiality section of the informed consent, it was clearly mentioned that confidentiality will be strictly maintained at all levels, and in reporting the results, no personal information will be shared, and data will be reported in aggregate form. Specific permission was obtained for the publication of participants' data.", "appendix": "References\n\nHae-Rim NAM, Yue SH, Hwan-Seok PARK, et al.: Artificial marble composition and production method for artificial marble using same. U.S. Patent Application 15/762,049; 2018.\n\nEsswein EJ, Breitenstein M, Snawder J, et al.: Occupational exposures to respirable crystalline silica during hydraulic fracturing. J. Occup. Environ. Hyg. 2013; 10(7): 347–356. PubMed Abstract \| Publisher Full Text\n\nRees D, Murray J: Silica, silicosis and tuberculosis. (State of the art). Int. J. Tuberc. 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PubMed Abstract\n\nNdlovu N, Nelson G, Vorajee N, et al.: 38 years of autopsy findings in South African mine workers. Am J Ind Med. 2016; 59(4): 307–314. PubMed Abstract \| Publisher Full Text\n\nZosky GR, Hoy RF, Silverstone EJ, et al.: Coal workers’ pneumoconiosis: an Australian perspective. Med J Aust. 2016; 204(11): 414–418. PubMed Abstract \| Publisher Full Text\n\nBlackley DJ, Halldin CN, Laney AS: Continued increase in prevalence of coal workers’ pneumoconiosis in the United States, 1970–2017. Am J Public Health. 2018; 108(9): 1220–1222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIftikhar B, Khan MH, Hussain H, et al.: Relationship between silica dust exposure and chronic obstructive pulmonary disease in workers of dust generating industries of district Peshawar. 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PubMed Abstract \| Publisher Full Text\n\nGruffydd-Jones K, Jones MM: NICE guidelines for chronic obstructive pulmonary disease: implications for primary care.2011.\n\nBerdal G, Halvorsen S, van der Heijde D, et al.: Restrictive pulmonary function is more prevalent in patients with ankylosing spondylitis than in matched population controls and is associated with impaired spinal mobility: a comparative study. Arthritis Res. Ther. 2012; 14(1): R19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRabe KF, Hurd S, Anzueto A, et al.: Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am. J. Respir. Critic. Care Med. 2007; 176(6): 532–555. PubMed Abstract \| Publisher Full Text\n\nButt IM, Mustafa T, Rauf S, et al.: Underlying data for ‘Pulmonary function parameters among marble industry workers in Lahore, Pakistan’. Figshare. 2021. Reference Source\n\nOphir N, Shai AB, Korenstein R, et al.: Functional, inflammatory and interstitial impairment due to artificial stone dust ultrafine particles exposure. Occup Environ Med. 2019; 76(12): 875–879. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed QR, Sau SK, Kar SK, et al.: The Effect of Marble Dust on Different Pulmonary Parameters in Marble Factory Workers. Natl. J. Integrated Res. Med. 2011; 2(3): 40.\n\nShaik A, Afroze MKH, Latha M, et al.: Lung function test in quarry workers. Int. J. Innovat. Res. Dev. 2015; 4(1): 50–55.\n\nJaakkola MS, Sripaiboonkij P, Jaakkola JJK: Effects of occupational exposures and smoking on lung function in tile factory workers. Int. Arch. Occup. Environ. Health. 2011; 84(2): 151–158. PubMed Abstract \| Publisher Full Text\n\nGholami A, Sajedifar J, Dehaghi BF, et al.: Lung function and respiratory symptoms among mine workers in the Eastern part of Iran. Russ. Open Med. J. 2018; 7(3): 1–4.\n\nSheta S, El Laithy N: Brick kiln industry and workers’ chronic respiratory health problems in mit ghamr district, dakahlia governorate. Egypt J. Occup. Med. 2015; 39(1): 37–51. Publisher Full Text\n\nJaber HM, Mohamed MS, El-safty MA, et al.: Pulmonary problems among stone cutting workers in West Bank, Palestine. Med. J. Cairo Univ. 2015; 83(1): 769–775.\n\nRafeemanesh E, Majdi MR, Ehteshamfar SM, et al.: Respiratory diseases in agate grinding workers in Iran. Int. J. Occup. Environ. Med. 2014; 5(3): 350–130. PubMed Abstract \| Free Full Text\n\nLuoto JA, Elmståhl S, Wollmer P, et al.: Incidence of airflow limitation in subjects 65–100 years of age. Eur Respir J. 2016; 47(2): 461–472. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammed J, Maiwada SAA, Sumaila FG: Relationship between anthropometric variables and lung function parameters among primary school children. Ann. Niger. Med. 2015; 9(1): 20.\n\nRao VMM, Rameswarudu M: Peak expiratory flow rate in healthy school children. Natl. J. Physiol. Pharm. Pharmacol. 2017; 7(4): 363–365.\n\nSezgi C, Abakay Ö, Önder H, et al.: The respiratory functions and radiologic findings of the marble factory workers. J. Clin. Exp. Invest. 2012; 3(2): 250–254. Publisher Full Text\n\nVyas S: A study of pulmonary function tests in workers of different dust industries. Int. J. Basic App. Med. Sci. 2012; 2(2): 15–21.\n\nDostbil Z, Polat C, Karakus A, et al.: Evaluation of the nasal mucociliary transport rate by rhinoscintigraphy in marble workshop workers. Toxicol. Ind. Health. 2011; 27(9): 826–830. PubMed Abstract \| Publisher Full Text\n\nRathod SB, Sorte SR: Effect of Duration of Exposure to Silica Dust on Lung Function Impairment in Stone Crusher Worker of Marawatdha Region. Int. J. Cur. Res. Rev. 2013; 5(4): 121–126.\n\nSheikh JA, Khan ZA, Khan T, et al.: Pulmonary function among stone quarry workers in India: The effect of duration of exposure, smoking status and job profile on pulmonary function tests. J. Health Social Sci. 2018; 3(2): 137–146. Publisher Full Text\n\nNwibo AN, Ugwuja EI, Nwambeke NO, et al.: Pulmonary problems among quarry workers of stone crushing industrial site at Umuoghara, Ebonyi State, Nigeria. Int. J. Occup. Environ. Med. 2012; 3(4): 178–185. PubMed Abstract\n\nNandini C, Malleshappa K, Vastrad BC, et al.: Study of lung function in granite workers based on duration of exposure. Natl. J. Physiol. Pharm. Pharmacol. 2018; 8(10): 1374–1376. Publisher Full Text" }	[ { "id": "95979", "date": "29 Oct 2021", "name": "Shamim Mohammad", "expertise": [ "Reviewer Expertise Respiratory diseases", "mental health", "and health inequities" ], "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 \"Pulmonary function parameters among marble industry workers in Lahore, Pakistan\" is a methodologically sound case-control study. The researchers have taken all the necessary steps to ensure adherence to the application of basic protocols of research. The study finds the negative impact of the long duration of silica dust exposure on the lungs of the marble industry workers. Findings are in line with similar studies conducted around the world on occupational exposure to dust, particularly stone or marble dust. However, a brief justification about the selection of the research site, the absence of female participants in the study, and the incentive provided to the participants, if any, will strengthen the generalizability of the study further.\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": "94844", "date": "22 Nov 2021", "name": "Ejaz Mahmood Ahmad Qureshi", "expertise": [ "Reviewer Expertise Public Health", "Environmental and Occupational 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\nCOMMENTS\nIt is one of the best efforts made by the authors to conduct such a study on workers of the marble industry. As reported by the authors, the study setting where this study was carried out is thickly populated, located in a residential area, hence named cottage industry where marble crushing/cutting activities have been going on in houses since long ago. Being located in a thickly populated place, lots of people are directly and indirectly exposed to silica dust. It would have been a remarkable effort if particulate matter (PM), both PM 2.5 and PM 10 were also measured as mentioned in observation 3.\nSecondly, as silica particles (being heavy in weight) are suspended in the atmosphere for a short distance around these plants, it would have been a great effort if a pulmonary function test was performed in people living at least 50 meters from the marble industry as noted in observation 1. Respiratory symptoms in these people should also be recorded.\nIn addition to that, one of the limiting factors of this study was its short duration. It would have been better if this study was carried out in a larger time period, 6 months or one year, as the weather conditions can affect the outcome of the result. It is usually seen that in hot weather, workers try to stay and work at the point where some fans are plying with the result that dust spread is more in such cases and chances of getting affected is more than in winter season. Technology being used in the marble industry is also very important. In those places where wet technology is used for cutting marbles, the spread of dust particles is far less than in those places where marble is cut in dry conditions.\nRegarding confounding factors like the presence of pollutants around the marble industry, it is pertinent to mention that lots of pollutants, like NOX, SOX, CO2, etc, are emitted from other sources, like vehicles, burning of waste, etc., which is very common practice, especially in an industrial area. These pollutants might have augmented bad effects on the lungs of workers of the marble industry and people residing nearby and so should have been measured. In addition to that, few workers were smokers as the result of this study reported, but also many people might have been addicted to other substances prepared locally like opium, niswar, battery, etc., which can also enhance the respiratory symptoms and decrease lung function test. These workers should have been included in the study.\nLooking at the prevailing situation in the industrial area in general and study setting in particular, it is the need of an hour that Environmental Monitoring, namely Environmental Impact Assessment (EIA) for larger marble cutting units and Environmental Examination (EE) for small industries, cottage units by the Environmental Protection Department (EPD) should be done. Although these measures have been in place but unfortunately not strictly followed.\n\nCOMMENTS ABOUT QUALITY OF PAPER\nAre sufficient details of methods and analysis provided to allow replication by others?\nMethods of data collection have been given in detail and were quite comprehensive. A list of inclusiveness and exclusiveness is also complete. One can say that this has increased the understanding of the matter even for a layman. Another good thing is the comprehensiveness of data analysis. So, I will say that sufficient details of methods and analysis have been provided and it has added the knowledge of the reader significantly. It is also a very good source of reference for other studies.\nThe status you would give this article:\nI can say with confidence that the article is scientifically valid in its current form. The experimental design, including controls and methods, is adequate; results are presented accurately and the conclusions are justified and supported by the data. Revisions/modifications suggested are minor in nature, and are considered to be non-essential.\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/10-938
https://f1000research.com/articles/10-937/v1	17 Sep 21	{ "type": "Research Article", "title": "A hybrid recommender system based on data enrichment on the ontology modelling", "authors": [ "Lit-Jie Chew", "Su-Cheng Haw", "Samini Subramaniam", "Lit-Jie Chew", "Samini Subramaniam" ], "abstract": "Background: A recommender system captures the user preferences and behaviour to provide a relevant recommendation to the user. In a hybrid model-based recommender system, it requires a pre-trained data model to generate recommendations for a user. Ontology helps to represent the semantic information and relationships to model the expressivity and linkage among the data. Methods: We enhanced the matrix factorization model accuracy by utilizing ontology to enrich the information of the user-item matrix by integrating the item-based and user-based collaborative filtering techniques. In particular, the combination of enriched data, which consists of semantic similarity together with rating pattern, will help to reduce the cold start problem in the model-based recommender system. When the new user or item first coming into the system, we have the user demographic or item profile that linked to our ontology. Thus, semantic similarity can be calculated during the item-based and user-based collaborating filtering process. The item-based and user-based filtering process are used to predict the unknown rating of the original matrix. Results: Experimental evaluations have been carried out on the MovieLens 100k dataset to demonstrate the accuracy rate of our proposed approach as compared to the baseline method using (i) Singular Value Decomposition (SVD) and (ii) combination of item-based collaborative filtering technique with SVD. Experimental results demonstrated that our proposed method has reduced the data sparsity from 0.9542% to 0.8435%. In addition, it also indicated that our proposed method has achieved better accuracy with Root Mean Square Error (RMSE) of 0.9298, as compared to the baseline method (RMSE: 0.9642) and the existing method (RMSE: 0.9492). Conclusions: Our proposed method enhanced the dataset information by integrating user-based and item-based collaborative filtering techniques. The experiment results shows that our system has reduced the data sparsity and has better accuracy as compared to baseline method and existing method.", "keywords": [ "Information Retrieval", "Ontology", "Recommender System", "Collaborative Filtering", "Content-based System", "Hybrid Recommender System" ], "content": "Introduction\n\nA Recommender System (RS) is a system that can provide item recommendation to a user based on their personalized interest. The attention for RS has increased dramatically over the past decade in various industries and domains such as e-commerce and online video streaming. There is a crucial need for having a system that can filter the numerous data around us as we are living in the area of the Internet with humungous data transactions and exchanges daily. With a properly implemented RS, the user will get a personalized recommendation based on the preferences, interest, rating, search results, the similarity between other users and so on. There are various successful use cases where RS helps in increasing the revenue of industrial, especially on online businesses. E-commerce companies such as eBay1 and Amazon2 have made use of RS to promote their products to the targeted customer. On the other hand, online video streaming company such as Netflix3 and YouTube4 have also implemented multiple types of RS in their system.\n\nGenerally, there are two types of RS: (1) content-based filtering (CB), and (2) collaborative filtering (CF). The CB RS provides recommendations to a user by using user preferences or history while the CF RS generates the recommendations based on the relationship between the user and item. These two methods have their advantages and shortcomings. As such, to combine the advantages and eliminate the shortcoming of each specific method, a new group of RS named hybrid RS has emerged. According to a recent survey in 20205, most of the recently proposed RS techniques fall under this group. Besides that, the most proposed hybrid RS combine at least one CF method in their system. CF method can be further classified as memory-based and model-based CF. A memory-based CF suggest item based on the similarity between user or item while the model-based CF builds the model by learning the interaction between user and item. There are a few researchers who focus on enhancing the model performance by fine-tuning the parameter and method in the model development process. However, the accuracy of the model built depends on the quality of the data6.\n\nOn the other hand, ontology helps to structure the data in a way that the entities are connected within the database7. Thus, the relationship between each entity is preserved. Semantic similarity can be easily calculated by various method that the ordinary method may not be able to discover. Ontology has been proven to help in increasing the accuracy of the RS and decrease the cold start issues8,9. With ontology, Manuela et al.10 reduced the fake neighbours’ problem cause by the CF method. Tarus et al.11 proposed an E-learning RS based on RS and the accuracy is better than using only CF without ontology. Shaikh et al.12 proposed an ontology-based RS in an e-commerce website. User behaviour on the website has been captured as implicit feedback to the RS. Gohari and Tarokh13 proposed a hybrid method that using ontology to structure the data. User-based (UB) CF and item-based (IB) CF were used to generate the recommendation. Bagherifard et al.14 proposed a hybrid approach that utilizing ontology in CB and CF hybrid RS. In their approach, the user has been clustered before calculating pass to CB and CF. This reduces the compute time of the CF RS. Celyan et al.15 proposed SEMCBCF, which is an ontology hybrid RS that extended from CBCF16, which is a CF RS without ontology integrated. The semantic similarity between items was calculated in their proposed system. The weighted average algorithm was used to combine the different similarity value. Nilashi et al.17 proposed an ontology hybrid recommendation that using IB and UB CF together with the clustering method to reduce overgeneralization. On another separate research, Liu and Li18 proposed an ontology CF RS based on Singular Value Decomposition (SVD). By employing the ontology as the data representation, the data sparsity has been decreased and the empty value of the user-item matrix was filled up based on IB CF. Inspired by their work, we proposed to address on enriching the data representation by means of ontology enrichment to give a more accurate recommendation. The summary of the recent publications has been done in Table 1.\n\nIn our proposed method, we focus on how to enrich the data information with ontology in order to increase the accuracy of the model-based RS. We proposed a method to enrich the user-item rating matrix by using the semantic similarity calculated from ontology. We added a UB RS to the item-based RS to generate the predicted rating that used to fill the user-item matrix to improve on the accuracy. In addition, our proposed approach will also reduce the main problem that usually faced in the model training, which is the data sparsity issue. With the predicted rating filled in the original user-item matrix, it can fill up the unknown value thus reduce the sparsity and increase the model training result. The experiment evaluations demonstrated that we have achieved higher accuracy and decrease the data sparsity problem of the original matrix.\n\n\nMethods\n\nInsipred by the data enrichment method proposed by Liu and Li18, we extended the work and proposed a hybrid method that used ontology to model the data. The semantic similarity between each attribute will be calculated by using the ontology structure. The semantic similarity will be used in the rating prediction in IB CF and UB CF. The flow diagram of our proposed method is illustrated in Figure 1. The proposed method consists of four parts:\n\n1. Crawling extra movie information from IMDB and construct the ontology\n\n2. Unknown Rating prediction by IB and UB CF\n\n3. Combine predicted ratings and forms a filled user-item rating matrix\n\n4. Model-based CF.\n\nWe have selected the MovieLens 100K dataset as this is the standard dataset used for benchmarking purpose. This dataset contains 100K rating records with 1682 movie data and 943 user profile details. However, the movie information of the MovieLens dataset is limited. To have more details for the movie, we crawled the extra information from the IMDB website such as movie country, classified, director, actors, and so on. After all the data had been crawled, we constructed the ontology representation for the dataset (see Figure 2). In the ontology representation, all the attributes nodes were connected with each other via the relationship edges. The two main nodes were User and Movie connected through their related profile node.\n\nThe semantic similarity of the dataset can be easily counted from the ontology constructed above. We used the IB CF and UB CF together to predict the unknown value from the original user-item matrix. The IB CF calculate the semantic similarity by considering the relationships between items. We used the Jaccard similarity index in calculating semantic similarity. Jaccard similarity measures the similarity by taking the percentage of the intersection of two sets of data. The formula is depicted in Equation (1).\n\nWhere J(A, B): the Jaccard similarity index between data A and data B.\n\nFrom the process above, we got the movie-movie similarity by each feature of the movie (see Table 2). We then combined all the movie-movie similarity by a weighted average algorithm, where the weight variables were decided by experiment evaluation to get the best combination.\n\nAfter completing the IB similarity calculation, we were able to predict the unknown rating values in the user-item matrix. The theory of the prediction is finding the related movie ratings rated by the specific user. The formula used is shown in Equation (2).\n\nWhere i: the movie rated by the user, a: rating, u: user, m: movie\n\nThe algorithm first took all the movie rated by the specific user and compare to the similarity calculated. It then summed up the predicted value by using the weighted average method where the weight is the similarity of the movie to that specific movie. The predicted value put in a temporary matrix which was later combined with UB RS.\n\nIn the UB CF, we applied similar methods from the IB CF above. First, we calculated the similarity of each user features then combined it with a weighted average algorithm. With the user-user similarity calculated, we then predicted the empty movie rating by finding similar users. The similar users’ rating to that specific movie was combined by the weight algorithm.\n\nOnce the two IB CF and UB CF methods were completed, the two predicted rating were then combined by using the weighted average algorithm to get the final predicted rating for filling the empty original user-item matrix. After the filling process was completed, it was then passed to the model-based CF to construct the model. The CF model used in this paper was SVD. SVD decomposed the matrix into two lower dimensionality matrix and extracted the latent features. It is a famous method used in the model-based CF.\n\n\nResults\n\nIn the evaluation, we have compared the result from the baseline model that based on SVD method alone to predict rating and an existing method that uses the IB CF to enrich the original user-item matrix.\n\nThe proposed system was developed using Jupyter Notebook 6.4.0 in Python 3.6 and Linux environment (Ubuntu 18.04). The Neo4j database has been used to store the data as the it is a graph database that our ontology representation will maintain in the data model. We applied the Root Mean square error (RMSE) algorithm to determine the accuracy of the system. It is a common approach to determine the predictive accuracy of the model19. It gives a relatively high weight to large errors. The smaller the RMSE value, the more accurate the model is.\n\nSeveral experiments have been done to decide the weight variable used in combining the IB CF and UB CF. Various weightage variables ranging from 0.3 to 0.7 have been tested. Figure 3 shows that the best accuracy is achieved with a weightage of 0.5.\n\nA similarity threshold was applied in the system to prevent destroying the original information of the original matrix when filling the empty value. Figure 4 shows that the accuracy of the model was affected by the similarity threshold. Overall, our proposed method had the lowest RMSE value across the similarity threshold testing (see Figure 5).\n\nThe experiment evaluations indicated that our proposed approach had the lowest RMSE value. With the unknown rating filled by IB and UB CF before passing to the model-based CF, the data sparsity also decreased from 0.9542% to 0.8435%.\n\n\nDiscussion\n\nFrom the experimental results in the earlier section, we observed that adding the IB CF method to enrich the original data helped to increase the accuracy of the model-based CF RS. It helped to boost the information of the original matrix while not destroying the original information. The added user-item CF method allowed the system to get more accurate similar user and items. However, we still want to know if our proposed method works in other model-based CF RS. Hence, we change the SVD model to the SVD++ model as the enhanced proposed method and re-run the experiment. SVD++ is an extended work from SVD, which achieve better accuracy by optimizing the algorithm to consider implicit feedback20. In our experiments, the results in Figure 6 below show that the enhanced ptoposed method with SVD++ outperforms any other method we used above with the enriched data. This helps to verify that our method can be applied to not only SVD, but any other model-based CF RS.\n\nFrom all the results above, it shows that our proposed method can increase the accuracy of the model-based CF RS. By adding the UB CF method to the existing method proposed by Liu and Li18 that employed only the IB CF method, we can achieve better accuracy than the existing method. This is due to the added UB CF method which allows the system to find the related item by user demography, whereas the IB CF method is not able to do it.\n\n\nConclusions\n\nIn this paper, we reviewed the current ontology based RS and proposed a data enrichment method which uses ontology in a hybrid RS. The proposed method increases the model-based CF RS input data quality by adding the UB CF to the existing IB CF method. Both methods use the structure of ontology to calculate the semantic similarity and, subsequently, fill the unknown rating values of the original user rating matrix. Experiment results indicated that the data sparsity problem has been minimized and the accuracy of the RS system has been increased.\n\nSeveral improvements can be conducted in future including algorithm optimization. The current offline model building algorithm takes time to process and can be optimized as parallel processing to improve the processing time. Besides, the semantic similarity calculation can be changed to the level-based calculation to fully utilise the benefits of having ontology in the system.\n\n\nData and Source Code Availability\n\nZenodo: chewljie/dataset-enrichment-RS: V1.0 Initial Release, https://doi.org/10.5281/zenodo.5418122\n\nThis project contains the following underlying data:\n\n• MovieLens 100K. (https://grouplens.org/datasets/movielens/100k/)\n\n• Extra movie details from OMDb API.\n\n(https://www.omdbapi.com/)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBen Schafer J, Konstan J, Riedi J: Recommender systems in e-commerce. Proceedings of the 1st ACM conference on Electronic commerce. 1999; 158–166. Publisher Full Text\n\nLinden G, Smith B, York J: Amazon.com recommendations: item-to-item collaborative filtering. IEEE Internet Comput. 2003; 7(1): 76–80. Publisher Full Text\n\nGomez-Uribe CA, Hunt N: The Netflix Recommender System: Algorithms, Business Value, and Innovation ACM Trans Manag Inf Syst. 2016; 6(4): 1–19. Publisher Full Text\n\nCovington P, Adams J, Sargin E: Deep Neural Networks for YouTube Recommendations. Proceedings of the 10th ACM Conference on Recommender Systems. 2016; 191–198. Publisher Full Text\n\nChew LJ, Haw SC, Subramaniam S: Recommender System for Retail Domain: An Insight on Techniques and Evaluations. Proceedings of the 12th International Conference on Computer Modeling and Simulation. 2020; 9–13. Publisher Full Text\n\nHeinrich B, Hopf M, Lohninger D, et al.: Data quality in recommender systems: the impact of completeness of item content data on prediction accuracy of recommender systems. Electron Mark. 2019; 31: 389–409. Publisher Full Text\n\nMiddleton SE, De Roure D, Shadbolt NR: Ontology-based Recommender Systems. Handbook on Ontologies. Berlin, Heidelberg: Springer Berlin Heidelberg, 2004; 477–498. Publisher Full Text\n\nAlmabdy S: Comparative Analysis of Relational and Graph Databases for Social Networks. 1st Int Conf Comput Appl Inf Secur ICCAIS. 2018; 2: 509–512. Publisher Full Text\n\nSieg A, Mobasher B, Burke R: Improving the effectiveness of collaborative recommendation with ontology-based user profiles. Proc 1st Int Work Inf Heterog Fusion Recomm Syst Het Rec 2010 Held 4th ACM Conf Recomm Syst Rec Sys 2010. 2010; 39–46. Publisher Full Text\n\nMartín-Vicente MI, Gil-Solla A, Ramos-Cabrer M, et al.: A semantic approach to improve neighborhood formation in collaborative recommender systems. Expert Syst Appl. 2014; 41(17): 7776–7788. Publisher Full Text\n\nTarus J, Niu Z, Khadidja B: E-Learning Recommender System Based on Collaborative Filtering and Ontology. Int J Comput Inf Eng. 2017; 11(2): 400–405. Publisher Full Text\n\nShaikh S, Rathi S, Janrao P: Recommendation System in E-Commerce Websites: A Graph Based Approached. 2017 IEEE 7th International Advance Computing Conference (IACC). 2017; 931–934. Publisher Full Text\n\nGohari FS, Tarokh MJ: A New Hybrid Collaborative Recommender Using Semantic Web Technology and Demographic data. Int J Inf Commun Technol Res. 2016; 8(2): 51–61. Reference Source\n\nBagherifard K, Rahmani M, Nilashi M, et al.: Performance improvement for recommender systems using ontology. Telemat Informatics. 2017; 34(8): 1772–1792. Publisher Full Text\n\nCelyan U, Birturk A: Combining Feature Weighting and Semantic Similarity Measures for Hybrid Movie Recommender System. 5th SNA-KDD Work. ’11, San Diego, CA USA, 2011.\n\nMelville P, Mooney RJ, Nagarajan R: Content-boosted collaborative filtering for improved recommendations. Proc Natl Conf Artif Intell. 2002; 187–192. Reference Source\n\nNilashi M, Ibrahim O, Bagherifard K, et al.: A recommender system based on collaborative filtering using ontology and dimensionality reduction techniques. Expert Syst Appl. 2018; 92: 507–520. Publisher Full Text\n\nLiu W, Li Q: Collaborative Filtering Recommender Algorithm Based on Ontology and Singular Value Decomposition. Proc - 2019 11th Int Conf Intell Human-Machine Syst Cybern. IHMSC. 2019; 2: 134–137. Reference Source\n\nSilveira T, Zhang M, Lin X, et al.: How good your recommender system is? A survey on evaluations in recommendation. Int J Mach Learn Cybern. 2019; 10(5): 813–831. Publisher Full Text\n\nWang S, Sun G, Li Y: SVD++ recommendation algorithm based on backtracking. Inf. 2020; 11(7): 369. Publisher Full Text" }	[ { "id": "94869", "date": "04 Oct 2021", "name": "Rajkumar Kannan", "expertise": [ "Reviewer Expertise Information retrieval", "Web mining", "machine learning", "deep learning" ], "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\nAuthor Conclusion\nIn this paper, the authors reviewed the current ontology based RS and proposed a data enrichment method which uses ontology in a hybrid RS. The proposed method claims to increase the model-based CF RS input data quality by adding the UB CF to the existing IB CF method. Both methods use the structure of ontology to calculate the semantic similarity and, subsequently, fill the unknown rating values of the original user rating matrix. Experiment results indicated that the data sparsity problem has been minimized and the accuracy of the RS system has been increased.\nReviewer comment\nDetails, such as, number of objects created, relationships, download link of the ontology, that is created and leveraged are not presented and justified. Comparison with just one dated approach is not adequate either. Hence, the paper is not recommended for indexing at this present stage.\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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "94868", "date": "21 Oct 2021", "name": "Dana Sulistyo Kusumo", "expertise": [ "Reviewer Expertise Recommender system", "information architecture", "software engineering" ], "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 data enhancement of hybrid recommender system using ontology to model user and item relationship. This can address the cold start problem of the recommender system because it can provide information when a new user or item first comes to the recommender system. This work extends Liu and Li1 that employed only the item-based collaborative filtering method. However, the writing of this paper can be improved in the following aspects. The authors can add equations and formulas for steps in the Methods section, so that others can easily evaluate and replicate this work. As the focus of this paper is ontology extension from the previous research, please add a detailed explanation about the function of data enrichment and the ontology modeling in the Methods section. I suggest to explain how the ontology structure can be used in different cases and the scope of complexity of the ontology structure. In addition, please add more discussion about the data enrichment and use of ontology in the Discussion section.\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?\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": "97017", "date": "22 Nov 2021", "name": "Heru Agus Santoso", "expertise": [ "Reviewer Expertise Information retrieval", "ontology", "machine learning" ], "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 has a good chance of being accepted, but requires explanation in detail:\nPlease check new references, there are other types of recommended systems such as knowledge-based, hybrid etc.\n\nThe study focuses on enriching information of data using ontology, explain: a. Method/technique how to crawl and construct the ontology; b. Technique to predict rating using IB and UB; c. How to combine predicted rating and user-item rating form.\n\nExplain clearly, the method to improve accuracy by carrying out semantic similarity using ontology.\n\nRMSE is not algorithm (see the Result), is there relation with the use of ontology? If so, how can it improve the performance of RS?\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-937
https://f1000research.com/articles/10-936/v1	17 Sep 21	{ "type": "Research Article", "title": "The impact of testing and treatment on the dynamics of Hepatitis B virus", "authors": [ "Olajumoke Oludoun", "Olukayode Adebimpe", "James Ndako", "Michael Adeniyi", "Oluwakemi E. Abiodun", "Babatunde Gbadamosi", "Olukayode Adebimpe", "James Ndako", "Michael Adeniyi", "Oluwakemi E. Abiodun", "Babatunde Gbadamosi" ], "abstract": "Despite the intervention of WHO on vaccination for reducing the spread of Hepatitis B Virus (HBV), there are records of the high prevalence of HBV in some regions. In this paper, a mathematical model was formulated to analyze the acquisition and transmission process of the virus with the view of identifying the possible way of reducing the menace and mitigating the risk of the virus. The models' positivity and boundedness were demonstrated using well-known theorems. Equating the differential equations to zero demonstrates the equilibria of the solutions i.e., the disease-free and endemic equilibrium. The next Generation Matrix method was used to compute the basic reproduction number for the models. Local and global stabilities of the models were shown via linearization and Lyapunov function methods respectively. The importance of testing and treatment on the dynamics of HBV were fully discussed in this paper. It was discovered that testing at the acute stage of the virus and chronic unaware state helps in better management of the virus.", "keywords": [ ": Positivity and boundedness of solutions", "Equilibria of solutions", "Next generation matrix", "Linearization", "Lyapunov functions", "local and global stabilities." ], "content": "1. Introduction\n\nHepatitis is an inflammation/scarring of the liver that contributes to various health complications, including death. It occurs due to an immune system attack by the virus in the liver and damages this vital organ of the body in the process (Ciupe et al., 2014). The hepatitis B virus (HBV) can survive outside the body for at least seven days. If the virus enters the body of someone who is not protected by vaccination during this time, it can still cause infection. The average incubation time for HBV is 75 days, but it can range from 30 to 180 days. Within 30 to 60 days of infection, the virus may be detected, persist, and grow into chronic hepatitis B (CDC, 2019). Hepatitis B is most common in the Western Pacific region with prevalence rate 6.2% and Africa with prevalence rate of 6.1%, with the Americas region (0.7%) having the lowest prevalence (WHO, 2019).\n\nIn highly endemic areas, the most common form of transmission of hepatitis B is from mother to child at birth (vertical transmission) or through horizontal route (contact with infected blood), particularly from infected children to uninfected children during the first five years of life. Chronic infection develops in infants infected by their mothers or before the age of five. It is often transmitted through transdermal or mucosal contact of infected persons to infected blood and different body fluids, such as spittle, catamenial, vaginal and spermatic fluids and, to a lesser degree, perspiration, breast milk, tears, and urine. In particular, hepatitis B can be transmitted through sexual contact in unvaccinated men who have sex with men (MSM) and heterosexual people who have multiple sexual partners or have contact with sex workers. However, adult infection contributes to chronic hepatitis in less than 5% of cases. This transmission may similarly ensue when needles and syringes are reused, whether in healthcare settings or among drug users. Furthermore, an infection can occur during medical, surgical, and dental procedures, such as tattooing or using razors and other similar objects contaminated with infected blood (Mpeshe and Nyerere, 2019).\n\n\n2. Mathematical formulation\n\nSome chronic carriers are unaware of their status and as such transmit the virus unknowingly and also at higher risk of cirrhosis and makes treatment less effective (Niederau, 2014, Mcpherson et al., 2013, Cohen et al., 2011, Piorkowsky, 2009, Lin et al., 2009, Meffre et al., 2004).\n\nIn view of this, this model is developed to factor the aforementioned set of people. In the model, the population is divided into the following different groups: the susceptible, the acute, the chronic unaware carriers, the chronic aware carriers, the treated chronic aware and the recovered individuals.\n\nThe total population at time t, denoted by Nt is divided into the six subgroups corresponding to different epidemiological status: susceptible individualsSt, acute At, unaware chronically infected Cut, aware chronically infected Cat, treated Tct, and removed/recovered class Rt. The model equation is subject to the initial conditions,\n\nFigure 1 represents schematically the epidemiology of HBV infected model. The different disease stages are reproduced by the different circle and the arrows indicate the way individual progress from one stage to the other. It is assumed that at time, t, susceptible individuals, S, enter the population at a constant rate, Π. For all classes, individuals die at a constant natural mortality rate, μ.HBV chronically infected individuals (Cut, Cat)have an additional death rate due to HBV, ⅆc (Zhang and Zhang (2018)). It is assumed that HBV infected individuals on treatment, Tct do not transmit HBV infection. Susceptible individuals, St, may acquire HBV infection when in contact with individuals in A,Cu, and Ca,populace at a rate, λ (force of infection associated with HBV), where\n\nParameter β represents the probability that a contact will result in an HBV infection while α1,α2>1 respectively account for modification parameter of chronic HBV-infected individuals.\n\nA proportion of the acute HBV-infected individuals, σ, spontaneously clear the virus, then return to being susceptible. The HBV acutely infected individuals develop to chronic without been aware if no testing at a rate, γ. The acutely infected and chronic unaware individual progress to chronic aware stage with a testing ν1,ν2 respectively and moved to treatment stage after testing at the rateδ. ω is the recovery rate of treated infected individual with full immunity.\n\nThese assumptions lead to the system of equations in (3)\n\nwhere λ=βA+α1Cu+α2CaN\n\nFor the system of equations (3) to be epidemiologically meaningful, it is important to prove that all solution with non-negative initial conditions will remain non-negative.\n\nLemma 1: The initial values of the parameters are\n\nThen the solution of the model StAtCutCatTctRtNt is positive for all t≥0.\n\nProof\n\nConsidering the first equation in (3),\n\nHence, S≥0\n\nwith respect to the second equation in (3);\n\nHence, A≥0. Same goes for the other compartments\n\nClearly, the above state variables are positive on bounding plane ℝ+6.\n\nFor the boundedness the following calculation follows:\n\nSimplifying:\n\nIntegrating gives:\n\nIt follows that the solutions of the model system (3) are positive and bounded in the region\n\nIt follows from Lemma 1 that it is sufficient to consider the dynamics of system (3) and the model can be considered to be epidemiologically well-posed.\n\nThe disease-free equilibrium of the equation (3) exists and is given by:\n\nThe endemic steady states are calculated here which is done by setting system of equation in (3.3.3) to zero and setting S=S∗,A=A∗,Cu=Cu∗,Ca=Ca∗,Tc=Tc∗,R=R∗ so that\n\nwhere\n\nThe basic reproduction number (Ro) which is the number of secondary infections caused by an infectious individual is determined by the next generation matrix which is given by ρFV−1\n\nwhere:\n\nTheorem 1: E0 is locally asymptotically stable if R0 < 1 and unstable if R0 > 1.\n\nProof: The resulting matrix from the linearized model is dXdt=AX\n\nThe resulting Jacobian matrix at E0 is\n\nFrom (15), λ1=−μ,λ2=−ω−μ,λ3=−μ\n\nand the resulting quadratic equation is:\n\nNow, λ1, λ2,λ3 < 0 since the values are assumed positive. If R0 < 1, E0 is stable and unstable when R0 < 1.\n\nThe global behavior of the equilibrium system (3) is analyzed here in this section.\n\nTheorem 2: For system (3), the disease-free equilibrium E0 is asymptotically stable globally if R0<1.\n\nProof: Considering the Lyapunov function defined as:\n\nAt DFE, S=N so that (20) becomes:\n\nExpanding and simplifying (21) gives:\n\nFrom Equation (23), it can be deduced that the DFE is globally stable since R0 < 1.\n\nTheorem 3: If R0>1, then the endemic equilibrium is locally asymptotically stable.\n\nProof:\n\nThe endemic equilibria of system (3), denoted by S∗A∗Cu∗Ca∗Tc∗R∗, can be rewritten as:\n\nFrom (25), λ1=−μ,λ2=−ω+μ,λ3=−dc+μ+ν2, then;\n\nfrom (26);\n\nThe result of the determinant of the Jacobian matrix is of the form:\n\nwhere\n\nBy the Routh–Hurwitz criterion governing the polynomials of order 3, we have the following:\n\n1. a2.a3 are positive\n\n2. a1a2>a3\n\nFrom equation (27), 1 and 2 are satisfied.\n\nTherefore, endemic equilibrium is locally asymptotically stable.\n\nTheorem 4: The equations of the model have a positive distinctive endemic equilibrium whenever R0 > 1, which is said to be globally asymptotically stable.\n\nProof: Considering the Lyapunov function defined as:\n\nwhere L takes it derivative along the system directly as:\n\nAt equilibrium,\n\nwhere,\n\nP1≤0 whenever\n\nand P2≤0 whenever\n\nThus, dLdt≤0 if the condition in (35) and (36) holds.\n\ntherefore, by LaSalle asymptotic stability theorem (LaSalle, 1976), and Oke et al. (2020) the positive equilibrium state dLdt is globally asymptotically stable in the positive regionR+6.\n\n\n3. Numerical computation\n\nThe numerical study is carried out using maple software embedded code for the Runge-Kutta of fourth order. Here, the subsequent default values are assumed for the embedded parameters taken from theoretical studies in literatures γ=0.9,β=0.008,σ=0.59,dc=0.00693,μ=0.00693,ω=0.1,ν1=0.002,ν2=0.002,α1=0.0016,α2=0.0016,δ=0.0085,Π=0.07. The values remain unchanged throughout* the computations except otherwise indicated.\n\nThe effects of varying the testing rate of the acute individuals υ1, testing rate of chronic individuals (υ2) and treatment rate of chronic individuals (δ) on the population dynamics are shown in Figures 2 to 7. From Figures 2 and 3, an increase in the parameters values reduces susceptible and acute populations thereby reducing the spread HBV due to low interaction between the host immune system and the virus. Therefore, the appearance of HBV and the pathogenesis reduces, which in so doing, lessens the potential injury on the liver. Hence, the liver is shielded from hepatocellular carcinoma over time. The rate of chronic unaware and chronically aware individuals is examined in Figures 4 and 5. The parameter variations show a significant decrease in the chronic unaware population which implies that testing at that stage is a great tool for reducing the disease transmission. The transmission process dies down as the time progresses; this discourages liver inflammation as a result of lowering the infected individuals. Meanwhile, the chronic population in Figure 5, depicts a high significant influence of the acutely infected and chronically unaware infected individuals over time. A chronic infection phase is found at the time range 10<t<20, as such, the individuals are exposed to liver carcinoma or cirrhosis. Hence, the chronic population diminishes as the parameters are increased.\n\nBehavioral dynamics of susceptible population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nBehavioral dynamics of acute population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nBehavioral dynamics of chronic unaware population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nBehavioral dynamics of chronic aware population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nBehavioral dynamics of treated population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nBehavioral dynamics of recovered population when varying testing rate for acute and chronic individuals and treatment for chronic individuals.\n\nIn Figures 6 and 7, the impact of varying the testing rate of the acute individuals υ1, testing rate of chronic individuals (υ2) and treatment rate of chronic individuals (δ) on the treated and the recovered population are presented. The treated population increases with parameters variation along the rising time (t) as a result of long time effect of parameter values. The recovery rate is enhanced as observed in Figure 7 due to significant simulation of surface antibodies of Hepatitis B. This is in conformity with the works of Pang et al. (2010) and Ullah et al. (2019). This result implies that an intensification in testing at all infectious states and rise in treatment of chronic individual will bring about a reduction in the HBV transmission process which is a response to the WHO goal for 2030 that concentrating efforts on awareness program and campaign will sure bring about a decrease or eradication in the transmission process of the virus (WHO, 2020).\n\n\n4. Conclusion\n\nA deterministic model of hepatitis B testing was developed and investigated, which included testing in the chronic unaware state as well as testing in all infectious states. The model has disease-free and endemic equilibria. The basic reproduction number was calculated using the next generation matrix method. The equilibria's local and global stability were discussed and shown to be asymptotically stable. The testing and treatment rate effects were thoroughly discussed.\n\n\nData availability\n\nNo data are associated with this article.\n\n\nGrant information\n\nNone declared.\n\n\nCompeting interests\n\nNone declared.", "appendix": "References\n\nCiupe SM, Ribeiro RM, Perelson AS: Antibody Responses during Hepatitis B Viral Infection. PLoS Comput Biol. 2014; 10(7): e1003730. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen C, Holmberg SD, McMahon BJ, et al.: Is chronic hepatitis B being undertreated in the United States? J Viral Hepatol. 2011; 18(1):377–383. PubMed Abstract \| Publisher Full Text\n\nLaSalle JP: The Stability of Dynamical Systems, Regional Conference Series in Applied Mathematics Philadelphia, Pa,USA: SIAM; 1976.\n\nLin SY, Chang ET, So SK: Stopping a silent killer in the underserved Asian and pacific islander community: a chronic hepatitis B and liver cancer prevention clinic by medical students. Asian Pac J Cancer Prev. 2009; 10(9):383–386. PubMed Abstract\n\nMcPherson S, Valappil M, Moses SE, et al.: Targeted case finding for hepatitis B using dry blood spot testing in the British-Chinese and South Asian populations of the North-East of England. J Viral Hepatol. 2013; 20(1):638–644. PubMed Abstract \| Publisher Full Text\n\nMeffre C, Le Strat Y, Delarocque-Astagneau E, et al.: Prevalence of hepatitis B and hepatitis C virus infections in France in 2004: social factors are important predictors after adjusting for known risk factors. J Med Virol. 2010; 82(2), 546–555. PubMed Abstract \| Publisher Full Text\n\nMpeshe SC, Nyerere N: Modeling approach to assess the transmission dynamics of Hepatitis B infection in Africa. Int J Advance App Math Mechanic. 2019; 6(3): 51–61.\n\nNiederau C: Chronic hepatitis B in 2014: great therapeutic progress, large diagnostic deficit. World J Gastroenterol. 2014; 20(33):11595–11617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOke IS, Ojo MM, Adeniyi OM, et al.: Mathematical modeling of malaria disease with control strategy. Comm Math Biol Neurosci. 2020; 43(1), 180–188. Publisher Full Text\n\nPang J, Cui JA, Zhou X: Dynamical behavior of a hepatitis B virus transmission model with vaccination. J Theoretical Biol. 2010; 265(4):572–578. PubMed Abstract \| Publisher Full Text\n\nPiorkowsky NY: Europe’s hepatitis challenge: defusing the “viral time bomb”. J Hepatol. 2009; 51(1):1068–1073. PubMed Abstract \| Publisher Full Text\n\nUllah S, Khan MA, Gómez-Aguilar JF: Mathematical formulation of hepatitis B virus with optimal control analysis. Optim Control Appl Methods. 2019; 40(3):529–544. Publisher Full Text\n\nZhang J, Zhang S: Application and Optimal Control for an HBV Model with Vaccination and Treatment. Discrete Dyn Nat Soc. 2018; 20(7):69–73. Publisher Full Text" }	[ { "id": "94651", "date": "06 Oct 2021", "name": "Sulyman Olakunle Salawu", "expertise": [ "Reviewer Expertise Mathematical Modeling and flow dynamic" ], "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\nReviewer Reports on the impact of testing and treatment on the dynamics of Hepatitis B virus. The impacts of different parameters were examined. The manuscript is well written and presented logically. The abstract captures accurately the whole work and the introduction is sound. However, I have the following comments and queries for the authors:\nThe authors should explain what prompted the choice of method of solution used over other techniques.\n\nThe significance and motivation of the study are not clear. The authors should clarify these.\n\nThere is a need to reference equation (3) to establish its correctness and source.\n\nSome grammatical mistakes should be checked through.\n\nWhy have the authors chosen to engage in this study?\n\nA likely extension of the study should be stated or recommended.\n\nThe manuscript should be indexed subject to minor correction.\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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "94650", "date": "17 Dec 2021", "name": "Segun oke", "expertise": [ "Reviewer Expertise Mathematical modelling", "Mathematical Oncology", "Optimal control", "Epidemiology", "etc" ], "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 worked on the impact of testing and treatment on the dynamics of Hepatitis B virus. Although the manuscript is well presented and gives new insight into the Hepatitis B virus, especially for unvaccinated men who have sex with men (MSM) and heterosexual people who have multiple sexual partners or have contact with sex workers.\nIn their paper, a mathematical model was formulated to analyse the acquisition and transmission process of the virus with the view of identifying the possible way of reducing the menace and mitigating the risk of the virus. The models’ positivity and boundedness were demonstrated using well-known theorems. Equating the differential equations to zero demonstrates the equilibria of the solutions i.e., the disease-free and endemic equilibrium. The next Generation Matrix method was used to compute the basic reproduction number for the models. Local and global stabilities of the models were shown via linearization and contrition of Lyapunov function methods respectively. The authors concluded by showing disease-free, endemic equilibria. The basic reproduction number was calculated using the next-generation matrix method. The equilibria's local and global stability were discussed and shown to be asymptotically stable. The testing and treatment rate effects were thoroughly discussed. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.\nSome minor comments:\n\nThe authors need to review more literature to back up their claims.\n\nThe introduction is too short and is not well cited. For example:\n\"In highly endemic areas, the most common form of transmission of hepatitis B is from mother to child at birth (vertical transmission) or through horizontal route (contact with infected blood), particularly from infected children to uninfected children during the first five years of life. vaginal and spermatic fluids and, to a lesser degree, perspiration, breast milk, tears, and urine. In particular, hepatitis B can be transmitted through sexual contact in unvaccinated men who have sex with men (MSM) and heterosexual people who have multiple sexual partners or have contact with sex workers\" there is no citation here.\n\nThe authors need to add epidemiological interpretation and recommendations to the manuscript.\n\nThe conclusion needs to be rewritten.\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?\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? No", "responses": [] } ]	1	https://f1000research.com/articles/10-936
https://f1000research.com/articles/10-935/v1	17 Sep 21	{ "type": "Research Article", "title": "Association between maternal antenatal care visits and newborn low birth weight in Bangladesh: a national representative survey", "authors": [ "Md. Sabbir Ahmed", "Sumaia Sahrin", "Fakir Md Yunus", "Sumaia Sahrin", "Fakir Md Yunus" ], "abstract": "Background: Low Birth Weight (LBW) is a global health concern for childhood mortality and morbidity. The objectives of this study were to assess the association between the number of Antenatal Care Visits (ANC) and LBW among Bangladeshi newborns, and to identify the demographic and socio-economic predictors of LBW.\n\nMethods: Our present cross-sectional study is based on the secondary data of the Bangladesh Demography and Health Survey (BDHS) 2014. Complete data of 4,235 (weighted) mother-child pairs were included in the analysis.\n\nResults: The overall prevalence of LBW among newborns were found to be 19.3% (95% CI: 17.8-20.9). Among the mothers who received antenatal care services 1-3 times during pregnancy, 35% had less possibility of having LBW babies [COR = 0.65, 95% CI: 0.50-0.85]. The association remained significant after adjusting the analysis with the sex of the newborn, administrative regions (division), maternal educational status, mother’s weight status and fathers’ occupation [AOR = 0.74, 95% CI: 0.55-0.99]. Additionally, the sex of the newborn, division, maternal education, maternal weight status, and fathers’ occupational status were found to be significantly associated with LBW.\n\nConclusion: Increasing the coverage of antenatal services and enabling mothers to receive quality antenatal services may substantially contribute to reducing the prevalence of LBW in Bangladesh.", "keywords": [ "Low Birth Weight", "Global Health", "Antenatal Care", "Bangladesh" ], "content": "Introduction\n\nLow birth weight (LBW) in newborn babies is defined as a birth weight of less than 2500 grams (< 5.5 lbs).1 LBW may occur due to intra-uterine growth restriction, pre-term birth (i.e., the baby is born before 37 weeks of gestation) or a combination of both.2 Higher prevalence of LBW is a global health concern for childhood mortality and morbidity.3 LBW may cause diarrhea, respiratory illness and mortality in childhood, and chronic noncommunicable diseases in adulthood.4–6 In addition, LBW has significant associations with cognitive development and decision making.7 Every year approximately 20 million babies suffer from LBW globally (i.e., 15-20% of total births).8 There is a high discrepancy in the incidence and prevalence of LBW across high income and low-income countries. About 95.6% of the total number of LBW births occur in low- and middle-income countries.9,10 Research also shows that the rate of LBW is double in South Asia regions compared to the global percentage.10 According to a most recent study prevalence of LBW was reported 14.5% among Bangladeshi newborns.3\n\nAntenatal care (ANC) is a broad aspect of healthcare including medical procedures and care provided to mothers during pregnancy and is significant to maintaining a healthy pregnancy state and ensuring safe childbirth.11 Low utilization of prenatal and maternal health services can adversely affect birth outcomes. ANC is one of the most effective interventions to protect maternal and child health and if implemented properly it can reduce maternal perinatal mortalities.12–14 Several studies conducted in developing countries demonstrated that improvement of ANC can significantly reduce the burden of LBW.15–18 The World Health Organization (WHO) recommends that every woman should receive at least four ANC visits during pregnancy.19 However, according to 2019 Multiple Indicator Cluster Survey (MICS), only 37% of Bangladeshi pregnant women are receiving at least four ANC services.20\n\nIn a previous study, several socio-economic factors were identified as predictors of LBW in Bangladesh, including educational status of the mother, educational status of the family head, number of ANC visits, amount of ANC assistance, location of delivery, skilled birth attendance, mode of delivery, and wealth index.9 According to the latest Bangladesh Demography and Health Survey (BDHS) report, 27% of newborns with a LBW had mothers who did not receive any ANC services during pregnancy.21 As no significant improvement was noted in reducing LBW rate, it is now considered a priority public health problem. Therefore, further identification of LBW is useful to identify the areas where progression has been made, where additional support is needed, and to implement evidence-based interventions to reduce Bangladesh’s burden of LBW. However, little research has been conducted to identify the association between ANC visits and LBW in Bangladesh. To fill the existing knowledge gap, our present study aimed to assess the association between the number of ANC visits and LBW among the Bangladeshi newborns. Furthermore, this study identified the demographic and socio-economic predictors of LBW (such as; sex of the newborns, maternal education, maternal weight status, etc.) Our study findings will generate evidence which Bangladeshi policymakers and public health managers can use to design interventions packages to ensure ANC coverage to reduce newborns’ LBW in Bangladesh.\n\n\nMethods\n\nStudy design and data source: Our present cross-sectional study is based on the secondary data of Bangladesh Demography and Health Survey (BDHS) 2014 conducted by National Institute of Population Research and Training (NIPROT) in collaboration with ICF International and Mitra and Associates.22 This national representative survey is conducted every three to five years. BDHS data were collected from household (HH) level by following a two-stage stratified cluster sampling method based on the enumeration areas (EAs) and HH samples. In the first stage, EAs were selected and HH were selected from each EA in the second stage. Details of the sampling process, data collection procedures, and the questionnaire used are available in the final report of the 2014 BDHS.22 In our study we used the women’s data file and extracted data only for those women who gave birth in recent years and have complete information on infant’s size at birth.\n\nStudy variables: Predictor variables such as sex of the newborn, mode and location of the delivery, place of residence seven administrative divisions, HH wealth index, mother and father’s education and occupation were included. We also included data from the BDHS dataset on mother’s weight (kg) and number of antenatal care visits (no visits, 1-3 visits, and ≥ 4 visits). The outcome variables of our study (i.e., LBW) were based on maternal recall on perceived birth size of the newborn at birth. The BDHS did not measure the actual birth weight of the newborns, so we could not define LBW according to the conventional cut-off value (i.e., birth weight < 2500 grams). During the survey, mothers were asked; ‘When (name of the child) was born, was he/she very large, larger than average, average, smaller than average, or very small?’.22 For our analysis, we categorized responses very small and smaller than average as ‘LBW babies’, and those with very large, larger than average, and average as ‘normal weight babies’. We adopted this method from a previous study where authors used DHS data and validated that a mother’s perception of birth size is a good proxy for birth weight in large nationally representative surveys.23 A similar method was also used in an earlier study.24\n\nStatistical analysis: The dataset was cleaned (removing missing cases, coding and recoding variables) before formal data analysis. Descriptive statistics were performed to assess the frequency and percentage of the study variables. A chi-square test was performed to compare the prevalence of birth weight and to assess the factors associated. We performed a logistic regression model (unadjusted and adjusted) to assess the association between the study variables and LBW. All statistical tests were considered significant at p<0.05 level. Data were analyzed by using Stata v14.2 (StataCorp, College station, TX, USA). We used the sampling weight provided by the BDHS in the dataset to ensure the acceptability of our results at a national level. We considered the stratified survey design by using Stata ‘svy’ command.\n\nEthical approval: As this study was based on a secondary source of data, authors did not require any IRB approval. However, Ethical approval for BDHS surveys have been obtained from ICF international and informed consent was received from the participants before data collection. The lead author received authorization from the DHS program for using the relevant dataset for this analysis. Details of ethical approval and data privacy are available at: https://dhsprogram.com/methodology/Protecting-the-Privacy-of-DHS-Survey-Respondents.cfm.\n\n\nResults\n\nSocio-demographic characteristics: Complete data of 4,235 (weighted) mother-child pairs were included in our study. Among the newborns the majority were male (52.9%), delivered by vaginal delivery (75.8%) and delivered at home (61%). Among the participants 74.2% belonged to rural areas and 21.8% belonged to HH with poor wealth index. Among the mothers; almost half (48.2%) completed secondary education and 16% of them were either overweight or obese. Of the participants, 20.9% of the mothers did not receive any antenatal care services during pregnancy, whereas only 31.2% of them received four and/or more ANC services (Table 1).\n\nPrevalence of LBW by socio-demographics: The overall prevalence of LBW among the newborns was found to be 19.3% (95% CI: 17.8-20.9). A higher prevalence was observed among the females (22%), delivered by vaginal delivery (20%) at home (20.4%). Prevalence of LBW was varied across the seven administrative divisions in Bangladesh. Sylhet division had the highest burden of LBW (27.8%), whereas Rangpur division experienced the lowest prevalence (12.7%) in Bangladesh. A higher prevalence of LBW was also observed among the children whose mothers did not have any formal education (26.4%), were underweight (25.1%) and did not receive any antenatal care service during pregnancy (25.1%). The prevalence was also higher in those whose fathers were not employed (42.5%) (Table 1).\n\nFactors associated with LBW: We found a significant positive association of LBW with (i) sex of the newborn (χ2 = 17.41, p = 0.003); (ii) division (χ2 = 41.98, p ≤ 0.001); (iii) maternal education (χ2 = 27.92, p ≤ 0.001); (iv) maternal weight status (χ2 = 30.23, p ≤ 0.001); (v) fathers’ occupational status (χ2 = 13.87, p = 0.045); and (vi) maternal antenatal care visits during pregnancy (χ2 = 23.15, p ≤ 0.001). However, we did not find any association between LBW and the mode of delivery, location of delivery, place of residence, wealth index, maternal occupation, and father’s educational status (Table 1).\n\n* Significant p value (p < 0.05).\n\n1 Chi-square test.\n\nAssociation between study variables and LBW: The association between socio-demographics and LBW are presented in Table 2. Female newborns had a 1.4 times higher likelihood of being LBW [AOR = 1.42, 95% CI: 1.13-1.78] compared to male newborns after adjusting sex of the newborn, division, mother’s education, mother’s weight status, and fathers’ occupation. Newborns living in Sylhet division had a higher likelihood [AOR = 1.82, 95% CI: 1.32-2.49] of being LBW compared to those who came from Barisal division. We found that the higher the maternal education, the lower the chances of having LBW newborns. Furthermore, we noted that overweight or obese mothers were 42% less likely [AOR = 0.58, 95% CI: 0.42-0.81] to have a LBW baby compared to those who were underweight. We found that the occupation of the father plays a major role in a newborn’s birth weight. Those fathers who were employed had a 71% less chance of having a LBW newborn compared to those who were unemployed [AOR = 0.29, 95% CI: 0.11-0.75] (Table 2).\n\n* Significant p value (p < 0.05).\n\n1 Adjusted with sex of the newborns, division, mother’s education, mother’s weight status, and fathers’ occupation.\n\nAssociation between antenatal care visits and LBW: Among the mothers who received antenatal care services 1-3 times during pregnancy had 35% less possibility of having LBW babies compared with who did not receive any service at all [COR = 0.65, 95% CI: 0.50-0.85]. The association remained significant after adjusting with sex of the newborn, division, maternal educational status, mother’s weight status and fathers’ occupation [AOR = 0.74, 95% CI: 0.55-0.99]. Mothers who received four or more antenatal care services had a 37% less possibility of having LBW babies [COR = 0.63, 95% CI: 0.49-0.81]. However, the association was not significant in the adjusted model [AOR = 0.81, 95% CI: 0.61-1.09] (Table 2).\n\n\nDiscussion\n\nWe investigated the association between ANC and LBW among newborns in Bangladesh. We found that the number of ANC visits during pregnancy are significantly associated with LBW of the newborn. Additionally, we found one in every five newborns in Bangladesh are born with LBW. Being female, residing in Chittagong and Dhaka division, having a mother with no formal education, having an underweight mother, and a father who is unemployed were identified as independent predictors of LBW in Bangladeshi newborns.\n\nThis paper indicated that the number of ANC visits have a significant relationship with the birth weight of newborns. This finding is consistent with earlier studies conducted in different developing countries such as Colombia, Mexico, Nepal, and Brazil.15–18 Regular ANC visits give the opportunity for health care workers to manage the pregnancy and enable mothers to receive a variety of services such as treatment of pregnancy-induced hypertension, tetanus immunization, and micronutrient supplementations etc.25 These measures play a vital role in improving pregnancy and neonatal outcomes.26 However, some studies also suggest that both adequacy and quality of ANC services are crucial for mothers to get the full beneficial effects of ANC visits and protect maternal and newborn health.27 The quality of ANC is measured by three factors: the number of visits, timing of initiation of care, and inclusion of all recommended components of care.28 According to our results, receiving ANC services influences the reduction of LBW more than the number of visits. We observed that mothers who received 1-3 visits had 35% less chance of having a LBW baby and the chances increased only 2% for those who received four or more ANC services which was the recommended amount according to WHO.19 The reasons behind this finding is not clear. One possible explanation could be mothers who are receiving any ANC services are provided with adequate services and messages. As several socio-economic factors restrict mothers from receiving four or more ANC services there should also be more information available during their first or second visit.29 After adjusting the potential cofounders, we found that mothers who received 1-3 visits had 26% less chance of having a LBW newborn and the chances decreased by 7% for those who received four or more ANC services. Although the government of Bangladesh created several initiatives (such as launching community clinics) to increase the coverage of ANC services and ensure 4+ ANC visits for mothers, still one in every three mothers are not receiving any ANC visits during their pregnancy which is an alarming statistic.20 Being a lower-middle income country, where there is a lack of resources and inadequate health services, improving health service seeking behavior remains a major challenge in Bangladesh.30 Because our study findings indicated ensuring ANC visits can significantly reduce the burden of LBW rather than a higher number of visits, ensuring 100% coverage of at least one ANC visit should be the first and foremost priority of the government of Bangladesh in reducing LBW prevalence. The focus can then turn to ensuring four or more visits; however, the quality of the visit is essential.\n\nIn our study, we found that female newborns had 1.4 times higher odds of being LBW compared to male newborns. Similar findings were reported in previous studies.31,32 However, a study in Zimbabwe found opposite results showing that female newborns were less likely to be born with LBW.33 Little is known about the sex differences in the likelihood of being LBW. Newborns from Chittagong and Dhaka division had a higher possibility of being LBW. Being the capital (Dhaka) and commercial capital (Chittagong) city of Bangladesh, adequate healthcare facilities and civic benefits are more assessable there compared to other regions. However, increased industrialization and environmental pollution in those regions may adversely affect the birth outcome, which is supported by previous findings.34,35\n\nFurthermore, we found an inverse relationship with mothers’ education and LBW of the newborn which is consistent with the earlier findings.36 It has been suggested that lower levels of education may limit understanding of the importance of ANC.29 Similarly, to educational status, the possibility of having a LBW baby has been shown to significantly decrease with the increased weight status among mothers. This would mean that the nutritional status of the mother is crucial for delivering a healthy newborn. Intra-uterine growth can be restricted due to the poor nutritional status of the mother which may lead to LBW of the baby as mother and fetus need to compete for optimal nutrition.37 We also found that newborns whose fathers are employed had a lower risk of LBW compared to those whose fathers are unemployed. Being employed (indicating economic stability) usually means a father can care for his pregnant partner by providing nutritious foods, allowing adequate time for rest and quality health care which ultimately contributes to lowering the risk of LBW.\n\nOur present study has its limitations. A major limitation is that the BDHS data used relied on mothers’ perception of child’s size to define the LBW. However, earlier evidence suggests that mothers’ perception is a valid proxy for birth weight.23 Secondly, causality cannot be explained since data was cross-sectional in nature. Maternal recall bias may also underestimate or overestimate the findings. We advise caution while interpreting the results. Future large scale observational studies are recommended to establish the causality. Additionally, variables were limited to common observable characteristics and the study is not able to describe the entire story of the LBW in Bangladesh. A mixed method study (quantitative followed by qualitative) may be useful. However, the study highlighted the modifiable socio-demographics which play a major role in newborns birth weight. Regardless, this study highlighted the importance of 100% coverage of ANC services in reducing LBW in Bangladesh through nationally representative data which is the major strength of the study. As this study is based on a national representative sample results can be generalized at national level.\n\n\nConclusion\n\nMaternal antenatal care visits have a significant role in newborn birth weight in Bangladesh. Increasing the coverage of antenatal services and enabling mothers to receive quality antenatal services may substantially contribute in reducing the burden of low birth weight in Bangladesh.", "appendix": "Acknowledgements\n\nThe authors wish to acknowledge the authority of the National Institute of Population Research and Training (NIPORT), Bangladesh, for providing the dataset used in this study.\n\n\nData availability\n\nData used in this study are available online from the ‘Individual Recode data file’ of the Bangladesh 2014 DHS, available from the Demographic and Health Survey (DHS) website. Access to the dataset requires registration and is granted only for legitimate research purposes. A guide for how to apply for dataset access is available at: https://dhsprogram.com/data/Access-Instructions.cfm.\n\n\nReferences\n\nOrganization WH: WHO handbook for guideline development. World Health Organization; 2014.\n\nAcharya D, Singh JK, Kadel R, et al.: Maternal factors and utilization of the antenatal care services during pregnancy associated with low birth weight in rural Nepal: analyses of the antenatal care and birth weight records of the MATRI-SUMAN trial. Int J Environ Res Public Health. 2018; 15: 2450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed MS: Mapping the prevalence and socioeconomic predictors of low birth weight among Bangladeshi newborns: evidence from the 2019 Multiple Indicator Cluster Survey. Int Health. 2021. Publisher Full Text\n\nNegrato CA, Gomes MB: Low birth weight: causes and consequences. Diabetol Metab Syndr. 2013; 5: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRisnes KR, Vatten LJ, Baker JL, et al.: Birthweight and mortality in adulthood: a systematic review and meta-analysis. Int J Epidemiol. 2011; 40: 647–661. PubMed Abstract \| Publisher Full Text\n\nWilcox AJ: On the importance—and the unimportance—of birthweight. Int J Epidemiol. 2001; 30: 1233–1241. PubMed Abstract \| Publisher Full Text\n\nChristian P, Murray-Kolb LE, Tielsch JM, et al.: Associations between preterm birth, small-for-gestational age, and neonatal morbidity and cognitive function among school-age children in Nepal. BMC Pediatr. 2014; 14: 58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhincup PH, Kaye SJ, Owen CG, et al.: Birth weight and risk of type 2 diabetes: a systematic review. JAMA. 2008; 300: 2886–2897. PubMed Abstract \| Publisher Full Text\n\nKhan JR, Islam MM, Awan N, et al.: Analysis of low birth weight and its co-variants in Bangladesh based on a sub-sample from nationally representative survey. BMC Pediatr. 2018; 18: 100. Publisher Full Text\n\nWorld Health Organization: Global nutrition targets 2025: Low birth weight policy brief.2025: 2014.\n\nEkabua J, Ekabua K, Njoku C: Proposed framework for making focused antenatal care services accessible: a review of the Nigerian setting. ISRN Obstet Gynecol. 2011; 2011. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFotso J-C, Ezeh AC, Essendi H: Maternal health in resource-poor urban settings: how does women’s autonomy influence the utilization of obstetric care services? Reprod Health. 2009; 6: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBilenko N, Hammel R, Belmaker I: Utilization of antenatal care services by a semi-nomadic Bedouin Arab population: evaluation of the impact of a local maternal and child health clinic. Matern Child Health J. 2007; 11: 425–430. PubMed Abstract \| Publisher Full Text\n\nOladapo OT, Osiberu MO: Do sociodemographic characteristics of pregnant women determine their perception of antenatal care quality? Matern Child Health J. 2009; 13: 505–511. PubMed Abstract \| Publisher Full Text\n\nPinzón-Rondón ÁM, Gutiérrez-Pinzon V, Madriñan-Navia H, et al.: Low birth weight and prenatal care in Colombia: a cross-sectional study. BMC Pregnancy Childbirth. 2015; 15: 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nServan-Mori E, Sosa-Rubí SG, Najera-Leon E, et al.: Timeliness, frequency and content of antenatal care: which is most important to reducing indigenous disparities in birth weight in Mexico? Health Policy Plan. 2016; 31: 444–453. Publisher Full Text\n\nKhanal V, Zhao Y, Sauer K: Role of antenatal care and iron supplementation during pregnancy in preventing low birth weight in Nepal: comparison of national surveys 2006 and 2011. Arch Public Heal. 2014; 72: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nda Fonseca CRB, Strufaldi MWL, de Carvalho LR, et al.: Adequacy of antenatal care and its relationship with low birth weight in Botucatu, São Paulo, Brazil: a case-control study. BMC Pregnancy Childbirth. 2014; 14: 255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarroli G, Villar J, Piaggio G, et al.: WHO systematic review of randomised controlled trials of routine antenatal care. Lancet. 2001; 357: 1565–1570. PubMed Abstract \| Publisher Full Text\n\nBangladesh Bureau of Statistics (BBS) and UNICEF Bangladesh: Progotir Pathey Bangladesh, Multiple Indicator Cluster Survey 2019. Survey Findings Report. Dhaka, Bangladesh: 2019.\n\nNational Institute of Population Research and Training (NIPORT), Mitra and Associates and II: Bangladesh Demographic and Health Survey: Key Indicators 2014. Dhaka, Bangladesh Calverton, Maryland, USA Natl Inst Popul Res Train (NIPORT). Mitra Assoc ICF Int. 2014.\n\nNational Institute of Population Research and Training (NIPORT): Mitra and Associates and ICF International. Bangladesh Demographic and Health Survey 2014.Dhaka, Bangladesh, and Rockville, Maryland, USA: 2016.\n\nChannon AAR: Can mothers judge the size of their newborn? Assessing the determinants of a mother’s perception of a baby’s size at birth. J Biosoc Sci. 2011; 43: 555–573. PubMed Abstract \| Publisher Full Text\n\nKabir A, Rashid MM, Hossain K, et al.: Women’s empowerment is associated with maternal nutrition and low birth weight: evidence from Bangladesh Demographic Health Survey. BMC Womens Health. 2020; 20: 93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsundep NN, Carson AP, Turpin CA, et al.: Determinants of access to antenatal care and birth outcomes in Kumasi, Ghana. J Epidemiol Glob Health. 2013; 3: 279–288. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarroli G, Rooney C, Villar J: How effective is antenatal care in preventing maternal mortality and serious morbidity? An overview of the evidence. Paediatr Perinat Epidemiol. 2001; 15: 1–42. PubMed Abstract \| Publisher Full Text\n\nTran TK, Gottvall K, Nguyen HD, et al.: Factors associated with antenatal care adequacy in rural and urban contexts-results from two health and demographic surveillance sites in Vietnam. BMC Health Serv Res. 2012; 12: 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrganization WH: Far more pregnant women getting antenatal care. Far more pregnant women Get. antenatal care. 2004.\n\nRahman A, Nisha MK, Begum T, et al.: Trends, determinants and inequities of 4+ ANC utilisation in Bangladesh. J Heal Popul Nutr. 2017; 36: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed SM, Adams AM, Chowdhury M, et al.: Changing health-seeking behaviour in Matlab, Bangladesh: do development interventions matter? Health Policy Plan. 2003; 18: 306–315. PubMed Abstract \| Publisher Full Text\n\nTshotetsi L, Dzikiti L, Hajison P, et al.: Maternal factors contributing to low birth weight deliveries in Tshwane District, South Africa. PLoS One. 2019; 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtuahene M, Mensah D, Adjuik M: A cross-sectional study of determinants of birth weight of neonates in the Greater Accra region of Ghana. Matern Heal Neonatol Perinatol. 2015; 1: 23. Publisher Full Text\n\nHoque ME, Towobola OA, Monokoane T: Comparison of adverse pregnancy outcome between teenage and adult women at a tertiary hospital in South Africa.Mashamba TJ; 2014.\n\ndos Reis MM, Guimarães MT, Braga ALF, et al.: Air pollution and low birth weight in an industrialized city in Southeastern Brazil, 2003-2006. Rev Bras Epidemiol. 2017; 20: 189–99. PubMed Abstract \| Publisher Full Text\n\nKaur P, Sachdeva R, Kochhar A: Impact of industrialization on nutritional and health status of pregnant women and their obstetric outcome. Stud Ethno-Medicine. 2010; 4: 149–155. Publisher Full Text\n\nMuula AS, Siziya S, Rudatsikira E: Parity and maternal education are associated with low birth weight in Malawi. Afr Health Sci. 2011; 11. PubMed Abstract \| Free Full Text\n\nKader M, Perera NKPP: Socio-economic and nutritional determinants of low birth weight in India. N Am J Med Sci. 2014; 6: 302. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "94632", "date": "26 Oct 2021", "name": "Irteja Islam", "expertise": [ "Reviewer Expertise Public Health", "Maternal and Child Health", "Child Mental Health", "Health Services 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\nThanks for conducting such important research. Though I have a few observations and feedback as follows:\nEven when a study is exploratory, the introduction should end with clearly articulated hypotheses and the grounds for those hypotheses based on previous findings. I found the rationale weak and need more evidence in the background.\n\nThere should be recall and response bias. The authors did not mention what measures they have undertaken to control bias.\n\nThe authors did not mention whether they used Survey weights or not.\n\nFinally, some model fit information would be very helpful. The sample size is large, but the predictive power of the models is unclear.\n\nA stronger call is needed for future research in the discussion and conclusion section.\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": [] }, { "id": "163676", "date": "21 Feb 2023", "name": "Rozeta Sokou", "expertise": [ "Reviewer Expertise Neonatology" ], "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 authors aimed to assess the association between the number of antenatal care (ANC) visits and Low birth weight (LBW) among the Bangladeshi newborns. Furthermore, this study identified the demographic and socio-economic predictors of LBW.\nThis is very interesting study bat the fact that the BDHS data used relied on mothers’ perception of child’s size to define the LBW is a major limitation of the study which may represent a major systematic bias.\nMinor comments:\nIntroduction “LBW may cause diarrhea….” This sentence need to be modified. There is well known that the LBW is a global public health challenging problem, identified as one of the major determinants of infant morbidity accounted for 15–20% of newborns deaths across the globe and that it contributes markedly to the overall burden of childhood death. Low birth weight is related to a range of both short and long-term sequels such as prematurity. I am I wondering how common is diarrhea in this population.\nDiscussion Authors mentioned that “After adjusting the potential cofounders, we found that mothers who received 1-3 visits had 26% less chance of having a LBW newborn and the chances decreased by 7% for those who received four or more ANC services. Although the government of Bangladesh created several initiatives (such as launching community clinics) to increase the coverage of ANC services and ensure 4+ ANC visits for mothers, still one in every three mothers are not receiving any ANC visits during their pregnancy which is an alarming statistic.” According to the results after adjustment for the confounder factors for the mothers who received ≥4 visits the statistical significance was lost please explain this finding.\n“Because our study findings indicated ensuring ANC visits can significantly reduce the burden of LBW rather than a higher number of visits, ensuring 100% coverage of at least one ANC visit should be the first and foremost priority of the government of Bangladesh in reducing LBW prevalence” how they come to this conclusion? on which data they are based?\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-935
https://f1000research.com/articles/10-933/v1	16 Sep 21	{ "type": "Policy Brief", "title": "Integrating evidence-based interventions in clinical settings in Jordan: a policy brief", "authors": [ "Mohammad Alzaatreh", "Obay A. Al-Maraira", "Nazih Abu Tabar", "Mohammad R. Alsadi", "Huthaifah Khrais", "Hamzeh Y Abunab", "Khaled M. Kasasbeh", "Mohammad Almaani", "Malek Khalil", "Obay A. Al-Maraira", "Nazih Abu Tabar", "Mohammad R. Alsadi", "Huthaifah Khrais", "Hamzeh Y Abunab", "Khaled M. Kasasbeh", "Mohammad Almaani", "Malek Khalil" ], "abstract": "Background: Evidence-based nursing practice (EBNP) is considered a major and very important global paradigm shift. Unfortunately, most healthcare providers and researchers embrace the concept of evidence-based practice (EBP) without integrating this concept in clinical settings. The current situation of EBP and new practice guidelines utilization in Jordan are scarce. This policy brief aimed to discusses the process of utilizing nursing EBP in clinical settings in Jordan. Methods: The authors adopted an action plan utilizing a systematic approach to develop and implement specific strategies and policies to integrate EBP in clinical settings in Jordanian hospitals. We present an experience of one country in terms of introducing a policy brief to establish an EBP policy accompanied by developing an EBP unit in the hospital's country. Results: A comprehensive description of this policy is provided with reference to the eminent steps of policy analysis and evaluation. In fact, EBP policies and clinical practice guidelines should keep a live document and revise regularly or as needed. Overall, the authors suggest the development of a unit for EBP to deal with issues related to clinical practice guidelines. Conclusions: Expected outcomes for the introduction of the EBP unit and its policy include increase research utilization and accelerated adoption of new evidence, increase the quality of care provided, increase patient, staff, and managers satisfaction, and decrease staff workload by reducing complications associated with medical errors and outdated interventions.", "keywords": [ "Policy Brief", "EBP", "Nursing", "Healthcare Professionals", "Clinical Practice Guidelines", "Jordan." ], "content": "Introduction\n\nEvidence-based practice (EBP), evidence-based medicine (EBM), or evidence-based nursing practice (EBNP) are all substitutes to what is known about providing medical care or taking a medical decision using the best available knowledge or research findings. 1 EBP is considered a major and fundamental global paradigm shift. 2 Generally, EBP is generated by identifying the problem, searching alternatives, evaluating the options, and therefore generating the evidence based on available experimental research and linking it to the practice and policy domain to ultimately improve the quality of healthcare and patients outcomes. 3,4 The term EBP is widely used throughout all the healthcare disciplines; such as nursing, medicine, physical therapy, and other fields. 5 EBP helped interdisciplinary healthcare teams towards achieving better healthcare outcomes. 4 Unfortunately, the utilization of EBP is limited. Most healthcare providers and researchers embrace the concept of EBP without actual integration in clinical environment. 4 Also, there is a gap in nurses’ knowledge of the research steps and their abilities in utilizing EBP in the clinical environment. So, this requires innovative solutions beyond the traditional ways of education. 6\n\nPersonal and system-level challenges in the implementation of EBP contributed to the persistent research to practice gap in healthcare delivery. 7 Research utilization (RU) into EBP is struggling with many barriers. One major barrier is that nurses, in general, lack the interest, time, and/or motivation to be involved in the research process due to its complexity. 8 Additionally, nurses are ill-prepared at the undergraduate level to understand, conduct, appraise, and consume research results. 9,10 Hence, it’s not possible for nurses who lack the fundamentals skill of research to conduct such a complicated task. 11 Nurses will not identify a clinical problem and access a medical database searching for alternative medical options targeting the main concern. After that, nurses should critically evaluate the result for any evidence based on its feasibility, appropriateness, and effectiveness and ultimately generate evidences. 4 For these reasons, healthcare organizations and nursing associations should be responsible for connecting or collecting experts who can collect, evaluate, and generate evidence, and reflect it in the form of protocols, practice guidelines, and ultimately regular policies.\n\nIn Jordan, the problem with the current situation of EBP is that utilization of the evidence is limited to a few initiatives through clinical practice, and a few attempts to distribute it through conducting educational lectures, seminars, and journal clubs, without implementing any policy that would organize the integration of newly approved clinical related evidence that promotes and enhances the quality of care.\n\nThe key solution to improve adherence to EBP and increase RU is to adopt it at the organizational level rather than relying on individual initiative. 4 Otherwise, nurses will continue to rely on tradition, authority, limited clinical experience, and intuition as sources of information, turning a blind eye to the new updates in the field of health sciences and nursing science. This will be reflected directly in the quality of care they provide and the outcome they are aiming for.\n\nThe purpose of this policy brief is to introduce a policy about how to implement the updated EBP research findings into clinical practice guidelines, protocols, and procedure manuals. This paper presents a structured process from O’Grady (2020) 12 that provides policymakers with the background and the existing status of EBP utilization, necessary to be more familiar with the problem of low utilization of EBP and new guidelines. Also, it discusses the contextual factors such as socio-cultural, economic, and moral concerns, ethical, legal, and political factors, and the target audience for this issue. It then describes the proposed solutions and the expected outcomes. Subsequently, it suggests an implementation methodology 13 includes the preparation for launching the EBP unit, incorporating the EBP within the institution's mission statement, EBP operational process, analysis of potential risks, and implementation timeline 14 and evaluation method.\n\n\nPolicy context\n\nEBP is taught at various courses in nursing curriculum at the undergraduate level and after graduation, with the majority of nurses attending many lectures regarding EBP at hospitals in Jordan. These courses have been aimed at ensuring nurses are fully aware of the importance of EBP. However, EBP is not reflected in their practice because nurses lack the knowledge, skills, and resources required to recognize, appraise, and implement interventions based on new and existing evidence. Moreover, the organizational attempts to promote EBP culture and practice are scarce. 15 Indeed, the positive attitudes toward EBP prepared nurses to implement EBP but unfortunately, no actual steps have been taken to integrate it into clinical practice.\n\nEBP is intended to produce the best care with minimal cost. Jordan is categorized as a lower-middle-income country. and the average health expenditure in Jordan is 9.8% of gross domestic product. 16 The public sector serves more than 60% of the general population in Jordan. Implementing EBP is associated with decreases in length of hospitalization, reduced postoperative complications and hospital-acquired infections, and thus, a decrease in economic burden for health. Unfortunately, the public sector is lacking attempts to integrate EBP in clinical practice. However, some individual initiatives to adopt EBP from the private sector achieved significant outcomes such as a decrease in hospital-acquired infection rate and increase hand hygiene compliance. As an example, new evidence such as an educational interventional program to increase hand hygiene practices in one private hospital in Jordan was implemented and resulted in decreased hospital-acquired infection by 5.6% and decrease the length of stay by three to six days. 17\n\nThe seriousness of not implementing EBP is considered as significant as the ethical issues attending to its application. While utilizing the EBP, we shall use it judiciously. 18 EBP helps to bridge the gap between healthcare providers and quality care, which is considered our ethical responsibility towards patients. 19\n\nThe concept of EBP is not well accepted by managers and policymakers unless it optimizes outputs and translates into financial benefits and money. 20 The language of money and effectiveness is an acceptable means to justify service provision. Some research findings might be legitimized as evidence-based if available research supports a favorable outcome. This approach will have the consequences of continuously neglecting implementation and, therefore, widening the gap between evidence and practice. 21\n\nThe stakeholders for this policy in Jordan comprise the Ministry of Health (MOH), Jordanian Council of Nursing (JNC), and Jordanian Nurses and Midwifery Council (JNMC). All healthcare providers, including nurses, are supposed to participate in policy development and implementation. Besides, the continuing education department and quality assurance units should be involved in the process for follow-ups on implementing the policies in the hospitals. Nurse educators and clinical nurse specialists should educate nurses and nursing students about the research process’s role in identifying the problem and proposing appropriate solutions. Health accreditation bodies such as the HealthCare Accreditation Council (HCAC) in Jordan should play a major role in promoting policy implementation in all healthcare settings to enhance quality.\n\nThe policy issue statement is to develop a method to utilize EBP results in developing clinical practice guidelines into the nursing practice milieu in Jordan.\n\nThe policy goal and objectives are first to establish and implement specific strategies and policies to enable professional nurses to provide the best nursing care in Jordan based on clinical practice guidelines derived from the best available evidence. The second is to develop educational and training programs for all professional nurses regarding the significance of embracing the concept of EBP in current clinical practice. Lastly, to establish a reviewing process using a common standard for appraisal of the evidence that is carried out regularly.\n\n\nEvaluation of policy options and alternatives\n\nEvaluation criteria for the policy and each alternative must reflect the potential to achieve the policy goals and objectives, decrease cost, consideration of different populations, political, social administrative-technical feasibility, and sustainability. The evaluation process will be based on modified Kraft and Furlong's (2019) evaluation criteria of policy options 22 as described in Table 1.\n\nThis is an incremental approach for change to increase RU by focusing on presenting the best evidence by identifying, synthesizing, evaluating, and disseminating evidence to professional nurses. This is mainly a personal initiative rather than an organizational one. Such an approach includes journals club (a meeting that involves a group of people discussing articles related to critical topic), better access to the electronic database, medical educational courses, and related workshops. Although it might be a good approach toward better RU, its a personal initiatives that don’t guarantee the sustainability of this evidence. Also, it needs advanced skills in appraising evidence. 23 However, this alternative might achieve policy goals. The application of this alternative will indirectly lower the cost while getting the benefit of implementing this policy. This alternative can also be politically feasible, but it is more difficult to achieve. This alternative is also administratively acceptable as it includes minor changes in the current situation of RU. Furthermore, this alternative is socially acceptable as it protects healthcare professionals and will ensure that the patients are receiving optimal care.\n\nTransforming integrated EBP into routine clinical nursing practice is an organizational effort and needs to be supervised by the managerial board and specialized unit or a committee. 4 Therefore, developing a policy that acts as a pathway for academics and clinicians, particularly at the bedside, who are usually facing clinical problems to propose and legitimize any new evidence is a means to solve this problem. This alternative will regulate the process of selecting any new evidence or guidelines and describes the process of endorsing it to the EBP unit that is responsible for appraising and integrating the newly adopted practice in the policy and practice manual, and the process of disseminating it.\n\nThe EBP unit should include members mainly from nursing as they constitute the vast majority of medical practitioners and also, a representative from other disciplines such as medicine, physiotherapy, pharmacy, laboratory, and respiratory therapy. Also, it will include a consultant and representative from JNC and JNMC. The criteria for the consultant, who must be an expert in the field of EBP research, including that they must; (1) hold a Ph.D. in nursing or in a medicine-related field, (2) have published at least four articles concerning EBP in peer-reviewed journals, and (3) have served as a reviewer in at least two peer-reviewed journals. The criteria for the members in the EBP unit includes that they must; (1) hold at least a masters-level qualification in a nursing or medicine-related field, (2) have published at least one article concerning EBP in peered review journals, (3) attended at least one course related to research ethics, and (4) have at least one year experience in clinical practice. A steering committee should be designed to recruit members for the EBP unit. An official announcement for the EBP unit vacancies should be circulated internally and posted on the website of the institution of implementation. Primarily, this committee will be developed to set the guidelines of the healthcare practices at hospitals based on what constitutes a legitimate source of evidence for healthcare practices. Setting the unit operational bylaws, conducting educational sessions, awareness sessions for hospital employees, clients, and stakeholders, and selecting a model for evidence-based decision making will be also the functions of the EBP unit.\n\nThis option can achieve the goal of a new policy and is socially acceptable. Implementing this alternative will indirectly lower the cost of compensation for healthcare professionals and patient health costs by decreasing medical errors and ensuring the quality of care that outweighs the cost of policy application. Further, this option will ensure sustainability. However, it is politically infeasible as this is challenging to achieve because it needs significant and frequent changes in medical procedures that medical practitioners used to do.\n\nTable 2 presents the final results of the comparison between the alternatives. The evaluation tool consists of eight criteria; the authors evaluated each criterion using a Likert scale ranging from 1-5. (Score 1 indicates very low likelihood, 2 – low likelihood, 3 – moderate likelihood, 4 – high likelihood, 5 – very high likelihood). As the authors of this paper represent different sectors including academic, clinical, and administrative sectors, and hold a Ph.D or are a Ph.D. student in nursing with at least three years of clinical experience, they were responsible for the evaluation. They were requested to independently rate each alternative and to submit it to the primary investigator. Then the primary investigator reviewed all the submitted comparisons and calculated the mean scores displayed in Table 2. The results indicate that the alternative of developing the EBP unit obtained the highest score.\n\nThe authors of this paper suggest the development of a unit for EBP as described in alternative Two. This unit has the main duties of identifying clinical problems and clinical practices utilized to solve the problems, in addition to the clinical practice protocols and guidelines used during routine clinical nursing practice within the hospitals to treat patients’ conditions. However, medical practitioners are encouraged to participate in this process by identifying the clinical problem and searching the literature for any evidence-based practice or guidelines. They are developing a patient, intervention, comparison, outcome, and time (PICOT) 1 question to ensure they are identifying the clinical problem. The P includes types of patients/patient populations. Consideration should include sex, ethnicity, and patients with particular healthcare problems. We include interventions or specific methods or treatments of interest. C is a comparison of alternatives in treatment or interventions to the problem. This may also include new or alternative ways to achieve the same outcome. The O is looking at the desired outcome. This must be precise and brief when developing your question. The T is for timing. This can be optional but may be relevant to the particular clinical question. 1,20 Next, they should endorse it officially to the EBP unit for evaluation. Evidence-based practice units will facilitate this process by providing clinicians’ scientific resources to search for up-to-date clinical interventions for different patient cases. Then, the EBP unit in cooperation with related hospital administrators should critically evaluate the outcomes of such interventions in terms of feasibility, appropriateness, meaningfulness, effectiveness, patient satisfaction, and quality of care. Furthermore, EBP unit members will conduct regular meetings with clinical teams and field experts to verify generated evidence. Ultimately, once proven successful against the identified criteria, newly generated evidence and related practices should be reflected in protocols, policies, and practice guidelines and officially disseminated to all related hospital departments and teams.\n\nExpected outcomes for introducing the EBP unit and its policy include positive reflections on administrative, clinical, financial, staff retention, and patient satisfaction. The key performance indicators (KPIs) include; positive patient experience in receiving healthcare services, top management satisfaction, enhanced employee satisfaction and retention, reduction of waste of the hospital resources, decreased length of stay, reduced postoperative complications, and hospital-acquired infections which are where we expect to get the better number after the introduction of the policy and unit. On the other hand, “Magnet organizations have an ethical and professional responsibility to contribute to patient care, the organization, and the profession in terms of new knowledge, innovations, and improvements”. 24 Magnet accreditation helps nurses be more involved in the process of decision-making. They can participate in committees such as the EBP committee. Participating in such a committee will enhance the professional growth and improved patient outcomes. 25\n\n\nPolicy implementation\n\nIn order to execute the introduction of an EBP policy and unit, a proposed methodology is presented in this section. It includes the adopted theoretical framework for implementation, the preparation for launching the EBP policy and unit, incorporating the EBP within the hospital’s mission statement, EBP operational process, analysis of potential risks, and implementation timeline.\n\nTo initiate the proposed change, the process should be started by inviting the selected members of the EBP unit committee for a meeting. The first meeting agenda will focus on introducing the idea to the selected members, getting their buy-in and approval to participate after presenting the current status highlighting the problems and issues faced with EBP, the proposed change, plan of implementation, proposed budget, evaluation of outcomes plan, and potential gains and benefits that will be achieved in introducing the EBP unit.\n\nAfter the collective approval of the unit members, the EBP unit will be officially launched and announced to the hospital employees. As described by Kouzes and Posner (2012) this kick-start meeting should “model the way” by the presented plans, and “inspire a shared vision” with all unit committee members and “encourage the hearts” to get the change approved, supported, and motivated toward success. 26\n\nOne of the important aspects of the staff member complies with EBP guidelines can be because EBP unit decisions will affect the whole inter-professional process of providing healthcare. This change can be viewed as a loss of authority on decision making, but it can be mitigated through the suggested awareness sessions and inviting all related team members to participate in journal clubs. 27,28\n\nLastly, to ensure that the implementation will be incremental, the timeline for project activities is suggested to introduce the project ( Table 3). Actual activities will pass over the six phases as follows: approval for establishing of this policy and unit, launching event, committee meeting and assigning coordinator, awareness sessions, piloting for one use case, and hospital-wide implementation. It is expected that the pilot phase will be conducted within a specified period, while the hospital-wide implementation process will last for about three months as outlined in Table 3.\n\n\nPlan for evaluation\n\nTo evaluate the outcomes of the policy, the authors suggest developing groups of KPIs to measure the implementation. Pre- and post-implementation of EBP unit measures will be appraised. However, there are four main domains of KPIs: administrative-related KPIs, clinical-related KPIs, patient satisfaction KPIs, and financial-related KPIs.\n\nExamples of administrative-related KPIs are the workload reports (i.e., the number of patients seen by a provider, number of procedures performed), hospital management satisfaction, and healthcare providers’ satisfaction in utilizing the best available evidence for clinical purposes, and percentage of waste reduction in terms of resources. Examples of clinical-related KPIs have included patients’ length of stay in the hospital, numbers of reported medication errors, and percentage of post-treatment complications. Also, by using surveys and questionnaires, patients’ satisfaction with the average time between admission and diagnosis, and the average time between diagnosis and choosing the best medical treatments will be measured. At last, examples of financial-related KPIs include the cost-effectiveness of implementing EBP, periodical net profit, and budget variance. However, the retrieved data from previous KPIs must be analyzed and presented regularly to top management.\n\nThe expected positive outcomes of the EBP unit will enhance the adoption and sustainability of the EBP functions. Celebrating the achievements (i.e., improved KPIs scores) would be another strategy for sustainability and enhancing the spirit of teamwork, the sense of achievement among healthcare team members. Besides, this alternative is subjected to modifications based on regular policy evaluation.\n\n\nImplication for nursing and health policy\n\nEBP is the keystone of nursing, but marketplace changes call for more collaboration to implement EBP. Current staff can become EBP champions and role models for newer nurses to implement a shared vision for implanting EBP across disciplines. As nurses, we receive many medical questions from the new nurses regarding the best intervention and patients and their families regarding their conditions. Some of these questions we cannot answer without examining the literature and researching the key components necessary to help us, our patients, and patients’ families make sound decisions. Using new evidence to underpin practice without assessing its effectiveness is shortsighted. Nursing colleges and universities are obligated to encourage student nurses to embrace and implement evidence-based practice from the onset of their clinical practice.\n\n\nConclusion\n\nThis paper presented an experience of one country, Jordan, regarding policy issue analysis to establish an EBP policy accompanied by developing a unit in the hospital’s country. The paper presented background information about the significance of introducing EBP in providing routine patient care, and then it goes through a comprehensive assessment of the current situations. The paper then moves to describe the change initiative, plan, and methods of implementing EBP along with an evaluation plan and sustainability strategies. EBP is proven to be the need to achieve the set mission and vision to excel as leaders at the local and regional levels of healthcare.\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\nLoBiondo-Wood G, Faan PR, Haber J, Faan PR: Nursing Research E-Book: Methods and Critical Appraisal for Evidence-Based Practice . Elsevier Health Sciences; 2021.\n\nCamargo FC, Iwamoto HH, Galvão CM, et al.: Models for the implementation of evidence-based practice in hospital based nursing: A narrative review1. Texto & Contexto-Enfermagem. 2018; 26. Publisher Full Text\n\nKristensen N, Nymann C, Konradsen H: Implementing research results in clinical practice-the experiences of healthcare professionals. BMC Health Serv Res. 2015; 16(1): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitchell GJ: Implications of holding ideas of evidence-based practice in nursing. Nurs Sci Q. 2013; 26(2): 143–151. PubMed Abstract \| Publisher Full Text\n\nFlorczak KL: Evidence or Clinicians or the Person: Who Should Be at the Center? Nurs Sci Q. 2017; 30(1): 17–20. PubMed Abstract \| Publisher Full Text\n\nMcEachan R, Taylor N, Harrison R, et al.: Meta-analysis of the reasoned action approach (RAA) to understanding health behaviors. Ann Behav Med. 2016; 50(4): 592–612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen L, Knighton AJ, Wolfe D, et al.: Implementing evidence-based clinical practice in the critical care setting. Qual Manag Health Care. 2020; 29(2): 123–125. PubMed Abstract \| Publisher Full Text\n\nDuncombe DC: A multi-institutional study of the perceived barriers and facilitators to implementing evidence-based practice. J Clin Nurs. 2018; 27(5-6): 1216–1226. PubMed Abstract \| Publisher Full Text\n\nAl-Ghabeesh SH, Abu-Moghli F, Salsali M, et al.: Exploring sources of knowledge utilized in practice among Jordanian registered nurses. J Eval Clin Pract. 2013; 19(5): 889–894. PubMed Abstract \| Publisher Full Text\n\nHorntvedt M-ET, Nordsteien A, Fermann T, et al.: Strategies for teaching evidence-based practice in nursing education: a thematic literature review. BMC Med Educ. 2018; 18(1): 1–11. Publisher Full Text\n\nNaderkhah Z, Kalhor R, Azmal M, et al.: The evaluation of level of knowledge, attitude and practice of evidence-based practice and its barriers among nurses working in selected Iranian hospitals. J Biology Today’s World. 2016; 5(9): 163–168. Publisher Full Text\n\nO’Grady ET: The policy process. In: Mason DJ, Perez A, McLemore MR, et al.: Policy & Politics in Nursing and Health Care-E-Book. Elsevier Health Sciences; 2020; pp. 52–64.\n\nLewin K: Field theory in social science: selected theoretical papers (Edited by Dorwin Cartwright.).1951;\n\nKeepnews DM: Developing a policy brief. Policy, Politics, & Nursing Practice. 2016; 17(2): 61–65. Publisher Full Text\n\nAbuRuz ME, Hayeah HA, Al-Dweik G, et al.: Knowledge, attitudes, and practice about evidence-based practice: a Jordanian study. Health Science J. 2017; 11(2): 1. Publisher Full Text\n\nMah’d Alloubani A, Taktak WFJ, Hussein AA, et al.: Improving Compliance of Hand Hygiene Practices among Intensive Care Unit Employees in AL-Istishari Hospital in Jordan: Rabadi HN.2014.\n\nChristiansen C, Lou JQ: Ethical considerations related to evidence-based practice. Am J Occup Ther. 2001; 55(3): 345–349. PubMed Abstract \| Publisher Full Text\n\nAhuja RB: Ethical practice of evidence-based medicine: A review for plastic surgeons. Indian J Plast Surg. 2013; 46(01): 011–017. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGibbs L, Gambrill E: Evidence-based practice: Counterarguments to objections. Res Social Work Practice. 2002; 12(3): 452–476. Publisher Full Text\n\nRycroft-Malone J: The politics of the evidencebased practice movements: Legacies and current challenges. J Res Nurs. 2006; 11(2): 95–108. Publisher Full Text\n\nKraft ME, Furlong SR: Public policy: Politics, analysis, and alternatives. Cq Press; 2019.\n\nGrol R, Grimshaw J: From best evidence to best practice: effective implementation of change in patients' care. Lancet. 2003; 362(9391): 1225–1230. PubMed Abstract \| Publisher Full Text\n\nWolters Kluwer: At the Core of Magnet Innovations and Outcomes\|Wolters Kluwer hwwcee-ia-t-c-o-m-i-a-o.\n\nWise NJ: Maintaining Magnet Status: Establishing an Evidence-Based Practice Committee. AORN J. 2009; 90(2): 205–213. PubMed Abstract \| Publisher Full Text\n\nKouzes JM, Posner B: The leadership challenge: How to make extraordinary things happen . Atlanta, GA: Better World Books; 2012.\n\nGallagher-Ford L: Leveraging shared governance councils to advance evidence-based practice: the EBP Council journey. Worldviews Evid Based Nurs. 2015; 12(1): 61–63. PubMed Abstract \| Publisher Full Text\n\nBednarski D: Shared governance: Enhancing nursing practice. Nephrol Nurs J. 2009; 36(6): 585.\n\nEmanuel V, Day K, Diegnan L, et al.: Developing evidence-based practice among students. Nurs Times. 2011; 107(49/50): 21–23." }	[ { "id": "99178", "date": "02 Dec 2021", "name": "Tonje Lossius Husum", "expertise": [ "Reviewer Expertise Ethics in mental health care and medical ethics in general." ], "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 the opportunity to review this article. The article is about developing an action plan utilizing a systematic approach to develop and implement specific strategies and policies to integrate EBP in clinical settings in Jordanian hospitals. The topic is an important topic in contemporary times, and It’s an important task to do research on how to implement EBP in clinical settings.\n\nThe concept of evidence-based care is not new, but I think that all the questions and the science about how to implement EBP in clinical settings has been an underdeveloped and underprioritized area. This research contributed with new important and concrete knowledge about how it is possible to work with implementing EBP in a clinical setting.\nThis study is conducted in a somatic hospital setting. To work according to and to implement EBP in clinical settings is also an important matter in mental health care. In the future I hope it will be more focus and scientific development in this area to.\nThe abstract is informative and well written, so is the paper itself. It has good structure and is easy to follow.\nThe authors could maybe also add something about ethical challenges when implementing EBP, and something about inhibitors and promoters in implementing the suggested plan for EBP. Also, the authors could maybe add a risk analysis in implementing the suggested model.\nAltogether an important work and scientific development concerning implementing EBP in clinical settings which hopefully will inspire other research groups to do research in the topic of implementation science.", "responses": [ { "c_id": "7544", "date": "06 Dec 2021", "name": "mohammad mohammad", "role": "Author Response", "response": "Dear Dr. Tonje Lossius Thanks for the time and effort you spent in reviewing this paper. Also, thanks for your comments. In the revised version, I will include one more step after approving any evidence that is obtaining ethical approval. The next step after this brief is the actual implementation. We are planning to pilot this brief in one educational hospital." } ] }, { "id": "96863", "date": "20 Dec 2021", "name": "Latefa Ali Dardas", "expertise": [ "Reviewer Expertise Mental 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 work presents a significant and timely addition to the literature and seems to have promising implications to enhance the quality of health practices. The authors have proposed an action plan utilizing a systematic approach to guide the process of integrating evidence-based practice (EBP) in clinical settings in Jordanian hospitals, and I cannot agree more with authors that most healthcare providers and researchers embrace the concept of EBP without actual integration in clinical environment.\nWithin the Jordanian context, it became evident that there is a significant and dramatic increase in the number of published research studies. Nevertheless, minimal changes in practice have been seen in response to these published findings. It is therefore important to tap issues that hinder the integration of EBPs in the clinical setting.\nThe authors have chosen a very important and influential factor in this process, which is the lack of biding policies that can guide how to implement the updated EBP research findings into clinical practice guidelines, protocols, and procedure manuals. In doing so, authors provided a comprehensive overview of the policy and the context of its implementation in a way that is accessible to a general reader.\nAuthors provided interesting contextual information related to the ‘cultural’, ‘economical’, ‘ethical’, ‘legal’, and ‘political’ aspects in Jordan, highlighting the unique description of each and building upon to propose solutions and alternatives.\nThe provided alternatives were made in a way that reflects the potential to achieve the policy goals and objectives, decrease cost, consideration of different populations, political, social administrative-technical feasibility, and sustainability.\nRecommendations made are clear, balanced, and justified on the basis of the presented arguments.\nOverall, this article includes novel and significant information that can be useful in guiding efforts to promote and support an evidence-based practice.\n\nDoes the paper provide a comprehensive overview of the policy and the context of its implementation in a way which is accessible to a general reader? Yes\n\nIs the discussion on the implications clearly and accurately presented and does it cite the current literature? Yes\n\nAre the recommendations made clear, balanced, and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-933

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