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https://f1000research.com/articles/9-252/v1	09 Apr 20	{ "type": "Systematic Review", "title": "Nitride oxide synthase 3 and klotho gene polymorphisms in the pathogenesis of chronic kidney disease and age-related cognitive impairment: a systematic review and meta-analysis", "authors": [ "Atma Gunawan", "Jonny Karunia Fajar", "Fredo Tamara", "Aditya Indra Mahendra", "Muhammad Ilmawan", "Yeni Purnamasari", "Dessy Aprilia Kartini", "Eden Suryoiman Winoto", "Efriko Septananda Saifillah", "Dewi Sri Wulandari", "Pratista Adi Krisna", "Ema Dianita Mayasari", "Tri Wahyudi Iman Dantara", "Ramadi Satryo Wicaksono", "Djoko Wahono Soeatmadji", "Fredo Tamara", "Aditya Indra Mahendra", "Muhammad Ilmawan", "Yeni Purnamasari", "Dessy Aprilia Kartini", "Eden Suryoiman Winoto", "Efriko Septananda Saifillah", "Dewi Sri Wulandari", "Pratista Adi Krisna", "Ema Dianita Mayasari", "Tri Wahyudi Iman Dantara", "Ramadi Satryo Wicaksono", "Djoko Wahono Soeatmadji" ], "abstract": "Background: While it has been known that the development of chronic kidney disease (CKD) and age-related cognitive impairment involves several mediators, the evidence in clinical practice only reveals nitride oxide synthase (NOS) and klotho. However, the evidence for this topic is conflicted. The aim of this study was to assess the role of NOS and klotho single nucleotide polymorphisms (SNPs) in the pathogenesis of CKD and age-related cognitive impairment. Methods: We performed a meta-analysis during October to December 2019. Paper collection was performed in major scientific websites, and we extracted information of interest from each paper. Data were analyzed using a Z-test with either random or fixed effect model. Results: Our initial assessment identified NOS3 G894T, NOS3 T786C, NOS3 4b/4a, klotho (KL) G395A, and KL C1818T as the gene candidate for our meta-analysis. Our pooled calculation revealed that NOS3 G894T was associated with the risk of both age-related cognitive impairment and CKD. Increased susceptibility to age-related cognitive impairment was observed in the GG genotype, and increased risk of CKD was found in patients with a single T allele and TT genotype for NOS3 nucleotide 894. For NOS3 4b/4a, increased risk of CKD was only found in 4a4a genotype. For NOS3 T786C, we failed to show the association with both CKD and age-related cognitive impairment. Subsequently, for KL G395A, A allele and GA genotype were found to correlate with increased susceptibility to CKD, while its correlation to age-related cognitive impairment was failed to clarify. For KL C1818T, our analysis failed to find the correlation with the risk of CKD. Conclusions: Our results reveal that the NOS3 G894T gene polymorphism has a crucial role in the pathogenesis of both CKD and age-related cognitive impairment.", "keywords": [ "Nitride oxide synthase", "klotho", "chronic kidney disease", "age-related cognitive impairment" ], "content": "Introduction\n\nAging had remained a challenging topic since the last three decades1. Some large scale studies have been developed to clarify the precise mechanism of how aging affects the body2,3 and also how to prevent this circumstance4. This is because aging is a normal condition in human life. This means that this proccess has to occur in everyone. Recently, to avoid this circumstance, studies have concerned to elucidate aging because aging is known to correlate with age-related diseases including cardiovascular disease5, stroke6, dementia7, and chronic kidney disease (CKD)8. Of these, CKD was considered the more serious disease because it was proven to associate with high risk of mortality and poor quality of life9. It is widely known that patients with stage V CKD should be treated with regular dialysis and or even renal transplantation10.\n\nCKD is a frightening disease for most populations11. The investigation regarding the better treatment option for this disease had not provided significant development in developing countries. In the context of aging, this might involve several mediators, including estrogen, androgen, L-arginine, nitride oxide synthase (NOS), and klotho12. Of these, only NOS and klotho have been well reported in genetic levels and in clinical settings in the context of aging and CKD. Other mediators, during this time, were only proposed as theory or hypothesis. The absense of direct clinical investigation regarding those mediators in the context of aging and CKD led to these mediators being considered as correlated with aging and CKD. The lack of studies investigating aging in clinical practice might be due to the fact that the definition of aging is complex, and it can be difficult to determine the appropriate scope of aging. However, aging is widely to correlate with age-related cognitive impairment13. For this reason, in our present study, our investigation only concerned age-related cognitive impairment. Furthermore, investigating the role of NOS and klotho gene polymorphisms in the case of age-related cognitive impairment and CKD was logical and crucial for better understanding concerning the development of aging and CKD. Moreover, due to conflicting reports regarding this topic, a meta-analysis study was required to elucidate the real association.\n\nOur current study, therefore, aimed to perform a meta-analysis concerning the role of NOS and klotho gene polymorphisms in the case of age-related cognitive impairment and CKD. Our present study might provide better understanding on which allele or genotype of NOS and klotho gene polymorphisms are associated with the risk of age-related cognitive impairment and CKD.\n\n\nMethods\n\nDuring the study time frame (October-December 2019), a meta-analysis was conducted to assess the correlation between NOS and klotho gene polymorphisms and the risk of CKD and age-related cognitive impairment. To attain our purpose, we collected papers from PubMed, Embase, Cochrane, and Web of Science. Moreover, to determine the association and effect estimates, data on allele and genotype frequency from selected papers were used to calculate the odds ratio (OR) and 95% confidence interval (95%CI). The protocols in our current study include paper selection, data extraction, quality assessment, and statistical analysis referred to our previous studies14–18, and we also used the checklist of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) to guide the protocols in our study19. A completed PRISMA checklist for the current study is available (DOI: https://doi.org/10.6084/m9.figshare.12016782)98.\n\nTo obtain the papers, the following criteria should be met to include the papers in our study: (1) assessing the correlation between NOS and klotho gene variants and the risk of CKD and age-related cognitive impairment; and (2) providing sufficient data for calculation of OR and 95%CI. Furthermore, we excluded the papers if the following reasons were met: (1) irrelevant topic, (2) review, (3) non-standard data presentation, (4) deviation from Hardy-Weinberg equilibrium, and (5) double publication. We managed reference list using EndNote v8 (Thompson Reuters, Eagan, Minnesota) to remove instances of double publication.\n\nPapers assessing the association between NOS and klotho gene polymorphisms and the risk of CKD and age-related cognitive impairment were searched in major scientific websites (PubMed, Embase, Cochrane, and Web of Science) up to 5 December 2019. In searching the articles, we restricted the publication language to English. Moreover, to perform a holistic searching, we applied the keywords adapted from medical subject headings (MeSH): [\"chronic kidney disease\" or \"chronic renal failure\"] and [\"aging\" or \"age-related cognitive impairment\"] and [\"nitride oxide synthase\" or \"NOS\"] and [\"klotho\"]. If we found double publication data, we only included article with the larger sample size. Subsequently, for data extraction, the following information of interest was extracted: (1) first author name; (2) publication year; (3) sample size of case and control, and (4) genotype frequencies of case and control groups.\n\nTo assess the quality of each study, a Methodological Index For Non-Randomized Studies (MINORS) scoring system was applied20. The MINORS score ranged from 0 to 24 and consisted of 12 items. Each item was assessed as 0 if the item was not reported, 1 if the item was inadequate reported, and 2 if the item was adequate reported. Each study was interpreted as having low quality if the score was less than or equal to 12, moderate if the score was less than or equal to 16 and more than 12, and high quality if the score was more than 1620.\n\nOur initial searching identified five single nucleotide polymorphisms (SNPs) for included in our meta-analysis: NOS3 4b/4a, NOS3 G894T, NOS3 T786C, klotho (KL) G395A, and KL C1818T. For age-related cognitive impairment, the SNPs were NOS3 G894T, NOS3 T786C, and KL G395A. For CKD, the SNPs were NOS3 4b4a, NOS3 G894T, NOS3 T786C, KL G395A, and KL C1818T. In each SNP, data analysis was performed in all alleles and genotypes models to assess the correlation and effect estimates. For NOS3 4b/4a, the genetic models were 4b vs. 4a; 4a vs. 4b; 4b4b vs. 4b4a+4a4a; 4b4a vs. 4b4b+4a4a; and 4a4a vs. 4b4b+4b4a. For NOS3 G894T, the genetic models were G vs. T; T vs. G; GG vs. GT+TT; GT vs. GG+TT; and TT vs. GG+GT. For NOS3 T786C, the genetic models were T vs. C; C vs. T; TT vs. TC+CC; TC vs. TT+CC; and CC vs. TT+TC. For KL G395A, the genetic models were as follows: G vs. A; A vs. G; GG vs. GA+AA; GA vs. GG+AA; and AA vs. GG+GA. For KL C1818T, the genetic models were C vs. T; T vs. C; CC vs. CT+TT; CT vs. CC+TT; and TT vs. CC+CT.\n\nThe association and effect estimation between NOS3 and KL gene variants and the risk of CKD and age-related cognitive impairment were determined using a Z-test. The p value of less than 0.05 was considered statistically significant. Moreover, to determine effect estimates, the calculation of pooled OR and 95%CI was performed. Prior to determining the association and effect estimation, to assess the consistency in our meta-analysis, data were analyzed for heterogeneity and potential publication bias. For assessing the heterogeneity, we applied a Q-test. A p-value of less than 0.10 was considered to indicate heterogeneity and data were analyzed using random effect model. Conversely, we used fixed effect model if the p value was more than 0.01. Moreover, for testing the potential for publication bias, Egger's test was employed. A p-value of less than 0.05 was considered as indicating publication bias. All analyses in our present study were performed using Review Manager [Revman Cochrane, London, UK] version 5.3. The cummulative calculation was presented using a forest plot.\n\n\nResults\n\nOur final paper selection identified 21 papers21–41 assessing NOS3 G894T gene polymorphisms in age-related cognitive impairment; three papers21,42,43 assessing NOS3 T786C gene polymorphisms in age-related cognitive impairment; five papers44–48 assessing KL G395A gene polymorphisms in age-related cognitive impairment; ten papers49–58 evaluating NOS3 4b/4a gene polymorphisms in CKD; seven papers54–56,58–61 evaluating NOS3 G894T gene polymorphisms in CKD; three papers49,54,62 assessing NOS3 T786C gene polymorphisms in CKD; six papers63–68 assessing KL G395A gene polymorphisms in CKD; and three papers65–67 assessing NOS3 C1818T gene polymorphisms in CKD. This number of papers were searched in PubMed, Embase, Cochrane, and Web of Science; and papers were selected in accordance with inclusion and exclusion criteria. In the initial searching, we identified 10,858 papers. Of those, 10,787 papers were excluded because of irrelevant topic. Moreover, 13 papers were also excluded because of review (seven), not providing required data for calculation of OR and 95%CI (four), and being of low-quality (two). A flowchart describing eligibility pathway in our study is provided in Figure 1.\n\nFor age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, such as: NOS3 G894T, NOS3 T786C, and KL G-395A. Of those, the correlation was only found in NOS3 G894T gene variant. Conversely, we failed to clarify the correlation between the risk of age-related cognitive impairment and NOS3 T786C and KL G395A gene polymorphism. For NOS3 G894T, we found that increased risk of age-related cognitive impairment (Figure 2A) was observed in GG genotype of NOS3 G894T gene polymorphism (OR [95%CI] = 1.14 [1.01 - 1.30], p = 0.0320). On other hands, reduced risk of age-related cognitive impairment (Figure 2B) was found in GT genotype of NOS3 G894T gene variant (OR [95%CI] = 0.86 [0.75 - 0.97], p = 0.0170). The summary of the association between age-related cognitive impairment and the gene polymorphisms in NOS3 and KL is given in Table 1.\n\n(A). GG vs. GT+TT; (B). GT vs. GG+TT.\n\nSNP, single nucleotide polymorphism; NS, number of studies; OR, odd ratio; pHet, p heterogeneity; pE, p Egger.\n\nFor NOS gene polymorphisms in CKD patients, we identified three SNPs, NOS3 4b4a, NOS3 G894T, and NOS3 T786C. For NOS3 4b4a (Figure 3A), our results found that only the 4a4a genotype was associated with increased risk of CKD (OR [95%CI] = 2.09 [1.43 - 3.06], p < 0.0001). For NOS3 G894T, we found that the T allele (Figure 3B) and TT genotype (Figure 3C) were, by 1.65 and 2.08-fold, respectively, associated with increased risk of CKD. Conversely, the G allele and GG genotype were associated to decreased risk of CKD. Moreover, for NOS3 T786C, our findings failed to confirm the correlation in CKD patients.\n\nA). 4a4a vs. 4b4b+4b4a of NOS3 4b/4a; B). T vs. G of NOS3 G-894T; C). TT vs. GG+GT of NOS3 G-894T.\n\nFurthermore, for klotho gene polymorphisms in CKD patients, only two SNPs were compatible for our analysis, KL G-395A and KL C1818T. For KL G395A, we included six papers consisting of 550 cases and 1131 controls. Of those, G allele and GG genotype were observed having protective effect against CKD, and A allele (Figure 4A) and GA genotype (Figure 4B) were found susceptible for CKD. Moreover, for klotho C1818T, we failed to show the correlation in CKD patients. The summary of the correlation between the risk of CKD and the gene polymorphisms in NOS3 and KL is described in Table 2.\n\n(A). A vs. G of KL G-395A; (B). GA vs. GG+AA of KL G395A.\n\nSNP, single nucleotide polymorphism; NS, number of studies; OR, odd ratio; pHet, p heterogeneity; pE, p Egger.\n\nIn the case of age-related cognitive impairment, for NOS3 G894T gene polymorphism, except for TT genotype, the evidence for heterogeneity was found in all genetic models, and therefore we applied random effect model to analyze the data. For NOS3 T786C, we found no evidence for heterogeneity, and therefore fixed effect model was used to analyze the data. For KL G395A, the evidence for heterogeneity was observed in all genetic models, except for the GA genotype. Therefore, we used random effect model to analyze the data. We provided the summary of heterogeneity analysis concerning this topic in Table 1.\n\nIn the case of CKD, for NOS3 4b/4a, evidence for heterogeneity was found in all genetic models, except for the 4a4a genotype. Therefore, we applied random effect model to analyze the data. Conversely, for the 4a4a genotype, because it was proven to have no heterogeneity, the analysis was performed using fixed effect model. Subsequently, for NOS3 G894T, due to the lack of evidence for heterogeneity, we used a fixed effect model to analyze the TT genotype. On other hands, for other genetic models, a random effect model was applied to analyze the data. Moreover, for NOS3 T786C, due to having the evidence for heterogeneity, we used a random effect model to analyze T and C alleles, the TT genotype, and the TC genotype. For the CC genotype, the association was determined using fixed effect model. Furthermore, for KL G395A, all genetic models were analyzed using a random effect model. For KL C1818T, a fixed effect model was used to analyze the correlation in all genetic models. We summarize the evidence of heterogeneity concerning the association between the risk of CKD and the gene polymorphisms of NOS3 and KL in Table 2.\n\nWe applied Egger's test to assess the potency of publication bias among studies. Our analysis revealed that, in the case of age-related cognitive impairment, the publication bias was found in TT genotype of NOS3 G894T and all genetic models of NOS3 T786C. Subsequently, in the case of CKD, we found that no publication bias was observed in all genetic models of NOS3 4b4a, NOS3 G-894T, NOS3 T-786C, and KL G-395A. However, publication bias was found in the C allele, T allele, CC genotype, and CT genotype of KL C1818T. The summary of Egger’s test in our study is presented in Table 1 for the case of age-related cognitive impairment and Table 2 for the case of CKD.\n\n\nDiscussion\n\nOur current study assessed the correlation between age-related cognitive impairment and the gene polymorphisms in NOS3 (NOS3 G894T and NOS3 T786C). Our results revealed that age-related cognitive impairment was not related to the gene polymorphism of NOS3 T786C. On other hands, we found that the GG genotype was found to correlate with susceptibility to age-related cognitive impairment, and GT genotype was found to have a protective effect against age-related cognitive impairment. Our findings were consistent with those of a previous study69. They also found that GG genotype of NOS3 G894T was proven to associate with increased the susceptibility to age-related cognitive impairment. The evidence had confirmed that the polymorphism of NOS3 G894T had been shown to correlate with NO basal production and NOS3 enzyme activity70. Moreover, elevated NOS3 expression was also found to correlate with increased mitochondrial function in neurons71. Therefore, it made sense that the NOS3 G894T gene polymorphism was associated with age-related cognitive impairment as reported in our study. On other hands, we also reported the NOS3 gene polymorphism in the case of CKD. Our results identified three SNPs available for the calculation of meta-analysis. However, the association with the risk of CKD was only observed in 4b/4a and G894T NOS3 gene polymorphisms. For 4b/4a, our findings revealed that the 4a4a genotype was associated with increased risk of CKD. For the G894T gene polymorphism, we found that the T allele and TT genotype were observed to correlate with increased risk of CKD. Previous meta-analysis regarding this topic had been conducted72,73. However, our current study provided the update, and our current results were consistent with previous studies. Additionally, supporting our results, a previous study confirmed that the TT genotype and T allele of the G894T polymorphism, but not 4b/4a, were associated with the lower level of NO in circulation74 and enzyme activity75. Furthermore, lower NO levels compared to control was found in patients with CKD76, although increased levels of NO were observed in CKD patients after dialysis77. The similarity of the dominant role of the NOS3 G894T gene polymorphism, both in age-related cognitive impairment and CKD, might explain the bridging mechanism between aging and CKD with NO involvement, in the context of gene-disease and gene-gene interactions.\n\nThe precise mechanism of NO in age-related cognitive impairment and CKD is undefined. However, some speculation may be proposed. Briefly, NO plays a significant role in cell growth and renal vasculature. It is widely known that NO plays as a vascular vasodilator. Additionally, NO may also inhibit the growth of mesangial cell and matrix production. The decreased level of NO in aging may cause to renal vasoconstriction, sodium retention, and increased matrix production and mesangial fibrosis78. Moreover, NO isoforms are observed at higher levels in the medullary region than other regions. On other hands, in the renal cortex, the levels of NO isoforms are reduced. Therefore, they may contribute to the reduced perfusion of renal cortex in the elderly79. The precise pathway of decreased level of NO in elderly remains confusing. However, several mechanisms have been proposed. First, oxidative stress is known to increase with age. It may stimulate to decrease the key factors for normal NO production, for example tetrahydrobiopterrin80. Second, L-arginine is known to be key for the production of NO. The availability of this substrate may decline with advance age. While L-arginine is not an essential amino acid, a study had reported that the level of L-arginine was observed decreased in older rats. This indicates that L-arginine may play a crucial role as an essential amino acid in advance age, and therefore sufficient dietary intake may be required to maintain the NO production78. Moreover, L-arginine level in circulation was also found to be significantly lower in patients with CKD than controls, and it was consistent with the level of NO76. This suggested the pivotal role of L-arginine and NO in aging and CKD. Third, it is known that NOS is degraded by asymmetric dimethyl arginine (ADMA). Previous study in a rat model revealed that ADMA levels were observed higher in advance age. This suggests that elevated ADMA level may increase the degradation of NOS and cause lower NO production81. Supporting this explanation, a study found that ADMA levels in circulation were higher in CKD patients than control, was contrary to the levels of NO and L-arginine76. This explanation might bridge the mechanism between NO, aging, and CKD as reported in our present study.\n\nWhile klotho was considered as one of the important mediators in aging, our findings failed to confirm the association between the KL G395A gene polymorphism and risk of age-related cognitive impairment. However, due to limited sample size, further investigation to assess this correlation was required. On other hands, correlating to CKD, our searching strategy identified KL G395A and C1818T as available for meta-analysis calculation. Our analysis confirmed that the association with CKD was only found in klotho G-395A gene polymorphism. We revealed that the A allele and GA genotype were correlated with increased risk of CKD. Until now, we have failed to obtain a systematic review or meta-analysis in the topic of either klotho in aging or in CKD. Therefore, a direct comprehensive comparison was unable to perform. However, it had been reported that α-klotho protein was related to the G395A polymorphism82, and α-klotho protein in circulation was also proven by a large scale meta-analysis study to have positive correlation with renal function83. It means that the lower level of klotho protein, the lower the renal function. Therefore, it might explain the results of our study confirming that the G395A gene polymorphism was correlated with the risk of CKD.\n\nThe theory explaining the exact mechanism between klotho, aging, and CKD is complicated and may involve genes, proteins, and target organ damage. At the genetic level, KL is expressed in limited tissues and cell types, and the highest expression is observed in distal convoluted tubules in the kidney and choroid plexus in the brain84. Therefore, the klotho protein exists in two forms. One is the trans-membrane form expressed primarily in renal tubular cells, and the other is the secreted form circulating in the blood85. Klotho protein level has been shown to correlate with human longevity86. However, in advance age, the level of klotho protein is decreased87, and this decreased level is associated with increased oxidative stress, proinflammatory cytokine production, and activation of endothelin signal transduction88. Furthermore, the interaction between klotho and CKD is complex. It may involve specific signaling axis, defined as the klotho-fibroblast growth factor-23 (FGF23) signaling axis. Briefly, when the body has excessive amounts of phosphate, FGF23 is secreted. Subsequently, in the kidney, FGF23 may promote phosphate excretion into urine and suppress vitamin D synthesis. Consequently, it may induce negative phosphate balance. However, in this circumstance, FGF23 requires klotho to bind and activate FGF receptors. After FGF receptor activation by klotho, FGF23 binds to its receptor89. There are four type of receptors for FGF23, such as FGFR1, FGFR2, FGFR3, and FGFR4. However, FGFR1 is the dominant receptor playing in this signaling pathway90. When the binding between FGF23 and FGFR1 occurs, it may activate extracellular signals - regulated kinase (ERK) and serum/glucocorticoid-regulated kinase (SGK) signals. Furthermore, the phosphorylation of the Na+/H+ exchange regulatory cofactor (NHERF)-1 by SGK-1 was established to down-regulate membrane expression of sodium phosphate co-transporter NaPi-2a. Consequently, it may cause increasing urinary phosphate excretion91. On other hands, the binding between FGF23 and FGFR1 may also suppress the expression of 1α-hydroxylase, the enzyme responsible for the production of 1.25(OH)2D. Therefore, it may participate to systemic mineral homeostasis and regulate the excretion of phosphate92. In this context, if the level of klotho is decreased, it may lead to lower level of FGF23 and stimulate to hyperphosphatemia, one of the pathological states widely observed in CKD93. Moreover, it was also reported that the level of klotho protein was found to decline and it was also accompanied by renal insufficiency in patients with CKD94. Additionally, the gene-interaction studies also revealed that the decline of klotho level in subjects with CKD involved specific phenotypes, suggesting that klotho was independently involved in the pathogenesis of CKD84,94. This explanation might be a benchmark for the results of our study that klotho is an important mediator involved in the development of aging and CKD.\n\nOur results have identified SNPs potentially involved in the pathogenesis of age-related cognitive impairment and CKD. Therefore, our current findings might help to elucidate the precise mechanism of aging and CKD in the perspective of clinical evidence and gene-disease interaction. Despite the limitations of our study, our findings might be considered as \"a candle in the darkness,\" meaning that our findings might be as the initial step to develop further investigation for the management of aging and CKD. However, more studies on this topic are required to establish further due to some limitations, especially the wide context of aging that may make it difficult to conduct analysis and also may lead to high potency for bias.\n\nIn our current study, several limitations were noted. First, some factors that might influence NOS3 and klotho level including multiple sclerosis95, asthma96, chronic obstructive pulmonary disease97, and cardiovascular disease5 were not controlled for. Second, due to relatively small sample size, our findings should be interpreted with caution, considering the potency for bias. Third, most of study design in our included studies were cross-sectional. Thus, further studies with involving better study design might be required.\n\n\nConclusion\n\nOur present study has identified that NOS3 G894T plays an important role in the pathogenesis of both age-related cognitive impairment and CKD. On other hand, while we have found an association between KL G395A gene polymorphism and the risk of CKD, its correlation with age-related cognitive impairment has not been clarified. Our current study may contribute to better understanding regarding the role of NOS3 and KL in the pathogenesis of age-related cognitive impairment and CKD.\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 ‘Nitride oxide synthase 3 and klotho gene polymorphisms in the pathogenesis of chronic kidney disease and age-related cognitive impairment: a systematic review and meta-analysis’. https://doi.org/10.6084/m9.figshare.1201678298.", "appendix": "Acknowledgements\n\nWe thank to DSKF Publishing Campus & Lembaga Pengelola Dana Pendidikan (LPDP) Republik Indonesia.\n\n\nReferences\n\nCurb JD, Guralnik JM, LaCroix AZ, et al.: Effective aging. Meeting the challenge of growing older. 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Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12016782.v1" }	[ { "id": "66026", "date": "06 Jul 2020", "name": "Hongliang Zhang", "expertise": [ "Reviewer Expertise nephrology", "neurology", "neuroimmunology", "neuroimaging", "neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed a meta-analysis to assess the role of NOS and klotho single nucleotide polymorphisms (SNPs) in the pathogenesis of CKD and age-related cognitive impairment. They identified SNPs potentially involved in the pathogenesis of age-related cognitive impairment and CKD. They concluded that NOS3 G894T gene polymorphism has a crucial role in the pathogenesis of both CKD and age-related cognitive impairment. Overall, the study is well designed and conducted. The PRISMA checklist was well implemented.\nMajor concern:\nA hypothesis is missing and validation is lacking. Association does not necessarily mean a causal relationship. Confounding factors, mediators, and compensatory factors can be implicated in the role of NOS and klotho single nucleotide polymorphisms (SNPs) in the pathogenesis of CKD and age-related cognitive impairment.\n\nThe definition and diagnostic criteria seem lacking for age-related cognitive impairment. Of note is that age-related cognitive impairment is not a disease. Ref 13 fails to give a definition.\n\nMinor points:\n\" and being of low-quality (two).\" in the paper selection should be specified.\n\n\"a candle in the darkness\" is kind of exaggerated. It is simply a meta-analysis. The data should not be over-interpreted.\n\nThe language should be polished. \"frightening\" appears frightening in scientific writings. \" 13 papers were also excluded because of review(s)\" should be rephrased. Please use \"we found that...... \" instead of \"our results found that...... \".\n\n\"For age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, such as: NOS3 G894T, NOS3 T786C, and KL G-395A.\" can be rephrased to \"For age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, including NOS3 G894T, NOS3 T786C, and KL G-395A.\"\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? No\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? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "6456", "date": "19 Mar 2021", "name": "Jonny Fajar", "role": "Author Response", "response": "1. A hypothesis is missing and validation is lacking. Association does not necessarily mean a causal relationship. Confounding factors, mediators, and compensatory factors can be implicated in the role of NOS and klotho single nucleotide polymorphisms (SNPs) in the pathogenesis of CKD and age-related cognitive impairment. Response: the potential confounding factors, mediators, and compensatory factors that might affect the final findings of our study have been provided in the limitations. 2. The definition and diagnostic criteria seem lacking for age-related cognitive impairment. Of note is that age-related cognitive impairment is not a disease. Ref 13 fails to give a definition. Response: In the second paragraph of introduction, we did not provide the definition and diagnostic criteria of age-related cognitive impairment. We only described the problems in the context of aging and age related cognitive impairment. 3. \" and being of low-quality (two).\" in the paper selection should be specified. Response: We have revised the sentence. 4. \"a candle in the darkness\" is kind of exaggerated. It is simply a meta-analysis. The data should not be over-interpreted. Response: We have revised the sentence. 5. The language should be polished. \"frightening\" appears frightening in scientific writings. \" 13 papers were also excluded because of review(s)\" should be rephrased. Please use \"we found that...... \" instead of \"our results found that...... \". Response: We have revised the sentence. 6. \"For age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, such as: NOS3 G894T, NOS3 T786C, and KL G-395A.\" can be rephrased to \"For age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, including NOS3 G894T, NOS3 T786C, and KL G-395A.\" Response: We have revised the sentence." } ] }, { "id": "68104", "date": "10 Aug 2020", "name": "Sang Won Park", "expertise": [ "Reviewer Expertise Acute kidney injury", "preclinical study" ], "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 performed a meta-analysis for an association study of NOS3 and klotho polymorphisms with CKD and age-related cognitive impairment (ACI). Several NOS3 and klotho polymorphic nucleotide sites were analyzed in 48 selected papers. The correlation was found only in NOS3 G894T with ACI. On the other hand, the correlation was found in NOS 4b4a, NOS3 G894T, and KL G395A with CKD. The authors conclude that NOS3 G894T has a crucial role in the pathogenesis of both CKD and ACI. I have a major concern as follows. The purpose of this paper is not clear. Which is to reveal common polymorphisms in ACI and CKD to associate these two pathologies, to find polymorphisms correlated in CKD and aging, by using ACI as a marker of aging, or other purposes? However, ACI is not a good marker of aging in a meta-analysis because ACI has pathological complexity. According to the purpose, the parameter selection of polymorphic genes should be different. NOS3 functions in the cardiovascular system and the NOS3 polymorphisms are susceptible to diseases, such as hypertension, atherosclerosis, stroke, and other complications. Klotho functions in the endocrine FGF-mediated metabolic processes, such as regulating insulin secretion, feeding, and renal reabsorption of calcium and phosphate; therefore, it is associated with diabetes, CKD, and other metabolic disorders. In this regard, a meta-analysis to reveal the correlation between NOS3 and ACI should be performed separately to the meta-analysis to reveal the correlation between Klotho with CKD. Without a clear purpose, correlation studies of NOS3 and Klotho in both ACI and CKD are not clinically important. Please clarify your purpose of the study and revise your introduction and conclusions with appropriate references. You may be required additional meta-analysis on other gene polymorphisms related to ACI and CKD.\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? Partly", "responses": [ { "c_id": "6457", "date": "19 Mar 2021", "name": "Jonny Fajar", "role": "Author Response", "response": "Our study aimed to perform a meta-analysis concerning the role of NOS and klotho gene polymorphisms in the case of age-related cognitive impairment and CKD. In our paper, we only tried to identify the potential SNPs having the role in the development of CKD and the development of age related cognitive impairment. We did not propose the possible causal correlation between age related cognitive impairment and CKD. They are two distinct condition. We only tried to identify the similar SNPs between CKD and age related to cognitive impairment, and in the future, this similarity might be used in the concept of gene-gene interaction." } ] } ]	1	https://f1000research.com/articles/9-252
https://f1000research.com/articles/10-52/v1	28 Jan 21	{ "type": "Opinion Article", "title": "From Yasashii Nihongo in non-disaster times towards a plurilingual language education approach: an outlook from the perspective of “reasonable accommodation”", "authors": [ "Hideaki Ito", "Alexander Tokarev", "Alexander Tokarev" ], "abstract": "In order to address labor shortages, starting April 2019 the Japanese government introduced two new visa categories, and it can be expected that the growing number of foreign residents living and working in Japan will be increasing further in the foreseeable future. Within this context, the notion of Yasashii Nihongo or Simplified Japanese has been gaining attention over recent years. Originally designed as a tool for transmitting information in disaster-related situations and proposed for disaster mitigation purposes, at present it is being advocated as a means of communication to be used in non-disaster situations as well. The authors argue that ultimately Yasashii Nihongo for non-disaster situations may be just a means to an end. Seen from the perspective of “reasonable accommodation”, a concept prevalent in the domain of disability studies, they assert that by de facto creating a new linguistic category making it a tacit prerequisite to communicate in “Japanese only”, Yasashii Nihongo is but a concept geared towards the language majority (speakers using Japanese as their first language) and is potentially serving no other purpose than to alleviate the psychological burden of having to speak in a language other than Japanese, thus potentially leading to a new form of discrimination towards language minorities. Offering an alternative approach for improving multicultural communication aimed at establishing a communicative space based on openness, equality, and mutual respect for each other’s cultural, linguistic and ethnic identities, the authors propose the introduction of language education based on the notion of plurilingualism, as outlined in the Common European Framework of Reference for Languages (CEFR) by the Council of Europe.", "keywords": [ "Yasashii Nihongo", "plurilingualism", "multicultural coexistence", "reasonable accommodation", "disability" ], "content": "Introduction\n\nAt present, there are a growing number of foreign residents living in Japan. There has been for a number of reasons also an increase in children who have need of formal Japanese language education (JLE), and all things to do with JLE have become an increasingly popular topic over the recent years and are covered by Japanese media outlets on a daily basis. On April 1, 2019, the Japanese Government established a new visa category for foreign personnel. It is, therefore, to be expected that the number of foreign residents in Japan will be increasing further in the foreseeable future as well. Communication barriers resulting from difficulties understanding the Japanese language, however, have been becoming noticeable already, and in this context, Simplified Japanese or Yasashii Nihongo has been steadily gaining attention.\n\nYasashii Nihongo denotes a variety of the Japanese language that has been simplified so that foreigners can understand it better. Originally, the notion of Yasashii Nihongo was introduced by the Sociolinguistics Department of Hirosaki University in 1999 as a response to the Great Hanshin earthquake of 1995 in Kobe, when crucial disaster-related information failed to reach people who didn’t speak English or Japanese. Seen within this context, such Yasashii Nihongo is also referred to as “Yasashii Nihongo for Disaster Mitigation”. Recently, a derivational form of Yasashii Nihongo has become prominent which is also used in daily life and is no longer reserved for disaster-related situations only. Various attempts or movements to utilize Yasashii Nihongo in non-disaster times as a tool of communication within the context of Japan’s multicultural coexistence policy1 (tabunka kyosei) can be observed (Iori 2009). However, can a construct that had been originally developed with the “primary goal of accurately transmitting information to persons affected by natural disasters” (Sato 2016: 256) and goes back to an adopted variety of the Japanese language specifically designed for purposes of disaster mitigation that “aims to offer safety and empowerment to victims of natural calamities” (Sato 2016: 272) be really considered an appropriate and efficient means of communication that could help successfully foster a truly multicultural society? Sato (2016) states that Yasashii Nihongo designed for disaster mitigation may run risk of being treated as a joke when applied to everyday life and even be a source of discrimination and potentially lead to conflict in other contexts as well; he concludes that a disaster situation after all represents a very different kind of communicative context altogether than that of a non-disaster situation. Iori (2009) explains why Yasashii Nihongo is nonetheless being increasingly used in non-disaster contexts as follows: “While we recognize that the multilingualization is an ideal to aim for, it is also a truism that de facto you need to be able to communicate in Japanese.” This prerequisite stems from an alleged “half-way resignation” towards a society where at present multilingualization may not always be deemed a viable option.\n\nAs Iori (2009) states correctly, from the standpoint of guaranteeing access to information, devising a means of communication able to provide information to a large number of people is obviously of great importance. However, creating a new linguistic category of Yasashii Nihongo to be used in non-disaster situations may not necessarily be the best option for that and might potentially even lead to discrimination against persons who are a language minority. Regarding this matter, Iori cites Tanaka (1989: 24) who touches upon the subject and illustrates how dialects are often perceived as inferior structures to standard Japanese and elaborates that this “inferiority doesn’t necessarily have to do with language in and of itself, but is rather construed as such by society which happens to see it that way.” According to Iori (2019: 129), Yasashii Nihongo has been devised to function as a variety of Japanese that is easier to understand; Iori asserts that the more it spreads, the faster Yasashii Nihongo should be able to lose its stigma of being a “second-rate language”. The present authors would like to raise the question if that is realistic, as potentially this could very well only lead to a new kind of language discrimination in the form of a mental shift within certain persons in society from criticizing someone simply because they don’t understand Japanese to criticizing someone because they are not even capable of understanding simplified Japanese. In the opinion of Tanaka (1989: 24), “the important thing isn’t to create a new or different kind of Japanese, but instead to improve on the user-friendliness of the already existing language”. This suggests that Tanaka, too, seems to believe that it shouldn’t be about creating a new linguistic category, but rather about “rethinking” the language that is being used at present. In this paper, the authors would like to reevaluate the category of Yasashii Nihongo used for non-disaster situations and discuss what qualities a successful means of communication should possess in contemporary Japan where the number of foreign residents has been steadily growing.\n\n\nWhy Yasashii Nihongo for non-disaster situations isn’t receiving more traction\n\nIn the past few years, a number of guidebooks on Yasashii Nihongo have been published by various municipal authorities throughout Japan. Among other things they contain rules and formalized suggestions on how to rewrite or verbally paraphrase content, so it is easier to understand. On December 3, 2020 when we performed a Google search using the keywords “Yasashii Nihongo + iikae (verbal paraphrasing)”, we received around 257,000 hits, and about 182,000 results for “Yasashii Nihongo + kakikae (rewriting)”.\n\nFrom these numbers alone we can conclude that at least prima facie there seems to be a considerable amount of information dealing with those topics. In 2018, a study on Yasashii Nihongo which was carried out in K City (Ibaraki Prefecture) by one of the authors, 180 Japanese residents aged 10-80 years were asked if they knew what Yasashii Nihongo was. Only 5 respondents (2.8%) said “I know what it is and I am using it consciously”, while 9 respondents (5%) replied with “I know what it is, but I am not using it.” A total of 31 persons (17.2%) answered “I have heard about it, but I couldn’t' tell what it is.”, while 135 respondents (75%) replied with “I don’t know”. This suggests that more than 90% of the residents who took part in the survey did not know much about Yasashii Nihongo at all. Of course, the scope of the aforementioned study is rather limited so we do not attempt to make any comprehensive statements. However, first, we do wonder why Yasashii Nihongo isn’t very well known and second, why it isn’t being applied more frequently, even though there seems to be so much information available on this topic. While there may be a variety of reasons as to why Yasashii Nihongo isn’t more widespread, the authors believe that with future endeavors awareness regarding Yasashii Nihongo can be fostered further by various measures which would, however, go beyond the scope of this paper. What we would like to focus on here is why there seem to be persons who aren’t using it, even though they may be well aware of its existence.\n\nLet us therefore first briefly become acquainted with the rules of Yasashii Nihongo as laid out by the Sociolinguistics Department of Hirosaki University. On their official homepage, Yasashii Nihongo is subdivided into two distinct categories. Category I aims to support communicative contexts that occur within 72 hours after an outbreak of a natural calamity while Yasashii Nihongo under category II is to aid all types of communication processes situated in daily life even after 72 hours have passed. Below are presented the rules conceived by the Sociolinguistics Department of Hirosaki University for category I and category II Yasashii Nihongo respectively.\n\n\n\n(1) Avoid difficult words, use easy-to-understand language instead.\n\n(2) Use short sentences; keep to simple sentence structures. Use spacing between words and organize linguistic elements in such a way that the reader can recognize them easily.\n\n(3) Words that are frequently used in disaster outbreak situations as well as words that can be considered common knowledge in such contexts are not to be changed in any way; they are to be used as is.\n\n(4) If possible, do not use Katakana words and/or loan words.\n\n(5) Do not use Romanized Japanese script.\n\n(6) Do not use onomatopoeia or phonomimes.\n\n(7) Choose the Kanji you want to use carefully. Also, make sure not to overuse Kanji and to always add Furigana to the ones you do use.\n\n(8) Use a date and time notation that can be easily understood by foreigners.\n\n(9) Nominalized verbs may be difficult to understand. Please use verb structures instead.\n\n(10) Avoid using ambiguous expressions.\n\n(11) Do not use double negatives.\n\n(12) Try to keep sentence-final structures uniform and consistent.\n\n\n\n(1) Avoid difficult words, use easy-to-understand language instead.\n\n(2) Make sure to use spacing between words.\n\n(3) Use short sentences and keep sentence structures simple.\n\n(4) Use traditional commas and dots for punctuation: ‘、’ and ‘°’ respectively.\n\n(5) Be careful not to overuse Kanji and always add Furigana.\n\n(6) Use loan words cautiously.\n\n(7) Avoid using onomatopoeia, as it may be unfamiliar to foreigners and they may potentially have a hard time understanding what is meant.\n\n(8) Nominalized verbs are difficult to understand. Whenever possible, use verb structures instead.\n\n(9) Avoid double negatives.\n\n(10) Try not to use ambiguous expressions.\n\n(11) Do not use Romanized Japanese script.\n\n(12) Use crucial expressions and words as is, and paraphrase them in brackets (…).\n\n(13) Try to keep sentence-final structures uniform and consistent.\n\n(14) Use a date and time notation that is easily understood.\n\nOf course, those rules are by no means set in stone2, but even if they are to be understood as rough guidelines for use, just how many people would actually be able to fully implement these after skimming through the rulebook and how many would be willing to start using this kind of Yasashii Nihongo? We believe that this high level of formalization may potentially contribute to the “creation of a very different type of Japanese”, as Tanaka (1989) puts it. As has been stated in chapter 1, the 72 hours between the occurrence of a natural disaster and satisfactory access to information and aid can potentially determine the difference between life and death. When considering communication that occurs in disaster situations in a multicultural society, it is crucial to establish the extent to which information can be provided and shared quickly and efficiently between language users with only limited language competence. Yet, with regard to Yasashii Nihongo classified under category II, one might entertain the suspicion that at least for some persons speaking Japanese as their first language, a major motivation behind the use of (category II) simplified Japanese might just be the simple fact that this way they could avoid having to speak in a foreign language altogether and would be resorting to Yasashii Nihongo solely to lessen their psychological burden of having to communicate in a language other than Japanese. Also, persons speaking Japanese as their first language who may not have been proactively seeking to interact with persons using Japanese as a second language before, might not feel the need to make use of Yasashii Nihongo to begin with.\n\nEven when the use Yasashii Nihongo is embraced, there exists a certain potential of it coming off as patronizing, instilling an awareness that the language used on a daily basis should be changed, but also that this language should still be Japanese. This would only translate to another tacit constraint to rely on “Japanese only”, also for people using Japanese as a second language. Even in situations when it remains unclear if this would constitute an appropriate approach, Sato & Murata (2018) point out that by repeatedly presenting this as a de facto standard, the same \"standard\" runs risk of getting reproduced over and over again until it becomes fossilized as the only option of communication leading to a potential risk of language becoming an ideology.\n\nBesides overly complex rules of simplification, the reason for the limited use of Yasashii Nihongo may also lie in a mode of communication that excessively focuses on the question of “how Japanese should be simplified”.3 Such behavior runs risk of ultimately creating a language ideology completely negating the possibility of diverse communication. In that case, we believe one should strive to look for a more diverse way of communicating in society that would certainly include, but not be limited to simplified Japanese. Even when simplified Japanese is used, but someone speaking Japanese as a second language still has difficulties understanding something, the linguistic burden would remain the same. Psychologically, it might even be greater, as the recipient of Yasashii Nihongo might feel like they failed to comprehend something they should have, as it had been specifically simplified on their behalf. Wouldn’t it be a more equal approach if both parties shared the psychological burden of communicating in a foreign language amongst themselves equally by respecting each other’s linguistic identities and trying to communicate in a more flexible manner by using a greater number of potential languages instead of limiting themselves to just one?\n\nThis is of course by no means an appeal to boycott the use of Yasashii Nihongo for non-disaster situations and shouldn’t be interpreted as such. However, it also shouldn’t be about language form in and of itself. It’s rather about a subtle difference in the degree of consideration given: if you pay attention to the person you’re talking to and you get the impression that they’re having trouble following you for some reason, you might want to try and paraphrase what you’ve just said in a different manner. Or present the same language elements written in Kanji if you think this might facilitate the understanding process. Or you might want to try and say something in the other person’s language, even if you’re not completely sure that what you’re saying is correct. Ultimately, it all comes down to fostering an environment where people can freely utilize different communication strategies in a flexible manner. There should, however, be no need to make the matter so formalistic or create an entirely novel language category for that.\n\nAfter all, shouldn’t the angle behind the ambitions of Yasashii Nihongo be about embracing a multicultural society? If so, there should be no need to cling to the notion of having to communicate in Japanese only as appears to have been stubbornly the case up to now. Resorting to Yasashii Nihongo simply to ease one’s psychological burden of having to speak in a language other than Japanese may just have been the reason why it hasn’t been able to find more exposure. The focus should rather be on the question of what steps can be taken to facilitate efficient communication in a multilingual and multicultural society.\n\n\nReasonable accommodation\n\nUnder the Convention on the Rights of Persons with Disabilities (CRPD) ratified by Japan in 2007, “persons with disabilities include those who have long-term physical, mental, intellectual or sensory impairments which in interaction with various barriers may hinder their full and effective participation in society on an equal basis with others” (MOFA 2014: 6). Of course, it goes without saying that according to this definition it would be quite inappropriate to subsume foreign residents living in Japan under the same category as outlined in the CRPD. However, if we were to understand the latter part (“…interaction with various barriers may hinder their full and effective participation in society on an equal basis with others”) in a broader context and consult Ishikawa’s social model of disability4 (Ishizkawa 2008: 93) according to which “a disability constitutes a barrier that occurs in relation to society” and “a person with a disability is defined as someone who encounters such a disability”, at least partially certain similarities with regard to the various barriers foreign residents may be encountering in society come to mind - as seldom they may also be a linguistic and/or cultural minority. Seen from this perspective, the authors believe that referencing the provision of the CRPD in the context of this paper may merit a second thought. Its article introduces the notion of “reasonable accommodation” which refers to “necessary and appropriate modification and adjustments not imposing a disproportionate or undue burden, where needed in a particular case, to ensure to persons with disabilities the enjoyment or exercise on an equal basis with others of all human rights and fundamental freedoms.” (MOFA 2014: 7).\n\nPersons with a visual disability and persons with a hearing disability would probably have a very different reaction when handed a piece of information written on paper and asked to read it. It is clear that a one-size-fits-all approach to providing accommodation does not work, and yet due to attempts to cut down costs, accommodation that would be specifically tailored to the needs of each and every individual with a disability is oftentimes denied. Even if an “accommodating” gesture is provided along the way, such accommodation cannot be regarded as reasonable. Denying reasonable accommodation to persons with a disability considerably limits their ability to participate in society and according to the CRPD constitutes a form of discrimination on the grounds of disability5. Ishikawa (2008) elaborates on how oftentimes the notion of reasonable accommodation is misunderstood and provides the following example: if it’s hard to hear a public speaker talk clearly, because the microphone isn’t working properly or hasn’t been set up correctly, it would be obvious to assume that this would be either the fault of the lecturer or the organizer. Obviously, this issue has to be fixed and naturally, no one would even think to call this corrective measure as being considerate or especially accommodating as it is self-evident that the attendees have the right to be given access to information. For some reason the same situation is treated very differently, for example when a sign language interpretation is provided for people with a hearing disability. We are all context- and environment-dependent beings to varying degrees, but strangely the simple fact of respecting the needs of a minority is suddenly viewed as being particularly considerate or especially accommodating. It is not, and to rectify this kind of inequality in how accommodation is perceived and offered is what reasonable accommodation is all about (Ishikawa 2008: 94).\n\nWhile the Kyushu Federation of Bar Associations and Oita Prefectural Bar Association (2017) recognize that there are differences between the Japanese language skills of foreign workers and what constitutes a disability, they believe that given past violations against obligations to consider safety, it is meaningful for a similar concept of reasonable accommodation to be developed and introduced for foreign workers. Mashiko (2018) states that while approaches in special needs education do represent a different domain, structurally there may exist certain similarities to the situation of Japanese language learners in language education: every endeavor undertaken is ultimately about finding specific ways of meeting the diverse and multifaceted needs of each individual.\n\nThe important thing when discussing disability is above all to focus on the people with a disability: to listen to what they have to say about it themselves, find out what their desires are, what they need, how they perceive it, what they think about it, what their personal experience has been. Only then can support be efficiently provided and reasonable accommodation realized. Foreign residents living in Japan may also encounter various barriers in their everyday lives. And while their struggles and concerns are very much multifaceted and should hardly be generalized, the type of support and accommodation they have been provided up to now is marked by a high level of formalization and regulation. By implicitly making it mandatory for residents whose first language is other than Japanese to use Japanese in Japan, the majority also denies the minority support and aid in their respective first language(s), thus de facto dictating what kind of accommodation the minority is entitled to receive. This may just be actively creating a further barrier hindering the participation in society of a minority. Beacco (2018) notes that “often immigrants are obliged to learn the language of the host society, that is the national and/or official language of a language majority, not always solely based on practical motives, but also oftentimes out of purely ideological reasons”. Beacco (2018: 326) states that such a standing of inequality only establishes further disadvantages to do with whether you are able to speak the language of the host society or not and should be revised from the standpoint of reasonable accommodation. To an extent, it may be possible to “overcome” a disability and indeed taking measures to compensate for a disability in order to alleviate the degree of inconvenience (Ishikawa 2000) may appear to be a sensible approach. However, it can also be unhealthy if one is persistently reminded by one’s environment to demonstrate that they have been indeed actively trying hard to overcome their disability or having to repeatedly present such “proof of ability”, oftentimes at the cost of their own emotional well-being. Making a conscious effort to overcome one’s disability, at least in theory, should at some point contribute to the mitigation of inconvenience, but if that action has no intrinsic value, is potentially forced and serves solely to prove to a third party that one belongs indeed to the category “overcomer”, the efforts invested in such endeavors may appear out of proportion.\n\nIshikawa (2000: 598) points out that such a status quo would de facto translate to the notion of a sort of “moral obligation or moral responsibility that people with a disability must assume” regardless of whether they want to or not. Communication that is carried out under the postulated framework of “Japanese-only” likewise results in a tacit claim for “proof of ability” and is liable to produce a number of disheartened individuals who might potentially start blaming themselves for their perceived linguistic shortcomings. It is easy for a society geared towards a majority to demand convenience and comfortable solutions. However, the moment the minority group concedes to the demands of the majority and adapts in a way that the majority wants it to, the moment it has to adjust to a greater extent than it feels it actually needs to - the minority group becomes subjugated to the prescriptivism of the majority. Beacco (2018: 236) states that such linguistic behaviors run risk of becoming the de facto norm, when the host society interprets such concessions as a token of gratitude and acceptance, thus reminding the minority once more and reinforcing the notion that this is exactly the way they expect them to act. According to Nagase (2002), one historically prominent fallacy in disability studies has been the rejection of a multifaceted self; rather assumptions about the individual were based on the grounds of a narrowly defined notion of normalism, oftentimes possibly invoking a feeling of shame or guilt in persons with a disability as a consequence and potentially making them believe that they might be somehow at fault for their perceived shortcomings, and that they might be a burden to society (Nagase 2002: 144). However, it can be observed that recently there has been a paradigm shift. “I'm not inherently wrong for being someone with a disability, society is wrong for not accepting me the way I am.” (Nagase 2002: 145) is a way of reframing the situation. Abe (2010: 293f.) comments on this idea and states that of course, while there may be some things people with a disability might have trouble doing and while some might argue it were the society oftentimes “disabling” these individuals, he postulates that in certain contexts everyone would be affected similarly. Abe provides the following example to illustrate his point: “When you fail to comprehend the modus operandi of a particular discourse, when you are unable to understand certain words used therein, just about everyone would have a hard time following, and participating in a communication process like that.” Facing such adversity, it might be quite difficult to get a sense of belonging or to experience oneself as being a respected member of a group. The use of expressions that are easily understood is a key element in creating a communication culture that is characterized by equality and mutual respect (Abe 2010: 303). This does not only hold true for the Japanese language community, but for other sociolinguistic contexts as well. Once Japan has transformed into a genuinely multilingual society, it will become a commonplace occurrence in daily life to encounter a variety of expressions being used in languages other than Japanese. And when that happens all people who are predominantly only able to communicate in Japanese exclusively, might in turn face similar communication issues: they would then experience first-hand what it feels like to be facing a communication barrier simply because they don’t understand a certain language or languages. It goes without saying that such communication mishaps run risk of creating a major rift between all parties involved. To prevent the formation of this communication gap it is vital to create more opportunities within society for its participants to be able to learn other languages. This way, native speakers situated in the host society, as well as speakers speaking Japanese as a second language could become avid learners of foreign languages, communicating using a variety of languages, oftentimes intermingling two or more languages at a time.\n\n\nPlurilingualism-based education approaches\n\nAs has been stated in the previous sections regarding the use of Yasashii Nihongo for non-disaster situations in a multicultural and multilingual society, the authors believe the currently predominating focus on the philosophy of “Japanese-only” to be a rather suboptimal solution, especially if seen from the standpoint of reasonable accommodation. It would be more desirable to try and realize a more equal model of communication where several languages could be used in combination with each other and elements from a variety of languages be intermingled. The question remains: what measures could be taken to make this a reality? How could this concept be practically implemented? The authors believe the key may lie in the idea of plurilingualism as proposed in the Common European Framework of Reference (CEFR) published by the Council of Europe in 2001. Kawakami & Ozeki (2010:80) offer a definition of plurilingualism in relation to its entities: individuals are plurilingual if they possess knowledge and experience of a variety of languages (=and cultures) and are able to combine and utilize both in a flexible manner depending on the communicative context (e.g. the persons they are communicating with). Accordingly, language education that is based on such a notion of plurilingualism does not aim to foster native-like language proficiency; in fact it does not even need to operate using the terms native language or mother tongue and 'native culture' or 'mother culture', respectively. Rather, it welcomes learners with different experiences from different backgrounds, and it is about cultivating learners with the competence and ability to respond to various situations at different times by engaging in communicative acts, which is to mean the ability to take action by means of communication. According to Okumra et al. (2016:21), the more languages you know, the easier it becomes to be in control of the communication even when confronted with an unfamiliar situation.\n\nPlurilingualism is empowering. It enables people to live and take action independently as diverse members of society. This is why in Japan, too, it might be a good idea to avoid making it a tacit prerequisite to communicate in Japanese only or limit communication to one particular language in general. Instead, adopting an attitude of openness towards the language(s) of the interlocutor may have a much more desirable effect. For instance, when exchanging greetings and pleasantries, but also in other daily life contexts, one could try and mix Japanese with the first language of the person or persons one is talking to, so as all parties involved would share the weight of the linguistic and psychological burden of communicating in a foreign language equally, thus fully mobilizing each other’s individual language potentials. Ultimately, such a more diverse type of communication would be an essential contribution to making the sociolinguistic reality richer. For this, we believe it is necessary to establish a new culture of language education based on the notion of plurilingualism. Candelier (2018) proposes the notion of “pluralistic approaches” (approches plurielles), describing a didactic system based on a multilingual framework of education. He identifies four distinct pluralistic approaches and subdivides these into the following groups (ibid). One includes the so-called ‘awakening to languages’ method, when at least a number of the interventions undertaken in educational settings touch upon languages that are not necessarily formally taught at school. Within the model of ‘inter-comprehension between related languages’, several languages of the same linguistic family are studied in parallel. The ‘integrated teaching’ approach is geared towards empowering learners to develop links between elements from a certain number of languages that they had acquired previously in formal educational settings. Last but not least, ‘intercultural education’, possibly one of the better known models of the four, strives to offer insights from the perspective of multiple cultures simultaneously. In this paper, the authors propose the promotion of an ‘awakening to languages’, a pluralistic education approach that they believe is much needed within contemporary Japanese society to foster a new culture of communication based on the possibility of utilizing several languages simultaneously. Oyama (2016) states that even if the working language were Japanese, within a framework of ‘awakening to languages’ at school, educational activities aimed at discovering similarities between multiple languages would benefit all parties involved, as long as they are carried out free of any position of perceived sociolinguistic superiority. If languages that are usually not taught in primary or secondary education, such as Tagalog or Portuguese, were given a chance to be explored and experienced by carrying out activities at school based on the ‘awakening to languages’ approach, not only would the children from language minorities discover new confidence in their own respective languages and cultures, but this would also lead to a culture of more openness and acceptance towards diversity in general, helping to deconstruct possible preconceived notions on the part of the language majority (Oyama 2016: 18). While activities based on the ‘awakening to languages’ approach are rarely implemented in Japan, Oyama (2016) portrays its potential use cases and implications in his work in more detail. Traditionally, Japanese and English have been the two languages given utmost priority within contemporary Japanese society. In this paper, the authors argue that in the near future young professionals who will have had the chance to experience, learn about, but also learn from cultural and linguistic minorities will become an indispensable source of human potential, regardless of their nationality or the fact that Japanese has been their first language or not. To foster such positive awareness of diversity, the authors believe it is essential to take full advantage of the learning opportunities found within formal school education and community-based regional Japanese language classes and thus create further opportunities for young people to become more engaged in learning about and getting acquainted with various foreign languages. As has been stated previously, language education based on the notion of plurilingualism doesn’t necessarily aim to produce language speakers who - from a linguistic point of view- would be capable of dealing just with any situation imaginable as proficiently as a native speaker might. Rather, it is a form of language education that encourages openness and diversity and teaches to adapt to an increasingly diverse society in order to live together with people from various personal backgrounds in a more harmonious way. By participating in everyday activities drawing from a diverse landscape of various languages while not being limited exclusively to Japanese or another single language, a new mode of learning can emerge that would have the potential to overcome discrimination and prejudice for all parties involved, regardless of one’s first language being Japanese or another language. Such a ‘community of practice’ would ultimately foster a Japanese society more open to linguistic and cultural diversity.\n\nSeen from the context of natural calamities, it might be necessary to establish functional methods of guaranteeing access to information as a means of disaster mitigation, and Yasashii Nihongo for disaster situations would be of course a fitting solution for that. At least with regard to contexts occurring in daily life, however, it would be crucial for the future Japanese society if communication were to be carried out in such a way that all parties involved would share the weight of the linguistic and psychological burden of communicating in a foreign language equally while respecting the diversity of each other’s languages and cultures by having the chance to learn and get acquainted with each other’s respective languages.\n\n\nConclusion\n\nThe number of foreign residents living and working in Japan has been growing steadily. In this paper, we have devoted our attention to the communication between the language majority (speakers using Japanese as their first language) and language minority (foreign residents with a first language other than Japanese) in contemporary Japanese society. We have drawn attention to the potential perils that go hand in hand with making “Japanese only” a tacit prerequisite for communication in a multicultural society, and we illustrated the necessity of rectifying the underlying inequality behind differing degrees of accommodating processes present in daily life, as both the language majority and language minority being equal constituent members of society should have the right to be building it on an equal footing side by side. As a promising approach to creating a truly multicultural and multilingual environment, we have proposed the introduction of a model of language education based on the notion of plurilingualism while putting a special emphasis on that a certain degree of flexibility is always desirable when engaging in such communicative contexts. With regard to current developments within the status quo of Japanese society, awareness must be raised to establish a more equal form of intercultural communication lest the majority takes on an overly dominating role. A first step to prevent such development may lie in the language majority becoming self-conscious about being the majority and making an active endeavor to act mindfully of the voices belonging to those in the language minority. The authors hope that this paper has been insightful in raising awareness to the positive potential of a new plurilingualism-based model of multicultural and multilingual communication in contemporary Japan.\n\n\nData availability\n\nNo data are associated with this work.", "appendix": "References\n\nAbe Y: Shikiji no yunibaasaru dezain. [Universal Design for Literacy]. In: Kadoya H, Abe Y, editors. Shikiji no shakaigengogaku [The sociolinguistics of Literacy] Tokyo: Seikatsushoin Co., Ltd; 2010.284–342.\n\nBeacco JC: Yooroppa ni okeru seijin imin no gengoteki tougou ni tsuite. Shitsuno takai imin kyouiku no tame. [Linguistic integration of adult immigrants in Europe: For a better migrant education]. In: Matsuoka Y, Adachi Y (eds.): Ajia-Oushuuno imin wo meguru gengo seisaku. Kotoba ga dekireba subete wa kaiketsu suru ka? [Language policy on immigration in Asia and Europe: everything is good as long as youspeak the language?]Coco Shuppan; 2018. pp. 232–253.\n\nCandelier M: Imin no gakushuusha no ukeire shien housaku to shite no, gengo to bunka no tagenteki apuroochi. [A pluralistic approach to language and culture as apolicy of supporting immigrant learners]. In: Matsuoka Y, Adachi Y (eds.): Ajiaoushuu no imin wo meguru gengo seisaku. Kotoba ga dekireba, subete wa kaiketsu suru ka? [Language policy on immigration in Asia and Europe: everything is good as long as you speak the language?] Coco Publishing; 2018. pp. 283–298.\n\nCouncil of EuropeCommon European Framework of Reference for Languages: Learning, teaching, assessment. Strasbourg: Council for Cultural Co-operation, Education Committee, Modern Languages Division; 2001.\n\nHirosaki daigaku shakai gengogaku kenkyuushitsu [Sociolinguistics Department of Hirosaki University]Gensai no tame no “Yasashii Nihongo”. [Yasashii Nihongo for disaster mitigation]. 1999 Reference Source accessed 29 March 2019.\n\nIori I: Chiiki nihongo kyouiku to nihongo kyouiku bunpou: Yasashii Nihongo to iu shiten kara. [From the viewpoint of “Easy Japanese”: What can the pedagogical grammar of Japanese say to “grassroots” Japanese-language classrooms?]. In: Jinbunshizen kenkyuu [Hitotsubashi review of arts and sciences] Hitotsubashi: Hitotsubashi Daigaku Daigaku Kyouiku Kenkyuu Kaihatsu Sentaa; 2009 Vol. 3. . pp. 126–141.\n\nIshikawa J: Disability no seijigaku shougaisha undou kara shougaigaku he. [The Politics of Disability: From the Disability Movement to Disability Studies]. In: Shakaigaku Hyouron [Japanese sociological review] Tokyo: Nihon Shakai Gakkai; 2000 Vol. 50(4).pp. 586–602.\n\nIshikawa J: Hon wo yomu kenri wa minna ni aru. [Reading books is everyone’s right]. In: Chizuko U, Ookuma Y, Oosawa M, et al. (eds.): Care to iu shisou [The notion of care]. Tokyo: Iwanami Shoten; 2008. pp. 96–106.\n\nKawakami I, Ozeki F: “Idou suru kodomo” to shite seichou shitadaigakusei no fukusuu gengo nouryoku ni kansuru katari. Mizukara no gengonouryoku wo dou ishiki shi, jiko keisei suru no ka? [A narrative on the multilinguallanguage proficiency of students growing up as “children crossing borders”: How is language competence internally formed and how is it perceived?] In: Hosokawa H, Nishiyama N (eds.): Fukugengo fukubunka shugi to wa nani ka? Yoroppa no rinenjoukyou kara nihon ni okeru juyou bunmyakuka he [What is multilingualism and multiculturalism? From the situation and thought models in Europe towards the reception and conceptualization in Japan]. Tokyo: Kuroshio Publishing; 2010. pp. 80–92.\n\nKyushu Bar Association, Oita Prefectural Bar AssociationGouriteki hairyo gimuno oudanteki kentou. Sabetsu, kakusa nado wo meguru saibanrei no kousatsu wo chuushin ni. [Cross-sectional investigation on the responsibility to reasonableaccommodation. Examining court cases of discrimination and disparity]. Gendai Jinbunsha; 2017.\n\nLave J, Wenger E: Situated Learning: Legitimate Peripheral Participation. Cambridge; 1991.\n\nMashiko H: Gengo kyouiku gakushuu no chishiki shakaigaku guroobaruka niokeru“baberu no tou”to nihon rettou wo oou gengo ideorogii. [Language education,learning & sociology of knowledge: The tower of Babel of globalization and the languageideologies of the Japanese Archipelago.]. In: Sato S, Murata A (ed.): Jinruigaku shakaigakuteki shiten kara mita kako, genzai, mirai no kotoba no kyouiku. Gengo to Gengo kyouiku ideorogii [Past, present and future of language education seen from the standpoint of anthropology & sociology. Language and language ideology.] Sangensha Publishers Inc; 2018. pp. 27–58.\n\nMinistry of Internal Affairs and CommunicationChiiki ni okeru tabunka kyousei suishin puran. [Plan for the promotion of multicultural coexistence in local communities]. 2006 Reference Source accessed 27 November 2020.\n\nMinistry of Foreign Affairs of JapanShougaisha no kenri ni kan suru jouyaku. [Convention on the Rights of Persons with Disabilities]. 2014 Reference Source accessed 27 November 2020.\n\nNagase O: Shougaigaku. [Disability Studies]. In: Ichinokawa Y (ed.): Seimei rinri to wa nani ka? [An introduction to bioethics] Tokyo: Heibonsha Limited Publishers; 2002. pp. 144–150.\n\nOkumura M, Sakurai N, Suzuki Y: Nihongo kyoshi no tame no CEFR. [CEFR for Japanese language teachers]. Tokyo: Kuroshio Publishing; 2016.\n\nOyama M: Gengo he no mezame katsudou. Fukugengo shugi ni motozuku jugyouhou. [Activities following the awakening to languages approach:multilingualism-based teaching methods]. Kuroshio Publishing; 2016.\n\nSato K: Gaikokujin hisaisha no futan wo herasu “Yasashii nihongo”: Zaijuu ichinen no gaikokujin ni mo wakaru hyougen de tsutaeru. [Yasashii Nihongo as a means for mitigating the situation for victims of a natural calamity speaking a first language other than Japanese: conveying information utilizing expressions that would be easily understood by foreigners living in Japan for a period of one year]. In: Kimura Y, Nomura M (eds.): Wakariyasui Nihongo [Easy-to-understand Japanese]. Tokyo: Kuroshio Publishing; 2016. pp. 245–275.\n\nSato S, Murata A: Gengo komyunikeeshon kyouiku ni okeru jinruigaku shakaigakuteki apuroochi no igi. [The significance of socio-anthropological approaches in language and communication education]. In: Sato S, Murata A (ed.): Jinruigaku shakaigakuteki shiten kara mita kako, genzai, mirai no kotoba no kyouiku. Gengo to Gengo kyouiku ideorogii [Past, present and future of language education seen from the standpoint of anthropology & sociology. Language and language ideology.]Sangensha Publishers Inc; 2018. pp. 3–24.\n\nTanaka H, Mino H, et al.: Yasashii nihongo nyuusu no koukai jikken.[Open experiment with Yasashii Nihongo News]. NHK Giken R&D; 2013139. :pp. 20–29.\n\nTanaka K: Kokkago wo koete kokusaika no naka no Nihongo. [Beyond national language: Japanese amidst internationalization]. Tokyo: Chikuma shobo Ltd; 1989.\n\nUchinami A: Chiteki shougai no aru hitotachi to “kotoba” “wakariyasusa” to jouhou hoshou, gouriteki hairyo. [On language and comprehensibility, guarantees to information, reasonable accommodation and persons with learning disabilities]. Seikatsushoin Co., Ltd; 2018.\n\nUsami Y: “Gaikokujin ni wakariyasui bunsho” wo kaku tame no hairyo - “Yasashii Nihongo” no sakusei ruuru no kouka to sono katsuyou. [Various considerations in writing easily-understood documents from non-natives: For more effective utilization of The Rules for Writing “Easy Japanese”]. In: CAJLE Annual Conference Proceedings. Toronto: Canadian Association for Japanese Language Education; 2014. pp. 174–183.\n\n\nFootnotes\n\n1 The term tabunka kyosei (literally, 'many cultures living together') became prominent in 2006, when the Ministry of Internal Affairs and Communication (MIC) issued a plan for the “promotion of multicultural coexistence” in local communities. Its aim is among other things to create an environment where “persons of various nationalities, races and ethnic identities live together as constituent members of local communities while recognizing each other’s cultural differences and attempting to establish equal relationships.” (MIC 2006: 1).\n\n2 According to Usami (2014), there exists a certain creative freedom as to following the proposed guidelines and the sequence that its individual entries are listed.\n\n3 The act of simplifying Japanese to express an idea in simpler terms is not at all problematic in and on itself. Tanaka et al. (2013) presents an interesting showcase of Yasashii Nihongo used within the domain of news reporting and writing while Uchinami (2018) addresses the positive potentials Yasashii Nihongo can unfold when applied to empower persons with a cognitive disability.\n\n4 The hitherto predominant ‘medical model’ does not relate to society as such. Instead it’s structured around the notion of whether a functional disability is present in an individual and to what extent it can be rectified using medical countermeasures.\n\n5 While individuals should be careful to avoid being discriminatory as well, the scope of its anti-discrimination statute is limited to governmental institutions, administrative bodies and public entities only." }	[ { "id": "78440", "date": "16 Feb 2021", "name": "Goro Christoph Kimura", "expertise": [ "Reviewer Expertise sociolinguistics", "interlingual communication" ], "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 contemporary Japan, where the number of foreign residents has been steadily growing, Simplified Japanese or Yasashii Nihongo has been steadily gaining attention. The authors argue that Yasashii Nihongo may be useful in immediate disaster situations, but creating a new linguistic category of Yasashii Nihongo to be used in every-day situations may not necessarily be the best option and might potentially even lead to discrimination against persons who are a language minority, forcing them to use Japanese only. They point to the possibility that Yasashii Nihongo can be used as a pretext by some members of the linguistic majority in Japan not to use any foreign language and discriminate those who cannot “even” understand simplified Japanese. Thus, the psychological burden of having to communicate not in the first language is laid on the side of the migrants. According to the authors, the notion of “reasonable accommodation” which calls for attention to individual needs of those suffering from disability caused by the society, can be utilized also to the communication between Japanese native speakers and foreign residents. Instead of trying to create an entirely novel category of Japanese with fixed rules of simplification, the authors propose a plurilingual approach that allows using various languages flexibly. They argue that adopting an attitude of openness towards the language(s) of the interlocutor may have a much more desirable effect than adhering to the use of one language. To enhance such a plurilingual attitude, the authors propose the promotion of an ‘awakening to languages’ that seeks to create positive awareness of linguistic diversity.\nResearch on Yasashii Nihongo is largely dominated by uncritical supporters of this approach. So this critical study is a welcome and indeed needed contribution to the field of research. The alternative proposed in this paper, plurilingual communication based on language awareness, shows a desirable way out of the monolingualism and normalism to which an emphasis on Yasashii Nihongo as a codified form of simplified Japanese could lead. The arguments comparing the situation of speakers of Japanese as a second language with persons with disabilities are convincing and can contribute to further elaborate the emergent discussion on “disability-oriented language rights” (Kimura, 20191).\nProviding such inspiring insights, it is regrettable that the authors seem not to have followed the latest developments in the study on Yasashii Nihongo. First, they heavily rely on early forms of Yasashii Nihongo for disaster situations, which was inclined to set fixed rules, to criticize Yasashii Nihongo in general. Recent developments, however, tend to suggest improvements in various situations, rather than proclaiming fixed rules (see for example Iori (ed.) 2020). Second, it is doubtful whether a philosophy of “Japanese-only” is so prevalent in the discourse of Yasashii Nihongo. Often, information in Yasashii Nihongo is given as one option among various languages, as prominently exemplified by https://covid19-tagengo.com/. Iori (ed)(2020) mentions Yasashii Nihongo as an interlanguage for translation (p.56). Yoshikai (2020) even more directly situates Yasashii Nihongo as a tool to promote multilingualism.\nSome assertions are stated without discussing the plausibility. For example, the following sentence lacks a source supporting this argument: “Resorting to Yasashii Nihongo simply to ease one’s psychological burden of having to speak in a language other than Japanese may just have been the reason why it hasn’t been able to find more exposure.” The question “why there seem to be persons who aren’t using it, even though they may be well aware of its existence” is not pursued in this article, so still waits for an answer. The authors also question if it is realistic that Yasashii Nihongo turns into an accepted register in the use of Japanese. But they seem to be too optimistic with regard to the acceptance of plurilingualism. Why should the majority reluctant even to accommodate their Japanese language to the need of a speaker of Japanese as second language show more willingness to flexibly interact in the sense of plurilingualism? If raising awareness about language diversity could change the attitude, it could also be expected that awareness raising on Yasashii Nihongo as a strategy of interlingual communication would lead to its general acceptance. To me, enhancing plurilingualism as a general attitude in Japanese society seems even less realistic than hoping that Yasashii Nihongo will be accepted as a variant on its own right. The authors do not mention English as a relevant factor, but the over-reliance to and over-estimation of English in the Japanese society seems to impede Yasashii Nihongo as well as plurilingualism.\nThe survey by the authors about the familiarity with Yasashii Nihongo could be complemented by a partly similar survey published by the Agency for Cultural Affairs, showing that about 30% of the respondents answered to know Yasashii Niihongo (https://www.bunka.go.jp/koho_hodo_oshirase/hodohappyo/pdf/92531901_01.pdf).\nI found only one typo: (Ishizkawa 2008: 93) -> (Ishikawa 2008: 93)\nReferences:\nIori, Isao (2020) Yasashii Nihongo Hyogen Jiten, Maruzen.\n\nKimura, Goro Christoph (2019) Language rights, in: Patrick Heinrich and Yumiko Ohara (eds.): Routledge Handbook of Japanese Sociolinguistics, London & New York: Routledge, 389-403.\n\nYoshikai Akira (2020) Nyumon Yasashii Nihongo, Ask Publishing.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\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": "6427", "date": "19 Mar 2021", "name": "Hideaki Ito", "role": "Author Response", "response": "Thank you very much for your very constructive and useful comments. 1) Regarding the reference to the latest research: Thank you very much for your valuable comments on the references to the latest research. In Section 2, we mentioned Iori et al. (2020) and Yoshikai (2020), and stated that there is no need to create a new language category based on the notions of \"mindset\" and \"improving the accuracy of machine translation\". 2) Regarding the leap in argument and Optimism about Plurilingualism Education: Thank you for your insightful comments. As you pointed out, there were a lot of definitive expressions, so we changed the overall description to point out the possibility. As for the point that it may be a practical hurdle to promote plurilingualism rather than to accept Yasashii Nihongo, we have added an argument for using language as “Yasashii language\" in Section 4, rather than having a language category called Yasashii Nihongo. Also, we recognize that the over-reliance on English is an important point. We have touched on this point in the text, citing Kimura (2016). 3) About data from the Agency for Cultural Affairs, Government of Japan (2020): Thank you very much for your valuable data. In addition to this data, we have cited data from Ito et al. (2020) as reinforcement. 4) Regarding typos: Thank you for pointing this out. We have corrected it." } ] }, { "id": "79158", "date": "19 Feb 2021", "name": "Anthony J. Liddicoat", "expertise": [ "Reviewer Expertise language policy", "language 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\nThis article presents an opinion about the role of Yasashii Nihongo in contemporary Japan and argues that the dissemination of Yasashii Nihongo is potentially problematic because it is more predicated on the needs of Japanese speakers than those of immigrants/foreigners. It then advocates for a plurilingual approach in Japan based on ideas drawn from the CEFR and éveil aux langues. It links such approaches to the notion of reasonable adjustment and launches an interesting idea about what such a concept might mean in the context of language.\nOverall, this paper makes some interesting points. I would have liked to see a more critical discussion of the Hirosaki University’s descriptions for category one, which are generic and very open to interpretation. And takes an interesting position in relation to what constitutes ‘easy’ in relation to Japanese. The paper also focuses on Yasashii Nihongo as if it were a unique phenomenon rather part of a larger language movement (Plain English, français pour tous, Vereinfachtes Deutsch, Español Simplificado Internacional, etc.), with similar motivation of simplifying communication while preserving monolingual practice.\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": "6428", "date": "19 Mar 2021", "name": "Hideaki Ito", "role": "Author Response", "response": "Thank you for your valuable comments. 1) Regarding the discussion in category 1 and language movements: Thank you for your comments from a broader perspective beyond the domestic movement in Japan. In Section 2, we have added a note on Category 1, referring to the global movement and citing Yoshikai (2020) and Usuyama & Okamoto (2020)." } ] } ]	1	https://f1000research.com/articles/10-52
https://f1000research.com/articles/10-224/v1	19 Mar 21	{ "type": "Research Article", "title": "Risk factors for mortality in hospitalized patients with COVID-19 from three hospitals in Peru: a retrospective cohort study", "authors": [ "Cristian Díaz-Vélez", "Diego Urrunaga-Pastor", "Anthony Romero-Cerdán", "Eric Ricardo Peña-Sánchez", "Jorge Luis Fernández Mogollon", "Julio Darwin Cossio Chafloque", "Gaston Cristobal Marreros Ascoy", "Vicente A. Benites-Zapata", "Cristian Díaz-Vélez", "Anthony Romero-Cerdán", "Eric Ricardo Peña-Sánchez", "Jorge Luis Fernández Mogollon", "Julio Darwin Cossio Chafloque", "Gaston Cristobal Marreros Ascoy", "Vicente A. Benites-Zapata" ], "abstract": "Background: Peru was one of the countries with the highest COVID-19 mortality worldwide during the first stage of the pandemic. It is then relevant to evaluate the risk factors for mortality in patients hospitalized for COVID-19 in three hospitals in Peru in 2020, from March to May, 2020. Methods: We carried out a retrospective cohort study. The population consisted of patients from three Peruvian hospitals hospitalized for a diagnosis of COVID-19 during the March-May 2020 period. Independent sociodemographic variables, medical history, symptoms, vital functions, laboratory parameters and medical treatment were evaluated. In-hospital mortality was assessed as the outcome. We performed Cox regression models (crude and adjusted) to evaluate risk factors for in-hospital mortality. Hazard ratios (HR) with their respective 95% confidence intervals (95% CI) were calculated. Results: We analyzed 493 hospitalized adults; 72.8% (n=359) were male and the mean age was 63.3 ± 14.4 years. COVID-19 symptoms appeared on average 7.9 ± 4.0 days before admission to the hospital, and the mean oxygen saturation on admission was 82.6 ± 13.8. While 67.6% (n=333) required intensive care unit admission, only 3.3% (n=16) were admitted to this unit, and 60.2% (n=297) of the sample died. In the adjusted regression analysis, it was found that being 60 years old or older (HR=1.57; 95% CI: 1.14-2.15), having two or more comorbidities (HR=1.53; 95% CI: 1.10-2.14), oxygen saturation between 85-80% (HR=2.52; 95% CI: 1.58-4.02), less than 80% (HR=4.59; 95% CI: 3.01-7.00), and being in the middle (HR=1.65; 95% CI: 1.15-2.39) and higher tertile (HR=2.18; 95% CI: 1.51-3.15) of the neutrophil-to-lymphocyte ratio, increased the risk of mortality. Conclusions: The risk factors found agree with what has been described in the literature and allow the identification of vulnerable groups in whom monitoring and early identification of symptoms should be prioritized in order to reduce mortality.", "keywords": [ "SARS-CoV-2", "COVID-19", "Mortality", "Adults", "Latin America." ], "content": "Introduction\n\nCOVID-19 is a disease characterized by severe pneumonia, and was first registered in December 2019 in Wuhan, China.1 This disease has generated great impact worldwide, especially in Latin America.2 According to the data reported by the World Health Organization (WHO), the number of COVID-19 cases at the end of December 2020 exceeded 80 million around the world, while fatal cases amount to more than 1.7 million.2 However, in regions with emerging economies or with limited access to health services such as Latin America, this disease has had great social impact.3\n\nIt is known that approximately 80% of COVID-19 cases present a mild to moderate course; however, the remaining 20% present a severe to critical course, requiring hospital care and leading to a high risk of death.4 Thus, factors affecting the prognosis of this disease have been related to the severity of the clinical presentation and analytical markers. The analytical factors include a high neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio and lymphocyte-to-monocyte ratio,5 leukopenia, elevated creatinine and lactate dehydrogenase (LDH) levels and prothrombin time.6 On the other hand, demographic characteristics and medical history have been described to increase the risk of mortality due to COVID-19 and include advanced age, male sex, the presence of comorbidities, such as chronic obstructive pulmonary disease, hypertension, type 2 diabetes mellitus or coronary heart disease.6,7\n\nAlthough risk factors for the disease have been described in different populations around the world, there is little scientific evidence in the Latin American population and especially in Peru, which is one of the countries with the highest mortality from COVID-19 worldwide.8,9 In addition, deficiencies in the Peruvian health system, including a scarcity of intensive care unit (ICU) beds and mechanical ventilators, as well as low compliance with government measures to combat the spread of the pandemic, could increase mortality by COVID-19.8,10 Likewise, the massive use of therapies without scientific evidence in the hospital setting, as well as the high frequency of self-medication in the Peruvian population can also aggravate the severity and mortality by COVID-19.11\n\nDespite the government having taken measures to improve access to health services in Peru, the implementation of these strategies was slow during the first months of the pandemic. The impact of the flaws of the health system could have caused a higher mortality in the population. Taking this into account, it is relevant to explore the sociodemographic and clinical characteristics of the population hospitalized for COVID-19 in Peru and the incidence of mortality at the first stage of the pandemic. Therefore, the objective of this study was to evaluate and describe the risk factors for mortality from COVID-19 in patients from three hospitals in Peru hospitalized during the period from March to May 2020.\n\n\nMethods\n\nWe carried out a retrospective cohort study. The study population consisted of adults hospitalized during the period from March 18 to May 13, 2020 for the diagnosis of COVID-19 at the Hospital Almanzor Aguinaga Asenjo and Luis Heysen Incháustegui Hospital, located in Chiclayo and the Hospital Clínica EsSalud Chepén, located in Chepén, two cities in Peru. We included patients older than 18 years of age who were hospitalized with a diagnosis confirmed by serological or molecular tests for COVID-19, as well as suspected by a compatible clinical or radiological pattern plus an epidemiological link, despite having a non-reactive serological test for COVID-19. The exclusion criteria were patients under 18 years of age and pregnant women.\n\nThe hospitals included correspond to the Lambayeque social security care network in Peru (EsSalud) and are the reference hospitals with the highest complexity for the management of patients with COVID-19. From the first confirmed case of COVID-19 in Lambayeque in March 2020 until November 30, 2020, there was a total of 18,570 confirmed cases of COVID-19, 5,654 hospitalized cases and 2,579 deaths.\n\nThe sampling was non-probabilistic and we included all participants hospitalized during the study period who met the inclusion criteria.\n\nThe information on the participants included in the study was collected by two researchers from the EsSalud virtual medical records registry. The collection of all records was carried out independently by each of the data entry operators, to ensure adequate data collection and reduce erroneous records. Sociodemographic variables, medical history and laboratory markers were collected from hospitalized patients during the period from March 18 to May 13, 2020. Laboratory markers were collected during the first 24 hours of hospitalization. Mortality follow-up was in-hospital and the date of hospital admission was considered as the start of follow-up. The information was tabulated in a Microsoft Excel 2016 document, and quality control of the data was carried out by a researcher of our team.\n\nThis study included participants insured by Peruvian social security, who have socioeconomic characteristics that could differ from the national population; however, this study is one of the first reports carried out in Peru,12,53 one of the countries with the highest mortality rates during the first wave of the pandemic. In addition, certain laboratory markers had a significant percentage of missing values, however, we included in the multivariate analysis the most relevant markers for the association of interest.\n\nOutcome variable: In-hospital mortality\n\nMortality in hospitalized patients with a confirmed or probable diagnosis of COVID-19 was evaluated as an outcome variable. This was collected according to outcome recorded in the clinical history at the end of the follow-up (June 2, 2020).\n\nExposure variables\n\nSociodemographic variables\n\nThe demographic characteristics recorded were: age (<50, 50-59, ≥60 years), sex (female, male), comorbidities (obesity, type 2 diabetes mellitus, hypertension, asthma, cancer, chronic kidney disease). Likewise, we generated a variable that grouped these comorbidities into different categories (0, 1, 2 or more).\n\nSymptoms and epidemiological link\n\nThe time of disease of the patients (in days), symptoms (respiratory distress, cough, fever, sore throat, diarrhea, headache, nasal congestion, anosmia, ageusia) were included. In addition, contact with a confirmed case of COVID-19 (yes, no) was considered.\n\nBaseline vital functions\n\nBaseline vital function values were collected at admission, including temperature, respiratory rate, heart rate, and oxygen saturation.\n\nBaseline auxiliary exams\n\nThe following laboratory values were considered: hemoglobin (g/dL), leukocytes (leukocytosis was defined as a value greater than or equal to 10,000 cells/mm3), neutrophils (cells/mm3), lymphocytes (lymphopenia was defined as a value less than 0.8 cells/mm3), the NLR (categorized into tertiles), platelets (thrombocytopenia was defined as a value less than 150,000 cells/mm3), creatinine (mg/dL), urea (mg/dL), aspartate transaminase (AST) (U/L), alanine aminotransferase (ALT) (U/L), and LDH (U/L).\n\nTreatment received\n\nThe treatment administered to hospitalized patients was included, considering antibiotic therapy (azithromycin, cephalosporins, carbapenems, among others), corticosteroid therapy (methylprednisolone, dexamethasone, hydrocortisone, prednisone), antiparasitic drugs (hydroxychloroquine, ivermectin), anticoagulants (enoxaparin) and antivirals (lopinavir)/ritonavir).\n\nThe descriptive results of the categorical variables were presented using absolute and relative frequencies, while quantitative variables are shown using central tendency and dispersion measures. The comparison of proportions between the categorical covariates and the outcome was performed using the Chi-square test, while the Student's t test or Mann Whitney U test was used to evaluate differences with numerical covariates.\n\nThe Kaplan-Meier method was used to describe the survival function, and the log-rank test was used for the crude comparison of survival functions. A Cox regression analysis (crude and adjusted) was performed to evaluate independent risk factors for mortality in the study sample. The adjusted model included variables the association of which has been described in the literature.6,12 Crude and adjusted hazard ratios (HR) were calculated with their respective 95% confidence intervals (95% CI). Compliance with the proportionality of hazards assumption of the Cox model was verified and collinearity relationships were evaluated in the adjusted model. All analyses were conducted with the statistical package STATA v14.0.\n\nThis study was carried out following the guidelines of the Declaration of Helsinki of 1964 and its subsequent amendments. The virtual medical records were reviewed without affecting the social, psychological and physical integrity of the study participants. We did not request the signing of an informed consent because the data was anonymized and we did not violate the integrity of the participant. In addition, this study was evaluated and approved by the Research Ethics Committee for COVID-19 of EsSalud, Peru (N°42-IETSI-ESSALUD-2020).\n\n\nResults\n\nA total of 493 hospitalized adults were analyzed, 72.8% (n=359) of whom were male with a mean age of 63.3 ± 14.4 years. Likewise, 62.5% (n=308) were 60 years of age or older, 25% (n=123) had hypertension, and 16.5% (n=81) were obese. The median length of hospital stay was five days (IQR: 3-9), and symptoms appeared on average 7.9 ± 4.0 days before admission to the hospital, the most common being respiratory distress, followed by cough and fever, while only 3.7% (n=18) reported having had contact with a confirmed case of COVID-19. Likewise, the mean oxygen saturation upon hospital admission was 82.6 ± 13.8; 83.8% (n=413) of the cases were confirmed, but only 3.4% (n=14) of the confirmed cases were diagnosed by a real-time polymerase chain reaction (RT-PCR) test. 61.1% (n=258) of the participants had leukocytosis, and 39.6% (n=167) had lymphopenia. While 67.6% (n=333) required ICU admission, only 3.3% (n=16) were actually admitted to this unit, and 60.2% (n=297) of the sample died. In addition, there were 1.99 deaths per 100 person-days at risk. Table 1 shows the bivariate analysis of the study variables and mortality.\n\nAntibiotic therapy was administered to 98.8% (n=487) of the participants, including a combination of azithromycin + cephalosporins in 78.2% (n=381). Likewise, 65.7% (n=324) of the participants received corticosteroid treatment, with methylprednisolone being the preferred treatment in 64.8% (n=210), followed by dexamethasone with 27.2% (n=88). Additionally, 76.7% (n=378) of the sample received hydroxychloroquine, and 26.0% (n=128) received ivermectin, while 79.3% (n=391) were prescribed enoxaparin. Likewise, in the bivariate analysis, statistically significant differences were found between the types of corticosteroids received, having received enoxaparin, and mortality (Table 2).\n\nA better survival curve was found in participants admitted to hospital with a higher oxygen saturation (≥92% vs. 91-86% vs. 85-80% vs. <80%), which was statistically significant (p<0.001) (Figure 1). Likewise, the survival curve was better in younger patients (<50 years vs. 50-59 vs. ≥60) and in those who received dexamethasone (dexamethasone vs. others) during hospitalization (Figures 2 and 3). These differences were statistically significant (p<0.001).\n\nIn the crude Cox regression analysis, it was found that age greater than or equal to 60 years (crude hazard ratio [cHR]=2.52; 95% CI: 1.93-3.28), having two or more comorbidities (cHR=1.59; 95% CI: 1.19-2.12), oxygen saturation between 85-80% (cHR=2.70; 95% CI: 1.81-4.04) or less than 80% (cHR=5.25; 95% CI: 3.69-7.46), as well as the intermediate (cHR=2.16; 95% CI: 1.52-3.07) and high NLR tertile (cHR=3.40; 95% CI: 2.42-4.78) were associated with a higher risk of mortality in hospitalized COVID-19 patients in Peru. In the adjusted regression analysis, age greater than or equal to 60 years (adjusted hazard ratio [aHR]=1.57; 95% CI: 1.14-2.15), having two or more comorbidities (aHR=1.53; 95% CI: 1.10-2.14), oxygen saturation between 85-80% (aHR=2.52; 95% CI: 1.58-4.02), less than 80% (aHR=4.59; 95% CI: 3.01-7.00), as well as being in the intermediate (aHR=1.65; 95% CI: 1.15-2.39) and higher tertile NLR (aHR=2.18; 95% CI: 1.51-3.15) remained associated with a higher risk of mortality (Table 3).\n\n\nDiscussion\n\nThis study included 493 patients hospitalized in three hospitals in Peru. We found that approximately six out of 10 patients died during follow-up, and while about seven out of 10 required admission to the ICU, only 3.3% were actually admitted to this unit. In addition, it was found that being an older adult, having an oxygen saturation level of 85% or less at admission, and having a high NLR value were associated with a higher risk of mortality. In addition, approximately eight out of 10 hospitalized patients received hydroxychloroquine and nine out of 10 were prescribed azithromycin.\n\nIt was found that about six in 10 hospitalized patients died, which is a high frequency compared to the 28.3%, 21.7% and 14.6% reported in patients hospitalized for COVID-19 from Wuhan,6 New York13 and Madrid,14 respectively. Likewise, the mortality found in this study was higher than the 39.6% and 38% described in studies carried out in Brazil15 and Mexico,16 respectively. This could be due to patients having received hospital care on average 7.9 days after the onset of symptoms, with a subsequently higher risk of severe disease and mortality. Likewise, it was of note that 73.8% of the patients arrived at the hospital with an oxygen saturation lower than 92%, hypoxia being a risk factor for mortality.17 On the other hand, despite 67.5% of the patients requiring admission to the ICU, only 3.3% were actually admitted, which could explain the high mortality and the shorter hospital stay in the group that died. In the present study, approximately 25% of the deaths occurred within the first 24 hours of hospitalization, suggesting that the population arrived late to medical care and adequate early monitoring of symptoms, which could avoid complications, was not carried out.18\n\nIn this study, older adults were found to have a higher risk of mortality. This situation is consistent with what has been described in previous studies,6,19 and could be explained by greater dysregulation of immune function and immunosenescence in older adults, as well as a higher prevalence of comorbidities.20 Certain comorbidities, such as hypertension, diabetes mellitus and chronic kidney disease, are treated with angiotensin converting enzyme inhibitors and angiotensin II receptor blockers, increasing the risk of severe disease and mortality.19 However, the role of the immune system in the pathophysiology of COVID-19 in this age group is still under study.\n\nFurthermore, elevated NLR values were found to be associated with an increased risk of mortality. Inflammation plays a relevant role in the pathophysiology of COVID-19 and allows establishing the prognosis of patients. Within the response of the innate immune system to a respiratory infection, there is a proliferation of neutrophils at the alveolar level, which could generate collateral damage and cytotoxicity. Furthermore, the release of anti-inflammatory cytokines could lead to lymphocyte apoptosis, producing lymphocytopenia.21,22 In this way, an elevated NLR has been described as an indicator of severe inflammation progression, which could lead to complications such as sepsis, multi-organ failure, and acute respiratory distress syndrome.23 In previous studies, it has been described as a prognostic marker for COVID-19, and its usefulness is highlighted due to its low cost, easy implementation and practicality.24\n\nHypoxemia was found to be a risk factor for mortality in the study sample, which is similar to results in previous studies.25,26 Approximately four out of 10 hospitalized patients arrived at the hospital with oxygen saturation lower than 86%, indicating that these patients arrived late at the hospital and correlates with the mean time of disease onset that exceeded seven days. Thus, the high proportion of patients with hypoxemia could be associated with the high mortality in the sample. Likewise, patients who arrive at the hospital with a higher degree of hypoxia require more intensive care, oxygen support, access to the ICU and mechanical ventilation,17 which in Peru is limited27 and could explain the high incidence of mortality. It should be noted that hypoxia has been associated with inflammation, which with the proliferation and elevation of cytokine levels, increases the already established lung damage and worsens the prognosis.28\n\nWe found an association between having two or more comorbidities and a higher risk of mortality. The main comorbidities in the population were hypertension, diabetes mellitus and obesity, which have been described as predictors of severity and worse prognosis in previous studies.29–31 The pro-inflammatory role of obesity has been mentioned, which induces diabetes mellitus and oxidative stress, affecting cardiovascular function.32 Likewise, a greater abdominal circumference increases breathing difficulty, which can restrict ventilation by decreasing the excursion of the diaphragm.33 It should be noted that diabetes mellitus and obesity alter immune response to viral infections.33\n\nNearly all of the participants received antibiotic therapy, the main drug being azithromycin. However, less than 7% of patients hospitalized for COVID-19 were reported as having bacterial coinfection.34 Likewise, the use of azithromycin in conjunction with hydroxychloroquine gained relevance based on initial favorable reports in March 2020,35 both drugs being approved for use in Peru as of April 2020.36 However, later, the use of these drugs was rejected internationally, due to their null positive effect37,38 and the increased risk of mortality.39 On the other hand, another drug frequently used was ivermectin, which began to gain relevance within the scheme of the Ministry of Health of Peru (MINSA) due to an in vitro study published during the study period.40 However, this drug was consolidated in Peru over the following months after promoting its use through self-medication41 and even subdermal application.42 The use of these medical therapies based on studies with biases or design flaws35,43 could have caused people to have false security and to have not gone to the hospital on the appearance of symptoms, thereby leading to disease progression and an increased risk of death or the development of severe illness. On the other hand, corticosteroids were administered in two out of three people, with a predominance of methylprednisolone, while dexamethasone, which was reported to reduce mortality in hospitalized COVID-19 patients in June,44 was not the most widely used.\n\nThe health system in Peru is fragmented and is currently overwhelmed.45 Although progress has been made in universal insurance for the population,46 the designated health budget is 2.3% of the annual gross domestic product.47 Moreover, despite the low budget assigned, it is not fully executed annually,47 which could further explain the shortcomings of the health system. In Peru, there were approximately 0.2 ICU beds per 100,000 inhabitants before the pandemic,27 which, in addition to the deficit of oxygen and hospital beds, could explain the high mortality that led Peru to occupy the first place in mortality per 100,000 inhabitants for several months.48 Likewise, the infodemic and high prevalence of self-medication are two latent problems in the Peruvian population.11,49 Both of these behaviors could increase the mortality of COVID-19 by providing false security and by people not attending health services in a timely manner, leading to a worse prognosis. The same occurs with the use of corticosteroids in the early and mild stages of the disease.50 On the other hand, the high rate of job informality and poverty in Peru has also aggravated the situation and could explain the poor adherence to quarantine and the failure to implement community mitigation strategies.51 This was reflected in a high mortality rate, especially in vulnerable groups.52\n\nThis study has limitations: 1) The population included in the study were patients insured by social security, which is made up of salaried workers and their families, which may not be representative of the entire country due to its socioeconomic characteristics; 2) There was a high percentage of missing values in certain relevant laboratory markers, which limited their evaluation in the multivariate model. However, relevant and useful markers described in the literature were included; 3) There are laboratory markers of immune response such as cytokines that could not be measured in the present analysis. Despite these limitations, this study represents one of the first reports from Peru,12,53 a country that became the global epicenter of the pandemic and leaves many lessons to be dealt with in the future to improve the failures of the health system and management of evidence-based disease.\n\n\nConclusions\n\nThe risk factors found are consistent with what has been described in the literature and allow the identification of vulnerable groups in whom monitoring and early identification of symptoms should be prioritized. Likewise, the findings of our study describe what happened during the first stage of the pandemic in Peru, highlighting the late arrival to receiving medical attention, as well as the lack of ICU beds, leading to a high incidence of mortality. In addition, the number of ICU beds, hospital beds and access to oxygen in the population should be improved in order to reduce mortality. Finally, evidence-based treatment schemes must be implemented to combat the infodemic and self-medication in the population of Peru.", "appendix": "Acknowledgments\n\nWe would like to thank the staff of the Hospital Almanzor Aguinaga Asenjo, Luis Heysen Incháustegui and the EsSalud Chepén Hospital Clinic for the logistical support provided. In addition, we would like to thank the Universidad Científica del Sur, for the financial support in the payment of the article processing charge.\n\n\nData availability\n\nFigshare: Database including information of COVID-19 patients from Peru. DOI: https://doi.org/10.6084/m9.figshare.14170955.v1. 54\n\nThis project contains the following underlying data:\n\n- .xls file containing the information of COVID-19 patients from Peru.\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\nReferences\n\nLi X, Wang W, Zhao X, et al.: Transmission dynamics and evolutionary history of 2019-nCoV. J Med Virol. 2020; 92(5): 501–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrganization WH, organization Wh: Coronavirus disease (COVID-2019) situation reports.2020.\n\nMeneses-Navarro S, Freyermuth-Enciso MG, Pelcastre-Villafuerte BE, et al.: The challenges facing indigenous communities in Latin America as they confront the COVID-19 pandemic. Int J Equity Health. 2020; 19: 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGandhi RT, Lynch JB, del Rio C: Mild or moderate COVID-19. N Engl J Med. 2020. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang I, Lim MA, Pranata R: Diabetes mellitus is associated with increased mortality and severity of disease in COVID-19 pneumonia–a systematic review, meta-analysis, and meta-regression. Diabetes Metab Syndr. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollaborators GO: Health effects of overweight and obesity in 195 countries over 25 years. N Engl J Med. 2017; 377(1): 13–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKass DA, Duggal P, Cingolani O: Obesity could shift severe COVID-19 disease to younger ages. Lancet (London, England). 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLansbury L, Lim B, Baskaran V, et al.: Co-infections in people with COVID-19: a systematic review and meta-analysis. J Infect. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGautret P, Lagier J-C, Parola P, et al.: Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial. Int J Antimicrob Agents. 2020; 105949. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinisterio de Salud del Perú: N 193-2020-MINSA. Documento Técnico: Prevención, Diagnóstico y Tratamiento de personas afectadas por COVID-19 en el Perú.2020.\n\nConsortium WST: Repurposed antiviral drugs for COVID-19—interim WHO SOLIDARITY trial results. N Engl J Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernandez AV, Roman YM, Pasupuleti V, et al.: Hydroxychloroquine or chloroquine for treatment or prophylaxis of COVID-19: a living systematic review. Ann Intern Med. 2020. PubMed Abstract \| Publisher Full Text\n\nSoto-Becerra P, Culquichicón C, Hurtado-Roca Y, et al.: Real-world effectiveness of hydroxychloroquine, azithromycin, and ivermectin among hospitalized COVID-19 patients: results of a target trial emulation using observational data from a nationwide healthcare system in Peru. Azithromycin, and Ivermectin Among Hospitalized COVID-19 Patients: Results of a Target Trial Emulation Using Observational Data from a Nationwide Healthcare System in Peru.2020.\n\nCaly L, Druce JD, Catton MG, et al.: The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res. 2020; 104787. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZavala-Flores E, Salcedo-Matienzo J: Medicación prehospitalaria en pacientes hospitalizados por COVID-19 en un hospital público de Lima-Perú. Acta Médica Peruana. 2020; 37(3): 393–5. Publisher Full Text\n\nRamal-Asayag C, Espinoza-Venegas LA, Celis-Salinas JC, et al.: Úlcera dérmica por ivermectina subcutánea en el tratamiento de COVID-19. Rev Soc Peru Med Interna. 2020: 88-. Publisher Full Text\n\nPodder CS, Chowdhury N, Sina MI, et al.: Outcome of ivermectin treated mild to moderate COVID-19 cases: a single-centre, open-label, randomised controlled study. IMC J Med Sci. 2020; 14(002). Publisher Full Text\n\nGroup RC: Dexamethasone in hospitalized patients with Covid-19—preliminary report. N Engl J Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAtun R, De Andrade LOM, Almeida G, et al.: Health-system reform and universal health coverage in Latin America. Lancet. 2015; 385(9974): 1230–47. PubMed Abstract \| Publisher Full Text\n\nMezones-Holguín E, Amaya E, Bellido-Boza L, et al.: Cobertura de aseguramiento en salud: el caso peruano desde la Ley de Aseguramiento Universal. Rev Peru Med Exp Salud Publica. 2019; 36: 196–206. PubMed Abstract \| Publisher Full Text\n\nGarcía E: Comex: Perú gasta en salud por debajo del promedio en América Latina. Diario Gestión. 2019. Reference Source\n\nSolari LM: Peru–The Role of the National Government in Combatting the COVID-19 Pandemic. Good Public Governance in a Global Pandemic. 465.\n\nUrrunaga-Pastor D, Benites-Zapata VA, Mezones-Holguín E: Factors associated with self-medication in users of drugstores and pharmacies in Peru: an analysis of the National Survey on User Satisfaction of Health Services, ENSUSALUD 2015. F1000Res. 2019; 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasin L, Navalesi P, Zangrillo A, et al.: Corticosteroids for Patients With Coronavirus Disease 2019 (COVID-19) With Different Disease Severity: A Meta-Analysis of Randomized Clinical Trials. J Cardiothorac Vasc Anesth. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnalytica O: COVID-19 will have wide-ranging impacts in Peru. Emerald Expert Briefings. (oxan-db).\n\nAguirre-Amaya K, Palomares-Custodio M, Quispe-Vicuña C, et al.: COVID-19 Mortality in Peruvian Older Adults: A Chronicle of a Health Crisis Foretold? J Frailty Aging. 2020: 1–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenites-Goñi H, Vargas-Carrillo E, Peña-Monge E, et al.: Clinical characteristics, management and mortality of patients hospitalized with COVID-19 in a reference hospital in Lima, Peru. Clinical characteristics, management and mortality of patients hospitalized with COVID-19 in a reference hospital in Lima, Peru.2020.\n\nUrrunaga-Pastor D: Database_v1. figshare. Dataset. 2021. Publisher Full Text" }	[ { "id": "84549", "date": "25 May 2021", "name": "José Fernando Gómez Montes", "expertise": [ "Reviewer Expertise Geriatrics", "Falls", "Frailty Geriatric syndromes" ], "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 comment This is a retrospective cohort study about of the risk factors for mortality in patients hospitalized for COVID-19 in three hospitals in Peru in 2020. The topic of the manuscript is appropriate for the Journal. It is of high interest to investigators and clinicians. Their findings provide valuable information about risk factors associated with deaths in developing countries; these results can be considerably useful for epidemiological description of the disease in terms of person-level risk. Minor essential revisions are necessary.\nMinor essential revisions\nTittle:\n\nThe title is accurate and sufficiently descriptive of the content.\n\nThe title is consistent with the presented problem and reflects the main message of the study.\nAbstract:\nAbstract: Concise and specific.\n\nMain objective of the review is presented.\n\nAbstract highlights the contribution of this work.\nIntroduction:\nThe background of the study is clear and helpful to readers unfamiliar with the subject.\n\nThe purpose of the article is clearly presented.\n\nInformation is clearly provided.\nMethods:\nThe approach and study design are appropriateness.\n\nMore information about serological and molecular test for Covid-19 is desirable.\n\nWhat are the requirements to be hospitalized in ICU? Please provide what ICU admission criteria were used.\nDiscussion:\nThe discussion is relevant.\n\nConclusions are logically valid and justified by evidence about the main theme proposed.\nReferences:\nThere were 54 and all are appropriate and relevant. However check references carefully; for example references 16.27.36.47.48.50. 51.53.54 are no complete; please provide them according to journal requirements.\n\nTables and figures:\nThree tables and three figures are shown. All of them clear and well designed.\nThanks for letting me review this manuscript. This could be a nice paper. Level of interest: An article whose findings are important to those with closely related research interests. Quality of written English: Well. Statistical review: No. Declaration of competing interests: I declare that I have no competing interest.\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": "83302", "date": "15 Jun 2021", "name": "Miguel Oswaldo Cadena Sanabria", "expertise": [ "Reviewer Expertise Internal Medicine", "Geriatrics", "Medical Education" ], "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 present study includes clinical and sociodemographic data in a cohort of Peruvian adults with COVID-19 and describes the factors associated with mortality. The authors demonstrated the concordance of the variables and medical conditions associated with higher mortality. It is a population with high mortality, less than 80% of the patients received steroids according to the recovery study protocol; however, given that the population was evaluated between March to May 2020, there was still uncertainty in effective treatments. Remarkably, the male gender was not associated with a higher risk of dying from COVID-19. There is adequate methodology and coherence with the results and conclusions.\nIt should be noted that only 3.4% of the cases were confirmed with a positive RT-PCR test. Serological diagnoses (IgM/IgG) tend to be later and may be related to an increased risk of mortality. Today, antigenic tests are available, which also allow a faster diagnosis.\nThe Cox regression model is adequate for the purpose of the study. The authors adequately recognize biases and limitations, as well as the number of losses in paraclinical variables.\nThis study is based on a hospital population with a census sample (it included all the cases that met the inclusion criteria). This limits the external validity or generalizability of the results. Mortality is oversized since it does not represent the risk of lethality in the general population with COVID-19. Another aspect associated with higher mortality could have been the limitation in admission to the intensive care unit, only 3.3% of the patients were admitted. These key aspects are recognized by the researchers and are part 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? 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/10-224
https://f1000research.com/articles/10-88/v1	10 Feb 21	{ "type": "Research Article", "title": "Under-reported COVID-19 cases in South Asian countries", "authors": [ "Soban Qadir Khan", "Imran Alam Moheet", "Faraz Ahmed Farooqi", "Muhanad Alhareky", "Faisal Alonaizan", "Imran Alam Moheet", "Faraz Ahmed Farooqi", "Muhanad Alhareky", "Faisal Alonaizan" ], "abstract": "Background: The purpose of the study was to compare trends in the progression of COVID-19 among South Asian countries with more developed Western countries. Methods: COVID-19 data from South Asian countries were used for this observational study. Data were taken up to April 21, 2020 from the outbreak of the COVID-19. Four of the seven countries met the inclusion criteria and were included in the analysis. Results: An exponential increase in the average number of weekly cases was reported after the fifth week following the first case. The correlation between reported cases and tests was found to be strong and significant (r=0.90, p=0.037). However, on average, 315.25 tests per million population were performed, which was at least 12 times lower than the number of tests performed in countries with a large number of COVID-19 cases. Conclusions: At present, the number of confirmed cases from South Asia was found to be significantly lower than in Western countries. However, this could be due to the smaller number of tests performed. Hence, an increase in the strength of performing diagnostic tests is highly recommended. Strict measures are required to make the people of these countries follow the instructions of social distancing and comply with preventive measures.", "keywords": [ "COVID-19", "coronavirus disease", "SARS-CoV-2", "South Asia", "pandemic" ], "content": "Introduction\n\nAny outbreak of an infectious disease or a natural disaster on a large scale, which spreads over a large geographical area leading to morbidity and mortality, is known as a pandemic. Evidence suggests that the likelihood of pandemics has increased over the past century because of an increase in global travel, urbanization, and greater exploitation of the natural environment1. Consequences of pandemics are multidimensional, have an impact on global health, socioeconomic conditions, and political implications2. The recent outbreak of coronavirus disease (COVID-19) was reported in the Huanan Seafood Market in Wuhan, China. China is therefore considered as the epicenter of the disease. However, in the current scenario, some European countries and the United States of America (USA) have become new epicenters of the disease. Individually, these countries have more than twice the number of reported cases when compared to China. Meanwhile, their death toll is at least five times greater than in China3. During the second week of March, the COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO)4.\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, belongs to the same family of corona viruses that includes the Middle East respiratory syndrome virus (MERS-CoV)5. Signs and symptoms of COVID-19 include respiratory symptoms, fever, cough, and shortness of breath. These breathing difficulties can worsen over time, and the disease can lead to complications such as: pneumonia, severe acute respiratory syndrome, kidney failure, and even death. People with low immunity and underlying systemic diseases are more prone to SARS-CoV-2 infection6. This is why the highest death rates are reported in the elderly population among those who were infected by COVID-197.\n\nVarious precautionary methods have been adapted by countries to control and stop the spread of COVID-19. A few such measures include hygiene maintenance, public awareness, partial lockdown, complete lockdown, and even the imposition of curfews8. Currently, social distancing is the only recognized way to prevent the spread of the virus. Hence, countries are making decisions based on their circumstances and the experiences of other countries. Based on data extracted from the WHO database, the progression of the disease and deaths in various regions and countries differ9. Statistics show a high number of reported COVID-19 cases and deaths in some European countries and in the USA as well. The first confirmed case of COVID-19 in some South Asian countries like India and Sri Lanka was reported on 15th of February 2020 while in Pakistan and Bangladesh, the first confirmed case was reported on 26th February and 8th March respectively. The trend in average weekly increases in reported cases remained untested. Therefore, the objective of the current study was to find the trend in the progression of COVID-19 among South Asian countries compared to more developed Western countries.\n\n\nMethods\n\nThis observational study was conducted between 20th April and 22nd April 2020. The study included COVID-19 data available from worldometer®10. Data was extracted from the date of the first COVID-19 case reported up until 21st April 2020. Data was extracted for all South Asian countries that were affected by the current outbreak. Only countries with at least 100 reported cases by 21st April 2020 were included in the study. Hence, a total of four out of the seven countries were included in the study: India, Pakistan, Sri Lanka, and Bangladesh.\n\nThe variables taken from the data source were: (1) total reported cases, (2) total deaths, (3) total recovered, (4) number of cases with outcome, (5) number of serious/critical cases, (6) total cases per one million population, (7) total tests performed, and (8) total tests performed per one million population. Furthermore, a few more variables were calculated using the extracted data and variables. The total outcome was calculated by adding the total number of deaths and the total number of recoveries. Percentage of deaths was calculated by using the equation [total deaths / (total deaths reported + total recovered) x 100%], the percentage of recovered cases was calculated as [total recovered/ (total deaths reported + total recovered) x 100%], the percentage of critical cases as “number of critical cases/active cases × 100%”, and the ratio of the number of cases tested by dividing the total number of cases by the total number of tests performed. The weekly number of cases reported after the first case reported until 21st April 2020 was also extracted from the data source. For the descriptive comparison of the present study’s findings of countries with a high number of COVID-19 cases, some of the statistics are summarized in Table 110.\n\nThe statistical package for social sciences (SPSS v. 23) was used for the analysis. Descriptive statistics included the calculation of averages and standard deviations as well as line graphs to present the number of weekly reported cases in each country. In inferential statistics, a simple linear regression was used between total cases (dependent variable) and total tests performed (independent variable). The Wilcoxon signed-ranks test was used to analyze the weekly increase in COVID-19 cases.\n\n\nResults\n\nThe total number of reported COVID-19 cases in South Asian countries by 21st April 2020 were 31,565 of which 5,526 (17.5%) recovered. The number of reported deaths was 901. Among South Asian countries, India had the highest number of positive COVID-19 cases (18,658; 59.1%), followed by Pakistan (9,216; 29.2%), Bangladesh (3,382; 10.7%), and Sri Lanka (309; 1%) (Table 2). The percentage of reported deaths was highest in Bangladesh (55.84%), followed by India (15.32%), Sri Lanka (6.54%), and Pakistan (8.5%).\n\nFigure 1 shows the exponential growth in the number of reported cases among the South Asian countries after the fourth to fifth week since the start of the disease. A sharp increase was observed in reported cases in India from the sixth week onwards. In Pakistan, the number of reported cases was also found to increase at the start of the fourth week. However, the spread of the virus was not as rapid in Bangladesh and Sri Lanka, as was found in the Indian and Pakistani populations. A comparison of the average number of reported cases in each week with the previous week revealed that the average increase in cases was not statistically significant except between fifth and sixth week (z-value -2.02, p-value 0.043). Figure 2 presented descriptive comparison of the average number of confirmed cases between two consecutive weeks.\n\nOn average, 315.25 tests per million population were performed in the countries included in the current study. A correlation between the total reported cases and the total number of tests performed was found. The data revealed a very strong, direct, and statistically significant correlation (r=0.90, p=0.037). The Bangladeshi population had the highest percentage of tested positive cases in relation to the total number of tests performed (12.71%), followed by Pakistan (8.24%), Sri Lanka (4.78%), and India (4.65%).\n\nThe regression model between the number of tests performed (independent variable) and total number of cases reported (dependent variable) demonstrated a very strong R-square value 0.925 with significance of the model (p =0.025). The predicted values of constant and slope for the model were 1442.4 and 0.045, respectively. Hence, the regression equation can be written as\n\nNo. of cases reported = 1442.4 + 0.045 (No. of tests performed)\n\n\nDiscussion\n\nCurrent data suggest that recent pandemics’ origins are associated with a zoonotic mode of transmission from animals to humans11. Animal to human transmission was presumed to be the main route of transmission for SARS-CoV-2, since the first reported case of COVID-19 was linked with direct exposure to the Huanan Seafood Market in Wuhan, China. Nevertheless, the subsequent cases that were reported did not follow this mechanism12. Therefore, it was concluded that SARS-CoV-2 could also be transmitted through aerosol, human to human (symptomatic/asymptomatic), and surface to human contact13.\n\nAs of 21st April 2020, there had been over 2.5 million reported cases of COVID-19 in 210 countries across six continents. At the beginning of this pandemic, China (82,758) was affected the most by the disease; however, later the USA (792,938), Spain (204,178), Italy (181,228), France (155,383), Turkey (90,980), and Iran (83,505) had the most COVID-19 cases worldwide. The number of reported cases has since started to increase in South Asian countries (India, Pakistan, Sri Lanka, and Bangladesh). The initial cases reported in South Asian countries were thought to be caused by travelers returning from other COVID-19 affected countries. Although, by the date (21st April, 2020), the total number of reported cases in South Asian countries is not as high as it was in the USA, Italy, Spain, France, and Iran (Table 1). However, the weekly growth in the number of reported cases (up to the eighth week) in South Asian countries is quite similar to the increase in the number of reported cases in the USA, France, and the United Kingdom (UK).\n\nBased on the preparedness index formulated by Greenhill and Oppenheim, which defines the ability of a country to curtail any pandemic14, the spread risk of this pandemic is higher in South Asian developing countries than in developed countries15. A few of the factors that contribute to a higher spread risk of the pandemic include population density, susceptibility to infection, patterns of movement driven by travel, trade, and migration, the speed and effectiveness of public health surveillance and response measures, and the socioeconomic status of the country15. Three out of four countries included in the current study fall under the top ten most populated countries in the world16-18. Furthermore, the per km2 population in Bangladesh, India, and Pakistan is more than any country listed in Table 1. In addition, a large number of people in these countries live in slums16-18, which makes it difficult to maintain social distancing and to adopt preventive measures. Furthermore, poor education and extreme poverty are other factors that make it more difficult to follow social distancing instructions, or early disease identification of symptoms of COVID-19. A report from Pakistan showed an increasing number of cases where people died due to COVID-19 before reaching a hospital19.\n\nTo date, there has been a total of 2,505,858 reported cases of COVID-19 worldwide, with a lower number of reported cases (31,608) and reported deaths (901) in South Asian countries compared to other regions of the world. There have been many hypotheses related to this lower reported COVID-19 cases and deaths. Some of the theories included stronger immunity, warmer weather, childhood BCG vaccinations, and exposure to anti-malaria medications. From the data collected from the worldometer® website, the authors believe that the lower number of reported cases in the South Asian countries could be due to the lower number of diagnostic tests performed for COVID-19 virus compared to countries that have reported a higher number of COVID-19 cases. India (18,658) and Pakistan (9,216) have the most COVID-19 cases in South Asian countries, with 291 and 506 tests performed per million population, respectively. The number of tests (per million) performed in India and Pakistan is significantly lower than in the USA (12,167), Spain (19,896), Italy (23,122), France (7,103), and the UK (7,386). Even countries in Asia with the highest number of COVID-19 cases, Turkey (7,991) and Iran (4,203), have a higher number of tests performed per million population. In South Asia, on average, 315.25 tests per million population were performed; this is at least 12 times lower than the number of tests performed in the epicenters of COVID-19.\n\nAccountability for preparedness in these countries is diffuse, and many countries that are at the greatest risk have the most limited capacity to manage and mitigate pandemic risk. In addition, these countries need to perform the virus diagnostic tests in greater numbers to get an accurate picture of the pandemic. Based on the data, one could suggest that the low number of reported cases but with high percentage increases for South Asian countries could be a ticking time bomb waiting to explode, and this region could be the next highlighted region of this current pandemic.\n\n\nConclusions\n\nAlthough the current number of reported cases and reported deaths from South Asia suggested that the spread of COVID-19 is not as high as it was in many other countries. However, a comparison of statistics and population characteristics does not portray a good picture for the future. Therefore, the following conclusions can be drawn:\n\n1. One side of the picture is a lower number of reported cases and deaths, but the other side of the picture suggests a large number of cases that are prevailing in the society that are unidentified and undiagnosed. Hence, identifying the spread of the disease by increasing the number of diagnostic tests is highly recommended.\n\n2. Governments in these countries are required to take strict measures such as partial or complete lockdowns in order to maintain proper social distancing, since a high population density coupled with low education levels and low disease awareness could lead to a new disease epicenter.\n\n\nData availability\n\nHarvard Dataverse: Low Reported COVID-19 Cases in South Asian Countries: A Luck of Nature or A Ticking Time Bomb, https://doi.org/10.7910/DVN/QLTVRW20.\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 information\n\nImran Alam Moheet was affiliated with Azra Naheed Dental College, Superior University, Pakistan at the time this paper was written but is currently at Baqai Dental College, Baqai Medical University, Pakistan. Faraz Ahmed Farooqi was affiliated with Department of Academic Affairs, Imam Abdulrahman Bin Faisal University, Saudi Arabia at the time this paper was written but is currently affiliated with Department of Dental Education, Imam Abdulrahman Bin Faisal University, Saudi Arabia.", "appendix": "Acknowledgements\n\nA previous version of this article is available on Research Square: https://doi.org/10.21203/rs.3.rs-33960/v2\n\n\nReferences\n\nJones KE, Patel NG, Levy MA, et al.: Global trends in emerging infectious diseases. Nature 2008; 451(7181): 990–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadhav N, Oppenheim B, Gallivan M, et al.: Disease control priorities: improving health and reducing poverty. In: 3rd ed. Washington (DC): The International Bank for Reconstruction and Development/The World Bank; 2017 Nov 27. Chapter 17.\n\nWorld Health Organization: Coronavirus disease 2019 (COVID-19): situation report, 85.2020Reference Source\n\nWorld Health Organization: WHO director-general’s opening remarks at the media briefing on COVID-19.11 Mar 2020Reference Source\n\nCDC: Coronavirus Disease (COVID-19): Situation Summary.Reference SourceAccessed 14 Apr 2020.\n\nAbdulamir AS, Hafidh RR: The possible immunological pathways for the variable immunopathogenesis of COVID-19 infections among healthy adults, elderly and children. Electron J Gen Med 2020; 17(4): em202. Publisher Full Text\n\nDowd JB, Rotondi V, Adriano L, et al.: Demographic science aids in understanding the spread and fatality rates of COVID-19. medRxiv 2020 Jan 1. Publisher Full Text\n\nKaplan J, Frias L, Johnsen M: A third of the global population is on coronavirus lockdown—here's our constantly updated list of countries and restrictions. Business Insider News 14 Apr 2020Reference Source\n\nWorld Health Organization: Coronavirus (COVID-19).Reference Source Accessed 19 Apr 2020.\n\nWorldometer coronavirus cases.Reference Source Accessed 21 Apr 2020.\n\nWoolhouse ME, Gowtage-Sequeria S: Host range and emerging and re-emerging pathogens. Emerg Infect Dis 2005 Dec; 11(12): 1842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCascella M, Rajnik M, Cuomo A, et al.: Features, evaluation and treatment coronavirus (COVID-19). In StatPearls Treasure Island (FL):StatPearls Publishing; Accessed 8 Mar 2020.\n\nLi Q, Guan X, Wu P, et al.: Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med 2020 Jan 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenhill KM, Oppenheim B: Rumor has it: the adoption of unverified information in conflict zones. Int Stud Q. 2017 Sep 1; 61(3): 660–76. Publisher Full Text\n\nSands P, El Turabi A, Saynisch PA, et al.: Assessment of economic vulnerability to infectious disease crises. Lancet 2016 Nov 12; 388(10058): 2443–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarsh DR, Kadir MM, Husein K, et al.: Adult mortality in slums of Karachi, Pakistan. J Pak Med Assoc 2000 Sep; 50(9): 300–6. PubMed Abstract\n\nJohnson K: Census: 1 in 6 India city residents lives in slums.22 Mar 2013Reference Source Accessed 21 Apr 2020.\n\nAngeles G, Lance P, Barden-O’Fallon J, et al.: The 2005 census and mapping of slums in Bangladesh: design, select results and application. Int J Health Geogr 2009 Dec; 8(1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhatti MW: Why are Karachi’s hospitals getting more DOAs, near-death patients? The News (International) 15 Apr 2020Reference Source Accessed 22 Apr 2020.\n\nQadir Soban: Low Reported COVID-19 Cases in South Asian Countries: A Luck of Nature or A Ticking Time Bomb Harvard Dataverse, V1, UNF:6:evfkN8AHLCnyrXwm5EbQOg== [fileUNF] 2021. Publisher Full Text" }	[ { "id": "79264", "date": "15 Feb 2021", "name": "Khalid Aziz Ansari", "expertise": [ "Reviewer Expertise Acute and Chronic lung diseases", "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\n02/07/2021: Added detail of a competing interest which recently came to light about this co-authored article.\nThis is interesting study and following areas need a little more revision.\nIntroduction section: There are some missing references. (\"The recent outbreak...epicentres of the disease, Currently, social distancing is the only recognized way to prevent the spread of the virus. Hence, countries are making decisions based on their circumstances and the experiences of other countries...of other countries\").\nMethod: To add more clarity, it needs a little more justification on why countries with more than 100 cases were selected.\nDiscussion: This section need major revision in relation to organisation. It does not clearly reflects the results of the study and the impact of present study findings in the these countries. For example, which of your study findings suggest that this disease is zoonotic?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6380", "date": "17 Mar 2021", "name": "soban khan", "role": "Author Response", "response": "Dear Khalid Aziz, Thank you for your time you gave to review this article. I appreciate your comments which will help to improve presentation of our work. We have modified the manuscript to address your comments. Many Thanks." } ] }, { "id": "79263", "date": "24 Feb 2021", "name": "Sumanth Kumbargere Nagraj", "expertise": [ "Reviewer Expertise Evidence-based healthcare", "qualitative evidence synthesis", "Randomised controlled trials in Oral healthcare", "Research priority setting" ], "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 observational study based on the data taken up to 21st April 2020. During this period South Asian countries mentioned in this study were already under lockdown (Bangladesh: from 22 March to 30 May; India: 24 March to 31 May and Sri Lanka: 26 March to 11 May). I do not understand why the authors conclude by suggesting lockdowns as their only suggestion. In contrast to their suggestion, today, these are the countries which are doing better compared to the countries which have or are still under lockdowns.\n\nThe authors have not considered the issues related to the population of India or Pakistan, the handful testing centres these countries had in the month of April with limited number of test kits and the availability of PPE kits in these four countries. Comparing a population of 1.3 billion (India) or 216 million (Pakistan) to countries with fraction of its population and suggesting lockdown looks futile. The economic impact of lockdown is another aspect which needs to be weighed before opting for a lockdown in these countries.\n\nHowever, the article highlights the importance of social distancing and preventive measures that needs to be implemented in these countries which are the most economic preventive measures.\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?\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": "79261", "date": "24 Feb 2021", "name": "Junaid Ahmed", "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 the opportunity to review this article. This article is related to the most pressing health crises faced by mankind i.e COVID-19. The article is well written and adheres to the formatting of the journal.\nFew points need clarification,\nIn the methodology, the authors state that only data from South Asian countries have been included. However, the data from other developed countries have also been included for comparison.\n\nDiscussion:\na) Kindly avoid restating the results in the discussion section.\nb) The authors should discuss the variations in the COVID-19 testing policies that the countries adopted initially , as it is variable from country to country and can alter the number of tests. Also, in the discussion the authors should elaborate on the timings and implementation of the country wide lockdown in the countries, that could have affected the rate of spread of COVID-19.\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": [] } ]	1	https://f1000research.com/articles/10-88
https://f1000research.com/articles/9-915/v1	05 Aug 20	{ "type": "Research Article", "title": "Morphological and genetic evaluation of the thumbprint emperor, Lethrinus harak (Forsskål, 1775) in the Pacific and Indian Oceans", "authors": [ "Muhammad Afrisal", "Yukio Iwatsuki", "Andi Iqbal Burhanuddin", "Muhammad Afrisal", "Yukio Iwatsuki" ], "abstract": "Background: The Lethrinidae (emperors) include many important food fish species. Accurate determination of species and stocks is important for fisheries management. The taxonomy of the genus Lethrinus is problematic, for example with regards to the identification of the thumbprint emperor Lethrinus harak. Little research has been done on L. harak diversity in the Pacific and Indian Oceans. This study aimed to evaluate the morphometric and genetic characters of the thumbprint emperor, L. harak (Forsskål, 1775) in the Pacific and Indian Oceans. Methods: This research was conducted in the Marine Biology Laboratory, Faculty of Marine Science and Fisheries, Hasanuddin University, and Division of Fisheries Science, University of Miyazaki. Morphometric character measurements were based on holotype character data, while genetic analysis was performed on cytochrome oxidase subunit I (COI) sequence data. Morphometric data were analysed using principal component analysis (PCA) statistical tests in MINITAB, and genetic data were analysed in MEGA 6. Results: Statistical test results based on morphometric characters revealed groupings largely representative of the Indian and Pacific Oceans. The Seychelles was separated from other Indian Ocean sites and Australian populations were closer to the Pacific than the Indian Ocean group. The genetic distance between the groups was in the low category (0.000 - 0.042). The phylogenetic topology reconstruction accorded well with the morphometric character analysis, with two main L. harak clades representing Indian and Pacific Ocean, and Australia in the Pacific Ocean clade. Conclusions: These results indicate that geographical and environmental factors can affect the morphometric and genetic characteristics of L. harak.", "keywords": [ "Lethrinus harak", "morphometric characters", "principal component analysis", "genetic distance", "Cytochrome Oxidase Subunit I" ], "content": "Introduction\n\nThe Lethrinidae, and in particular the genus Lethrinus, comprise many fish species of importance in commercial and subsistence fisheries around the worldp1. One important fisheries target species, the thumbprint emperor, Lethrinus harak (Forsskål, 1775), is widely distributed throughout the Indo-West Pacific from the Red Sea and East Africa to Samoa, and from north Japan to northeastern Australia2. This fish tends to be found living solitary or in small schools over shallow sandy areas and coral rubble, among mangroves, in lagoons and channels, and in seagrass areas inshore and adjacent to coral reefs, at depths ranging from 1m to 20m3,4.\n\nSustainable fisheries management should be based on an understanding of fish biology and ecology, including the accurate determination of species and stocks5. Research on the life history, biology and genetics of L. harak has been conducted on populations in both the Indian and Pacific Oceans. Like the majority of Lethrinid species. L. harak is a relatively long-lived fish; however, although studies on several Pacific Ocean populations have provided information on the reproductive biology of L. harak in Japan, the Philippines and Indonesia6, many aspects of the reproductive biology of this species are still unknown7. Genetic studies on L. harak have reported the possible presence of cryptic species in the southwest Indian Ocean (SWIO)8. Genetic information indicates the possible presence of two clusters, in the Indian Ocean and the Pacific Ocean. However, to date there are no studies evaluating morphological and genetic characters of L. harak across the Indian and Pacific Oceans.\n\nCombined analyses of morphometric characters and genetic variation are increasingly common. Such methods aim to evaluate the differences and similarities in the grouping of individuals and populations using measures such as similarity indices, genetic distances and cluster analysis9. Species identification based on morphometric and meristic characters should be supported by molecular identification methods, as identification based solely on morphometric-meristic characters can often lead to misidentification, taxonomic ambiguity, and fluctuations in the number of recognised species. Such studies have found strong indications of separated populations with sufficient differences to warrant the designation of new species10 or, conversely, to indicate that currently recognised species are in fact synonyms11. These characters can be related to food habits and homoplasy (different species develop, rather than inherit, similar traits), as some traits can be lost or emerge depending on the environment. Such phenomena are enabled by phenotypic and genotypic plasticity and can lead to the presence of cryptic diversity) and hidden species, as well as considerable variations in colour patterns and other features between different life-stages of the same species12.\n\nInformation on the population structure of L. harak based on morphometric and genetic characters is needed to evaluate the stocks of this fish. The genetic structure of the population can be studied based on mitochondrial DNA (mtDNA). Although mitochondria undergo rapid evolution, the cytochrome oxidase subunit I (COI) is a segment of mtDNA with low evolution (conserved region) and has been chosen as a widely used genetic marker in so-called COI barcoding13. This study aimed to evaluate the relationship between L. harak in the Pacific Ocean and the Indian Ocean based on morphological characters and genetic diversity using a standard COI mtDNA genetic marker.\n\n\nMethods\n\nThis research was conducted for three months (November 25, 2019 - February 24, 2020) under the Enhancing International Publication Program conducted by the Directorate for Human Resource Qualifications Directorate General of Resources for Science, Technology & Higher Education Ministry of Research, Technology and Higher Education of the Republic of Indonesia in collaboration with the University of Miyazaki. This study complied with relevant ethical regulations in Japan and Indonesia. In Indonesia, the doctoral research proposal of the first author was assessed and accepted by an academic panel. This assessment included compliance with institutional ethical guidelines in vigour at Universitas Hasanuddin. A letter of acceptance was issued by the Head of the Laboratory of Fisheries Sciences, Faculty of Agriculture, Miyazaki University, permitting the research activities at the University of Miyazaki's Division of Fisheries Science Museum (MUFS). The issuance of this acceptance was based on the research proposal, including compliance with institutional ethical guidelines in vigour at Miyazaki University. The use of the samples in this study did not require specific ethical approval for the following reasons: all fish specimens used were already dead when they were acquired; there was no use of live animal specimens; specimens obtained from fish port auction houses had been captured by legal fisheries and were obtained following all applicable regulations; the IUCN Red List assessment lists Lethrinus harak in the Least Concern category (not considered at risk of extinction); this study was based on field research (surveys) with no experimental component.\n\nSpecimens of L. harak were selected as paratypes at the Marine Biology Laboratory, Faculty of Marine Science and Fisheries, Universitas Hasanuddin (MSFUH), and the Division of Fisheries Science, Miyazaki University. Specimens MSFUH000591, MSFUH0000592, MSFUH0000593, collected from the Makassar Strait (-4°58’N, 119°17’17’’E), were obtained from the Paotere fish auction site, Makassar City, South Sulawesi, Indonesia. The samples were placed in a coolbox with ice for transport to the Marine Biology Laboratory, Universitas Hasanuddin, Makassar. The samples were cleaned under running water to remove any dirt and organic matter attached the exterior of the specimens. Each sample was placed on its right-hand side and sterilised using alcohol. A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. Each sample was numbered and labelled according to the Marine Science and Fisheries Universitas Hasanuddin (MSFUH) catalogue number system, preserved in 10% formalin and stored in the laboratory collection.\n\nThe seven specimens measured in Japan formed part of the pre-existing fish collection of the University of Miyazaki Division of Fisheries Science Museum (MUFS). These specimens had been collected from Yonashiro, Uruma, and Okinawa, Japan, with catalogue and sample numbers based on the cataloguing system of Miyazaki University Department of Fisheries Science (MUFS): MUFS 43290, MUFS 4417, MUFS 6025, MUFS 8819, MUFS 12632, MUFS 2098, and MUFS 6014. One specimen had been collected from Meitsu, Nago, Miyazaki, Japan (MUFS 16284) and one from the Philippines (MUFS 6136) (Figure 1).\n\nA. Specimen JN311937, paratype, SL 170mm, collected from Arumbai Fish Market (-3°68’N, 128°18’E), Ambon, Indonesia, 06 January 2016 (photographed by Limmon, reproduced from BOLD under a CC BY-NC-SA 3.0 license). B. Specimen MUFS43290, paratype, SL 131mm, from the MUFS (Miyazaki University, Fisheries Sciences) museum collection (photographed by the authors). C. Specimen MSFUH000591, paratype, SL 217mm, collected in the Makassar Strait (-4°58’N, 119°17’17’’E), 20 January 2019 (photographed by the authors – Muhammad Afrisal). D. Specimen HM423533, paratype, SL 219mm, collected from Lizard Island (-14°66’N, 145°44’E), Queensland, Australia, 07 September 2008 (photographed by the Australian Museum team, Sydney, modified from BOLD under a CC BY-NC-SA 3.0 license). E. Specimen HQ561476, paratype, collected in Mozambique (-22°84’N, 35°55’E), 15 November 2010 (photographed by Connell, modified from BOLD under a CC BY-NC-SA 3.0 license). F.Specimen MH331781, holotype, SL 200mm, collected from Thuwal, Mecca, Saudi Arabia (22°30’N, 39°09’N), 22 March 2017 (photographed by catta, modified from GBIF under a CC BY-NC 4.0 license). G.Specimen JQ350086, paratype, SL 190mm, collected from Nosy Tanikely-West, Quest (-13°48E, 48°24N), Antananarivo, Madagascar, 07 May 2008, (photographed by Planes et al., 2008, modified from BOLD (no rights reserved)).\n\nThe preceding samples were used in this study to represent the Pacific Ocean. The samples used to represent the Indian Ocean were downloaded from the Global Biodiversity Information Facility (GBIF) and The Barcode of Life Data System (BOLD). Measurements of these remotely sourced specimens were made using ImageJ version 1.52a software14, except for the interorbital width character, which could not be measured from the downloaded photographs. The morphological characters were compared with reference to data from three holotypes: MNHN 9087, holotype of L. azureus; RMNH 5758, holotype of L. rhodopterus; and BMNH 1873.3.160, holotype of L. bonhamensis15. The characters measured using callipers (accuracy 0.02 mm) included standard length, body depth, head length, pectoral length, pelvic length, orbital length, interorbital width, snout length, suborbital width, upper jaw length.\n\nDNA extraction was carried out using DNeasy Blood and Tissue Kit (Qiagen, Cat. No. 69504) following the manufacturer’s protocols. Fin clippings (2–3cm) were taken from the pectoral fin of each specimen using surgical scissors or a surgical scalpel. Each sample was preserved in a labelled 2ml tube filled with 95% ethanol.\n\nA sub-sample weighing 0.025mg was taken from each fin clipping sample and placed in a 2mL tube to which 180µL of ATL solution (tissue lysis buffer) and 20µL of proteinase K were added. The tubes were vortexed and incubated at 56°C for 1–3 hours or overnight. Then 200µL AL buffer (lysis buffer) solution and 200µL ethanol were added before vortexing and centrifuging at 8000rpm for 1 minute. The supernatant was transferred to a new tube, 500 µL AW1 (column wash buffer 1) was added, and the tube was centrifuged for 1 minute at 8000rpm. This step was repeated with AW2 (column wash buffer 2) and centrifuged at 14,000rpm for 1 minute. The column was transferred to a new 1.5mL tube and 100μL warm AE buffer was added, then centrifuged for 1 minute, incubated for 10–15 minutes, and centrifuged at 6000×g (8000rpm) for 1 minute. RNAse was added to the extracted DNA solution and incubated for 24 hours. The extracted DNA was prepared for use through dilution, at a ratio of 30µL DNA and 70µL ddH2O.\n\nAgarose gel was prepared by mixing 1.4g of agarose 0.8% in 180mL of 1x TAE buffer solution. A colouring agent (1.5μl GelRed) was added to the gel while still warm and liquid before pouring it into the mould. Aliquots of 2μL DNA template were pipetted and mixed with 1μl of DNA loading dye solution and loaded into the wells in the agarose gel. Electrophoresis was performed at 100 volts for 90 minutes. The results were visualised using an ultraviolet transilluminator (biostep Dunkelhaube, bi000052).\n\nThe DNA barcode analysis used a segment of mtDNA from the cytochrome b oxidase subunit 1 (COI) gene. The target sequences were amplified using the primer pair developed by Ward et al.16: Fish F1-5’ TCA ACC AAC CAC AAA GCA TTG GCAC 3 (forward) and Fish R1-5’ TAG ACT TCT GGG TGG CCA AGA ATCA 3 (reverse). Each reaction tube contained 5µL of PCR mix Hotstart (Cat. No. 203645, QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany), 3µL DNA template, 3µL ddH2O, and 0.675µL each of the forward and reverse primers. The PCR amplification procedure (Labcycler Gradient, 012-102, Sensoquest) comprised the following stages: initial denaturation at 95°C for 5 minutes; 35 cycles with denaturation at 94°C for 1 minute, annealing at a primer-specific temperature for 1 minute, elongation at 72°C for 1 minute; and final elongation at 72°C for 10 minutes. The amplified PCR products were stored in a freezer (-20°C) until the separation process. The annealing temperature followed a gradient of ±5°C compared to the reference temperature for the primer pair, in order to determine the optimal annealing temperature, resulting in a bright and sharp band under electrophoresis. Amplified PCR products with a length (in bp) consonant with the target gene segment were sent to a sequencing company (1st Base Asia, Singapore) in order to obtain DNA sequence data.\n\nPrincipal component analysis (PCA) was used to analyse the morphometric data, and was implemented in MINITAB version 1717. The quality of the DNA sequences obtained was evaluated using the software Sequence Scanner 2.0 (Applied Biosystems, USA). The sequence data obtained from this study were complemented by additional sequences downloaded from the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDJB) databases (accession numbers: HQ561476 Mozambique; KF489627 South Africa; JQ350086 Madagascar; MH331781 Saudi Arabia; JF952781 Japan; JN311937 Indonesia; HM423533 Australia). The forward and reverse sequence data were combined using the software Bioedit version 7.2.6.118. The sequences were trimmed and aligned using the ClustalX19 routine in MEGA 620. The phylogenetic topology reconstruction was implemented in MEGA 6 with the neighbour-joining method and genetic distance was analysed using the compute within groups mean distance option.\n\nAlthough PCA in this research was implemented in MINITAB, the free software JASP21 could be used as an alternative, although we could not guarantee total equivalence between the two software packages. Genetic analyses in this study were implemented using Sequence Scanner 2.0, ClustalX and MEGA 6. An alternative free open source software package which could perform the same functions is UGENE22, although once again we could not guarantee total equivalence between this software package and the software packages used.\n\n\nResults\n\nThe PCA analysis shows that the distribution of morphological characters of individual L. harak specimens (see Table 1) indicates a general pattern of close relationships within two population groups: those in the Pacific Ocean and those in the Indian Ocean (Figure 2). Holotype specimens used in this research (Bonham, New Ireland, Singapore) tend to be grouped to the right above the X axis (Figure 2).\n\nIn general, individuals from Pacific Ocean populations, i.e. from Japan (Okinawa, Miyazaki, and Ishigaki), Indonesia (Makassar and Maluku), the Philippines, and Australia, are predominantly grouped in the upper quadrants of the PCA graph (Figure 2).\n\nPopulations from the Indian Ocean were mostly spread across the lower left quadrant, i.e. South Africa, Mozambique, and Saudi Arabia, with the Seychelles being the only population placed (very low) in the bottom right quadrant. However, the specimens from Tanzania and Madagascar grouped together with the Pacific Ocean populations in the upper right quadrant and had morphometric characters similar to the holotype of L. harak.\n\nThe genetic distance analysis using the compute within groups mean distance method indicates that, in general, L. harak populations in the Pacific Ocean and the Indian Ocean have a close kinship relationship (Table 2). The molecular analysis in this study shows that the smallest genetic distance between populations in the Indian Ocean (South Africa and Mozambique) is 0.000, while the lowest genetic distance in the Pacific Ocean (between Japan and Indonesia) is 0.004. The lowest genetic distance between populations in the two oceans (Saudi Arabia and Indonesia) is 0.031. Meanwhile the largest genetic distance is 0.042 (between Madagascar and Japan, Makassar, and Australia), and is still included in the low category.\n\nPhylogenetic topology reconstruction was made to confirm the morphological grouping and genetic distance between the Pacific and Indian Oceans. The phylogenetic reconstruction (Figure 3) used the neighbour-joining method with outgroup sequence data obtained from GenBank for species in the genus Lethrinus found in eight populations in the Indian and Pacific Oceans.\n\nThe topology of this neighbour-joining phylogenetic reconstruction shows two L. harak population clusters. The fish from Mozambique, South Africa, Madagascar, and Saudi Arabia formed a clade with a bootstrap value of 100%. The second clade comprised populations from Indonesia (Makassar and one other location), Japan, and Australia with a bootstrap value of 99%.\n\n\nDiscussion\n\nThis confirms the view expressed by Baur and Leuenberger23 that morphometric data can be extremely useful for the study of variation in form due to geographical differences. In addition, morphometric data are frequently used in taxonomy and fish description24. In taxonomy, data on the holotype of a species are vital as a basis for comparison when observing other specimens. Data on the holotype can also be used to evaluate whether there are any changes in the morphometric characters of a given specimen. Such methods form the basis for establishing new fish species25.\n\nTheir proximity indicates similarities in morphometric characters of L. harak between populations experiencing similar environmental conditions. Conversely, environmental pressures will tend to drive adaptation through physiological and behavioural modifications. A species with the capacity to adapt through plasticity will produce a variety of phenotypes as well as genotypes in response to a given set of environmental conditions, in order to increase fitness, the ability of individuals to survive and reproduce26,27. Matthews28 found adaptations in body shape, colour and fins based on the environmental conditions in which fish live, as well as adaptations in head shape associated with feeding processes.\n\nGenetic effects and environmental parameters can both result in differences in growth and development processes, which will produce variations in fish body shape29. A relatively high level of geographical isolation coupled with limited geographical extent can result in significant differences in morphometric and genetic characters between stocks or populations of the same species due to a lack of gene-flow between populations30. This is supported by the identification of cryptic lethrinid species with similar morphological characters by Healey31, e.g. L. nebulosus and L. mahsena in the SWIO. This study found two clades, with the Seychelles separated from the other populations in the SWIO. The emergence of this phenomenon in the Seychelles could be related to high fishing pressure and habitat degradation. Wilson & Clarke32 report that increasing exploitation and environmental pressures can cause a decline in fish stock abundance, as well as reductions in the average size, and detrimental genetic selection which can reduce potential fecundity and mean spawning size, alter sex ratios, and interspecific balances, as well as reducing genetic diversity.\n\nThe genetic distance can indicate the level of kinship between populations. Koh et al.33 state that the lower the genetic distance between individuals, the closer their kinship and vice versa. According to Nei34, genetic distance values can be categorised as low (0.010 - 0.099), moderate (0.1-0.99), or high (1.00-2.00). Such phylogenetic analyses can be used to construct detailed relationships between species and estimate the divergence between members of a family descended from a common ancestor35.\n\nThis pattern indicates a divergence in genetic characteristics between L. harak populations in the Pacific and Indian Oceans. Williams et al.36 found a genetic break between the Pacific and Indian Oceans in several invertebrate taxa. The topology of their phylogenetic reconstruction grouped northwestern Australia with the Indian Ocean for most taxa, although some taxa showed an affinity with Pacific Ocean populations, which they considered may be due to the oceanographic conditions around nine million years ago. Meanwhile, as in our study, eastern Australia populations were more closely related to Pacific Ocean populations.\n\nThe characteristics of the water masses and oceanographic currents in the two oceans influence the distribution and population connectivity of marine organisms, and may affect their physiology and exert differential selection pressures, thus potentially influencing genetic characteristics in several ways. Surface currents are a key factor in larval dispersal37. As a protogynous hermaphrodite, L. harak undergoes a process of gonad differentiation associated with sex change from the female to terminal male phase6,38. As a fish with a pelagic spawning reproductive strategy, L. harak releases pelagic eggs which drift with other plankton37–41. The density of L. harak eggs is similar to or slightly less than that of sea water, so that they float at or close to the sea surface40. After hatching the larvae remain planktonic for a period of time, which can vary from a few hours to several months41. It is therefore likely that surface currents significantly affect the distribution and genetic population structure of L. harak.\n\nThe Pacific low latitude western boundary currents (LLWBCs) are one of the factors that can influence genetic structure in the Indian Ocean42. In the Pacific Ocean there are at least four currents affecting the distribution of marine organisms: the New Guinea Coastal Current (NGCC), the Halmahera Eddy (HE), the North Equatorial Coastal Current (NECC) and the North Equatorial Current (NEC)43. The main source of the water masses underpinning current circulation in the LLWBCs system are the South Equatorial Current (SEC) and the North Equatorial Current (NEC)44. A proportion of the water masses travelling westwards in the equatorial currents join the Kuroshio current flowing northward towards Japan, while some flows south in the Mindanao current or flows south to join the East Australia Current (EAC). A branch of the Mindanao current enters the Sulawesi Sea and flows through the Makassar Strait, while another branch flows east along the north coast of Sulawesi to the Halmahera Sea45. These complex currents are further complicated by seasonal variations and local geography, and could enable gene-flow between several of the sites in our study.\n\nDespite the clear demarcation of the two clades found in our study, the genetic distances between L. harak from the Pacific and Indian Oceans was relatively small, unlike the large differences found by Carvalho10 between the Pacific and Atlantic populations of Haemulon steindachneri. These differences were considered sufficiently large to indicate that the two putative populations of H. steindachneri were in fact two different species, thus indicating speciation after the closing of the Isthmus of Panama. In contrast, despite the reduced connectivity between the Indian and Pacific Oceans described by Williams et al.36, their data indicate some continuing gene-flow between the Pacific and Indian Oceans, although oceanographic flow patterns45 indicate that gene-flow is more likely from the Pacific to the Indian Ocean than the reverse. Our results are consonant with such a continued (albeit reduced) gene-flow.\n\n\nConclusion\n\nThe Lethrinus harak populations in the Pacific and Indian Oceans appear to form two major clades based on morphometric and genetic characteristics. One clade includes populations from South Africa, Mozambique, Madagascar and Saudi Arabia, while the other includes populations in Japan (Okinawa, Miyazaki, and Ishigaki), Indonesia (Makassar and Maluku) and the Philippines. Two populations exhibit intermediate (Australia) or unique (Seychelles) morphometric characters; however, based on the genetic reconstruction topology, the Australian population grouped with the Pacific L. harak clade.\n\n\nData availability\n\nPartial cytochrome C oxidase subunit I (COX1) gene mitochondrial DNA sequence of L. harak (specimen voucher MSFUH0000591) from Makassar waters on GenBank, Accession number MT551656.", "appendix": "Acknowledgements\n\nWe thank Nurijrana, Meilani, Aida Shinsei, Ono Soichiro, Chihiro Fujii, and Kumagai Tadakumo for their assistance with the research and Abigail Moore for proofreading the manuscript.\n\n\nReferences\n\nCarpenter KE, Allen GR: FAO species catalogue. Vol.9. 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Publisher Full Text" }	[ { "id": "68802", "date": "26 Aug 2020", "name": "Jayasankar Pallipuram", "expertise": [ "Reviewer Expertise Aquaculture", "fish genetics", "molecular taxonomy" ], "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\nNo justification is being given for using alternative software used for genetic analysis – without guaranteeing total equivalence, two different softwares are used [page 5] Though sample details are furnished, it hasn’t been clearly mentioned anywhere as to exact number of samples subjected to DNA barcoding. However, from the report the number seems to be insufficient to interpret the results. If DNA were extracted from museum-preserved samples, the methodology used should have been specifically mentioned. The protocol is certainly different from that used for fresh/frozen samples. In the methodology it’s mentioned “Amplified PCR products with a length (in bp) consonant with the target gene segment were sent to a sequencing company (1st Base Asia, Singapore) in order to obtain DNA sequence data.” Though we accept that in majority of studies DNA sequencing is outsourced, such kind of statement as given here would dilute the scientific seriousness and originality. The authors haven’t even briefly described the actual methodology of DNA sequencing as intended and executed. In discussion the authors have described endlessly on influence of water currents and other environmental parameters in stock distribution and separation. However, the present study has no such data to substantiate those concepts. This has resulted in just copying others’ observations and presenting the same, without having any study attempted in those lines by the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "74360", "date": "19 Nov 2020", "name": "Femy Mahmud Sahami", "expertise": [ "Reviewer Expertise Goby fish" ], "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 conclusion in abstracts is still too general. The research novelty needs to be explicitly highlighted, particularly the differences of L. harak in the Pacific and Indian oceans based on morphometric and genetic aspects.\n\nThe word “alcohol” was mentioned twice in the following sentences: Each sample was placed on its right-hand side and sterilised using alcohol. A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. Please consider replacing “alcohol” with “ethanol” to avoid misinterpretation.\n\nThe method described as: A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. This sentence needs to elaborate in detail. Please connect it with the different-samples explanation in the DNA extraction section.\n\nIt is not clear how many samples were taken in Makassar for morphometric and molecular analysis purposes. Samples with the code MSFUH000591, MSFUH0000592, MSFUH0000593 were not specified what analysis was applied. Only the sample coded MSFUH000591 was mentioned in the results section. Likewise, in Table 1, there were stated only 3 Makassar samples with the code MSFUH000591, MSFUH915, and MSFHU917. Where were the other samples (coded MSFUH0000592 and MSFUH0000593) descriptions?\n\nIn the method mentioned: One specimen had been collected from Meitsu, Nago, Miyazaki, Japan (MUFS 16284) and one from the Philippines (MUFS 6136) (Figure 1). However, these two samples were not shown in Figure 1. What is the purpose of writing “(Figure 1)” at the end of the sentence?\n\nWhat do “preceding samples” mean in the following sentence “The preceding samples were used in this study to represent the Pacific Ocean”\n\nTwo sentences seem somewhat ambiguous in measuring morphology, as follows: Measurements of these remotely sourced specimens were made using ImageJ version 1.52a software14, except for the interorbital width character, which could not be measured from the downloaded photographs. Then in the following sentence, it was stated that: The characters measured using callipers (accuracy 0.02 mm) included standard length, body depth, head length, pectoral length, pelvic length, orbital length, interorbital width, snout length, suborbital width, upper jaw length. It might be necessary to clarify which samples were measured using ImageJ and measured manually using a caliper.\n\nThe second sentence's meaning in the DNA extraction sub-section is not clear: Fin clippings (2–3cm) were taken from the pectoral fin of each specimen using surgical scissors or a surgical scalpel. Each sample was preserved in a labelled 2ml tube filled with 95% ethanol. Are the specimen referred to samples from Makassar or some other areas? As in the 6th sentence in the Fish Specimen sub-section stated as: A fin-clipping of around 1.8 cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol.\n\nIf possible, replace the subsection “Statistical Analysis” with “Data Analysis” since there were also explaining genetic analysis.\n\nIn the second paragraph in the sub-section Morphological Characters, it mentioned “Indonesia (Makassar and Maluku)”, while in Figure 1 it was stated that: Specimens of Lethrinus harak. A. Specimen JN311937, paratype, SL 170mm, collected from Arumbai Fish Market (-3°68’N, 128°18’E), Ambon, Indonesia, 06 January 2016. What mentioned here was Ambon, not Maluku. The words Maluku and Ambon need to be consistent as even though Ambon is in Maluku, this will lead to confusion. Also, Figure 2 mentioned only Makassar and Indonesia, while Maluku was not mentioned. In Table 1 and Table 2, the word Maluku was not mentioned either, but in conclusion, it was written in Indonesia (Makassar and Maluku). Please make it consistent whether Indonesia (Maluku) or Indonesia (Ambon).\n\nIt needs consistency in writing. It should be written in capital letters at the beginning of each word in the stands: The Pacific low latitude western boundary currents (LLWBCs).\n\nThe writing of citations is still inconsistent. Some are written with the author's name along with the serial number in the bibliography, and some are written directly without writing the author's name.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6148", "date": "04 Dec 2020", "name": "Muhammad Afrisal", "role": "Author Response", "response": "Thank you for being our manuscript reviewer. We will revise your suggestions for writing our manuscript." } ] } ]	1	https://f1000research.com/articles/9-915
https://f1000research.com/articles/10-216/v1	16 Mar 21	{ "type": "Research Article", "title": "COVID-19 related misconceptions among older adults in Bangladesh: findings from a cross-sectional study", "authors": [ "Sabuj Kanti Mistry", "ARM Mehrab Ali", "Uday Narayan Yadav", "Nafis Md. Irfan", "Binod Rayamajhee", "Melinda N. Stanners", "ARM Mehrab Ali", "Uday Narayan Yadav", "Nafis Md. Irfan", "Binod Rayamajhee", "Melinda N. Stanners" ], "abstract": "Background The COVID-19 has been appeared as the most remarkable global calamity of this century. Just as the COVID-19 spread throughout the world, so as the rumour and misconceptions related to it. The present study aimed to explore the prevalence of COVID-19 related misconceptions and its correlates among the older adults in Bangladesh.\nMethods The study followed a cross-sectional design and was conducted among 1032 participants aged 60 years and above from Bangladesh. Information was collected on 14 different locally relevant misconceptions related to the spread, prevention, and treatment of COVID-19, scored each misconception as one, and obtained a cumulative score, ranging from 0 to 14, with a higher score indicating a higher level of misconceptions. A multiple linear regression model explored the factors associated with misconceptions.\nResults The most common misconceptions were, all returning migrants carrying COVID-19 (45.5%), wearing personal protective equipment (PPE) in outdoors (80.1%), not going to the funeral of people died of COVID-19 (45.2%), its prevention by nutritious food (57.6%) and drinking water (39.4%), and doctor can cure COVID-19 (49.9%). Misconceptions were higher among the participants who were living alone, whose family members were not responsive to their needs during COVID-19, and who received COVID-19 related information from Radio/TV and health workers. These misconceptions were less likely among those who were aged 70-79 years, who had pre-existing non-communicable chronic conditions, who were overwhelmed by COVID-19, and who felt themselves at highest risk of COVID-19.\nConclusion Overall, we found that misconceptions were prevalent among the older adults in Bangladesh. Government and other relevant stakeholders should take immediate actions to address the prevalent misconceptions through using appropriate channels, media, and message delivery systems, and applying evidence-based risk communication methods.", "keywords": [ "COVID-19", "Misconceptions", "Older adults", "Bangladesh" ], "content": "Introduction\n\nThe human population is shrouded in a global health crisis due to the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. As of the 24th of January 2021, more than 96 million people have been infected, and more than two million people have lost their lives2. Furthermore, this unprecedented pandemic has substantially changed the way we live across the globe, with deleterious societal and financial outcomes likely to take many years to return to normal3. The ongoing COVID-19 pandemic has affected each part of the world, leaving no country untouched4.\n\nHowever, certain population groups are more vulnerable than others to the deadliest effects of COVID-19. Mortality rates from the USA show that over 80% of COVID-19-related deaths occur among patients ≥65 years5, with similar figures reported globally, demonstrating that older adults are especially vulnerable to complications and death resulting from COVID-196,7. The severity of the SARS-CoV-2 infection is influenced by the age of a patient, and disproportionately affects adults over 65 years8, meaning that older people are at a higher risk of morbidity and deaths due to COVID-196. Older adults with comorbidities like diabetes, chronic obstructive pulmonary disease, organ receiver/donor, hypertension, heart and kidney diseases are at a significantly higher risk of SARS-COV-2 severe infection and increased case fatality rates9.\n\nBangladesh, in South Asia, reported its first COVID-19 patient on 8th March 202010, when a traveller returned from Italy is believed to have brought the virus into the country11. Since then more than 500,000 infected cases have been reported as of 11th January 2021, with 7,781 deceased due to COVID-192. This triggered a nationwide response to curb the pandemic that has been in operation since early March 202012. However, the absence of travel restrictions and allowance of mass gatherings for religious functions are responsible for the recent growth in COVID-19 cases13. Like many other countries, Bangladesh is also experiencing growth in the older population, with more than 3.2 million people, or 7.7% of the total population, aged 60 years or above; a proportion that is projected to increase three-fold to 21.9% by 205014. As age is a critical risk factor related to COVID-19, Bangladeshi older adults are disproportionately vulnerable to COVID-19; although nearly 7% of total COVID-19 patients are aged ≥60 years, this group faces a mortality rate of almost 39%15.\n\nJust as SARS-CoV-2 has spread all over the world, so too has misinformation. Misleading or false information has led to individuals putting themselves and others at risk by believing or acting upon falsehoods, not engaging in protective measures, and self-medicating using toxic medicines. Common misconceptions about the pandemic include: the idea that face masks do not filter SARS-CoV-2, that COVID-19 is like seasonal flu, and that only old older people are affected by the virus16. Electronic media serves as the dominant platform for circulating fake news and misinformation about the public health crisis17, creating what the Director-General of the World Health Organisation has called an ‘infodemic’18. Social stigma is closely associated with believing false information about how SARS-CoV-2 spreads, while gossip quickly circulates rumours and myths19. Nearly 59% of Bangladeshi have access to the internet, through which they can access rumours and fear about COVID-1920.\n\nAlthough some recent studies carried out in Bangladesh focused on knowledge, attitude, and fear of COVID-1912,21, none have focused on COVID-19 misconceptions among older adults. Given the heightened vulnerability of older adults to COVID-19-related morbidity and mortality, understanding what older adults believe is of paramount importance in facilitating effective interventions to educate and protect the older adult population. Therefore, the aim of this study was to explore the misconceptions about the COVID-19 pandemic, and associated predictors, amongst older people in Bangladesh.\n\n\nMethods\n\nThis cross-sectional study was conducted remotely through telephone interviews in October 2020. We utilized our pre-established registry, developed through merging the contact information of households from different research projects accomplished by ARCED Foundation during 2016–2020 as sampling frame. There were around 9000 households with verified contact information in the registry that included households of different demographic characteristics (urban and rural both) and income groups, and covered all eight administrative divisions of Bangladesh.\n\nAnticipating 50% prevalence of misconception among older adults (as prevalence is unknown) with a 5% margin of error, at the 95% level of confidence, 90% power of the test, and 95% response rate, a sample size of 1096 was calculated. However, only 1032 eligible participants responded to the study with an overall response rate of approximately 94%. Based on the population distribution of older adults by geography in Bangladesh, we adopted probability sampling proportionate to the number of older adults to ensure representative selection of older adults from each division14. This was deemed to address the potential sources of bias that may results through sampling process (selection bias). The inclusion criterion was being aged 60 years and above, with exclusion criteria including severe mental health problems (clinically proved schizophrenia, bipolar mood disorder, dementia/cognitive impairment), a hearing disability, or inability to communicate.\n\nOutcome measure. The primary outcome measure of this study was the level of misconceptions related to COVID-19, which was captured through the use of a 14 item misconceptions scale. World Health Organization compiled a list of the most common COVID-19 related misconceptions22, we included some of those which were contextually relevant. The survey also included other locally relevant misconceptions, including transmission through mosquito bites, and transmission only to people who practice socially unacceptable activities. Each item was nominally coded as a true/false statement where a correct response scored zero, and each misconception scored one. The scores were summed to generate a misconception score for each participant. The cumulative score of the 14-items ranged from zero to 14, with a higher score indicating a higher level of misconception.\n\nExplanatory variables. Explanatory variables considered in this study were age (categorized as 60–69, 70–79, and ≥80 years), sex (male/female), marital status (currently married/single), literacy (yes/no), family size (≤4 and more than 4), family income in Bangladeshi Taka (BDT) (<5000, 5000–10000, >1000), residence (urban/rural), current occupation (employed/unemployed or retired), living arrangements (living with other family members/living alone), source of COVID-19 related information (TV/radio, health workers, and friends/family/neighbors), problem in memory or concentration (no problem/low memory or concentration), walking distance to the nearest heatlh centre (<30 min/≥30 min), presence of any non-communicable chronic conditions (yes/no), feeling concerned about COVID-19, feeling overwhelmed by COVID-19 (yes/no), frequency of communication with friends and family during COVID-19 (less than previous/same as previous), feeling at a highest risk of COVID-19 (yes/no), and family members not responsive to their needs during COVID-19 (yes/no).\n\nSelf-reported information on non-communicable chronic conditions such as arthritis, hypertension, heart diseases, stroke, hypercholesterolemia, diabetes, chronic respiratory diseases, chronic kidney disease, and cancer were collected.\n\nA pre-tested semi structured questionnaire in the Bengali language was used to collect the information through telephone interview. Data collection was accomplished electronically using SurveyCTO mobile app (Version 2.70.6, SurveyCTO CATI feature, Dobility, Inc., Washington, DC, USA) (https://www.surveycto.com/) by ten research assistants, recruited based on previous experience of administering health surveys using an electronic platform. The research assistants were trained extensively by SKM, AMI and UNY for three days through Zoom meeting (Version 5.4.2, Zoom Video Communications, Inc., San Jose, CA, USA) before the data collection commenced.\n\nThe English version of the questionnaire was first translated to Bengali language and then back translated to English by two researchers to ensure the contents' consistency. The questionnaire was then piloted among a small sample (n=10) of older adults to refine the language in the final version which ensures the face validity of the tool. The contents of the questionnaire were approved by the participants from the pilot study without requiring any changes in wording or sentence structure. This was undertaken to address any potential bias that may arise from using the tool. Moreover, Cronbach’s alpha for the misconception items was 0.87, which indicates acceptable reliability of the tool. The final Bengali version of the tool was administered for collection of the information.\n\nThe distribution of the variables was assessed through descriptive analysis. The Chi-square test was performed to compare the prevalence of misconceptions and stigma within different gender groups at a 5% level of significance. To explore the factors associated with misconceptions among the participants we executed a linear regression model. We used backward elimination criteria with the Akaike information criterion (AIC) to select the final model. Briefly, the backward elimination algorithm starts with a full model (model with all variables) and drops one by one variable from the model based on the statistical significance of that variable. In this case, we reported the adjusted beta-coefficient and 95% confidence interval (95% CI). We also performed the model diagnostics, such as normality of the residuals, and multicollinearity in the model. All the analysis was performed using the statistical software Stata (Version 14.0)\n\nThe study protocol was approved by the institutional review board of Institute of Health Economics, University of Dhaka, Bangladesh (Ref: IHE/2020/1037).\n\nVerbal consent was sought from the participants before administering the survey. Participation was voluntary, and participants did not receive any compensation. As the interview was conducted remotely using telephone, verbal consent was sought instead of written consent and consent of each participant was noted in the questionnaire. This verbal consent was approved by the ethics committee (Ref: IHE/2020/1037).\n\n\nResults\n\nA total of 1032 older Bangladeshi adults aged 60 years and above participated in the study (Figure 1). Table 1 describes the sociodemographic and lifestyle characteristics of participants, reflecting a higher proportion of participants aged 60–69 years (77.8%), male (65.5%), married (81.4%), illiterate (58.3%), residents of rural areas (73.9%), and living with other family members (92.3%). The most common source of COVID-19 related information was radio/television (83.04%), followed by friends/family/neighbors (71.2%), and a very small number also received information from health workers (9.3%). The majority of participants reported that they were concerned about (71.3%) and overwhelmed by (63.2%) COVID-19. Reduced social interaction during the pandemic was reported by 42% participants and around 59% had pre-existing non-communicable chronic conditions.\n\n*Multiple responses\n\nParticipant misconceptions related to the spread, prevention, and treatment of COVID-19 are presented in Table 2. Nearly half of the participants (45.5%) had the misconception that all returning migrants carried COVID-19. The most commonly held misconceptions related to the prevention of COVID-19 included wearing personal protective equipment (PPE) outdoors (80.1%), not going to the funeral of people who died of COVID-19 (45.2%), and its prevention by nutritious food (57.6%) and drinking water (39.4%). Meanwhile, the most notable misconception regarding the treatment of COVID-19 was that doctors can cure COVID-19 (49.9%) (Table 2).\n\nThe final model, based on the lowest AIC, retained the variables shown in Table 3. Hence, the model is adjusted for all the variables in Table 3. The q-q plot of the residuals of the model (Figure 2) shows that the data is normally distributed, while VIF values of less than 10 for each variable (Table 4) reflects the absence of multicollinearity. Average misconception scores were higher among participants who were living alone (β: 0.94, 95% CI: 0.43 to 1.45); who were literate (β: 0.43, 95% CI: 0.18 to 0.68); whose family members were not responsive to their needs during COVID-19 (β: 0.99, 95% CI: 0.71 to 1.28); who received COVID-19 related information from radio/TV (β: 0.89, 95% CI: 0.55 to 1.21); and who received COVID-19 related information from health workers (β: 0.60, 95% CI: 0.25 to 0.94). On the other hand, the misconception scores were lower among participants who were aged 70–79 years (β: -0.57, 95% CI: -0.87 to -0.26); who had pre-existing non-communicable chronic conditions (β: -0.43, 95% CI: -0.70 to -0.15); who were overwhelmed by the pandemic (β: -0.40, 95% CI: -0.69 to -0.10); and who felt themselves at highest risk of COVID-19 (β: -0.41, 95% CI: -0.68 to -0.14).\n\n\nDiscussion\n\nThe present study assessed the prevalent misconceptions about the spread, treatment and prevention of COVID-19 and its associated factors among older adults in Bangladesh. To the best of our knowledge this is the first study to document misconceptions about the COVID-19 pandemic among the older population of Bangladesh at the community level. Overall, we found that some misconceptions related to COVID-19 spread, treatment and prevention remain persistent to varying degrees among the participants. Evidence from similar epidemics (such as Middle East respiratory syndrome (MERS) and Swine flu (H1N1)23,24) show that misconceptions, myths and rumours always exist in the general population during and following an outbreak, and this is also true for the COVID-19 pandemic22,25.\n\nIn the present study, the most notable misconception was the mandatory use of PPE for everyone to go outside (80.1%). The importance of PPE in preventing the spread of infection is indisputable, but PPE is particularly important for health professionals who come in close contact with COVID-19 patients26. Over-emphasis of PPE as a mode of prevention of COVID-19 and widespread reporting in print media regarding the shortage of PPE in health services might have created the notion that one should wear PPE when outside to protect themselves from COVID-1911.\n\nMany participants in the present study believed that all returnee migrants carry COVID-19. This can possibly be attributed to the increase in number of COVID-19 cases in Bangladesh after the arrival of Bangladeshi citizens returning from abroad during this pandemic27. Also, many countries including Bangladesh have taken measures to restrict international travel and quarantine travellers upon arrival, which might have shaped the perception among the older adults that all returnee migrants carry COVID-1928. While the percentage was relatively low, some participants also reported about the belief that COVID-19 spreads through mosquito bites. This may be because many of the symptoms of COVID-19, such as fever and body ache, are like that of dengue, Chikungunya virus and malaria, which are spread through mosquito bites29. The myth of transmitting the virus through mosquito is also prevailing in neighboring countries (e.g. India) and other parts of the world30.\n\nOne of five participants also believed that COVID-19 was spread through testing for it. According to the Centers of Disease Control, one of the key steps for successful COVID-19 management is early test, trace (contact tracing) and quarantine of the cases31. This misconception is therefore of serious concern, as it may reduce the willingness of people to undergo testing, and eventually lead to community spread of the virus. This misconception may relate to uncertainty regarding accuracy of the test (due to false positives), false rumors on COVID-19 spread, poor health literacy of older adults, and reports of COVID-19 test scams (e.g. selling COVID-19 positive or negative results without actual laboratory analysis)32,33.\n\nSome of the participants also believed that COVID-19 is transmitted only to non-religious, less religious and people who practice socially undesirable activities, which is important to consider. Evidence from several countries suggests that religious stigma and orthodoxies resulted in mismanagement of COVID-19. For example, in India, cow dung or urine has been used as a treatment to cure COVID-19, in Latin America and Spain religious leaders have claimed vaccine development as the work of devil stating that it would involve cell lines from aborted fetuses34. Earlier, religious misinformation from Muslim clerics stating that the polio vaccine contains pork have reduced the success of vaccination in Pakistan35. Therefore, it is important to dissect these existing prevailing religious myths regarding the spread of COVID-19 among older population of Bangladesh and make sure that these myths are addressed by appropriate/accurate information dissemination and risk communication.\n\nThe present research supports recent studies that also documented misconceptions regarding prevention of COVID through good nutrition and drinking water30,36. Although the benefits of hydration, balanced diet and specific nutrients such as vitamin C and D in COVID-19 management is proven, to date there is no evidence that a specific nutrient or food can prevent COVID-19 infection37,38. Many participants also believed that smoking does not increase the risk of COVID-19, although emerging evidence suggests that smokers are more likely to develop severe COVID-19 symptoms and outcomes as compared to non-smokers39,40. This misconception may be attributed to poor health literacy41 and lack of updated information of COVID-19 among older adults in Bangladesh12.\n\nIn our study we found that half of the participants believe that doctors can cure COVID-19. This is also in line with the findings that around 14% of respondents had the misconception that antibiotics can treat COVID-19. The notion that doctors can cure every disease including COVID-19, and antibiotics can treat every disease is not surprising in developing countries. Recent evidence also suggests that there is unnecessary use of antibiotics as a treatment of COVID-19 in many places42. A study on hospitalized patients in New York, USA revealed that 71% of COVID-19 patients received antibiotics, while only 4% had bacterial co-infection43. Studies carried out in Australia36 and Iran44 also revealed a higher proportion of people inaccurately using antibiotics to treat COVID-19.\n\nThe present study also identified some correlates of misconceptions among the participants. It was found that the participants who were overwhelmed by COVID-19 and felt themselves at highest risk of COVID-19 had lower misconception scores, implying that anxiety, worry, and caution might have contributed in a positive way to drive them to acquire more accurate information, or made them more aware of the existing misconceptions45. Likewise, misconception scores were significantly lower among those who had pre-existing non-communicable chronic conditions. In general, complications and case fatality risk of COVID have been reported higher in people who had other chronic disease or co-morbid conditions46,47. There have been reports of increased hospitalization rate and death in elderly populations with pre-existing conditions around the globe48–50 and have been aired through various news agencies. This might have encouraged older adults with non-communicable chronic conditions to gather accurate information regarding COVID transmission, spread and treatment.\n\nWe also observed that living alone, and family members’ non-responsiveness to their needs during the pandemic, increases the misconceptions among older populations. This is important to consider because older people often rely on other family members not only for their medical care but also as trusted source of information51. We also observed that misconception scores were significantly lower among older adults aged 70–79 years compared to those aged 60–69 years. This suggests that relatively older adults are more open to receiving accurate information related to COVID-19, which is also supported by the findings of recent research52. Participants aged 70–79 years may have been open to receiving accurate information regarding COVID-19 due to awareness that COVID-19 related morbidities and mortality increases with age52. Therefore, accurate COVID-19 messaging should prioritize the older age groups. The present study found that literacy was not associated with more accurate knowledge, with a BBC report suggesting that literate people may be more prone to misinformation related to COVID-19 because of an overload of information, sharing before thinking, and override of reactions53.\n\nSurprisingly we haven’t observed reduced misconceptions scores in groups who received information from radio/TV compared to those who didn’t receive any information. This is a big concern from the public health perspective since it denotes that there is a mistrust in the content of information provided by the media. There is also evidence that COVID-19 myths and misconceptions can spread through the media54,55, which could explain distrust of the media as a source of accurate information. Also, misconceptions were higher among the participants who received information from health workers. There could be two possible reasons for this. Firstly, health workers may not be adequately trained on the issues related to COVID-19. Secondly, the participants may not trust the information provided by health workers even when it is accurate. Thus, health care workers need to be properly trained on accurate COVID-19 related information and on effective delivery to the community.\n\nThe misconceptions reported in this study present several public policy implications. The findings demonstrate that unscientific misconceptions regarding spread and prevention of COVID-19 persist among older people, supporting calls for extensive and accurate dissemination of information, as these misconceptions may lead to failure to follow recommended public health measures and practices in this vulnerable group. Furthermore, in a collectivist society where the household-head or the decision-maker is the older family member, misconceptions and misinformation among household heads prevent younger family members from accessing health care. For example, they may prevent their daughters, daughters-in-law, and grandchildren from seeking health services, including future immunization programs against COVID-19. Awareness programs designed for older people should utilize a range of different channels; telecommunications might play a pivotal role here, since older adults are less likely to make an in-person visit to a heath care provider. Finally, to reduce and control the infodemic, the government must monitor print and social media to ensure credible and correct information sharing based on evidence and should take legal action against the spread of any fake news, myths or rumors.\n\nOur study has several limitations. Although we collected nationwide data to reflect a representative view of misconceptions in Bangladesh, a larger sample would have allowed further stratification of the factors associated with misconceptions. Also, we prepared our sampling frame based on the available household-level information that we have in our repository, therefore presenting the possibility of selection bias towards previously engaged participants. The decision to use telephone interviews to collect data was made to maintain social distancing, but this presents a bias towards telephone owners (people without a telephone were excluded), and people comfortable with speaking on the telephone. Finally, although we conducted a thorough literature review to identify the most prevalent misconceptions regarding COVID-19 in Bangladesh, other misconceptions may exist that were not included in the current study.\n\n\nConclusion\n\nCertain misconceptions are held by many older people residing in Bangladesh regarding the spread, prevention, and treatment of COVID-19, which increases the likelihood of disease transmission and mismanagement of COVID-19 in this vulnerable group. These findings can provide insight into and support public health training and awareness activities in this population. Government and other agencies should co-ordinate education activities with consideration of the prevalent misconceptions, and focus on addressing the varied misconceptions, using appropriate channels, media, and message delivery systems, and applying evidence-based risk communication methods. A holistic health literacy intervention program should be included as part of infodemic management activities to address these misconceptions at individual, household, community, and health care levels.\n\n\nData availability\n\nDataverse. Replication Data for: Misconception of COVID-19 among older adults in Bangladesh. DOI: https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/AFSPZS\n\nThis project contains the following underlying data:\n\n- Data file 1. Misconceptions of COVID-19 among older adults in Bangladesh\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nDataverse, Replication Data for: Misconception of COVID-19 among older adults in Bangladesh. DOI: https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/AFSPZS\n\nThis project contains the following extended data:\n\n- Copy of the questionnaire in English and Bengali.\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 acknowledge the role of Zahirul Islam, Project Associate, Sadia Sumaia Chowdhury, Programme Manager, and Muntasir Alam, Research Assistant, ARCED Foundation for their support in data collection for the study.\n\n\nReferences\n\nWiebers DO, Feigin VL: What the COVID-19 crisis is telling humanity. Neuroepidemiology. 2020; 54(4): 283–286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: WHO Coronavirus Disease (COVID-19) Dashboard. 2020. Reference Source\n\nSeitz BM, Aktipis A, Buss DM, et al.: The pandemic exposes human nature: 10 evolutionary insights. Proc Natl Acad Sci U S A. 2020; 117(45): 27767–76. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Coronavirus disease (COVID-19) advice for the public: Mythbusters. 2020; [22/10/2020]. Reference Source\n\nEastwood K, Durrheim DN, Butler M, et al.: Responses to pandemic (H1N1) 2009, Australia. Emerg Infect Dis. 2010; 16(8): 1211–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlsahafi AJ, Cheng AC: Knowledge, attitudes and behaviours of healthcare workers in the Kingdom of Saudi Arabia to MERS coronavirus and other emerging infectious diseases. Int J Environ Res Public Health. 2016; 13(12): 1214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli S: Combatting Against Covid-19 & Misinformation: A Systematic Review. Hu Arenas. 2020; 1–16. Publisher Full Text \| Free Full Text\n\nLivingston E, Desai A, Berkwits M: Sourcing personal protective equipment during the COVID-19 pandemic. JAMA. 2020; 323(19): 1912–4. PubMed Abstract \| Publisher Full Text\n\nMannan KA, Farhana KM: The COVID-19 pandemic: Challenges and reality of quarantine, isolation and social distancing for the returnee migrants in Bangladesh. International Journal of Migration Research and Development. 2020; 6(1). Reference Source\n\nBodrud-Doza M, Shammi M, Bahlman L, et al.: Psychosocial and socio-economic crisis in Bangladesh due to COVID-19 pandemic: a perception-based assessment. Front Public Health. 2020; 8: 341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutsuddy P, Jhora ST, Shamsuzzaman AKM, et al.: Dengue situation in Bangladesh: An epidemiological shift in terms of morbidity and mortality. Can J Infect Dis Med Microbiol. 2019; 2019: 3516284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahoo S, Padhy SK, Ipsita J, et al.: Demystifying the myths about COVID-19 infection and its societal importance. Asian J Psychiatr. 2020; 54: 102244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCDC: Contact Tracing for COVID-19. 2020; [07/01/2021]. Reference Source\n\nSakib SMN: Bangladesh: Low COVID-19 testing rate raises concerns. 2020; [07/01/2021]. Reference Source\n\nAljazeera: Bangladesh arrests hospital owner over fake coronavirus results. 2020; [07/01/2021]. Reference Source\n\nFirst Draft: ‘An unquestionable truth’: Religious misinformation in the coronavirus pandemic. 2020; [07/01/2021]. Reference Source\n\nFirst Draft; Bhattacharjee S, Dotto C: Vaccine case study: Understanding the impact of polio vaccine disinformation in Pakistan. 2020; [07/01/2021]. Reference Source\n\nThomas R, Greenwood H, Michaleff ZA, et al.: Examining Australian's beliefs, misconceptions, and sources of information for COVID-19: A national online survey. medRxiv. 2020. Publisher Full Text\n\nCalder PC: Nutrition, immunity and Covid-19. BMJ Nutr Prev Health. 2020; 3(1): 74–92. 2020:bmjnph-2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Coronavirus disease (COVID-19): Food safety and nutrition. 2020; [07/01/2021]. Reference Source\n\nKalan ME, Ghobadi H, Taleb ZB, et al.: COVID-19 and beliefs about tobacco use: an online cross-sectional study in Iran. Environ Sci Pollut Res Int. 2020; 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGülsen A, Yigitbas BA, Uslu B, et al.: The effect of smoking on COVID-19 symptom severity: Systematic review and meta-analysis. Pulm Med. 2020; 2020: 7590207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrimo H, Ito H, Suzuki T, et al.: Reviewing the definition of “elderly”. Geriatr Gerontol Int. 2006; 6(3): 149–58. Publisher Full Text\n\nArshad M, Mahmood SF, Khan M, et al.: Covid -19, misinformation, and antimicrobial resistance. BMJ. 2020; 371: m4501. PubMed Abstract \| Publisher Full Text\n\nNori P, Cowman K, Chen V, et al.: Bacterial and fungal coinfections in COVID-19 patients hospitalized during the New York City pandemic surge. Infect Control Hosp Epidemiol. 2021; 42(1): 84–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSallam M, Dababseh D, Yaseen A, et al.: Conspiracy beliefs are associated with lower knowledge and higher anxiety levels regarding COVID-19 among students at the University of Jordan. Int J Environ Res Public Health. 2020; 17(14): 4915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkdeniz G, Kavakci M, Gozugok M, et al.: A survey of attitudes, anxiety Status, and protective behaviors of the university students during the COVID-19 outbreak in Turkey. Front Psychiatry. 2020; 11: 695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang J, Zheng Y, Gou X, et al.: Prevalence of comorbidities and its effects in patients infected with SARS-CoV-2: a systematic review and meta-analysis. Int J Infect Dis. 2020; 94: 91–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLancet T: COVID-19: a new lens for non-communicable diseases. Lancet. 2020; 396(10252): 649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernández-Galdamez DR, González-Block MÁ, Romo-Dueñas DK, et al.: Increased risk of hospitalization and death in patients with COVID-19 and pre-existing noncommunicable diseases and modifiable risk factors in Mexico. Arch Med Res. 2020; 51(7): 683–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalmer K, Monaco A, Kivipelto M, et al.: The potential long-term impact of the COVID-19 outbreak on patients with non-communicable diseases in Europe: consequences for healthy ageing. Aging Clin Exp Res. 2020; 32(7): 1189–1194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClark A, Jit M, Warren-Gash C, et al.: Global, regional, and national estimates of the population at increased risk of severe COVID-19 due to underlying health conditions in 2020: a modelling study. Lancet Glob Health. 2020; 8(8): e1003–e17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerkman LF, Sekher TV, Capistrant B, et al.: Social networks, family, and care giving among older adults in India. Aging in Asia: Findings from new and emerging data initiatives. National academies Press (US); 2012. Reference Source\n\nMekonnen HS, Azagew AW, Wubneh CA, et al.: Community’s misconception about COVID-19 and its associated factors among Gondar town residents, Northwest Ethiopia. Tropical Medicine and Health. 2020; 48(1): 99. Publisher Full Text\n\nBBC: Why smart people believe coronavirus myths. 2020; [07/01/2021]. Reference Source\n\nMheidly N, Fares J: Leveraging media and health communication strategies to overcome the COVID-19 infodemic. J Public Health Policy. 2020; 41(4): 410–420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin CY, Broström A, Griffiths MD, et al.: Investigating mediated effects of fear of COVID-19 and COVID-19 misunderstanding in the association between problematic social media use, psychological distress, and insomnia. Internet Interv. 2020; 21: 100345. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "82318", "date": "06 Apr 2021", "name": "Shervin Assari", "expertise": [ "Reviewer Expertise Public health expert", "global health expert", "social epidemiologist" ], "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 introduction is written on COVID. The paper, however, is not on COVID but COVID-related misconceptions. Almost nothing is mentioned in the introduction about COVID-related misconceptions, which is not acceptable.\nThe tables are not designed well. There is no descriptive table that describes all aspects of the participants and also shows the correlates of each COVID-related misconception item and correlates of overall measure. The current tables are very small and are not informative at all.\n\nWe also see some results that should be in fact in the methods section. Test of distribution of the variables should not be presented in the results. That is a part of the methods section.\n\nGiven all these issues, the paper needs major revision.\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? 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/10-216
https://f1000research.com/articles/9-1350/v1	19 Nov 20	{ "type": "Research Article", "title": "PET measured hypoxia and MRI parameters in re-irradiated head and neck squamous cell carcinomas: findings of a prospective pilot study", "authors": [ "Julian Rogasch", "Marcus Beck", "Carmen Stromberger", "Frank Hofheinz", "Pirus Ghadjar", "Peter Wust", "Volker Budach", "Holger Amthauer", "Ingeborg Tinhofer", "Christian Furth", "Thula C. Walter-Rittel", "Sebastian Zschaeck", "Julian Rogasch", "Marcus Beck", "Carmen Stromberger", "Frank Hofheinz", "Pirus Ghadjar", "Peter Wust", "Volker Budach", "Holger Amthauer", "Ingeborg Tinhofer", "Christian Furth", "Thula C. Walter-Rittel" ], "abstract": "Background: Tumor hypoxia measured by dedicated tracers like [18F]fluoromisonidazole (FMISO) is a well-established prognostic factor in head and neck squamous cell carcinomas (HNSCC) treated with definitive chemoradiation (CRT). However, prevalence and characteristics of positron emission tomography (PET) measured hypoxia in patients with relapse after previous irradiation is missing. Here we report imaging findings of a prospective pilot study in HNSCC patients treated with re-irradiation. Methods: In 8 patients with recurrent HNSCC, diagnosed at a median of 18 months after initial radiotherapy/CRT, [18F]fluorodeoxyglucose (FDG)-PET/CT (n=8) and FMISO-PET/MRI (n=7) or FMISO-PET/CT (n=1) were performed. Static FMISO-PET was performed after 180 min. MRI sequences in PET/MRI included diffusion-weighted imaging with apparent diffusion coefficient (ADC) values and contrast enhanced T1w imaging (StarVIBE). Lesions (primary tumor recurrence, 4; cervical lymph node, 1; both, 3) were delineated on FDG-PET and FMISO-PET data using a background-adapted threshold-based method. SUVmax and SUVmean in FDG- and FMISO-PET were derived, as well as maximum tumor-to-muscle ratio (TMRmax) and hypoxic volume with 1.6-fold muscle SUVmean (HV1.6) in FMISO-PET. Intensity of lesional contrast enhancement was rated relative to contralateral normal tissue. Average ADC values were derived from a 2D region of interest in the tumor. Results: In FMISO-PET, median TMRmax was 1.7 (range: 1.1-1.8). Median HV1.6 was 0.05 ml (range: 0-7.3 ml). Only in 2/8 patients, HV1.6 was ≥1.0 ml. In FDG-PET, median SUVmax was 9.3 (range: 5.0-20.1). On contrast enhanced imaging four lesions showed decreased and four lesions increased contrast enhancement compared to non-pathologic reference tissue. Median average ADC was 1,060 ×106 mm2/s (range: 840-1,400 ×106 mm2/s). Conclusions: This pilot study implies that hypoxia detectable by FMISO-PET may not be as prevalent as expected among loco-regional recurrent HNSCC. ADC values were only mildly reduced, and contrast enhancement was variable. The results require confirmation in larger sample sizes.", "keywords": [ "radiotherapy", "head and neck squamous cell carcinoma", "hypoxia", "FMISO", "positron emission tomography", "FDG", "PET" ], "content": "Introduction\n\nLocally advanced head and neck squamous cell carcinomas (HNSCC) that are not associated with human papillomavirus (HPV) infections have an unfavorable prognosis. Standard treatment consists of definitive chemoradiation (CRT) with radiation doses of around 70 Gray (Gy). Acute toxicity is considerable and dose limiting in this approach. Nonetheless, about 50% of patients present with local or regional recurrence after definitive CRT1. Re-irradiation is frequently chosen in cases with recurrence at the primary tumor site or regional lymph nodes. Due to radiation-induced long-term sequelae of the surrounding organs at risk, the re-irradiation dose is usually lower than in the primary setting. Consequently, tumors that are apparently radioresistant are treated with lower radiation doses than those applied in the primary situation. Not surprisingly, this approach is associated with a very unfavorable outcome. Especially in HPV negative tumors that are unresectable, re-irradiation is merely a palliative approach2.\n\nOne important factor of radioresistance is tumor hypoxia. Hypoxia is associated with a more aggressive tumor phenotype as shown by various studies of different tumor entities3–5. Additionally, when treating patients with photon radiotherapy, hypoxia leads to a decreased cytotoxic effect of irradiation due to the lower availability of oxygen radicals. This effect can be specified by calculating the oxygen enhancement ratio (OER). Usually the OER is between two and four, which further underlines the important and strong effect of hypoxia in radiotherapy6. One sophisticated method to measure hypoxia non-invasively is positron emission tomography (PET) with hypoxia specific radiotracers. The most commonly used hypoxia radiotracer is [18F]fluoromisonidazole (FMISO)7. PET measured hypoxia provides a substantial and independent prognostic value in patients undergoing primary CRT for HNSCC8–10. Re-oxygenation, measured by repeated hypoxia PET during treatment, seems to be of even greater prognostic relevance in HNSCC with hardly any local tumor control in case of residual tumor hypoxia during the second week of CRT11–14. Given these data, we postulated that PET measured tumor hypoxia should also play an important role in local recurrent HNSCC after prior radiotherapy. To improve patient outcome by increasing re-oxygenation during CRT, a prospective pilot study was initiated to evaluate the effect of fever-range whole-body hyperthermia (FRWBH) on the tumor microenvironment in patients with re-irradiation for HNSCC. FRWBH has been shown to increase tumor perfusion in preclinical models15,16. The rationale of the GKH-TMM trial was to increase tumor perfusion and subsequently reduce tumor hypoxia by adding weekly FRWBH to re-irradiation. The primary endpoint of the trial was feasibility of FRWBH, which will be published separately when mature outcome data of patients are available. Secondary endpoints included changes of hypoxia and perfusion between pre-treatment and second week of fractionated CRT. Due to the lack of PET detectable hypoxia prior to treatment, we were not able to calculate these planned secondary outcome parameters. Here we report the imaging findings of the pretherapeutic PET and MRI scans within this trial.\n\n\nMethods\n\nThe GKH-TMM, ARO-2018-3, study was registered at clinical trials (ClinicalTrials.gov identifier NCT03547388) and has been approved by the local Ethics committee (Charité Ethics committee, campus Virchow, EA2-047-18). All patients provided written informed consent to participate in the study and to publish results in a pseudonymized way.\n\nThe article complies with the reporting guidelines for observational studies (STROBE).\n\nThe GKH-TMM study was a prospective Phase-I study to evaluate the effect of FRWBH on the tumor microenvironment. Inclusion criteria for this study were as follows: unresectable local, regional or loco-regional recurrent non HPV-associated HNSCC with prior high-dose radiotherapy of the head and neck region (either as definitive or as adjuvant CRT or radiotherapy), time interval between previous radiotherapy and recurrence between 6 months and 5 years, complete whole-body staging without evidence of distant metastases by [18F]fluorodesoxyglucose (FDG)-PET/CT, Eastern Cooperative Oncology Group (ECOG) performance status between zero and two, and age between 18 and 75 years.\n\nThe study was designed as a pilot study with ten patients, who were recruited between April 2018 and March 2020. Eligible patients were asked to take part in the trial as a complementary method to routine treatment. The evaluation of tumor microenvironment was performed by pre-therapeutic FMISO-PET in combination with magnetic resonance imaging (MRI) with diffusion weighted imaging (DWI and ADC maps) and contrast enhanced high resolution imaging (post contrast T1w StarVIBE). FMISO-PET was scheduled prior to therapy and repeated at the end of the second week of CRT in case of evidence for pre-therapeutic hypoxia.\n\nEight out of ten patients had pre-therapeutic FMISO-PET images. In two patients, FMISO-PET could not be performed due to logistical reasons. Out of the eight patients, seven patients underwent integrated PET/MRI, while one patient had to be examined by PET/CT due to severe claustrophobia.\n\nAll patients underwent hyperfractionated re-irradiation with two fractions of radiotherapy (1.2 Gy) per day and a minimum of eight hours between each fraction. Total treatment dose was 66 Gy, prescribed to the macroscopic tumor lesions plus 5 mm safety margin. Concomitant chemotherapy was not specified in the protocol but should preferentially include cisplatin due to its increased efficacy at mildly increased temperatures17.\n\nPretherapeutic FDG-PET/CT was performed with a dedicated PET/CT scanner (Philips Gemini TF 16, Philips, Amsterdam, The Netherlands). Patients were required to fast for at least 6h prior to tracer injection, and a blood glucose level ≤130 mg/dl was validated. After intravenous injection of a median of 259 MBq [18F]FDG (interquartile range [IQR], 252 to 296 MBq/kg; median, 4.4 MBq/kg; IQR, 3.9 to 4.9 MBq/kg), static PET acquisition was performed after 80 min (IQR, 68 to 84 min) for 2 or 3 min per bed position from base of skull to proximal femora in supine position (matrix, 144 × 144). PET data were reconstructed iteratively using ordered subset expectation maximization (OSEM; BLOB-OS-TF) with 3 iterations and 33 subsets and time of flight (voxel size, 4.0 × 4.0 × 4.0 mm3) without resolution recovery (point spread function; PSF). Random correction, scatter correction and dead time correction were also included. Attenuation correction was performed based on a non-enhanced low-dose CT (slice thickness, 5 mm).\n\nPretherapeutic FMISO-PET/MRI was performed with a dedicated PET/MRI scanner (Biograph mMR, Siemens Healthcare, Erlangen, Germany). After intravenous administration of 208 MBq (IQR, 185 to 212 MBq) [18F]FMISO (median, 3.5 MBq/kg; IQR, 3.0 to 3.7 MBq/kg), static PET images of the relapse location in the head and neck region were acquired after 180 min (IQR, 172 to 221 min) in one bed position with an acquisition time of 15 min (matrix, 172 × 172). PET data were reconstructed iteratively using OSEM with 3 iterations, 21 subsets and a 3 mm Gaussian in-plane filter (voxel size, 4.17 × 4.17 × 2.03 mm3). Resolution recovery (point spread function) was not applied, and time of flight reconstruction was not available. Random correction, scatter correction and dead time correction were included. MR-based attenuation correction used the 3D CAIPIRINHA HiRes sequence (acceleration factor 5; voxel size, 1.3 × 1.3 × 3.0 mm3). Approximately five minutes after intravenous injection of 0.2 ml/kg of gadoteric acid (Dotarem, Guerbet, Roissy CdG Cedex, France; 0.5 mmol/ml; 4 ml/s, followed by a 25 ml bolus of saline solution), contrast-enhanced imaging was performed by T1w 3D in-plane radial sampling by a fat-suppressed spoiled-Gradient Echo core sequence (StarVIBE; TR, 4.91 ms; TE, 2.14 ms; flip angle (FA), 12°; field of view [FOV], 220 × 220 mm2; voxel size, 0.7 × 0.7 × 1.3 mm3). Diffusion-weighted imaging and apparent diffusion coefficient (ADC) maps of the tumor volume were acquired with a fat-saturated multi-shot readout-segmented echo-planar sequence (TR, 3840 ms; TE1, 61 ms; TE2, 100 ms; b1, 50 s/mm2; b2, 800 s/mm2; EPI factor 82; FA, 180°; FOV, 220 × 220 mm2; voxel size, 1.1 × 1.1 × 4.0 mm3); a pre- and post-contrast T1w Turbo Spin Echo (TSE) sequence (TR 692 ms; TE, 9 ms; FA, 142°; FOV, 180 × 180 mm2; voxel size, 0.6 × 0.6 × 3.0 mm3) and a T2w turbo -inversion recovery magnitude (TIRM) sequence (TR, 4510 ms; TE, 40 ms; FA, 140°; FOV, 200 × 200 mm2; voxel size, 0.6 × 0.6 × 3.0 mm3) were acquired. Parallel imaging was enabled by Generalized Autocalibrating Partially Parallel Acquisition (GRAPPA).\n\nIn a single patient with severe claustrophobia, pretherapeutic FMISO-PET/CT was performed instead of PET/MRI. After intravenous injection of 171 MBq [18F]FMISO (1.8 MBq/kg), PET acquisition was performed with the above-named PET/CT scanner (Philips Gemini TF 16) after 197 min for 15 min in a single bed position (matrix, 144 × 144). PET data were reconstructed with identical parameters as described for FDG-PET/CT imaging. Additionally, a venous-phase contrast enhanced, diagnostic CT (automated tube current modulation; maximum tube current-time product, 200 mAs; tube voltage, 120 kV; FOV, 395 × 395 mm2; voxel size, 0.77 × 0.77 × 3.0 mm3) was performed 100 s after intravenous injection of 120 ml of Imeron 350 (bolus rate, 2 ml/s; Bracco Imaging Deutschland GmbH, Konstanz, Germany).\n\nImage analysis was performed as previously published and as shortly described in the following passage using ROVER software (version 3.0.50h; ABX, Radeberg, Germany; available freely for research purposes on request) without any preprocessing of the final image data18. After co-registration of diagnostic FDG-PET/CT images and FMISO-PET/MRI or PET/CT images by the mutual information algorithm, correct alignment of the primary tumor volume was verified, and co-registration was corrected, if deemed necessary. The tumor (primary tumor recurrence or lymph node) was semi-automatically delineated in the PET images using a background-adapted threshold-based algorithm19,20. Central necrotic tumor areas or areas that were suspicious of tumor in MRI or CT were subsequently included manually to the primary tumor/lymph node volume. A reference region of interest (ROI) was delineated in the deep neck muscles contralateral to the primary tumor with a spheroid of 16 mm diameter. The ROIs of the tumor and of the muscle were transferred to the hypoxia PET for calculation of the maximum standardized uptake value (SUVmax; normalized to the body weight) of the tumor, of the tumor-to-muscle ratio (TMR) and for thresholding of hypoxic volumes, e.g. the commonly used hypoxic volume with uptake above 1.6 times of the average muscle uptake (HV1.6).\n\nThe intensity of lesional contrast enhancement was rated by an experienced radiologist (9 years of experience) relative to the corresponding contralateral normal tissue using either the contrast-enhanced T1w images (StarVIBE sequence; n=7 patients) or the contrast-enhanced CT data (n=1 patient). Intensity was rated on a three point Likert-type item as “less intense”, “comparable intensity” or “more intense” compared to the contralateral reference tissue. Mean ADC values of the tumor lesions were measured by the same radiologist using representative 2D region of interest placed in the tumor volume.\n\nStatistical analysis was performed using SPSS (version 26, IBM, Armonk, NY, USA). On the basis of the small sample size, non-normal data distribution was assumed, and descriptive data were expressed as median, IQR and range, unless otherwise specified.\n\n\nResults\n\nEight patients with available pretherapeutic FMISO-PET imaging are reported here (PET/MRI, n=7; PET/CT, n=1). Characteristics of patients and treatment are summarized in Table 1.\n\nabbreviations: OC = oral cavity, OP = oropharynx, HP = hypopharynx, def CRT = definitive chemoradiation, adj CRT = adjuvant chemoradiation, T = local recurrence, N = regional recurrence.\n\nAssessment of tumor hypoxia in static FMISO-PET images three hours post injection revealed absence of detectable hypoxia in almost all patients. The median FMISO TMRmax was 1.7 (IQR, 1.3 to 1.8; range, 1.1 to 1.8). Only two patients showed hypoxic volumes that were delineable using the 1.6-fold average background muscle activity threshold (HV1.6). The median HV1.6 of all 8 patients was 0.05 ml (IQR, 0 to 0.33 ml; range, 0 to 7.3 ml). No lesion showed a TMRmax >2.0. Notably, especially the largest recurrent lesions did not show any detectable hypoxia (HV1.6 = 0 ml).\n\nAll lesions showed unequivocal and extensive FDG uptake (median MTV, 11.2 ml; IQR, 6.0 to 16.8 ml; range, 1.0 to 51.7 ml). The median SUVmax was 9.3 (IQR, 8.4 to 13.6; range, 5.0 to 20.1), and the median SUVmean was 6.3 (IQR, 5.8 to 8.6; range, 3.0 to 12.6).\n\nDetails of FDG-PET and FMISO-PET parameters for all patients are shown in Table 2. Figure 1 shows exemplary FDG-PET/CT scans and FMISO-PET/MRI scans.\n\nAbbreviations: MTV = metabolic tumor volume, SUVmax = maximum standardized uptake value, SUVmean = mean standardized uptake value, TMRmax = maximum tumor-to-muscle ratio, HV1.6 = hypoxic volume with a threshold of 1.6 times the average background/ muscle uptake; ADC = apparent diffusion coefficient.\n\n*Only a single voxel above the threshold\n\nExamples of patients with local recurrence of a larynx cancer (left; patient #2), cervical lymph node metastasis (middle; patient #3) or oropharyngeal cancer relapse (right; patient #4), respectively. Fused FDG-PET/CT above, corresponding contrast-enhanced T1w MRI (StarVIBE) in the middle, and FMISO-PET below. In each patient, FDG uptake of the recurrent lesion is extensive and high, while specific FMISO uptake was absent (HV1.6 ≤1.0 ml).\n\nTumor contrast enhancement was less intense compared to the contralateral reference in four of eight patients (lymph node only, n=1; primary tumor recurrence ± lymph node, n=3; Table 2). In the remaining four patients, enhancement was more intense, at least in parts of the lesion, compared to the reference tissue (primary tumor ± lymph node, n=4).\n\nThe median average ADC value was 1,060 × 106 mm2/s (IQR, 1,025 to 1,211 × 106 mm2/s; range, 840 to 1,400 × 106 mm2/s).\n\n\nDiscussion\n\nHere we report pretrial data from the first trial that evaluated tumor hypoxia by specific hypoxia PET in previously irradiated, recurrent HNSCC. Data on hypoxia measured by PET tracers in the recurrent disease situation after radiotherapy is absent. To the best of our knowledge, there is only one study that investigated various tumor locations and histologies with hypoxia PET. In the mentioned study, the authors describe one patient with recurrent adenocarcinoma of the uterus who showed a relatively high uptake of FMISO. It is not clear if this patient was previously treated with radiotherapy/CRT or presented recurrence after initial surgery21,22.\n\nVery surprisingly, the incidence of hypoxia, determined by FMISO-PET, was extremely low in our study investigating recurrent HNSCC. The largest analysis of hypoxia PET images from five European centers established the hypoxia parameter TMRmax 2.0 as a suitable value to distinguish high- and low-risk treatment-naive patients. The prevalence of hypoxia (defined as TMRmax > 2.0) was relatively high in primary tumors. PET hypoxia was found in 48% of all patients; among those who received FMISO-PET, 61% showed hypoxic tumors/lesions18. In the present trial of previously irradiated HNSCC, none of the eight included patients showed hypoxic tumors according to these cut-off values, despite the fact that patients with relatively large recurrent lesions were included (see Table 2). This finding may indicate that microenvironmental mechanisms of tumor radioresistance might be very different between treatment-naïve and previously irradiated tumors. This also seems to be the case with genetic alterations23,24. Dose painting for re-irradiation does not seem feasible in FMISO-PET due to the low tracer uptake, i.e. low rates of hypoxia in this patient collective [25,26,p.33].\n\nIn addition to late static FMISO-PET images, dynamic acquisition from 0 to 40 min post injection has been proposed. Combining dynamic and static PET images for a voxel-wise 2-compartment model of [18F]FMISO accumulation, Thorwarth et al. were able to identify patients with treatment-naïve HNSCC with either favorable or very poor local tumor control after radiotherapy. The derived dynamic parameter MFMISO was superior to TMRmax in predicting local tumor control27. Dynamic FMISO-PET acquisition was not available in our patient collective. However, it is questionable if dynamic data would provide any added value in lesions that are negative on late FMISO images.\n\nIn addition to mostly absent hypoxia, ADC values were relatively high in all tumors with a median of >1,000 × 106 mm2/s, which corresponds to only mildly restricted diffusion. Only in one tumor (patient #7), the average ADC was below 1,000 × 106 mm2/s. These MRI findings are consistent with the literature28–30. Hwang et al. reported average ADC values of 1,200 × 106 mm2/s in patients with recurrent HNSCC after initial treatment compared to an average of 1,650 × 106 mm2/s in lesions corresponding to post-therapeutic changes30. Previous reports on the association between tissue hypoxia in different tumor entities and properties in DWI or corresponding ADC maps are inconsistent. Hino-Shishikura et al. reported a negative correlation between ADC values and the SUVmax and tumor-to-background ratios in brain tumors obtained with hypoxia-specific PET imaging31. Hompland et al. demonstrated a weak negative correlation between ADC values and hypoxic fraction assessed by pimonidazole tissue staining in malignant melanoma xenografts. Moreover, ADC values as high as 1,000 × 106 mm2/s were only observed in tumors with hypoxic volumes <10% of the total tumor volume32. In contrast, a positive correlation was reported in murine melanoma tumors (but imaging was performed ex vivo)33. Swartz et al. and Carmona-Bozo et al. found no correlation between hypoxia and ADC values in oropharyngeal and breast cancer lesions, respectively34,35.\n\nContrast enhancement (StarVIBE MRI sequence or contrast-enhanced CT) was atypically low in 4/8 lesions, and hypoxia was also absent in these tumors (Table 2). Among the 4/8 lesions with substantial contrast enhancement (at least in the tumor periphery), some showed small hypoxic proportions as determined by the FMISO HV1.6. Gerstner et al. found that high lesional cerebral blood flow in glioblastoma quantified by dynamic susceptibility contrast MRI correlated positively with the hypoxic volume in FMISO-PET. The authors postulated that abnormal tumor vasculature contributes to hypoxia36. This may explain the lack of substantial and extensive contrast enhancement in the current mostly non-hypoxic tumors. However, the current sample size was too small to conclude if there is a negative correlation between contrast enhancement and tumor hypoxia. Dynamic contrast MRI sequences were not evaluated in this study, and contrast kinetics – as described by 36 – could not be assessed.\n\nIn summary, the current pilot study implies that relevant hypoxia, which is detectable by static FMISO-PET, may not be as prevalent among recurrent lesions of HNSCC as expected. However, studies with substantially larger sample sizes would be required for a definite statement or more differentiated analyses (e.g., dependence of hypoxia on the type and localization of the recurrent lesion). This is also true for the investigation of a possible association of low tumor hypoxia with high ADC values and low contrast enhancement in these recurrent lesions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article (concerning absolute values of imaging parameters). Original imaging data cannot be made publicly available due to concerns regarding data protection since the imaging data contained several high-resolution methods with unique personal properties. German data protection is not only restricted by Ethics committees, but also by dedicated data protection agencies. One general concern of German data protection agencies, including Charité data protection commission, is that image information that is uploaded to public available repositories might be accessed by countries that do not comply with current data protection agreements. Therefore, pseudonymized data can be shared upon reasonable request by contacting the corresponding author. Access to the data will be granted after ethical approval and after ensuring that adequate data protection is adhered by the country of the applicant.", "appendix": "References\n\nBudach V, Tinhofer I: Novel prognostic clinical factors and biomarkers for outcome prediction in head and neck cancer: a systematic review. Lancet Oncol. 2019; 20(6): e313–e326. PubMed Abstract \| Publisher Full Text\n\nCacicedo J, Navarro A, Alongi F, et al.: The role of re-irradiation of secondary and recurrent head and neck carcinomas. Is it a potentially curative treatment? A practical approach. Cancer Treat Rev. 2014; 40(1): 178–189. PubMed Abstract \| Publisher Full Text\n\nHockel M, Schlenger K, Aral B, et al.: Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 1996; 56(19): 4509–4515. PubMed Abstract\n\nChang J, Erler J: Hypoxia-mediated metastasis. Adv Exp Med Biol. 2014; 772: 55–81. PubMed Abstract \| Publisher Full Text\n\nEisinger-Mathason TSK, Zhang M, Qiu Q, et al.: Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis. Cancer Discov. 2013; 3(10): 1190–1205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWenzl T, Wilkens JJ: Theoretical analysis of the dose dependence of the oxygen enhancement ratio and its relevance for clinical applications. Radiat Oncol. 2011; 6: 171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZschaeck S, Steinbach J, Troost EGC: FMISO as a Biomarker for Clinical Radiation Oncology. Recent Results Cancer Res. 2016; 198: 189–201. PubMed Abstract \| Publisher Full Text\n\nHorsman MR, Mortensen LS, Petersen JB, et al.: Imaging hypoxia to improve radiotherapy outcome. Nat Rev Clin Oncol. 2012; 9(12): 674–687. PubMed Abstract \| Publisher Full Text\n\nMortensen LS, Johansen J, Kallehauge J, et al.: FAZA PET/CT hypoxia imaging in patients with squamous cell carcinoma of the head and neck treated with radiotherapy: results from the DAHANCA 24 trial. Radiother Oncol. 2012; 105(1): 14–20. PubMed Abstract \| Publisher Full Text\n\nWelz S, Mönnich D, Pfannenberg C, et al.: Prognostic value of dynamic hypoxia PET in head and neck cancer: Results from a planned interim analysis of a randomized phase II hypoxia-image guided dose escalation trial. Radiother Oncol. 2017; 124(3): 526–532. PubMed Abstract \| Publisher Full Text\n\nZips D, Zö phel K, Abolmaali N, et al.: Exploratory prospective trial of hypoxia-specific PET imaging during radiochemotherapy in patients with locally advanced head-and-neck cancer. Radiother Oncol. 2012; 105(1): 21–28. PubMed Abstract \| Publisher Full Text\n\nLöck S, Perrin R, Seidlitz A, et al.: Residual tumour hypoxia in head-and-neck cancer patients undergoing primary radiochemotherapy, final results of a prospective trial on repeat FMISO-PET imaging. Radiother Oncol. 2017; 124(3): 533–540. PubMed Abstract \| Publisher Full Text\n\nWiedenmann NE, Bucher S, Hentschel M, et al.: Serial [18F]-fluoromisonidazole PET during radiochemotherapy for locally advanced head and neck cancer and its correlation with outcome. Radiother Oncol. 2015; 117(1): 113–117. PubMed Abstract \| Publisher Full Text\n\nHu M, Xie P, Lee NY, et al.: Hypoxia with 18F-fluoroerythronitroimidazole integrated positron emission tomography and computed tomography (18F-FETNIM PET/CT) in locoregionally advanced head and neck cancer: Hypoxia changes during chemoradiotherapy and impact on clinical outcome. Medicine (Baltimore). 2019; 98(40): e17067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWinslow TB, Eranki A, Ullas S, et al.: A pilot study of the effects of mild systemic heating on human head and neck tumour xenografts: Analysis of tumour perfusion, interstitial fluid pressure, hypoxia and efficacy of radiation therapy. Int J Hyperthermia. 2015; 31(6): 693–701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRich LJ, Winslow TB, Alberico RA, et al.: Enhanced tumour perfusion following treatment with water-filtered IR-A radiation to the thorax in a patient with head and neck cancer. Int J Hyperthermia. 2016; 32(5): 539–542. PubMed Abstract \| Publisher Full Text\n\nUrano M, Kahn J, Kenton LA: The effect of cis-diamminedichloroplatinum(II) treatment at elevated temperatures on murine fibrosarcoma, FSa-II. Int J Hyperthermia. 1990; 6(3): 563–570. PubMed Abstract \| Publisher Full Text\n\nZschaeck S, Löck S, Hofheinz F, et al.: Individual patient data meta-analysis of FMISO and FAZA hypoxia PET scans from head and neck cancer patients undergoing definitive radio-chemotherapy. Radiother Oncol. 2020; 149: 189–196. PubMed Abstract \| Publisher Full Text\n\nHofheinz F, Pötzsch C, Oehme L, et al.: Automatic volume delineation in oncological PET. Evaluation of a dedicated software tool and comparison with manual delineation in clinical data sets. Nuklearmedizin. 2012; 51(1): 9–16. PubMed Abstract \| Publisher Full Text\n\nHofheinz F, Langner J, Petr J, et al.: An automatic method for accurate volume delineation of heterogeneous tumors in PET. Med Phys. 2013; 40(8): 082503. PubMed Abstract \| Publisher Full Text\n\nTachibana I, Nishimura Y, Shibata T, et al.: A prospective clinical trial of tumor hypoxia imaging with 18F-fluoromisonidazole positron emission tomography and computed tomography (F-MISO PET/CT) before and during radiation therapy. J Radiat Res. 2013; 54(6): 1078–1084. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTachibana I, Nishimura Y, Hanaoka K, et al.: Tumor Hypoxia Detected by 18F-fluoromisonidazole Positron Emission Tomography (FMISO PET) as a Prognostic Indicator of Radiotherapy (RT) Anticancer Res. 2018; 38(3): 1775–1781. PubMed Abstract \| Publisher Full Text\n\nHedberg ML, Goh G, Chiosea SI, et al.: Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma. J Clin Invest. 2016; 126(4): 1606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith JD, Birkeland AC, Rosko AJ, et al.: Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma. Head Neck. 2019; 41(2): 423–428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkjøtskift T, Evensen ME, Furre T, et al.: Dose painting for re-irradiation of head and neck cancer. Acta Oncol. 2018; 57(12): 1693–1699. PubMed Abstract \| Publisher Full Text\n\nSaksø M, Primdahl H, Johansen J, et al.: DAHANCA 33: functional image-guided dose-escalated radiotherapy to patients with hypoxic squamous cell carcinoma of the head and neck (NCT02976051). Acta Oncol. 2020; 59(2): 208–211. PubMed Abstract \| Publisher Full Text\n\nThorwarth D, Welz S, Mö nnich D, et al.: Prospective Evaluation of a Tumor Control Probability Model Based on Dynamic 18F-FMISO PET for Head and Neck Cancer Radiotherapy. J Nucl Med. 2019; 60(12): 1698–1704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim S, Loevner L, Quon H, et al.: Diffusion-weighted magnetic resonance imaging for predicting and detecting early response to chemoradiation therapy of squamous cell carcinomas of the head and neck. Clin Cancer Res. 2009; 15(3): 986–994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeltenburg B, Driessen JP, Vasmel JE, et al.: Pretreatment ADC is not a prognostic factor for local recurrences in head and neck squamous cell carcinoma when clinical T-stage is known. Eur Radiol. 2020; 30(2): 1228–1231. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang I, Choi SH, Kim YJ, et al.: Differentiation of recurrent tumor and posttreatment changes in head and neck squamous cell carcinoma: application of high b-value diffusion-weighted imaging. AJNR Am J Neuroradiol. 2013; 34(12): 2343–2348. PubMed Abstract \| Publisher Full Text\n\nHino-Shishikura A, Tateishi U, Shibata H, et al.: Tumor hypoxia and microscopic diffusion capacity in brain tumors: a comparison of 62Cu-Diacetyl-Bis (N4-Methylthiosemicarbazone) PET/CT and diffusion-weighted MR imaging. Eur J Nucl Med Mol Imaging. 2014; 41(7): 1419–1427. PubMed Abstract \| Publisher Full Text\n\nHompland T, Ellingsen C, Galappathi K, et al.: DW-MRI in assessment of the hypoxic fraction, interstitial fluid pressure, and metastatic propensity of melanoma xenografts. BMC Cancer. 2014; 14: 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerša I, Bajd F, Savarin M, et al.: Multiparametric High-Resolution MRI as a Tool for Mapping of Hypoxic Level in Tumors. Technol Cancer Res Treat. 2018; 17: 1533033818797066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarmona-Bozo JC, Manavaki R, Woitek R, et al.: Hypoxia and perfusion in breast cancer: simultaneous assessment using PET/MR imaging. Eur Radiol. 2020. PubMed Abstract \| Publisher Full Text\n\nSwartz JE, Driessen JP, van Kempen PMW, et al.: Influence of tumor and microenvironment characteristics on diffusion-weighted imaging in oropharyngeal carcinoma: A pilot study. Oral Oncol. 2018; 77: 9–15. PubMed Abstract \| Publisher Full Text\n\nGerstner ER, Zhang Z, Fink JR, et al.: ACRIN 6684: Assessment of Tumor Hypoxia in Newly Diagnosed Glioblastoma Using 18F-FMISO PET and MRI. Clin Cancer Res. 2016; 22(20): 5079–5086. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75138", "date": "02 Dec 2020", "name": "Jacqueline Kelly", "expertise": [ "Reviewer Expertise Radiation therapy", "radiation 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\nIn this manuscript, the authors explore the novel and important question of hypoxia levels in recurrent HNSCC that has previously been irradiated with high-dose RT. Because hypoxia has been shown to be prognostic in the definitive HNSCC RT setting, and as re-RT is toxic and may be effective in only very select patient populations, the knowledge gained from this study could be used to select appropriate candidates who may be more likely to have a durable response to re-RT. Surprisingly, the incidence of hypoxia was extremely low in the present study, albeit in a very limited number of patients in a heterogeneous patient population.\n\nThe paper is well-written, with clear methodology, presentation of the results, and an appropriate discussion. In viewing Table 1, it appears initial treatment was uniform, with def CRT patients receiving 70.4 Gy and adj CRT receiving 63.9. Since these doses are clearly not achieved using the commonly used 2Gy/fraction, it may be worth mentioning the fractionation schedule used with the initial treatment course. Altered fractionation schedules have at least theoretical advantages relative to OER effects and thus this should simply be noted.\n\nI would also recommend in the conclusion reiterating that these findings apply to only the HPV(-) HNSCC population.\n\nOverall, a soundly conducted study, unfortunately with conclusions limited by a very low number of patients.\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": "6414", "date": "16 Mar 2021", "name": "Sebastian Zschaeck", "role": "Author Response", "response": "We thank the reviewer for her useful comments. We included a description of the initial treatment schedule (definitive and adjuvant CRT). Both regimes are applied according to clinical guidelines, using simultaenous integrated boost. However, the slight increase of single dose (2.13 or 2.2 Gray) is probably within the range that can be regarded normo-fractionated (1.8 to 2.0 or 2.2 Gray, according to definition), therefore we do not expect a major impact on OER. We added the limitation that these finding apply only to HPV negative HNSCC in the abstract and in the results section." } ] }, { "id": "79089", "date": "01 Mar 2021", "name": "Maria Picchio", "expertise": [ "Reviewer Expertise Molecular Imaging in 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\nThe present manuscript deals with a very interesting issue reporting preliminary data on the use of a PET radiotracer, 18F-FMISO, to measure hypoxia in head and neck squamous cell carcinoma patients with relapse after previous irradiation. Eight patients have been included, 7/8 studied by FMISO PET/MRI and 1/8 by using PET/CT.\nAs for strengths, the paper is very well written and methodologically correct. In addition, although 18F-FMISO is a well-established prognostic factor in head and neck squamous cell carcinomas treated with definitive chemoradiation, data on prevalence and characteristics of PET measured hypoxia in patients with relapse after previous irradiation are still missing. This paper report imaging findings of pretherapeutic PET and MRI scans within a prospective trial.\nAs weakness, the number of patients included is very limited and the population is heterogeneous. In addition, two different scanners have been used (7 PET/MRI and 1 PET/CT). The rationale and the use of FDG in this preliminary evaluation has not been definitely defined and commented.\nIt should be also clarified why 1.6-fold muscle has been considered to define hypoxic volume. In literature other values have been also reported (i.e. 1.2; 1.4)\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": "6416", "date": "16 Mar 2021", "name": "Sebastian Zschaeck", "role": "Author Response", "response": "We thank the reviewer for her helpful and positive comments As weakness, the number of patients included is very limited and the population is heterogeneous. In addition, two different scanners have been used (7 PET/MRI and 1 PET/CT). The rationale and the use of FDG in this preliminary evaluation has not been definitely defined and commented. We agree that the number of patients is limited and somehow heterogenous (especially regarding surgery). However, one has to bear in mind the low compliance of this group of HPV negative HNSCC and local relapse. Therefore it was already relatively difficult to recuit these patients into a trial with time consuming additional imaging - as a side note, only 1/3 of all screened patients could be recruited to this trial. Usually the same PET-MRI scaner was used, however due to severe claustrophobia one patient was scanned on the PET-CT. FDG is routinely used for re-irradiation in our department for staging and better target delineation. Additionally FDG PET should be used to define regions of FDG and hypoxia overlap within the macroscopic recurrent tumor. We added these information accordingly. It should be also clarified why 1.6-fold muscle has been considered to define hypoxic volume. In literature other values have been also reported (i.e. 1.2; 1.4) We fully agree that other thresholds have been reported. The 1.6 fold score is taken from the current meta-analysis. However, even this relatively high threshold led to scattered hypoxia volumes inside and outside the macroscopic tumor lesions. For better ilustration we included images of the two patients with (small) HV1.6 volumes inside the macrospopic tumor (#4 and #6) as supplementary information and hope that this visualizes that a true hypoxic volume can probably only defined in patient #6." } ] } ]	1	https://f1000research.com/articles/9-1350
https://f1000research.com/articles/10-215/v1	16 Mar 21	{ "type": "Data Note", "title": "The complete genome sequences of two species of seventeen-year cicadas: Magicicada septendecim and Magicicada septendecula", "authors": [ "Harold B. White", "Stacy Pirro", "Harold B. White" ], "abstract": "The genus Magicicada (Hemiptera: Cicadidae) includes the periodical cicadas of Eastern North America. Spending the majority of their long lives underground, the adult cicadas emerge every 13 or 17 years to spend 4-6 weeks as adult to mate. We present the whole genome sequences of two species of 17-year cicadas, Magicicada septendecim and Magicicada septendecula. The reads were assembled by a de novo method followed by alignments to related species. Annotation was performed by GeneMark-ES. The raw and assembled data is available via NCBI Short Read Archive and Assembly databases.", "keywords": [ "Genome", "assembly", "arthropoda", "insecta", "hemiptera" ], "content": "Introduction\n\nPeriodical cicadas in North America spend 13 or 17 years in the larval stage underground, and emerge in very large numbers for 4–6 weeks to mate and lay eggs. This strategy, known as “predator satiation” is intended to ensure that after all predators have eaten as much as possible, most cicadas will survive. (Williams & Simon, 1995). The emergence occurring in prime-numbered years is thought to be a mechanism to avoid competition between species for egg-laying sites and accidental cross-species mating as the emergence of the 13- and 17-year cicadas would only coincide once every 221 years (Tanaka et al., 2009).\n\nThe length of time spent in the larval stage is thought to be dependent on a single gene, although this has not yet been demonstrated at the genomic level (Cox & Carlton, 1991).\n\nComplete genome sequences for these two species will assist with studies on taxonomy, longevity, and the timing of long-term larval development.\n\n\nMethods\n\nWild caught specimens of Magicicada septendecim and Magicicada septendecula from a small premature emergence of Brood X (2017) collected in Newark, Delaware, USA were used in this study. DNA extraction was performed using the Qiagen DNAeasy genomic extraction kit for tissue, using the standard process. A paired-end sequencing library was constructed using the Illumina TruSeq kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina Hi-Seq platform in paired-end, 2 × 150bp format.\n\nThe resulting fastq files were trimmed of adapter/primer sequence and low-quality regions with Trimmomatic v0.33 (Bolger et al., 2014). The trimmed sequence was assembled by SPAdes v2.5 (Bankevich et al., 2012) followed by a finishing step using RagTag v1.0.0 (Alonge, 2020) to make additional contig joins based on conserved regions in related insect species: Rhopalosiphum maidis (GCA_003676215), Euschistus heros (GCA_003667255), and Aphis glycines (GCA_009928515). Default parameters were used for all assembly steps.\n\nAnnotation was performed using GeneMark-ES v2.0 (Lomsadze et al., 2005). Annotation was performed fully de novo without a curated training set and using default parameters.\n\n\nResults\n\nThe genome assembly for Magicicada septendecim yielded a total sequence length of 1,579,033,894 with an N50 value of 983 kb and 27,124 gene models.\n\nThe genome assembly of Magicicada septendecula yielded 1,585,977,997 with an N50 value of 281 kb and 28,651 gene models.\n\n\nData availability\n\nGenome data available from NCBI’s Short Read Archive (SRA):\n\nMagicicada septendecim, Accession number SRR6782667: https://www.ncbi.nlm.nih.gov/sra/SRR6782667\n\nMagicicada septendecula, Accession number SRR6792649: https://www.ncbi.nlm.nih.gov/sra/SRR6792649\n\nAssembled genomes available from NCBI’s Assembly database:\n\nMagicicada septendecim, Accession number GCA_011326945: https://www.ncbi.nlm.nih.gov/assembly/GCA_011326945.1/\n\nMagicicada septendecula, Accession number GCA_011763675: https://www.ncbi.nlm.nih.gov/assembly/GCA_011763675.1/", "appendix": "Author information\n\nHarold B. White is now retired from Department of Chemistry and Biochemistry, University of Delaware.\n\n\nReferences\n\nAlonge M: Ragtag: Reference-guided genome assembly correction and scaffolding. GitHub archive. 2020.\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012; 19(5): 455–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCox RT, Carlton CE: Evidence of genetic dominance of the 13-year life cycle in periodical cicadas (Homoptera: Cicadidae: Magicicada spp.). Am Midl Nat. 1991; 125(1): 63–74. Publisher Full Text\n\nLomsadze A, Ter-Hovhannisyan V, Chernoff YO, et al.: Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 2005; 33(20): 6494–6506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTanaka Y, Yoshimura J, Simon C, et al.: Allee effect in the selection for prime-numbered cycles in periodical cicadas. Proc Natl Acad Sci U S A. 2009; 106(22): 8975–8979. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams KS, Simon C: The ecology, behavior, and evolution of periodical cicadas. Annu Rev Entomol. 1995; 40(1): 269–295. Publisher Full Text" }	[ { "id": "81859", "date": "12 Apr 2021", "name": "Hu Li", "expertise": [ "Reviewer Expertise comparative genomics", "phylogenomics", "population 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\nThe authors presented the genome assembly of Magicicada septendecim and Magicicada septendecula using Illumina platform, which is of great help to the Magicicada evolution study. Using the RagTag method to correct, orient and scaffold the assembled sequence is a novel idea for the short reads assembly. However, there are still some limitations which might be improved.\nIn this manuscript, detailed information of sequencing samples counts, male or female, were not given. It also lacks the amount of data sequenced by Illumina platform, genome assembly and annotation completeness assessed by BUSCO, and repetitive sequence annotation.\nAdditionally, one of my major concerns is that, we usually use a long reads platform such as PacBio or ONT to sequence the genome; could short sequence be assembled completely? Although de novo method by using SPAdes and RagTag software in this study works well to assemble short sequences, when aligned to related species, contigs lower than 1 kb were filtered and some genome sequences might be lost.\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": "83461", "date": "12 May 2021", "name": "Shuai Zhan", "expertise": [ "Reviewer Expertise Insect 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\nThe fascinating life cycle of Periodical cicadas attracts public and scientific interests from around the globe. In this Data note, the authors report the genome references of two such cicadas species, Magicicada septendecim and M. septendecula. The available genomes would no doubt benefit the community in various areas. However, it is difficult to judge the actual quality of the yielded genomes, due to the incomplete method description and lacking of necessary quality evaluation. I think this is important for the community to determine whether the resource is reliable and to what extent.\nHere are some specific concerns:\nIt is unclear to me how the genome is generated, which is critical to assess whether the assembling approach is reasonable. The authors should provide key information such as how many libraries being generated for a single species and the insert size information for the library(s). It seems that only one paired-end library was constructed, thus I cannot imagine that how a single library would yield genome assemblies for two species and how the assembly could reach a scaffold level with the N50 size as long as hundreds of Kb.\n\nThe authors claimed that the assembling approach includes the alignment step to related species, such as some aphids. This is uncommon for de novo assembling a genome reference, particularly for an insect species, given the high level of divergence between families and species.\n\nThe current note only presents the genome size and N50 size, but lacks of most other key properties to assess the quality of a genome assembly, such as the expected genome size, the completeness, the redundancy ratio, the GC ratio, the potential contamination ratio, etc.\n\nThe current gene prediction method is unsatisfying and could be substantially improved by applying an integrated approach. Using GeneMark alone is uncommon for annotating a large eukaryotic genome. Instead, this gene predictor is more common to be used as partial ab initio evidence of a comprehensive gene annotation, which is usually achieved by combing transcriptome, homology of related species, and several lines of ab initio signatures (e.g. GeneMark and other predictors).\n\nI'm not sure whether 'data note' has a word limit or some special requirements, but the current form of the manuscript is obviously too dense. To allow replication by other researchers, the current method section should be provided with necessary details, such as the parameters for each software.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-215
https://f1000research.com/articles/10-213/v1	16 Mar 21	{ "type": "Research Article", "title": "A bibliometric study on the research outcome of Brazil, Russia, India, China, and South Africa", "authors": [ "Aparna I Narayan", "Bharti Chogtu", "Manthan Janodia", "Santhosh Krishnan Venkata", "Aparna I Narayan", "Bharti Chogtu", "Manthan Janodia" ], "abstract": "Background: Publication is one of the quantitative measures of countries' contribution to research and innovation. This paper attempts to understand the publication related information of the BRICS countries (Brazil, Russia, India, China, and South Africa). Methods: Detailed analysis of publications on the basis of collaboration, research area, number of publications, percentage of Gross Domestic Product (GDP) spent on research, and citation is presented in the paper. An attempt is also made to understand the relations between each of the parameters and the overall performance of the country. Results: Times Higher Education global ranking is considered as a measure to validate the claims of this paper. This study shows that among the BRICS nations, China with the highest percentage of GDP spent on research has also the highest number of researchers and publication output whereas South Africa excels in terms of number of international collaborative publications and publications in high impact journals. This article has highlighted the distribution of publications as per the subject area with India leading in the area of Computer Science. Discussion: Results showed a strong relationship between each of the parameters discussed on the research performance of a country.", "keywords": [ "BRICS", "Collaboration", "Scopus", "Field Weighted Citation Impact" ], "content": "Introduction\n\nThe BRICS (Brazil, Russia, India, China, and South Africa) countries are emerging as major influencers of geopolitical changes. BRICS was referred to for the first time in 2001 in a report titled Building Better Global Economic BRICS by Goldman Sachs economist Jim O’Neill. With the success of BRICS over the years, scientific literature has tried to have an improved understanding of these economies.1 In 2011 a total of 17.3% of research papers in science and engineering were published by BRICS countries compared to just 7.6% in 1995. This accounted for a 233.4% increase in publication output in 2011 compared to 1995.2 Globally, Brazil ranked 13th in terms of peer-reviewed papers produced between 2011 and 2016. Expenditure on research and development (R&D) as a percentage of Gross Domestic Product (GDP) was 1.17% in 2014 whereas citation impact has remained below average during the same period but improved between 2011 (0.73) and 2016 (0.86), an increase of 18%.3 Russia had set aside a budget of 70 billion roubles (approx. US $3 billion) in 2018 while doubling its scientific paper output between 2006 and 2016.4 A total of 74,697 papers (research, review articles, and conference papers) were published by the Russian Federation in Scopus-indexed journals with a compound annual growth rate (CAGR) of 8% between 2006 and 2016, more significantly between 2012 and 2016 with a CAGR of 15.2%, while 54,669 papers were published in Web of Science (WoS) journals with CAGR of 5.9% during 2006-2016 and 12.9% between 2012-2016.5 Russia and China are predominantly single funding agencies, whereas Brazil and India have diversified funding sources. It was also found that research publications from Russia with foreign funding received more visibility and are published in journals with an average mean impact factor 1.9 times greater than Russian-funded publications in WoS.6 Russia spends 1% of national income on R&D, whereas public institutions conduct about 75% of research.7 In recent years, China is second only to the United States (US) in terms of R&D investment. It spent around 2.08% of the national GDP on R&D in 2015. Chinese Academy of Sciences (CAS), Ministry of Science and Technology (MOST), and National Natural Science Foundation of China (NSFC) are the three major funding agencies in the country.8 Based on recent data, South Africa spent about 0.8% of GDP on R&D.\n\nThe government of South Africa also aims to increase this spending to 1.5% of GDP in the ensuing decade.9 In the five-year block of 2013-2017, researchers from South Africa increased their average growth rate in WoS publications by 13.12%, almost double that of the world average of 6.36%.10 South Africa as a country is ranked 18th in the world in social sciences and arts and humanities; 33rd in life sciences; 38th in physical sciences and 40th in technology out of 198 countries studied. However, the total number of papers published by South Africa during 1995-2016 is approximately 31,651 for social sciences and 37,847 for technology. Though by sheer number the technology domain has produced more papers, the ranking for this domain for SA is way below 18 for social sciences with a much lesser number of papers. Similarly, for the arts and humanities field, with 8,842 papers South Africa’s global rank was 18.11 The journal publication output in 2016 increased by 4.5% compared to 2015, which was still lower than 6.4% during 2014-15. The majority of papers from researchers were in the fields of science, engineering, and technology, which accounted for 58.44% of total publications.12 R&D expenditure for India tripled in the last decade; from Rs 24,117 crores in 2004-05 to Rs 85,326 crores in 2014-15. However, as a percentage of GDP, the R&D expenditure remained stagnant at 0.7% during the same period. Of total investment in R&D, the public sector has contributed between 0.4-0.5%, whereas 0.2-0.3% came from the private sector. Strikingly for India, the government is also a user of a research fund; apart from playing the role of the largest funder, the major chunk of R&D expenditure is used by the Central government. In addition, to increase nominal R&D expenditure, India’s share of total publication output also increased during 2009-2014 with a growth of 14% during the said period; an increase in the share of total global publications rose from 3.1% in 2009 to 4.4% in 2014. India was also ranked 6th in terms of total scientific publication output globally with a growth rate of 13.9% (Scopus) and 7.1% (Science Citation Index - SCI) compared to the world growth rate of 4.4% and 4.1%, respectively.13,14 The number of researchers per million population increased from 110 in 2000 to 218 in 2015.14\n\nBRICS represents about 42% of the world’s total population. 31.2% of science and engineering doctorates globally are produced by BRICS.15 BRICS countries together accounted for an impressive 233.4% growth in 2011 over 1995 in publishing science and engineering research papers, whereas the growth rate for other countries was 31.3%.16 Other than Russia, the output of BRICS countries increased for most frequently cited papers at a higher rate than top-cited countries globally.17 The co-authored papers among the BRICS countries are found to be countries with which they have “geographical” “cultural” and/or “historical” proximity.18 The intensity of intra-BRICS countries’ collaboration is also low.19 Further, the strength of each BRICS country is in the different scientific domain in terms of their research output according to the WoS database for the period 2008-2011. Research output from Brazil was primarily in agricultural, plant and animal sciences; for Russia it was physics, chemistry, mathematics and geosciences; India’s research outputs were in the fields of pharmacology and toxicology, agricultural sciences, material sciences, and chemistry; while China’s publications were in the fields of material sciences, chemistry, and physics. South Africa’s research output focused more on the fields of plant and animal sciences, immunology, environment/ecology as well as multidisciplinary research areas. Quantitatively, China is the largest in terms of publication output, whereas South Africa is the smallest among the five BRICS countries. Alternatively, based on Scopus data for the year 2015, China took the lead in the subject areas of mathematics, physics and astronomy, multidisciplinary science, and earth and planetary science. For Brazil, the strongest subject area according to Scopus is dentistry; for Russia, it is physics and astronomy; India is strong in the fields of pharmacology, toxicology, and pharmaceutics; and for South Africa, the strongest subject area is agricultural and biological sciences.19 Based on Scopus data, the total publication output of BRICS countries in 2015 was 29% with China contributing 18%, India 5%, Russia and Brazil 2.6%, and South Africa 0.72%.19 From the above discussion, it is clear that existing literature has discussed the need for analysis of research outcomes of a country. From the literature study, it can be seen that BRICS nations are the emerging economies where research publications are gaining over the years. With a motive to understand the research scenarios of BRICS nations this study compares number of researchers and the research outcomes of these nations in terms of research spent, citations, collaborative publications, distribution of publications across different subject areas and percentage of high impact publications.\n\nThe ranking of universities has always been a topic of debate, but it is a necessary mechanism to understand the performance of the universities across the globe. There are multiple ranking agencies that rate higher education institutions across the globe such as the QS world ranking, Times Higher Education (THE) world university ranking, Shanghai university ranking, Leiden ranking, etc. THE is one of the widely accepted ranking agencies that ranks the higher education institutions based on four basic parameters, which are teaching, research, international outlook, and industry interaction, out of which research contributes almost 62.5% of the overall scores, thus making it important for the proposed work to analyze the outcomes concerning research. Considering the emerging economies (BRICS), classification is made across the ranking to project the distribution of the number of universities across the entire scale. For this study we have classified the ranking window into five different ranges, which are 1 to 250, 251 to 500, 500 to 800, 801 to 1000, and more than 1000. Figure 1 shows the distribution of universities of BRICS nation across these five ranges for the 2018 ranking and Figure 2 shows the distribution for 2019 ranking. It can be seen that from Brazil, 32 universities have been featured in the 2018 ranking and the number has increased to 36 for the year 2019 ranking. From Russia 27 universities feature in the year 2018, and the number increased to 35 for the 2019 ranking. India had 42 universities ranked in 2018 and the numbers rose to 49 for the year 2019 ranking. There were 63 Chinese universities ranked in the year 2018 and 72 universities ranked for 2019 and finally, from South Africa, eight universities feature in 2018 ranking and nine universities for the year 2019. From the trends, it can be understood that in all the emerging countries the number of universities ranked has increased by a minimum of 10% percent or more as compared to its previous years. From the figures, it can be seen that for the year 2018, universities from South Africa, China, and Russia were featured in the bracket of 1-250 with China leading the group by seven universities, with no universities from India and Brazil in that bracket. In the bracket of 251-500, all the nations have representation, with Russia as the leading country with seven universities followed by China with five universities, then by South Africa with three universities and both Brazil and India with two universities each in this range. The 501-800 bracket is the range in which the maximum number of universities are placed; China leads the list with 32 universities, followed by India with 15 universities, Brazil with eight universities, Russia with five universities, and South Africa with three universities. In the next bracket of 801-1000, China leads by 16 universities followed by India with 13 universities, Brazil with 11 universities, Russia with five universities, and South Africa with one university. In the last bracket of universities ranked more than 1001+ India leads by 12 universities followed by Brazil with 11 universities, Russia with nine universities, China with three universities, and no university from South Africa is ranked in this bracket.\n\nThe 2019 ranking also showed a similar trend with some slight modification in the behavior, which is due to an increase in the number of universities. Again, in the bracket of 1-250 only universities from China, Russia, and South Africa are featured with seven, two, and one universities, respectively. The 251-500 bracket has Chinese universities in the lead with seven universities, followed by India with five universities, Russia with three universities, Brazil and South Africa with two universities each. Similarly to 2018, the year 2019 also shows a big number of universities in the 501-800 range with China having 28 universities, followed by India with 16 universities, Russia with nine universities, Brazil with five universities, and South Africa with three universities. The next bracket of 801-1000 also had an increase in number as compared to the previous year, with China having 22 universities, followed by India with 12 universities, Brazil with eight universities, Russia with four universities, and South Africa with one university. The last bracket of 1001+ ranking shows Brazil leading the list with 21 universities, followed by Russia with 18, India with eight universities, and China with eight universities, and lastly South Africa with one. From the comparison of the two years, it can be seen that India and China have improved in numbers and also quality with not much change from Brazil, Russia, and South Africa.\n\nTo further understand the ranking of universities we attempt to analyze the research outcomes of the countries, as research constitutes 62.5% weightage in the computation of scores for ranking the universities. Several parameters contribute to research outcomes; a detailed analysis is presented in the following section considering parameters such as the number of researchers, percentage of GDP spend on research, number of publications, citations, collaboration, and area of research.\n\n\nMethods\n\nTo further analyze the reasons for the same we have first looked at the comparison of researchers’ population across the countries (Source: https://data.worldbank.org/indicator/SP.POP.SCIE.RD.P6?view=chart), Research and Development spend by % GDP (Source: https://data.worldbank.org/indicator/GB.XPD.RSDV.GD.ZS), and publication data from Scopus/ SciVal. Data related to researcher population for the years 2013 to 2017 is extracted from the world bank data, for computation the average value over the year is taken. Similarly for computation of research spend by percentage GDP is also extracted from the world bank data for the years 2013 to 2017 by taking the average over the period, all the data were extracted from the source site on 18th December 2019. Publication related data corresponding to country output is extracted from the Scopus website on 18th December 2019. Parameters related to the citation outcome, subject wise breakup and international coauthored publication details are analyzed from the extracted data. SciVal tool from the Elsevier is used to obtained score related to citation impact, international collaboration as obtained on 18th Dec 2019. We also analyzed research output on three important parameters, namely researcher per million populations, total scholarly output, and expenditure on R&D as a percentage of GDP. The data on these three parameters were obtained from World Bank indicators. We compared these three parameters against the Field Weighted Citation Impact (FWCI) and its impact on collaborative publications with international authors. Based on three basic parameters, subject-wise analysis for BRICS countries’ research output was carried out.\n\n\nResults\n\nThe researcher population is one of the important factors which determines the research strength of the institution or country. To understand the outcome in terms of publication in Scopus indexed publications for five years (2013-17) for the researcher population across the BRICS nations, a graph is plotted in Figure 3. From the plot, it is clear that China (CHN) with the largest researcher population of 1.67 million produces the highest quantum of publication of the 2.8 million. India (IND) has a researcher population of 0.43 million, producing around 0.48 million publications. Russia (RUS) produces around 0.8 million publications with around 0.35 million researcher population. Brazil (BRA) with a researcher population of 0.18 million produces around 0.48 million publications and South Africa (RSA) with a researcher population of 0.026 million produces around 0.05 million publications.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nOne more important factor which constitutes the growth of research is the percentage of GDP spent on research. Figure 4 shows the comparison of the percentage of GDP spent on research by countries to the citation index of the country (here we consider the FWCI as a citation metric as it is the normalized value of citation across the various subject areas). China has around 2.1% of its GDP spent on research, followed by Brazil with 1.27%, Russia with 1.07%, South Africa with 0.77%, and India with 0.6%, with a country average FWCI of 0.89, 0.89, 0.72, 1.27, and 0.78, respectively. From the figure, it is clear that the quality of research and the GDP spent has a direct relation except for the scenarios in India.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nTo have a picture of the quantity and quality of research, a graph was plotted in Figure 5 comparing the population of the researchers to the country average FWCI. It can be seen that China produces the highest number of publications, whereas South Africa with the least number of publications has the best quality citation of 1.27. Brazil, India, and Russia’s research outcomes and quantity of publications lie in this range.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nFrom the above discussion, it can be seen that the quantity of research output, quality of research, researcher population, and research investment of the country are interrelated. To have a deeper understanding of the rest of the variables, the next section focuses on other parameters like the collaboration and subject area of the research.\n\nData for publications that are indexed in Scopus for the period of 2012-17 are analyzed for the BRICS nations with different research metrics like the number of publications published year on year during the period of evaluation, followed by the impact of collaborative publication. Publication behavior across the different subject areas is studied and discussed in detail in this section.\n\nYearly scholarly output in Scopus-indexed publications of BRICS countries is plotted in Figure 6. It is seen that China produces the highest number of publications, which is much higher than the summation of the output of all other BRICS nations. India produces the next highest number of publications, which is almost 20% in comparison to China. Brazil and Russia produce almost the same number of publications. It can be observed that Brazil produced higher numbers of publications in comparison to Russia prior to 2015, but post 2015 the publication output of Russia is higher as compared to Brazil. South Africa produces the least publications as compared to all the other countries, a trend which is almost consistent over the period studied (2012-17). The quantity of publications produced by India constantly increases over the period 2012-17. As expected for the time period in 2015, the publication output of China was mostly moving in a positive direction. This increase in number may be relative to the increased number of researchers and also the higher research investment.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nCollaboration in the publication would reflect the multicenter activity carried out by researchers. It is expected that research executed with multiple partners would have a relatively higher outreach and would lead to higher readership and citation. To understand this, Figure 7 shows the FWCI of BRICS nations in comparison to the percentage of publications with international collaboration for the time period 2012-17. From the figures, it is seen that South Africa has the highest percentage of international collaborative publication (as high as 45%) and also has the highest FWCI as compared to all BRICS nations. On the other hand, India has the lowest percentage of international collaborative publications and FWCI. China, Brazil, and Russia have a similar percentage of papers with international collaboration: 19%, 25%, and 28%, respectively. The FWCI of these nations is 0.9, 0.2, and 0.89, respectively. From Figure 7 it can be observed that India, Brazil, and South Africa have a similar trend. The behavior of Chinese and Russian characteristics is a little deviated, which may be due to other factors.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nAn attempt is made to understand the distribution of the international collaborative papers published by BRICS among different regions across the globe as shown in Figure 8. For the analysis, the world is divided into six regions, namely Africa, Asia Pacific, Europe, Middle East, North America, and South America. Figure 8 shows the distribution of BRICS collaborative publications among these regions. It is observed that the highest percentage of collaborative publications for Brazil, India, South Africa, and Russia happens from the European region with 58%, 40%, 70%, and 58% of international collaborative publications, respectively. China has the highest percentage of collaborative publications from North America (50%). For the rest of the countries the North American collaborative publication share among collaborative papers is second; Brazil with 43%, South Africa with 38%, Russia with 29%, and India with 38%. With China again a different characteristic is observed; the Asia Pacific collaborative publication share is second with a share of approximately 36%. Based on percentage share of collaborative publications, the Asia Pacific region is in third position for Brazil, South Africa, Russia, and India with 8%, 30%, 26%, and 35%, respectively. European collaborative publication share is seen to be the third largest share of publications for China with 20%. For Brazil, the rest of the publications are shared with South America, the Middle East and the African region, with a percentage share of 15%, 1%, and 0.8%, respectively. In the case of South Africa, the rest of the collaborative publications are from the African region with 23%, followed by South America and the Middle East with almost 8% each. Russia's collaborative publications are shared 9% with the Middle East, followed by 8% with South America and 7.2% with Africa. India's collaborative publications with countries in the Middle East is around 16%, followed by 9% with African countries, and 7.5% with the South American region. Lastly, China has around 2% of papers in collaboration with the Middle East, South America, and Africa. Except for Russia where a large skew is present for European collaborative publication (70%), for the rest of the countries it can be seen that they have a similar sharing percentage.\n\nOnce we have understood the collaborative publications with the other regions, it is also necessary to understand the significance of collaboration mutually inside the BRICS nations. To understand the same, collaborative publications and their impact in terms of FWCI are plotted in Figures 9 to 13. Figure 9 shows the collaboration of Brazil with other BRICS nations and its impact. Similarly, Figure 10 shows the relation of Russia with other BRICS nations, followed by India in Figure 11, China in Figure 12, and South Africa in Figure 13.\n\nBrazil: Brazil has around 0.98% of publications collaborating with India; these collaborative publications have a FWCI of 4.7. Around 0.76% of publications were co-authored with Chinese researchers, with a FWCI of 5.7. Coauthored publications with South Africa make up around 0.58%; these papers have a FWCI of 7.2. Collaborative publications with Russia make up around 0.42% of the total number of papers published by Brazil, with a FWCI of 7.1. It is observed that Brazil has a large number of collaborative works with India as compared to other BRICS nations and the highest impact is from collaborative work with South Africa.\n\nChina: China has around 0.85% of its total papers published with coauthors from BRICS nations. Russian collaborative work is at 0.27%, followed by India with 0.26%, Brazil with 0.18%, and South Africa with 0.11%. If FWCI is considered, publications with South Africa have a FWCI of 5.8, which is highest among the collaborations with BRICS nations. This is followed by collaborations with Brazil with a FWCI of 5.2 and Indian collaborative work with a FWCI of 4. Collaborative work with Russia has a FWCI of 3.7.\n\nIndia: India produces around 0.92% of its publications in collaboration with China, which is the highest among all other BRICS nations, and its collaborative papers have a FWCI of 3.98. Around 0.5% of publications are South African collaborations, with a FWCI of 4.6. Of the total number of Indian publications, 0.43% are Brazil collaborations with a FWCI of 6.2, which is the highest value in comparison to collaborative publications with other BRICS nations. India has around 0.42% of its collaborative publications with Russia; these papers have a FWCI of 5.2.\n\nRussia: Russia has around 4.13% of publications in collaboration with other BRICS nations. Russia has around 1.92% of its publications with China, and these papers have a FWCI of 3.7. The percentage of collaborations of Russia with India and Brazil is almost the same at around 0.97% and 0.96%, respectively. These papers have yielded a FWCI of 5.2 and 5.8, respectively. Russia has around 0.48% of its publications in collaboration with South Africa; these papers yield a FWCI of 7.2. It is seen that Russia has the highest collaboration with China and the papers with the highest FWCI are from South African collaborations, with a FWCI of 7.2.\n\nSouth Africa: South Africa has around 9.4% of its publications in collaboration with the rest of the BRICS countries, with India's collaboration being the highest at 3.48%, with a FWCI of 4.7. ChinaSouth Africa collaborative publications account for 2.55%, with the papers having a FWCI of 5.8. There are around 1.95% of papers published by South Africa in collaboration with Brazil, with a FWCI of 7.3. Around 1.495% of the publications are with Russian collaborators; these papers have a FWCI of 7.1.\n\nIt can be understood that from the above graphs in Figure 9 to Figure 13 that South Africa shares a larger percentage of its publication with other BRICS nations, which is around 9.4% of its total publications. On the other hand, China has the lowest percentage of collaborative publications with other BRICS nations of 0.85%. Collaborative publication with South Africa has a higher FWCI as compared to other BRICS nations.\n\nAlong with collaboration, one more important parameter that also affects the quality of the publication is the choice of journal. For the analysis, the publication of BRICS nations in the journals that are in the top 10th percentile by citation score is compared with the quality in terms of FWCI. Figure 14 shows the outcome of BRICS nations in the top 10th percentile journals. It was found that 24.8% of South African publications are in the top 10th percentile journals with the highest FWCI of 1.39. Russia has the lowest percentage of publications in the top 10th percentile journals and its FWCI is also lowest at 0.73. India has around 15.5% of its publications in top 10th percentile journals with a FWCI of 0.79. Brazil, with a FWCI of 0.89, publishes 19.1% of its publications in the top 10th percentile journals. China produces around 22.8% of its publications in the top 10th percentile journals with a FWCI of 0.9. From the above discussions, it is clear that collaborative publication and journal quality are factors that would affect the outcome of the publication. Along with these two parameters, one more factor affecting the outcome is the subject area of the journals where papers are published.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nThe choice of journal for publishing a research article is becoming very important as it relates to the outcome of the publication in terms of research visibility and citation. The selection of journals based on subject area is also an important parameter that needs to be considered. As metrics like FWCI are normalized parameters across a given subject area, the outcome of the publication would depend on the subject area. Based on the present-day scenario, journals are categorized into 300+ sub-subject areas by Scopus. For the present analysis, we are considering the broad classification as followed by THE, where all journals are divided into 11 main subject topics, which are ‘arts and humanities’, ‘business and economics’, ‘clinical, pre-clinical and health’, ‘computer science’, ‘education’, ‘engineering and technology’, ‘law’, ‘life sciences’, ‘physical sciences’, ‘psychology’ and ‘social sciences’. In this classification, the available 300+ sub-subject area journals are mapped to these 11 areas. It is to be noted that the journals can be overlapping with one or more classified areas as many journals publish work on multi disciplinarily or interdisciplinarity research areas. Table 1 shows the percentage distribution of publications of each country over the 11 subject areas. An analysis is also shown in Figures 15 to 25 of each subject area and the BRICS countries’ publication outputs, publication quantity, and quality of the publications.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nRSA: South Africa; BRA: Brazil; RUS: Russia; IND: India; CHN: China.\n\nFrom the table, it is seen that the split of publications across all the 11 subject areas is not equal and also varies by country. Brazil as a country produces a large share of publications in the subject areas of ‘clinical, pre-clinical and health’, ‘life science’ and ‘physical science’, and the least number of publications is seen in the subject area of ‘law’. In the case of China, a large share of publications is in the subject areas of ‘physical science’ and ‘engineering and technology’, and the least number of publications is from the subject areas of ‘law’, ‘education’, ‘psychology’, ‘arts and humanities’. India has a large share of publications in ‘physical sciences’, ‘engineering and technology’, and ‘clinical, pre-clinical and health’ subjects and lowest share of publications in the areas of ‘law’, ‘psychology’, ‘education’, and ‘arts and humanities’. Russia has a large share of publications in ‘Physical Sciences’, ‘Engineering and Technology’, with the lowest share of publications in the area of ‘psychology’ and ‘law’. South Africa has a large share of publications from ‘physical sciences’, ‘clinical, pre-clinical and health’, ‘life sciences’ and least share of publication in ‘law’ ‘psychology’ and ‘education’.\n\nArts and humanities: Figure 15 shows the output of BRICS nations in the subject area of ‘arts and humanities’ for the period 2012-17. China produces a large number of papers in ‘arts and humanities’, whereas the percentage share of South African papers is the highest. The FWCI of South African publications is the highest with a value of 1.1, followed by China with 0.99, Russia with 0.88, India with 0.8, and Brazil with 0.48. Publication of papers in the top 10th percentile journals is highest in China with 22%, followed by India with 17.6%, China with 15.5%, Russia with 10.2%, and Brazil 8.8%. It can be seen that China, with a higher percentage of papers in the top 10th percentile journals, also has a higher FWCI, and Brazil with less publications in the top 10th percentile journals has a lower FWCI.\n\nBusiness and economics: China produces a large number of papers in the subject area of ‘business and economics’. South Africa has a large share of its publications in ‘business and economics’. China also produces 28.4% of its publications in the top 10th percentile journals and has the highest FWCI of 1.13. Brazil has around 12% of its publications in the top 10th percentile journals with a FWCI of 0.64. South Africa produces around 10.2% of its publications in the top 10th percentile journals with a FWCI of 0.82. The FWCI of Russian publications is 0.83 with around 6.2% in the top 10th percentile journals. Around 7% of publications from India are in the top 10th percentile journals with a FWCI of 0.75, as evident in Figure 16.\n\nClinical, pre-clinical, and health: China produces the highest number of papers in the subject area of ‘clinical, pre-clinical and health’ which is around 470,000, but Brazilian publications have a higher percentage share in this subject area as compared to other countries as shown in Table 1. South African publications have the highest FWCI quality parameter of 1.78 with the highest percentage of publications in the top 10th percentile journals (25.2%). Brazilian publications have the next highest FWCI of 1.06, with 15.5% of its publications in the top 10th percentile journals. The FWCI of Chinese publications is 0.9, with 17.3% of publications in the top 10th percentiles journals. Publications from Russia have a FWCI of 0.86, with 8.9% of its publications in the top 10th percentile journals. India has a lower FWCI of 0.71 in comparison to other BRICS nations, with 8.6% of its publications in journals that are in the top 10th percentile. It is observed that countries with more publications in the top 10th percentile journals have a higher FWCI as shown in Figure 17.\n\nComputer science: It can be observed from Figure 18 that in the subject area of ‘computer science’, the publication number of China is the highest. India and China have a higher share of publications as compared to other BRICS nations. The behavior observed is unique and different as compared to other subject areas where we see that almost all nations have similar values of FWCI ranging 0.87 to 0.83. The percentage of publications in top 10th percentile journals is 11.5% for China, followed by Brazil with 11.2%, South Africa with 9.8%, India with 7.4%, and Russia with 3.3%.\n\nEducation: Publications from South Africa have a FWCI of 0.88, which is the highest of all the BRICS nations, and 9.4% of publications of South Africa are in the top 10th percentile journals. China has around 13.4% of its publications in the top 10th percentile journals, with a FWCI of 0.72. India, with 5.45% of its publications in the top 10th percentile journals, has a FWCI of 0.56. Russia, with 2.5% of its publications in the top 10th percentile journals, has a FWCI of 0.54, and Brazil also has around 2.55% of its publications in journals of the top 10th percentile as indicated in Figure 19.\n\nEngineering and technology: From Figure 20, it can be seen that the number of publications from China is the highest in the subject area of ‘engineering and technology’ in comparison to other BRICS nations. Publications from South Africa have the highest FWCI among other BRICS nations at 1.1, and their percentage of publications in the top 10th percentile journals is also highest at 24%. Brazil has around 23.8% of its publications in the top 10th percentile journals with a FWCI of 0.92, followed by India whose publications have a FWCI of 0.91 and has 19.6% of its publications in top 10th percentile journals. China has around 20.1% of its papers in the top 10th percentile journals, and the publications from China have a FWCI of 0.88. Russia, at 8.8%, has the lowest percentage of publications in the top 10th percentile journals and the lowest FWCI at 0.71.\n\nLaw: Law as a subject has a very small share of publications in all the BRICS nations as compared to the other subject areas, with South Africa having the highest share of 1.34% and China having the lowest share at 0.09%, although in terms of absolute number, the number of publications from China is higher in comparison to other BRICS nations. The quality of publications in terms of FWCI is also higher in papers produced by China (1.3), and it is also seen that around 50% of papers published are in journals that are in the top 10th percentile. The percentage of publications from India in the top 10th percentile journals is about 16.6% and the FWCI of papers published from India is 0.66. Papers published from Brazil have a FWCI of 0.55 and 19% of its publications are in the top 10th percentile journals. South Africa has around 16.6% of its publications in the top 10th percentile journals and the FWCI of South African publications is 0.52 for ‘law’. Russia has the lowest percentage of papers published in the top 10th percentile journals, which is 4.5%, and the FWCI of Russian papers in law has the lowest FWCI of 0.43 as compared to other BRICS nations as shown in Figure 21.\n\nLife science: From Table 1 it is understood that Brazil has a large share of publications in the subject area of life science, while China has a higher number of publications. South Africa has around 26.4% of its publications in top 10th percentile journals and the FWCI of the papers published is 1.2, which is highest in all BRICS countries. China has around 24.6% of its publications in the top 10th percentile of journals and the published papers have a FWCI of 1.01. Around 16.3% of the publications from Brazil are in journals which are in top 10th percentile, and published papers have a FWCI of 0.82. The FWCI of papers published from India is 0.7 with 11.7% of papers in the top 10th percentile journals. Of papers published from Russia, 11.3% are published in journals which are in the top 10th percentile, and papers published have a FWCI of 0.65, which is lowest among all the BRICS nations. It can be seen that in all these nations the quality of the publication is proportional to the number of publications in the top 10th percentile journals as displayed in Figure 22.\n\nPhysical science: In the area of physical science, as shown in Figure 23, China has the highest number of publications, while the share of publications in Russia is the highest. Publications from South Africa have the highest percentage of publication in the top 10th percentile journals (22.8%), with publication having a FWCI of 1.26. Around 22.3% of publications are in the top 10th percentile journals in China and publications have a FWCI of 1.01. Around 20.3% of papers Brazil produces are published in the top 10th percentile journals and papers have a FWCI of 0.95. The FWCI of papers published by India is 0.89, and they publish around 16.2% of their papers in top 10th percentile journals. Papers from Russia have a FWCI of 0.74 with 10.2% of its publications in the top 10th percentile journals.\n\nPsychology: Publications from China have around 26.7% of papers in the top 10th percentile journals and the FWCI of papers published is 1.17. In this subject area, 16.8% of South African papers were published in the top 10th percentile journals, and the FWCI of the papers published was 1.08. Publications from India have a FWCI of 0.76, with 9.8% of papers published in the top 10th percentile journals. Brazil published around 10.7% of its publications in the top 10th percentile journals, and publications from Brazil had a FWCI of 0.53, which is the lowest compared to other BRICS nations. Russia produced around 10.5% of its publications in the top 10th percentile journals and the FWCI of the papers published by Russia was 0.7, as shown in Figure 24.\n\nSocial sciences: From Figure 25, it is seen that South Africa publications have a FWCI of 0.96, with 16.5% of its publications in the top 10th percentile journals. In China around 26.6% of papers have been published in the top 10th percentile journals, and papers published have a FWCI of 0.9. Russia has 4.8% of its papers published in the top 10th percentile journals and papers have a FWCI of 0.75. The FWCI of papers published by India is 0.68 and around 11.3% of its publications are in the top 10th percentile journals. Brazil has the lowest FWCI at 0.58 and around 10.2% of their publications are in the top 10th percentile journals.\n\nThe publication numbers of China is the highest compared to other BRICS nations in all the 11 subject areas. The FWCI of publications from South Africa is highest in the subject areas of ‘arts and humanities’, ‘clinical, pre-clinical and health’, ‘education’, ‘engineering and technology’, ‘life sciences’, ‘physical sciences’ and ‘social sciences’. For the subject areas ‘business and economics’, ‘computer science’, ‘law’ and ‘psychology’, publications from China have the highest FWCI. From the above discussion, it is clear that the quality of publication depends on multiple parameters like collaboration, publication in quality journals, and subject area of publication.\n\n\nDiscussion\n\nResearch and innovation are the parameters that often decide the growth of an institute or organization or country. Publication output is one of the measures by which a country’s research can be quantified. An attempt was made in this paper to study the behavior of publications of the developing countries Brazil, Russia, India, China, and South Africa (BRICS). To validate the analysis of the work reported, the number and position of universities ranked in THE were treated as a unit of measure. It is evident from the publication that China which has the higher number of publications, research spend, researcher population, has a greater number of universities ranked. On the other hand, SouthAfrica with lesser publication outcome, research spend, etc. has lower number of universities ranked. But it can also be seen that the percentage of universities ranked higher due to higher quality parameters like higher FWCI, international collaboration, etc. Though the number of universities being ranked is higher due to higher researcher volume and publication the quality parameters reflect on the percentage of universities ranked higher. To understand the behavior of publication output, only Scopus-indexed sources were considered as THE considers only Scopus-indexed publications. The first parameter to be evaluated was the population of researchers and it was found that China has the largest population also produces the highest number of publications; the relationship between researcher population and quantity of publications was evident. The second parameter to be analyzed was the percentage of GDP spent on research and though it was expected that it would be proportional to the quality and number of publications, in reality, it was not so, leading to further analysis to find the other parameters contributing to the outcome. One such measure was collaborative publications, and from this analysis it was evident that South Africa with the most collaborative research had a higher impact than the rest of the BRICS nations. The pattern of collaboration was also studied to understand the geography of collaboration. Results show that South Africa, Russia, and Brazil have more collaboration with Europe, China with North America, and India showed broader collaboration.\n\nThe focus area of each of the countries is unique to its culture, geography, and relevance to society. An attempt was made to understand the impact of focus area in terms of the 11 basic subjects on the quantity and quality of research. The nations showed an inclination to a few research areas; for example, Brazil had a large focus toward clinical, preclinical, and life sciences. China and India had a large focus on engineering and physical sciences. Russia showed large publication numbers (more than 50% of its publications) in the areas of physical sciences, followed by engineering with 34%. South Africa showed equal priorities to clinical, life science, and physical science. It was also found that the fewest publications were from law. The paper tried to report the behavioral analysis of research in the BRICS nations.\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nFranco C, de Oliveira RH: Inputs and outputs of innovation: analysis of the BRICS: Theme 6–innovation technology and competitiveness. RAI Revista de Administração e Inovação. 2017 Jan 1; 14(1): 79–89. Publisher Full Text\n\nHasan SA, Luthra R: Comparative performance of India with other BRICS countries in publishing science and engineering research papers. Curr Sci. 2014 Jun; 25: 1654–7.\n\nCross D, Thomson S, Sibclair A: Research in Brazil: A report for CAPES by Clarivate Analytics.2018.\n\nSchiermeier Q: Russian science chases escape from mediocrity. Nature. 2018 Mar 15; 555(7696). PubMed Abstract \| Publisher Full Text\n\nMoed X, Akoev MA, Markusova VA: Trends in Russian research output indexed in Scopus and Web of Science. Scientometrics. 2018; 116: 1153. Publisher Full Text\n\nAbu KS, Asoka Siriwardena: Science Funding Research Output in BRIC Countries: A Scientometric Analysis.2015.\n\nCommander S, Plekhanov A: Diversifying Russia. chapter 07 EBRD.2012 Dec 13; 2: 66–77.\n\nNi X: China's research & development spend. Nature. 2015 Apr 29; 520(7549): S8–S9. PubMed Abstract \| Publisher Full Text\n\nWild S: South Africa pushes science to improve daily life. Nature. 2018 Sep 1; 561(7722): 158–60. PubMed Abstract \| Publisher Full Text\n\nSA Growth of Research Outputs Surpasses World Average, Science matters.November 2018; pp-13.\n\nInglesi-Lotz R, Pouris A: Does South African research output promote innovation? South African Journal of Science. 2018 Oct; 114(9-10): 1–3.\n\nQonde GF: Report On The Evaluation Of The 2016 Universities’ Research Output, Department of Higher Education and Training, MARCH 2018.\n\nRaman CV: Transforming Science and Technology in India. Economic Survey. 2017-18; Volume 1: pp. 119–130.\n\nYin RK: Qualitative research from start to finish. Guilford publications; 2015.\n\nHasan SA, Luthra R: Comparative performance of India with other BRICS countries in publishing science and engineering research papers. Current Science. 2014 Jun; 25: 1654–1657.\n\nSingh M, Hasan N: Trend in research output and collaboration pattern among BRICS countries: A scientometric study. In 2015 4th International Symposium on Emerging Trends and Technologies in Libraries and Information Services (IEEE). 2015 Jan 6.\n\nBornmann L, Wagner C, Leydesdorff L: BRICS countries and scientific excellence: A bibliometric analysis of most frequently cited papers. J Assoc Inform Sci Technol. 2015 Jul; 66(7): 1507–1513. Publisher Full Text\n\nFinardi U, Buratti A: Scientific collaboration framework of BRICS countries: an analysis of international coauthorship. Scientometrics. 2016 Oct 1; 109(1): 433–446. Publisher Full Text\n\nNascimento PA: Some trends in higher education and research in BRICS countries. BRICS AND AFRICA. 2014; 117." }	[ { "id": "81649", "date": "21 Apr 2021", "name": "Alka Ahuja", "expertise": [ "Reviewer Expertise Pharmaceutics", "Health outcomes", "Pharmaceutical Management", "Publication Ethics" ], "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 highlights comparison of BRICKS countries in terms of quality publications in various fields of Research. A very interesting study done in a meticulous and unbiased way based on THE data, which is globally accepted. More citations from 2020 could have been added. The figures summarize the findings very well. The comparison is based on development expenditure, publication quality, international collaborations etc. I recommend indexing of this article.\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": "85737", "date": "02 Jun 2021", "name": "Vipan K. Parihar", "expertise": [ "Reviewer Expertise My research is motivated by the need to better understand the pathophysiology of brain dysfunction. Specifically", "my interests include evaluating the sex differences in brain structure", "chemistry", "and function. The long-term goal of my research is to develop clinically applicable strategies that are efficient for enhancing brain function after injury", "disease", "or aging." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found this article interesting and insightful and particularly well written. The authors have done a noteworthy study to gauge the scientific input and its impact on development in BRICS countries. As such, for this manuscript, the authors have used multiple sources of data namely World Bank, Scopus, and also from Elsevier which is praiseworthy and is quite elaborate. This kind of bibliometric study is needed to bring the focus on core areas of development that will help in 21st-century nation-building. It also helps to show gaps, and improve research thrust and intensify research pursuits in those areas which lack focus which need some special stimulus to kick start research. These will help governments and policymakers to take note of the direction in which future research should be shaped.\n\nIn my opinion, if the authors would have correlated this data with some more relevant info on commercialization efforts (patents), the number of millionaires, HNWI, thrown in with data on AI, 5G/6G internet connectivity for good measure it would have been a comprehensive study although quite complicated which would need more time and detailed statistical analysis to pool the results and come to a meaningful conclusion.\nAnyway, I congratulate the authors for choosing this study impacting the BRICS.\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/10-213
https://f1000research.com/articles/10-212/v1	16 Mar 21	{ "type": "Research Article", "title": "The art and science of achieving zero COVID-19 transmissions in staff at a large community care facility in Singapore using implementation science: a retrospective analysis", "authors": [ "Weien Chow", "Elaine Lum", "Arif Tyebally", "Sze Ling Chan", "Lai Chee Lee", "Moi Lin Ling", "Hiang Khoon Tan", "Nigel CK Tan", "Weien Chow", "Arif Tyebally", "Sze Ling Chan", "Lai Chee Lee", "Moi Lin Ling", "Hiang Khoon Tan", "Nigel CK Tan" ], "abstract": "Background: The Singapore COVID-19 multi-ministry taskforce commissioned community care facilities (CCFs) as a hospital-sparing strategy amidst rising coronavirus disease 2019 (COVID-19) cases. An exhibition centre was repurposed within ten days as an ad-hoc 3200-bed medical facility (CCF@EXPO) to isolate and treat infected patients amidst concerns of potential COVID-19 transmissions to healthcare staff. This paper deconstructs the implementation of the CCF@EXPO infection prevention and control (IPC) innovation and elicits critical factors which enabled zero transmission of COVID-19 to staff during 100 days of operation using an implementation science framework. Methods: This study employed retrospective analysis using the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) framework. The CCF@EXPO IPC innovation comprised five key elements: (a) physical environment, (b) work practices, (c) tools and technology, (d) staff training, and (e) audits. Contextual assessment was conducted for baseline and 100th day of CCF@EXPO operations. Actions taken to improve scores between these timepoints were mapped against the Expert Recommendations for Implementing Change (ERIC) tool to surface key implementation strategies. Results: Positive shifts were observed in all constructs of the i-PARIHS framework, between baseline and 100th day. The largest shifts were in work practices, tools and technology, and staff training. Key implementation strategies used included: rapid Plan-Do-Study-Act (PDSA) cycles, identifying champions, team building, creating a culture of collaborative learning, multi-disciplinary teamwork, transparency in communications and decision-making, and skillful facilitation. Conclusions: Rapid PDSA cycles anchored by principles to ensure staff safety, was the key approach used in implementation of the CCF@EXPO IPC innovation. Retrospective analysis using the i-PARIHS framework is useful to elicit success factors and to inform preparedness planning of future pandemics.", "keywords": [ "COVID-19", "implementation science", "infection control", "safety management", "pandemics", "i-PARIHS." ], "content": "Introduction\n\nMany countries have repurposed pre-existing public venues or infrastructure as community care facilities (CCFs) to provide treatment for those infected with coronavirus disease 2019 (COVID-19), reserving hospital care for patients with serious sequelae.1-3 An inherent challenge in using non-healthcare public venues, is to ensure adaptations to layout, infrastructure, workflows, and work practices are designed to support infection prevention and control (IPC) for staff and patient safety. Reports of frontline healthcare professionals who contracted COVID-19 and lost their lives, shock and sadden.4-8 Although no official worldwide tally is available, conservative estimates are that between 7,000 to >20,000 healthcare professionals have died in the line of duty, as of October 2020.9,10\n\nIn Singapore, two waves of imported and community cases of COVID-19 in March/April 2020 were dwarfed by a sharp rise in the number of infected migrant workers housed in dormitories.11 The COVID-19 multi-ministry taskforce commissioned CCFs to be rapidly set-up as a hospital-sparing strategy in April 2020 amidst rising cases, to provide care for patients with mild symptoms.12 Given fatalities in healthcare professionals reported around the world due to COVID-19, ensuring staff safety in CCFs was a priority. We were tasked to set-up a CCF within ten days, repurposing halls in an exhibition centre (hereafter CCF@EXPO) to isolate and treat infected patients in the community.13 However, due to the speed required in setting up CCFs, a prospective theory-informed approach to implementation is uncommon. At the time of writing, we found no published studies using implementation science to plan, set-up, and operate a large-scale CCF in this pandemic.\n\nWe achieved our goal of zero transmission of COVID-19 to healthcare and non-healthcare staff working at CCF@EXPO, while providing high quality patient care. This paper is a retrospective analysis using implementation science to elicit critical factors in the achievement of the stated goal, and to inform similar endeavors in the future.14 Contextual barriers and implementation strategies used to address these are surfaced. Findings are reported in line with the Standards for Reporting Implementation Studies (StaRI) checklist.15\n\n\nMethods\n\nResearch ethics approval was provided by the SingHealth Centralized Institutional Review Board (CIRB), approval number 2020/2890. Participant consent was waived by the institutional review board.\n\nThe setting for this paper is the CCF@EXPO (Halls 7 to 10; 3200 beds) run by SingHealth, a major provider of public healthcare in Singapore.16 The CCF@EXPO comprised ten exhibition halls with a total capacity of 7873 beds, the largest CCF here (extended data file 131).\n\nWe highlight the most significant contextual challenges in setting up and managing the SingHealth CCF@EXPO, notwithstanding the ten-day lead time. At the organisational level, SingHealth was stretched in supporting other COVID-19 initiatives e.g. mass swabbing/testing and medical outposts at migrant worker dormitories with COVID-19 clusters.17 There was no existing blueprint for the set-up and management of large ad hoc medical facilities such as CCFs.\n\nInfrastructure adaptations were constrained by both the built environment and time. Critical infrastructural decisions made to enable good IPC practices include layout and workflow, areas for donning/doffing of personal protective equipment (PPE), negative air pressure for patient areas, and additional rest areas for staff to ensure safe distancing within teams and segregation between teams, prior to commencement of operations. There were challenges in rapidly building a resilient well-functioning team. Healthcare staff deployed to CCF@EXPO were from different institutions, diverse disciplines and specialties, and had not functioned as a team previously. Few had expertise in managing COVID-19 patients. There was a significant reduction in staff to patient ratio compared to pre-COVID-19 times.13 The need to engage with multiple external vendors/partners (e.g. bed and facility management, cleaning and security services) including training non-healthcare staff in PPE use, introduced added complexity. Patients at CCF@EXPO were mostly male migrant workers from South Asia, who spoke little or no English.\n\nThe intervention\n\nWe defined the intervention as ‘the isolation and treatment of COVID-19 patients not requiring hospital care, in a community care facility’, shown to be effective for virus containment elsewhere1-3 and part of a comprehensive strategy for pandemic management in Singapore.12\n\nThe Innovation\n\nThe innovation is a bundle of five elements focussed on IPC, hereafter referred to as the ‘CCF@EXPO IPC innovation’: (a) physical environment, (b) work practices, (c) tools and technology, (d) staff training, and (e) audits.\n\ni-PARIHS framework\n\nWe used the integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) as the framework for retrospective analysis. The i-PARIHS was selected for its real-world applicability to complex interventions which are multi-dimensional, iteratively implemented, and contextual; and for its emphasis on facilitation.18,19 Main constructs of the framework are: (a) characteristics of the innovation (the bundle of five elements described above), (b) recipients—the people affected by and who influence implementation (staff at CCF@EXPO), (c) inner context–local (CCF@EXPO), (d) inner context–organisational (SingHealth), (e) outer context (Ministry of Health/National level), and (f) facilitation—the role of facilitators and process of facilitation.18 Facilitation was led by two facilitators (WEC and HKT) appointed as joint medical directors for the SingHealth CCF@EXPO.\n\nThis was a retrospective case study investigating the end-to-end planning, set-up, operation, and stand-down of the SingHealth CCF@EXPO IPC innovation within a 100-day period from 23rd April to 31st July 2020, which achieved zero transmission of COVID-19 to healthcare and non-healthcare staff.\n\nPatients were not involved in the design and conduct of this retrospective analysis.\n\nQuantifying implementation\n\nWe developed an assessment rubric based on the i-PARIHS framework for a systematic retrospective deconstruction of the CCF@EXPO IPC innovation (EL, WEC).18,19 We adapted the i-PARIHS assessment questions into statements accompanied by a qualitative rubric for allocating scores (extended data file 231). The constructs assessed were those described in the preceding section (Definitions: the innovation and i-PARIHS framework). Face validity of the assessment rubric was ascertained (AT, NT, SLC, HKT) and the rubric revised (extended data file 231). Items were scored from 0 to 3. Higher scores (2 or 3) are desirable/signal fewer challenges, while lower scores (0 or 1) are undesirable/signal more challenges. Scores were allocated for baseline (commencement of operations) and 100th day, or pragmatically as close as possible to these two time points constrained by the availability of data.\n\nAssessments were independently completed by three assessors (AT, NT, SLC) representing meso, micro, and arms-length perspectives, respectively. Routine data and resources collected during the 100-day operation of CCF@EXPO were used for assessments: (a) anonymous staff surveys at various timepoints regarding any concerns and feedback pertaining to IPC or being deployed/working at CCF@EXPO (includes responses from the digital Kaizen board, Padlet (an online noticeboard), and pre-deployment survey), (b) IPC audit data (e.g. compliance with use of PPE, hand hygiene, environmental cleaning, food safety, waste management, etc.), (c) a comprehensive matrix summarizing key elements of the innovation (extended data file 331), and (d) on-site observations (AT, NT). As staff surveys and feedback were ongoing during 100 days of CCF@EXPO operations, we primarily used data from the pre-deployment survey (response rate 57%, 56/98) as baseline, and the end-of-deployment survey in the final month of operations. Fifty responses were received for the end-of-deployment survey; a response rate was not calculated as the survey was open to all staff ever deployed within the 100-day period, via word-of-mouth and through posters displayed on-site for those still deployed. In total, there were 488 staff ever deployed to CCF@EXPO in the stated period. Similarly, as IPC audits were conducted daily, data from the first fortnight (baseline) and from the last week of operations were used for assessment (100th day).\n\nDiscordant scores for baseline and 100th day were resolved via discussion. Where discordance remained, an average score was used for that item. Scores for items under each sub-construct were averaged and represented as radar diagrams, to serve as visual summaries of the gaps/barriers and strengths/enablers of the innovation (baseline) and any shifts following the use of various implementation strategies (100th day).\n\nEliciting implementation strategies\n\nWe mapped actions taken by facilitators to address contextual barriers at pre-commencement/baseline and iteratively throughout the implementation against the Expert Recommendations for Implementing Change (ERIC), a compilation of 73 discrete implementation strategies, to enable systematic reporting of strategies used.20\n\nDescriptive statistics summarised assessment scores for (a) characteristics of the innovation (the bundle of five elements), (b) recipients—the people affected by and who influence implementation (staff at CCF@EXPO), (c) inner context–local (CCF@EXPO), (d) inner context–organisational (SingHealth), and (e) outer context (Ministry of Health/National level). Assessment scores were analysed using Microsoft Excel (version 2101, build 13628.20448).\n\n\nResults\n\nThe assessments were conducted by three of the authors (AT, NT, SLC).31 Baseline scores for innovation characteristics indicated that ‘physical environment’, ‘work practices’, ‘tools and technology’, and ‘staff training’ needed to be addressed from an IPC perspective (Figure 1a and extended data file 431). The main pre-commencement/baseline challenges were (Table 1): inadequate facilities for PPE donning/doffing and inadequate space for staff to rest/have meal breaks (physical environment); hospital IPC workflows and practices required modifications to suit CCF@EXPO (work practices); missed opportunities to reduce dwell time in patient areas (suboptimal use of tools and technology); and training especially for non-healthcare staff in PPE use,21 safe workflows, and safe behaviours (staff training). At baseline, only basic IPC audits were in place.\n\na Main contextual barriers at baseline, unless otherwise stated.\n\nb Definitions from Waltz et al20, unless otherwise stated.\n\nAbbreviations: COVID-19: Coronavirus disease 2019; ERIC: Expert Recommendations for Implementing Change; i-PARIHS: integrated Promoting Action on Research Implementation in Health Services; IPC: Infection prevention and control; PPE: Personal protective equipment.\n\nSeveral key implementation strategies were used to address baseline challenges for innovation characteristics (Table 1), driven by Deming’s Plan-Do-Study-Act (PDSA) methodology.22 For example, Plan—assessing local needs for safer work practices (PPE donning/doffing, Figure 2); Do—developing enhanced practices (conduct training in the use and sequence of PPE donning/doffing, provide gowning stations, posters as visual reminders); Study—audit and feedback (PPE donning/doffing audit, ‘PPE buddy’ (staff were assigned a staff ‘buddy’ for verification of correct donning/doffing of PPE), mirrors); and Act to refine work practices further (trial of Smart mirror). The practice of using posters as a checklist was normalized. A no-blame culture of continual learning and looking out for one another was modelled and encouraged by facilitators and leaders. By the 100th day, PPE compliance had improved from 97% to 100% and from 93% to 98%, respectively, for healthcare and non-healthcare staff. These outcomes were more favourable when compared to another Singaporean study in a hospital setting.23 Assessments showed positive shifts in all five elements of innovation characteristics by the 100th day.\n\nFor recipients, baseline scores although favourable (Figure 1b and extended data file 431), could be strengthened given the challenge of caring for >3000 COVID-19 patients with lean human resources.13 The ability to change at the individual level remained stable between the two timepoints—individual staff understood the modifications in workflows and practices for delivery of patient care, and each had the ability, resources (e.g. PPE supplies), and support to enact these workflows and practices. However, positive shifts in scores from baseline to 100th day were observed for motivation to change at individual level, motivation to change at team level, and ability to change at team level.\n\nKey implementation strategies (Table 1) used to strengthen individual and team efficacy include: facilitators being skillful in identifying champions and domain leads, team building, and creating virtuous cycles centred on cultures of collaborative learning, multi-disciplinary teamwork, as well as transparency in communications and decision-making. Key strategies were enacted mainly through rapid PDSA cycles serving as vehicle and enabler of staff initiatives to improve processes, to identify and raise relevant issues impacting staff safety and patient care, as well as to develop, test, refine and own the solutions. Experiencing themselves as empowered agents of change individually and collectively was critical for rapid ground-up innovation in a pandemic. In the end, increased efficacy and agency of individuals and teams allowed out-of-box thinking beyond comfort zones. New tools and technologies, accompanied by new procedures/protocols refined via PDSA cycles, were adopted as routine work practices. For example, an autonomous robot (TEMI®, New York, USA) was used to provide medication counselling and tele-triage to reduce staff dwell time in patient areas, and fluorescent marker (Glo-Germ) was used for objective assessment of environmental cleaning during audits (Figure 3).\n\nBaseline scores for context were favourable (Figure 1c and extended data file 431), which signaled good alignment between culture, leadership, systems, policies, and strategic priorities at local, organisational, and national levels in Singapore. To maintain alignment, facilitators used implementation strategies such as identifying champions at various levels, engaging with executive/advisory boards (for clinical governance, resource allocation, accountability reporting), and developing partnerships (for CCF@EXPO operations or research). Favourable scores for context were maintained at the 100th day (Figure 1c and extended data file 431).\n\nFacilitation featured strongly as an implementation strategy that ensured success. The findings of the assessment indicate that these are seasoned/expert facilitators (extended data file 531). Facilitators used various communication techniques and channels (formal and informal means) to engage, involve, inform, update, encourage, acknowledge, gather support, and celebrate wins/milestones with staff and senior stakeholders (extended data files 3 and 631). A ‘flat organisational structure’ was intentionally adopted to enable open communication and ease of access to facilitators. This non-hierarchical structure was important and necessary for encouraging a constant flow of ground-up innovations and initiatives, especially as few in the team were experts in managing COVID-19 patients.\n\nImportantly, facilitators were given authority, autonomy, support, and resources to implement the CCF@EXPO IPC innovation. Facilitators displayed desirable attributes (16/18, 89%), performed essential tasks (11/13, 85%), and demonstrated a diverse range of required skill sets (14/15, 93%). Facilitators were skilled in addressing task, team, and individual needs (Table 2), and were able to iteratively move through the four phases of facilitation articulated in the i-PARIHS framework: (a) clarify and engage (identify the problem, get the right people together); (b) assess and measure (appraise the evidence, consider recipients and context); (c) action and implementation (use rapid PDSA cycles); and (d) review and share (audit/re-audit, timely feedback, sustain, spread).19 Apart from working with staff on the ground, facilitators actively engaged SingHealth organisational stakeholders and decision-makers at the National/Ministry level. Facilitators focused on goals, thus acted as translators and bridges across professional, semantic, syntactic, and pragmatic boundaries.\n\nCCF@EXPO: Community care facility repurposed from the Singapore exhibition centre; EXPO: Refers to the Singapore exhibition centre; PPE: Personal protective equipment.\n\n\nDiscussion\n\nThe SingHealth CCF@EXPO required rapid planning and set-up to deliver healthcare to >3000 COVID-19 patients. Clinician facilitators worked closely with experts in IPC to design a tightly woven set of strategies to overcome contextual challenges to ensure staff safety while delivering high quality patient care. To actualize the goal of zero transmission of COVID-19 to staff, the following principles were used as a basis for the planning, set-up, and operations of CCF@EXPO: (a) reduce opportunities for transmission by minimizing staff dwell time in patient areas; (b) reduce risks of transmission by ensuring that physical infrastructure, environmental cleaning, workflows and practices support IPC; (c) enhance staff protection by ensuring staff competency and adherence to correct PPE use; and (d) constant monitoring via audit and feedback mechanisms to quickly identify emerging issues that threaten staff or patient safety. We reflected these principles in the ‘characteristics of the innovation’ construct within the i-PARIHS framework.\n\nThis deconstruction demonstrates that the SingHealth CCF@EXPO IPC innovation was a piece of implementation research (IR) in action.24 The CCF@EXPO possessed all eight defining characteristics of IR—context specific, addressed challenges relevant to implementation, used fit-for-purpose methods (e.g. rapid PDSA cycles), demand driven, displayed multi-stakeholder and multi-disciplinary engagement at all levels, occurred in the real-world and in real-time, and focused on processes and outcomes.24\n\nThe i-PARIHS framework with its real-world applicability to complex multi-dimensional interventions, allowed articulation of what was an organic (and creative) implementation process. However, because the i-PARIHS is usually used prospectively to plan implementation studies, contextual assessments (as per its originators) are posed as questions which facilitators use at pre-implementation to ascertain, select, and tailor implementation strategies.19 For the retrospective analysis reported in this paper, we adapted the i-PARIHS assessment questions into statements accompanied by a qualitative rubric for allocating scores. While this unusual use of the framework may draw scholarly criticism, we believe that the assessment rubric allows transparent sharing of the methods used in arriving at assessment scores which informed the deconstruction. We offer the rubric as a resource which other clinicians and researchers may wish to adapt for use during this and future pandemics.14\n\nThe retrospective analysis has proven useful in informing our own ongoing efforts in keeping staff safe while treating/caring for COVID-19 patients in CCFs, improving IPC practices, and contributing to a contextually adaptable blueprint for rapid set-up and operations of future CCFs.\n\nWhile implementation may seem like a 3-D version of a logic model with inputs–activities–outputs, albeit within a particular context, far more needs to happen for successful implementation. Namely, skillful facilitation, which we showed was able to overcome contextual challenges of the CCF@EXPO IPC innovation. Facilitators used various leadership styles (servant-leader, participative, directive, transformational) as the situation demanded (nature of the problem, purpose, audience, desired outcome).25 Other major skill sets displayed by facilitators include emotional intelligence, project management, as well as change, risk, and conflict management.19,26\n\nBuilding on the emphasis that the i-PARIHS places on both the role and the process of facilitation,18,19 we highlight that the person or ‘being’ of facilitators is equally important, especially in this case where facilitators are also clinician leaders. In her analysis of healthcare leadership in groups and teams, Greenhalgh argues that a leader is “someone I can look up to, someone I am prepared to follow, whose judgment I trust, and … who has integrity (and attends to the moral dimension of our work). [ … ] to be able to articulate a vision on behalf of our team—and to talk about that vision in a way that inspires me. If my leader wants me to commit to something, I want them to show their own commitment first. […] I want my leader to generate and sustain team spirit, […] to have excellent personal qualities and to attend skillfully and wisely to the task, the individuals, and the team as a whole.”27\n\nThe CCF@EXPO facilitators embodied these qualities and demonstrated skills in line with leadership processes drawn from organisational sociology.28 They created structures to oversee and coordinate teamwork, afforded psychological safety in creating a culture of collaborative (and no-blame) learning, and promoted team stability (most evidently in their role-modelling of the in-house TLC framework). T (we work as a Team), L (we Learn from one another), and C (we Care for each other). Facilitators engaged staff not just to co-opt their service, but also to address real fears and concerns about personal safety amidst local reports of healthcare workers being infected,8 and safeguarding staff mental health (preventing mental fatigue, burn-out, and counterproductive states). Anonymous staff surveys at later stages of the implementation showed increased confidence in leaders, colleagues, work processes and practices. Camaraderie, teamwork, and friendship were dominant memes in late-stage staff surveys.\n\nTowards the end of CCF@EXPO operations, facilitators prepared staff psychologically for returning to base hospitals. Initiatives to smooth the transition included a perpetual online presence to collate photographs and personal stories from staff.29 An online celebratory event officiated by SingHealth leaders provided a symbolic milestone to thank the team and to mark the end of their service at CCF@EXPO. At stand-down, staff had evolved the TLC framework to represent T (Trust in each other), L (Love for one another), and C (having Confidence as a team) (extended data file 331). We propose that successful operation at the CCF@EXPO went beyond application of best practices and frameworks. The principles used by facilitators and IPC experts to achieve zero transmission of COVID-19 to staff, were enacted in a way that engendered mutual trust, love (sense of belonging, empathy, compassion), and care. These values fueled necessary team dynamics for successful implementation of the CCF@EXPO IPC innovation and remain important and relevant.\n\nKey learnings from this initiative include: (a) an understanding of contextual challenges during a pandemic, (b) using a wide range of implementation strategies, (c) always putting people first (recipients–staff), and (d) the importance of facilitation in the implementation of complex multi-dimensional interventions. While Singapore was better prepared post the 2003 severe acute respiratory syndrome (SARS) pandemic, the COVID-19 pandemic highlighted the need for additional planning and a comprehensive blueprint for establishing large-scale ad hoc medical facilities on short notice. This is to better manage the large number of COVID-19 cases without neglecting those who need acute management of non-COVID-19 illnesses in hospitals.\n\nLimitations of this study are inherent to retrospective research, with available data being the main constraint. The use of three assessors is not a limitation but a strength, as prospective implementation studies often use one or two facilitators for contextual assessments. It may be fruitful to investigate transferability of how we implemented the CCF@EXPO IPC innovation to other settings or other types of infectious diseases. Health workforce studies may examine the effect of CCFs on base hospitals in terms of service capacity and diversion of resources for healthcare delivery.\n\n\nConclusions\n\nThe goal of zero transmission of COVID-19 to staff at a large CCF was achieved via skilful facilitation amidst significant contextual challenges. The use of rapid PDSA cycles anchored by principles to ensure staff safety was instrumental for implementation success. The i-PARIHS framework is relevant and useful for deconstruction of rapid implementation in a pandemic. It can inform preparedness planning for future scenarios of volatility, uncertainty, complexity, and ambiguity.\n\n\nData availability\n\nOpen Science Framework: The art and science of achieving zero COVID-19 transmissions in staff at a large community care facility in Singapore using implementation science: a retrospective analysis. https://doi.org/10.17605/OSF.IO/98G5Q31.\n\nThis project contains the following underlying data:\n\n• Underlying data_assessment scores.xlsx\n\nOpen Science Framework: The art and science of achieving zero COVID-19 transmissions in staff at a large community care facility in Singapore using implementation science: a retrospective analysis. https://doi.org/10.17605/OSF.IO/98G5Q31.\n\nThis project contains the following extended data:\n\n• Additional File 1. List of Community Care Facilities in Singapore\n\n• Additional File 2. Assessment rubric adapted from the i-PARIHS framework for this study\n\n• Additional File 3. The 14PS matrix (High-level documentation and rationale for implementation strategies used)\n\n• Additional File 4. Consensus scores for baseline and 100th day\n\n• Additional File 5. Assessment of facilitation (Assessment of facilitation based on i-PARIHS framework)\n\n• Additional File 6. Communication channels to build collaboration, teamwork, and learning\n\n• Standards for Reporting Implementation Studies (StaRI) checklist for this study\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\nEL conceptualized the paper and methods, provided critical input into the use of implementation science, and developed the assessment rubric. WEC piloted the assessment rubric and developed the 14PS Matrix which provides high-level documentation and rationale for strategies used. NT and HKT obtained research ethics approval. LCL and MLL were instrumental in the development and implementation of IPC practices, including audits. Data was collected, collated, and provided by WEC, AT, LCL, HKT, and NT. WEC, AT, SLC, HKT, and NT provided critical review of the assessment rubric; EL refined the rubric. Assessment was conducted by AT, SLC, and NT; results were collated by SLC. EL and WEC had full access to the data in this study; and verified, analyzed, and interpreted the data. Radar diagrams were prepared by SLC. Photos were provided by LCL and WEC. EL prepared the figures, tables, and boxes. EL and WEC drafted and refined the manuscript. All authors reviewed the manuscript, provided critical input, and approved the final manuscript for submission.\n\n\nCompeting interests\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work. The CCF@EXPO was funded by the Singapore Ministry of Health (sponsor). The sponsor was not involved in the study design, data collection, analysis and interpretation, reporting of the study, or in the decision to submit this paper for publication.", "appendix": "Acknowledgements\n\nWe would like to thank the patients, SingHealth leadership and staff, locum and agency staff, as well as external partners SingEx, Resorts World Sentosa, and Certis Cisco for their contributions to the operation of the SingHealth CCF@EXPO.\n\n\nReferences\n\nChen S, Zhang Z, Yang J, et al.: Fangcang shelter hospitals: a novel concept for responding to public health emergencies. Lancet. 2020; 395(10232): 1305–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHer M: Repurposing and reshaping of hospitals during the COVID-19 outbreak in South Korea. One Health. 2020; 10: 100137. PubMed Abstract \| Free Full Text Free Full Text\n\nKaysin A, Carvajal DN, Callahan CW: The Role of Alternate Care Sites in Health System Responsiveness to COVID-19. Am J Public Health. 2020; 110(9): 1362–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharacteristics of Health Care Personnel with COVID-19 - United States, February 12-April 9, 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(15): 477–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIng EB, Xu QA, Salimi A, et al.: Physician deaths from corona virus (COVID-19) disease. Occup Med (Lond). 2020; 70(5): 370–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Nurses United: Sins of Omission: How Government Failures to Track Covid-19 Data Have Led to More Than 1,700 Health Care Worker Deaths and Jeopardize Public Health.2020Reference Source\n\nGreen A: Obituary: Li Wenliang. Lancet. 2020; 395(10225): 682. Publisher Full Text\n\nWong LY, Tan AL, Leo Y-S, et al.: Healthcare workers in Singapore infected with COVID-19: 23 January-17 April 2020. Influenza Other Respir Viruses. 2020; n/a(n/a). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nICN confirms 1,500 nurses have died from COVID-19 in 44 countries and estimates that healthcare worker COVID-19 fatalities worldwide could be more than 20,000 [press release].Geneva, Switzerland; 28 October 2020.\n\nMcCarthy N: Where Most Health Workers Have Died From Covid-19: Statista;3 September 2020. Reference Source\n\nSingapore Ministry of Health: COVID-19 Situation Report: Number of Cases.9 November 2020. Reference Source\n\nSingapore Ministry of Health: Comprehensive Medical Strategy for COVID-19.28 Apr 2020. Reference Source\n\nChia ML, Him Chau DH, Lim KS, et al.: Managing COVID-19 in a Novel, Rapidly Deployable Community Isolation Quarantine Facility. Ann Intern Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWensing M, Sales A, Armstrong R, et al.: Implementation science in times of Covid-19. Implementation Sci. 2020; 15(1): 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPinnock H, Barwick M, Carpenter CR, et al.: Standards for Reporting Implementation Studies (StaRI) Statement. BMJ. 2017; 356: i6795. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingapore Ministry of Health: Reorganisation of Healthcare System Into Three Integrated Clusters to Better Meet Future Healthcare Needs.2017 [updated 2019]. Reference Source\n\nThe Straits Times: Covid-19: Heroes of the storm.21 July 2020. Reference Source\n\nHarvey G, Kitson A: PARIHS revisited: from heuristic to integrated framework for the successful implementation of knowledge into practice. Implement Sci. 2016; 11(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarvey G, Kitson A: Implementing evidence-based practice in healthcare: a facilitation guide. New York, NY: Routledge; 2015.\n\nWaltz TJ, Powell BJ, Matthieu MM, et al.: Use of concept mapping to characterize relationships among implementation strategies and assess their feasibility and importance: results from the Expert Recommendations for Implementing Change (ERIC) study. Implement Sci. 2015; 10(1): 109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiow MH, Lee LC, Tan N, et al.: Personal protective equipment training for non-healthcare workers in the COVID-19 pandemic: effectiveness of an evidence-based skills training framework [manuscript in preparation].2020.\n\nInstitute for Healthcare Improvement: How to improve 2020.Reference Source\n\nWee LE, Sim JXY, Conceicao EP, et al.: Re: 'Personal protective equipment protecting healthcare workers in the Chinese epicenter of COVID-19' by Zhao et al. Clin Microbiol Infect. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTheobald S, Brandes N, Gyapong M, et al.: Implementation research: new imperatives and opportunities in global health. Lancet. 2018; 392(10160): 2214–28. PubMed Abstract \| Publisher Full Text\n\nGorman P: Managing multi-disciplinary teams in the NHS. McGraw-Hill Education:London, UK;1998.\n\nGabel S: Perspective: Physician Leaders and Their Bases of Power: Common and Disparate Elements. Acad Med. 2012; 87(2): 221–5. PubMed Abstract \| Publisher Full Text\n\nGreenhalgh T: Groups and Teams. In: How to implement evidence-based healthcare .Oxford, UK: John Wiley & Sons Ltd; 2018.\n\nEdmondson AC, Bohmer RM, Pisano GP: Disrupted Routines: Team Learning and New Technology Implementation in Hospitals. Admin Sci Quarter. 2001; 46(4): 685–716. Publisher Full Text\n\nSingHealth: EXPOnentially Better!2020. Reference Source\n\nPerry CK, Damschroder LJ, Hemler JR, et al.: Specifying and comparing implementation strategies across seven large implementation interventions: a practical application of theory. Implement Sci. 2019; 14(1): 32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChow W, Lum E, Tyebally A, et al.: The art and science of achieving zero COVID-19 transmissions in staff at a large community care facility in Singapore using implementation science: a retrospective analysis.2021, February 4. Reference Source" }	[ { "id": "82386", "date": "23 Apr 2021", "name": "Lisa Hall", "expertise": [ "Reviewer Expertise Implementation science", "infection control", "epidemiology", "evaluation" ], "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 was a pleasure to read. It was well laid out, and very comprehensive in its use of implementation science to unpack what contributed to the success of the intervention. I would suggest more information be provided in a few areas:\nReferences to previous literature looking at the use of implementation science in infection prevention and control initiatives. For example the REACH study1 used a similar approach to quantify implementation - were the findings similar or different? What can we learn about the use of these frameworks and tools in different infection control settings that could help with future work?\n\nMore detail on the intervention components themselves. For example, the training: Who was trained? how? when? was knowledge assessed? Audit feedback: what parts of the infection control program were audited, using what tools? The detail is spread throughout the paper and the appendices but it would be useful to have this pulled together in a table.\n\nIn areas where there were no barriers identified at baseline, perhaps the authors could discuss the contextual enablers that may be present?\n\nMore discussion on the generalisability of the intervention to other settings. What did you learn that would help with the scale up or spread to other sites, particularly outside of Singapore, where the resourcing and attitudes of management may not be as supportive.\n\nDo you believe this approach would be sustainable outside a pandemic when the incentives for compliance may not be as evident?\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": "6616", "date": "26 Apr 2021", "name": "Elaine Lum", "role": "Author Response", "response": "Dear Lisa, Thank you for reviewing our article and providing suggestions for improvement. We will incorporate these as best as we can, in the next version of the article. Warm regards, Elaine" } ] } ]	1	https://f1000research.com/articles/10-212
https://f1000research.com/articles/10-209/v1	15 Mar 21	{ "type": "Research Article", "title": "Development of a physiologically based pharmacokinetic (PBPK) model of psilocybin and psilocin from magic mushroom in rats and humans", "authors": [ "Prinya Musikaphongsakul", "Kimheang Ya", "Pakpoom Subsoontorn", "Manupat Lohitnavy", "Prinya Musikaphongsakul", "Kimheang Ya", "Pakpoom Subsoontorn" ], "abstract": "Background: Psilocybin (PB) is a psychoactive compound commonly found in magic mushroom (Psilocybe cubensis). PB is quickly converted by the body to psilocin (PI), which has a psychedelic effect through the activation of the 5-HT2A receptor in the brain. The objective of this study is to develop a physiologically based pharmacokinetic (PBPK) model of PB and PI in rats and humans for predicting concentrations of the psychoactive substance in the brain. Methods: Following a search in PubMed, three studies were retrieved and information concerning concentration-time profiles of PI were extracted from the selected studies. In the study in rats, PI was orally administered with a dose of 10.1 mg/kg. There were two studies in humans following a single intravenous dose of PB (1 mg) and oral dose of PB (0.224 mg/kg and 0.3 mg/kg). Berkeley Madonna software was used for computer coding and simulations. The developed PBPK model consisted of seven organ compartments (i.e. lung, heart, brain, fat, muscle, kidney, and liver). Results: The simulations show a good agreement between observed and simulated data, although results for oral administration in rats and humans showed under-predictions and results for intravenous administration in humans showed over-predictions. Conclusions: A PBPK model of PB and PI in rats and humans was developed and could predict concentration-time profiles of PI in plasma, particularly in the brain, following intravenous and oral administration of PB. This model may be useful for a safer dosage regimen of PB for patients with some disorders.", "keywords": [ "Psilocybin", "Psilocin", "Magic mushroom", "Gold cap mushroom", "PBPK model" ], "content": "Introduction\n\nMagic mushroom (Psilocybe cubensis), also called gold cap mushroom, can be found in tropical and subtropical areas around the world. Magic mushroom is a psychedelic mushroom that can cause euphoria, hallucinations (mental, auditory) and changes in perception1. Psilocybin (PB) and psilocin (PI) are the major psychoactive compounds in the mushrooms2.\n\nPB (4-phosphoryloxy-N,N-dimethyltryptamine) is a compound found in the mushroom in a psychologically inactive form. It can be orally absorbed (F = 0.5) following single oral administration in humans2. After being orally absorbed, PB is rapidly dephosphorylated by alkaline phosphatase and non-specific esterase to produce an active metabolite, namely PI (4-hydroxy-N,N-dimethyltrypt-amine)2,3. Since PI is structurally similar to serotonin, PI can bind and activate several receptors in the brain such as 5-HT2A, 5-HT1A, 5-HT1D, and 5-HT2C receptors3. Activation through the 5-HT2A receptor can lead to the psychedelic effects of the mushrooms4. PI can be glucoronidated by UDP-glucuronosyltransferase (UGT) 1A10 in the liver and intestinal mucosa5.\n\nThere have been studies of PB for treatment of some psychological disorders such as depression, suicidal attempts, obsessive-compulsive disorder (OCD), alcohol use disorder, tobacco use disorder and resistant depression5,6. However, knowledge of how PB and PI behave in the body is still incomplete, particularly information concerning PI concentration levels in the brain, the major target organ of the magic mushroom. As a result, dosing of PB and PI in humans can lead to inadequate outcomes. Physiologically-based pharmacokinetic (PBPK) modeling is a quantitative tool capable of predicting concentration-time profiles of chemicals including drugs, toxicants and natural products7–12. Any biologically relevant process related to the PK of the drugs can be incorporated into the model. To be able to quantify brain concentration levels of PI in human brains following PB administration, a PBPK model of PB and PI with a description of a brain compartment can be useful in understanding the disposition of PI. Therefore, the objective of this study was to develop a PBPK model of PB and PI in rats and humans.\n\n\nMethods\n\nPBPK model structure. A PBPK model structure consists of seven compartments including the lung, heart, brain, fat, muscle, kidney, and liver. All of the organ compartments are linked together by the blood circulation system. The lung is the site of an interchange of the arterial and venous blood. The brain is the major site of action of PI. PI is assumed to solely be eliminated from the body through the liver. Adipose tissue is the storage organ for PI in the body. Additional organs including the heart and kidney are included in the model regarding their role in the pharmacokinetics and pharmacodynamics of PI. The structure of our PBPK model of PB and PI is depicted in Figure 1.\n\nTissue model specification. A perfusion rate-limited and well-stirred tissue model hypothesis was assumed for all organs.\n\nModel parameterization and equations. Physiological parameters including organ blood flow (Q) and organ volume (V) were obtained from published literature13–15. Pharmacokinetic parameters including bioavailability and absorption rate constant were acquired from published literature16,17 and optimized using Berkeley Madonna Software. Physicochemical parameters including partition coefficient of each compartment were calculated using an approach from the literature18. A PBPK model was coded and performed using Berkeley Madonna Software version 8.3.18 (developed by Robert Macey and George Oster of the University of California at Berkeley). Replication of the simulation results from our PBPK model using an alternative and freely available software is possible; recently, Zurlinden T.J. et al.19 have developed a physiologically based pharmacokinetic model of a rifapentine model using the Python software (version 2.7.2) as their computational tool along with these numby, scipy and matplotlib packages20–23. All of the parameters used in the PBPK model are summarized in Table 1.\n\nNote: * Optimized value using Berkeley Madonna software.\n\nRoutes of PI administration. In this development of the PBPK model of PB and PI in rats and humans, there were two different routes of administration. Those included 1) intravenous (IV) administration and 2) oral administration. Differential equations (Equation 1 – Equation 3) describing the respective routes of administration are as follows:\n\n1) Intravenous administration:\n\n\n\nwhere Dose is an administered dose of PI (mg), BW is the body weight (kg), Time is the duration period of the injection (h), and Kc is the conversion rate constant.\n\n2) Oral administration:\n\n\n\n\n\nwhere AGU is the amount of PI in the gut lumen after an oral administration (μg), ka is the absorption rate constant (1/h), F is the bioavailability, and Kc is the conversion rate constant.\n\nOrgan compartments. In this PBPK model, there were seven organ compartments. Differential equations (Equation 4 – Equation 20) describing concentration-time profiles of PI in each of seven compartments are as follows:\n\n1) Lung:\n\n\n\n\n\nwhere ALU is the amount of PI in the lung (µg), QC is the cardiac output (l/h), CV is the concentration of PI in venous blood (µg/l), CALU is the concentration of PI in arterial blood leaving the lung (µg/l), CLU is the concentration of PI in the lung (µg/l), VLU is the volume of the lung (l), and PLU is the lung/blood partition coefficient.\n\n2) Heart:\n\n\n\n\n\nwhere AH is the amount of PI in the heart (µg), QH is the blood flow to the heart (l/h), CA is the concentration of PI in arterial blood (µg/l), CVH is the concentration of PI in venous blood leaving from the heart (µg/l), CH is the concentration of PI in the heart (µg/l), VH is the volume of the heart (l), and PH is the heart/blood partition coefficient.\n\n3) Brain:\n\n\n\n\n\nwhere ABR is the amount of PI in the brain (µg), QBR is the blood flow to the brain (l/h), CA is the concentration of PI in arterial blood (µg/l), CVBR is the concentration of PI in venous blood leaving from the brain (µg/l), CBR is the concentration of PI in the brain (µg/l), VBR is the volume of the brain (l), and PBR is the brain/blood partition coefficient.\n\n4) Fat:\n\n\n\n\n\nwhere AF is the amount of PI in the fat (µg), QF is the blood flow to the fat (l/h), CA is the concentration of PI in arterial blood (µg/l), CVF is the concentration of PI in venous blood leaving from the fat (µg/l), CF is the concentration of PI in the fat (µg/l), VF is the volume of the fat (l), and PF is the fat/blood partition coefficient.\n\n5) Muscle:\n\n\n\n\n\nwhere AMU is the amount of PI in the muscle (µg), QMU is the blood flow to the muscle (l/h), CA is the concentration of PI in arterial blood (µg/l), CVMU is the concentration of PI in venous blood leaving from the muscle (µg/l), CMU is the concentration of PI in the muscle (µg/l), VMU is the volume of the muscle (l), and PMU is the muscle/blood partition coefficient.\n\n6) Kidney:\n\n\n\n\n\nwhere AK is the amount of PI in the kidney (µg), QK is the blood flow to the kidney (l/h), CA is the concentration of PI in arterial blood (µg/l), CVK is the concentration of PI in venous blood leaving from the kidney (µg/l), CK is the concentration of PI in the kidney (µg/l), VK is the volume of the kidney (l), and PK is the kidney/blood partition coefficient.\n\n7) Liver:\n\n\n\n\n\n\n\nwhere ALI is the amount of PI in the liver (µg), QLI is the blood flow to the liver (l/h), CA is the concentration of PI in arterial blood (µg/l), CVLI is the concentration of PI in venous blood leaving from the liver (µg/l), CLI is the concentration of PI in the liver (µg/l), VLI is the volume of the liver (L), PLI is the liver/blood partition coefficient, LIMet is the amount of PI excreted via liver metabolism, Vmax is the maximum velocity of UGT1A10 (μmol/h), and Km is Michaelis-Menten constant of UGT1A10 (μM).\n\n8) Blood:\n\n\n\n\n\nwhere AV and AA are the amount of PI in the venous and arterial blood, QH, QBR, QF, QMU, QK, QLI are the blood flow rate into the heart, brain, fat, muscle, kidney, and liver (l/h), and CVH, CVBR, CVF, CVMU, CVK, CVLI are the concentration of PI in the venous blood leaving from the heart, brain, fat, muscle, kidney, and liver (μg/).\n\nFrom a search in PubMed from inception to July 2019, the search terms “psilocybin; psilocin; magic mushroom; pharmacokinetic” were used as keywords. Studies were included if they met the selection criteria as follows: pharmacokinetic studies of psilocybin or psilocin that were conducted in animals or humans and different of routes of administration. Studies were excluded if they conducted pharmacokinetic studies in unhealthy or pregnancy animals or humans. From the following information in the literature, three studies were retrieved and were subsequently used for further model development. The PBPK model was evaluated based on visual inspection of goodness of fit. A summary of the selected studies is as follows:\n\nIn the study by Chen et al.26, Sprague-Dawley rats (n = 10, BW = 220 – 250 g) were orally administered with a single dose of G. spectabilis extract, which is equivalent to 10.1 mg/kg of PI. Then, a serial of blood samples was collected at 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360, and 420 minutes after oral administration. Then, the samples were analysed for PI using ultra-performance liquid chromatography coupled to photodiode array detection (UPLC-PDA).\n\nIn the study by Hasler et al.17, nine healthy male volunteers (BW = 56 – 72 kg) were intravenously administered with a single dose of 1 mg PB. Subsequently, blood samples were collected at 0.75, 1.5, 2.5, 3.75, 5, 6.75, 10, 15, 20, 30, 60, and 120 minutes. In addition, six healthy male and female volunteers (BW = 53 – 88 kg) were orally administered with a single oral dose of 0.224 mg/kg PB. Blood samples were collected at 15, 30, 4.5, 60, 7.5, 90, 105, 120, 150, 180, 220, 300, 350, and 390 min after the oral PB administration. Then, the samples were analysed using high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD).\n\nIn Brown et al.16, 12 healthy volunteers were given a single oral administration of PB 0.3 mg/kg. Blood collections were performed at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 18, and 24 hours. Subsequently, the samples were analysed for PI concentration using a HPLC technique.\n\nPI concentration levels shown in figures for the selected studies were extracted using WebPlotDigitizer version 4.4 (Free Software Foundation, Inc., Boston, MA). Assessment of model qualification was based on visual inspection of the agreement between observed and simulated PI concentrations. Regression analysis between the simulated and observed PI levels was also performed in Microsoft Excel version 2019.\n\n\nResults\n\nFollowing a single oral administration of PI (10.1 mg/kg) in rats, the simulated results of PI concentration profiles in plasma from the developed model compared to observed data acquired from the study by Chen et al.26 is demonstrated in Figure 2A with the simulated PI concentration-time profiles in the brain27. Subsequently, simulated results of PI concentration-time profiles in the lung, heart, muscle, kidney, and liver are illustrated in Figure 2B.\n\n(A) Concentration-time profiles of psilocin (PI) in rat plasma and brain following a single oral administration of psilocin (PI) (10.1 mg/kg). Solid and dashed lines are concentration-time profiles of PI from the developed physiologically based pharmacokinetic (PBPK) model, whereas closed circles with error bars (S.E.) are experimental data acquired from Chen et al.26. (B) Simulated concentration-time profiles of PI following a single oral dose of PI (10.1 mg/kg) in rat’s tissues such as lung (blue solid line), fat (brown solid line), muscle (red solid line), liver (green solid line), kidney (purple solid line), and heart (black solid line).\n\nFollowing a single IV administration of PB (1 mg) in humans, simulated PI concentration-time profiles in plasma compared to the observed data acquired from Hasler et al.17 are demonstrated in Figure 3A. In addition, PI concentration-time profiles in the human brain were simulated and are presented in Figure 3A. Simulated PI concentration-time profiles of other tissues are illustrated in Figure 3B.\n\n(A) Concentration-time profiles of psilocin (PI) in human plasma and brain following a single intravenous administration of psilocybin (PB) (1 mg). Solid and dashed line are concentration-time profiles of PI from the developed physiologically based pharmacokinetic (PBPK) model, whereas closed circles with error bars (S.E.) are experimental data acquired from Hasler et al.17. (B) Simulated concentration-time profiles of PI following a single intravenous dose of PB (1 mg) in human tissues such as lung (blue solid line), fat (brown solid line), muscle (red solid line), liver (green solid line), kidney (purple solid line), and heart (black solid line).\n\nIn the selected studies, two different doses of PB (0.224 mg/kg and 0.3 mg/kg) were administered to humans via a single oral administration. Simulated results of PI concentration-time profiles in plasma following oral administration of PB (0.224 mg/kg and 0.3 mg/kg) compared to the observed data acquired from Hasler et al.17 and Brown et al.16 are presented in Figure 4A and Figure 5A, respectively. Simulated brain concentration-time profiles of PI for both doses (0.224 mg/kg and 0.3 mg/kg) are also presented in Figure 4A and Figure 5A, respectively. Then, from the developed model following oral administration of PB (0.224 mg/kg and 0.3 mg/kg), the simulated results of PI in organs of interest including the lungs, fat tissue, muscle, kidney, and liver are depicted in Figure 4B and Figure 5B.\n\n(A) Concentration-time profiles of psilocin (PI) in human plasma and brain following a single oral administration of psilocybin (PB) (0.224 mg/kg). Solid and dashed lines are concentration-time profiles of PI from the developed physiologically based pharmacokinetic (PBPK) model, whereas closed circles with error bars (S.E.) are experimental data acquired from Hasler et al.17. (B) Simulated concentration-time profiles of PI following a single oral dose of PB (0.224 mg/kg) in human tissues such as lung (blue solid line), fat (brown solid line), muscle (red solid line), liver (green solid line), kidney (purple solid line), and heart (black solid line).\n\n(A) Concentration-time profiles of psilocin (PI) in human plasma and brain following a single oral administration of psilocybin (PB) (0.3 mg/kg). Solid and dashed lines are concentration-time profiles of PI from the developed physiologically based pharmacokinetic (PBPK) model, whereas closed circles with error bars (S.E.) are experimental data acquired from Brown et al.16. (B) Simulated concentration-time profiles of PI following a single oral dose of PB (0.3 mg/kg) in human tissues such as lung (blue solid line), fat (brown solid line), muscle (red solid line), liver (green solid line), kidney (purple solid line), and heart (black solid line).\n\nOur model simulations in both species included different routes of administration including an oral dose of PI in rats (10.1 mg/kg), IV dose of PB in humans (1 mg), and different oral doses of PB in humans (0.224 mg/kg and 0.3 mg/kg). Key parameters including maximum concentration (Cmax), time to reach maximum concentration (Tmax), and area under the curve from time equals zero to the last time point (AUC0-t) from the developed PBPK model and experimental data in both rats and humans are demonstrated in Table 2.\n\nNote: Cmax, maximum plasma concentration; Tmax, time to reach maximum concentration; AUC, area under the curve.\n\n\nDiscussion\n\nTo our knowledge, this current PBPK model of PB and PI is the first model with the capacity to describe PI concentration-time profiles following an oral administration of PI in rats as well as an intravenous and oral administration of PB in humans. The developed PBPK model could describe PI concentration-time profiles in plasma from both species (Figure 2A, Figure 3A, Figure 4A and Figure 5A). Furthermore, the developed PBPK model could simulate PI concentration-time profiles in other tissues, particularly in the brain, which is the major target organ of the psychoactive compound in magic mushrooms.\n\nHowever, under-predicting of blood PI levels at early time points after PB oral administration was observed (Figure 4A and Figure 5A). These under-predictions could be a result of the conversion rate constants used in the current PBPK model of PB and PI. In this model, the conversion rate constant was assumed as a constant in a zero-ordered kinetic process. However, the enzymatic reaction by the esterase enzyme system in the blood could be either a first-ordered kinetic process or Michaelis-Menten process. Nonetheless, our model outputs after two hours post-administration could capture most of the data points acquired from the selected studies reasonably well (Figure 2A, Figure 4A, and Figure 5A).\n\nFollowing IV administration in human subjects, from time equals zero to about half an hour, model over-predictions were observed (Figure 3A). These over-predictions might be a result of 1) over-simplification from the well-stirred model hypothesis and 2) at the beginning of the IV administration, the administrated amount of PB could saturate the available enzymatic process for changing PB to PI in the body.\n\nOur developed PBPK model of PB and PI is capable of making predictions of PI concentration-time profiles in the brain of both rats and humans. However, data concerning brain PI concentration-time profiles are lacking. From our literature search, there was a study by Law et al.28. In this study, pregnant rats were intravenously administrated with PI. Subsequently, tissue samples (i.e. brain, lung, fat, muscle, kidney, liver, spleen, placenta, and fetus) were harvested at different time points and PI concentration levels in the tissue samples were analyzed. However, this study was conducted in pregnant rats which, due to potential changes as a result of the physiology of the pregnancy, may significantly influence the pharmacokinetics of PB and PI. Consequently, we could not use this dataset in the current model development. Thus, to have more confidence in the developed PBPK model, further PI concentration-time profiles in tissue organs including the brain are necessary. Therefore, a new study in rats intravenously and orally administered with PB with an additional design of tissue harvesting (e.g. brain, fat, muscle, kidney, and liver) is warranted. In addition, to further extend the model’s application and to make an adequate prediction between the administered dose of PB and PI and its psychoactive effects, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model for PB and PI in humans should be developed.\n\n\nConclusions\n\nA PBPK model of PB and PI in rats and humans was developed. The PBPK model could predict concentration-time profiles of PI following intravenous and oral administration of PB. In addition, concentration-time profiles of PI in the brain, the major target organ of PI, could be simulated. The PBPK model of PB and PI may be useful for a safer dosage regimen for patients with some disorders for which PB or magic mushrooms may be therapeutically effective.\n\n\nData availability\n\nZenodo: Heang60/PBPK-of-Magic-Mushroom: Coding for PBPK of Magic Mushroom. https://doi.org/10.5281/zenodo.453661727.\n\nThis project contains the following underlying data:\n\n- Raw data (Figures 2–5)_magic mushroom.xlsx (raw data underlying Figures 2–5)\n\nZenodo: Heang60/PBPK-of-Magic-Mushroom: Coding for PBPK of Magic Mushroom. https://doi.org/10.5281/zenodo.453661727.\n\nThis project contains the following extended data:\n\n- Code availability_magic mushroom.pdf (parameters and code for model reproducibility)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWong E, Patel JK, Langenfeld S: Chapter 4 - Drugs of abuse. In: Aronson JK, editor. Side Effects of Drugs Annual. 31: Elsevier; 2009; 33–55.\n\nPassie T, Seifert J, Schneider U, et al.: The pharmacology of psilocybin. Addict Biol. 2002; 7(4): 357–64. PubMed Abstract \| Publisher Full Text\n\nTylš F, Páleníček T, Horáček J: Psilocybin--summary of knowledge and new perspectives. Eur Neuropsychopharmacol. 2014; 24(3): 342–56. PubMed Abstract \| Publisher Full Text\n\nMadsen MK, Fisher PM, Burmester D, et al.: Psychedelic effects of psilocybin correlate with serotonin 2A receptor occupancy and plasma psilocin levels. Neuropsychopharmacology. 2019; 44(7): 1328–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaniel J, Haberman M: Clinical potential of psilocybin as a treatment for mental health conditions. Ment Health Clin. 2018; 7(1): 24–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNutt D: Psychedelic drugs-a new era in psychiatry? Dialogues Clin Neurosci. 2019; 21(2): 139–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLohitnavy M, Chitsakhon A, Jomprasert K, et al.: Development of a physiologically based pharmacokinetic model of paraquat. Annu Int Conf IEEE Eng Med Biol Soc. South Korea. 2017; 2017: 2732–2735. PubMed Abstract \| Publisher Full Text\n\nLohitnavy M, Lu Y, Lohitnavy O, et al.: A physiologically-based pharmacokinetic model of methotrexate incorporating hepatic excretion via multidrug-resistance-associated protein 2 (Mrp2) in mice, rats, dogs, and humans. Annu Int Conf IEEE Eng Med Biol Soc. South Korea. 2017; 2017: 2728–2731. PubMed Abstract \| Publisher Full Text\n\nMethaneethorn J, Naosang K, Kaewworasut P, et al.: Development of a Physiologically-Based Pharmacokinetic Model of Δ9-Tetrahydrocannabinol in Mice, Rats, and Pigs. Eur J Drug Metab Pharmacokinet. 2020; 45(4): 487–494. PubMed Abstract \| Publisher Full Text\n\nMethaneethorn J, Poomsaidorn C, Naosang K, et al.: A Δ9-Tetrahydrocannabinol Physiologically-Based Pharmacokinetic Model Development in Humans. Eur J Drug Metab Pharmacokinet. 2020; 45(4): 495–511. PubMed Abstract \| Publisher Full Text\n\nTran QB, Phenrat T, Lohitnavy M: Physiologically based pharmacokinetic modeling of hydrogen cyanide in humans following the oral administration of potassium cyanide and cyanogenic glycosides from food. Human and Ecological Risk Assessment: An International Journal. 2019; 1–16. Publisher Full Text\n\nYa K, Methaneethorn J, Tran QB, et al.: Development of a Physiologically Based Pharmacokinetic Model of Mitragynine, Psychoactive Alkaloid in Kratom (Mitragyna Speciosa Korth.), In Rats and Humans. J Psychoactive Drugs. 2020; 1–13. Publisher Full Text\n\nBrown RP, Delp MD, Lindstedt SL, et al.: Physiological parameter values for physiologically based pharmacokinetic models. Toxicol Ind Health. 1997; 13(4): 407–84. PubMed Abstract \| Publisher Full Text\n\nDavies B, Morris T: Physiological Parameters in Laboratory Animals and Humans. Pharm Res. 1993; 10(7): 1093–5. PubMed Abstract \| Publisher Full Text\n\nLuttringer O, Theil FP, Poulin P, et al.: Physiologically based pharmacokinetic (PBPK) modeling of disposition of epiroprim in humans. J Pharm Sci. 2003; 92(10): 1990–2007. PubMed Abstract \| Publisher Full Text\n\nBrown RT, Nicholas CR, Cozzi NV, et al.: Pharmacokinetics of Escalating Doses of Oral Psilocybin in Healthy Adults. Clin Pharmacokinet. 2017; 56(12): 1543–54. PubMed Abstract \| Publisher Full Text\n\nHasler F, Bourquin D, Brenneisen R, et al.: Determination of psilocin and 4-hydroxyindole-3-acetic acid in plasma by HPLC-ECD and pharmacokinetic profiles of oral and intravenous psilocybin in man. Pharm Acta Helv. 1997; 72(3): 175–84. PubMed Abstract \| Publisher Full Text\n\nPoulin P, Theil FP: Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distribution. J Pharm Sci. 2002; 91(1): 129–56. PubMed Abstract \| Publisher Full Text\n\nZurlinden TJ, Eppers GJ, Reisfeld B: Physiologically Based Pharmacokinetic Model of Rifapentine and 25-Desacetyl Rifapentine Disposition in Humans. Antimicrob Agents Chemother. 2016; 60(8): 4860–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPython Language Reference version 2. 7: Python Software Foundation: Python 435 Language Reference, version 2.7.\n\nSvd W, Colbert SC, Varoquaux G: The NumPy Array: A Structure for Efficient Numerical Computation. Computing in Science & Engineering. 2011; 13(2): 22–30. Publisher Full Text\n\nJones E, Oliphant T, Peterson P: SciPy: Open Source Scientific Tools for Python. 2001. Reference Source\n\nHunter JD: Matplotlib: A 2D Graphics Environment. Computing in Science & Engineering. 2007; 9(3): 90–5. Publisher Full Text\n\nNational Center for Biotechnology Information: PubChem Database. Psilocybine, CID=4980. 2020. Reference Source\n\nNational Center for Biotechnology Information: PubChem Database.Psilocybine, CID=10624. 2020. Reference Source\n\nChen J, Li M, Yan X, et al.: Determining the pharmacokinetics of psilocin in rat plasma using ultra-performance liquid chromatography coupled with a photodiode array detector after orally administering an extract of Gymnopilus spectabilis. J Chromatogr B Analyt Technol Biomed Life Sci. 2011; 879(25): 2669–72. PubMed Abstract \| Publisher Full Text\n\nKimheang Y: Heang60/PBPK-of-Magic-Mushroom: Coding for PBPK of Magic Mushroom (Version v1.0.0) [Data set]. Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4536617\n\nLaw F, Poon G, Chui Y, et al.: 14C-Psilocin tissue distribution in pregnant rats after intravenous administration. Functional Foods in Health & Disease. 2014; 4: 232–44. Publisher Full Text" }	[ { "id": "81816", "date": "31 Mar 2021", "name": "Michael Dzierlenga", "expertise": [ "Reviewer Expertise Pharmacokinetics and PBPK modeling." ], "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\nMusikaphongsakul et al. present the first PBPK model for psilocin (PI), the active metabolite of psilocybin (PB). The model is described as a model of PB and PI, but PB is not included in the PBPK as it is assumed to be instantaneously metabolized to PI. The model and its presentation are incomplete, and the manuscript is not yet ready to be indexed.\nThe key inadequacies are a disagreement between the way that the conversion from PB to PI is described and modeled, a poor fit to the experimental plasma time-courses at early times, lack of an analysis of the accuracy of the predicted partition coefficients, lack of consideration of urinary clearance of unmetabolized PI, lack of an analysis of the results of parameter optimization and the sensitivity of model predictions to those parameters, and a lack of distinction between PI in blood and plasma. These inadequacies need to be addressed before the model is suitable forindexing because they will help readers to understand the limitations of the model. As the paper stands, I have concerns that others might apply the model to predict brain concentrations without understanding how uncertain those measurements are.\nMajor Issues:\nI have concerns about the way that the conversion from PB to PI is implemented. The paper states that this conversion occurs “rapidly” after oral absorption. In the model, this is captured as an instantaneous and incomplete process, where Kc simply scales the dose. For example, in an oral experiment, the initial amount in the oral uptake compartment is the dose multiplied by F, the fraction bioavailable, and Kc. Kc is described in the paper as a “conversion rate constant” and as a “zero-ordered kinetic process”. This description does not agree with the model implementation.\nTo justify the way that Kc is included in the model as written, the paper needs to describe Kc as the fraction of PB that is converted to PI, provide justification that the conversion between PB and PI is incomplete, and justify that this conversion is instantaneous both in oral and IV exposure. The correct way to model a “conversion rate constant” and a “zero-ordered kinetic process” would be to track the amount of PB as a state variable and describe decreases in PB and increases in PI as rates in differential equations.\nThe only data which was used to inform the model was PI plasma data from 3 experiments. 5 parameters were optimized using this data by a visual fitting process. Visual fitting is not a recommended method but can be overlooked if the outcome of the fit is satisfactory. The model fit to the plasma time-courses were poor at early times, especially for the oral rat time-course and the IV human time-course. A poor model fit to data after optimization suggests that a better optimization scheme is need or that something about the model structure itself is insufficient.\nThe use only of plasma data to inform the model is problematic because there is no way to determine the accuracy of the predicted tissue-blood partition coefficients for PI. The authors mention that tissue specific concentrations do exist, but only for pregnant rats (Law et al. 2014; reference 28 in manuscript). Pregnant animals will have some PK differences relative to non-pregnant animals, but a comparison of that data to the model prediction may still prove fruitful. A quick look shows that that maternal blood concentration was roughly twice the maternal brain concentration in Law et al (a partition coefficient of roughly 0.5). The predicted brain:blood partition coefficient was 2, which is substantially different, and this discrepancy should be discussed and explained. Furthermore, the paper being reviewed states in the first paragraph of the methods that adipose tissue is a storage organ for PI. The predicted partition coefficient is around 0.15, which suggests that PI has a low affinity for adipose tissue compared to blood or other organs. Suggesting that most of the chemical is not stored in fat. Another way to evaluate whether the predicted partition coefficients were reasonable would be to calculate the volume of distribution of the model (as the weighted sum of organ volumes and partition coefficients) and compare that to Vd from PK studies (if available). The clearance rate of the model should also be compared to published clearance or half-life values (if available).\nKalberer et al. (1962)1 reported that approximately 25% of PI dosed is excreted unchanged in urine. Urinary clearance is not captured in the model, which assumes PI is metabolized prior to excretion. This is a flaw of the model that should be addressed. In addition, PI is described to be metabolized by UGT 1A10 in the liver and intestinal mucosa, but metabolism in the model only takes place in the liver, which seems to disagree with the description in the introduction. This may be especially important for oral dosage since metabolism in the intestine would increase initial first-pass metabolism.\nTable 1 shows the optimized parameters for rat and human. There needs to be some analysis of the parameter values that were reached after optimization, including a comparison between rat and human values. In addition, I think a sensitivity analysis is warranted to show the relative impact of the different parameters on the plasma and brain concentrations. Table 1 also has some typographical errors. Partition coefficients for lung and brain have the symbol for muscle. For human bioavailability and ka, there is some issue with the presentation of the human values. Also, molecular weight is put in columns for rat and humans when the molecular weight of PI and PB is species independent.\nThere needs to be some description of the ratio of PI between whole blood and plasma. It seems like organ blood flow and partition coefficients are for whole blood, but chemical concentrations are treated as plasma concentrations. I believe this assumes that the whole blood to plasma ratio is 1, for which evidence is not provided.\nMinor Issues:\nDose should have units of mg/kg in the equations in the methods section.\n\nAbsorption rate constant is formatted as Ka in the oral equation, but ka in Table 1.\n\nCv equations need a parenthesis: Cv = A/(V*P).\n\nLImet should be described as the rate of metabolism of PI, not the amount of PI metabolized. The amount metabolized is the integral of LImet.\n\nIn the Blood equation description, the unit at the end is “ug/” instead of ug/l.\n\nIn the first subheading of the Results section, “PB” is listed as the chemical, when PI is dosed.\n\nConcentration figures are not color-blind friendly. Ideally, a palette optimized for color-blindness or different line-types should be used.\n\nThe times for the Hasler experiment should be listed in increasing order.\n\nIn Zurlinden, et al. (reference 19 in the manuscript), the model was implemented and run in MCSim, not in Python. Data processing and analysis was performed in Python.\n\nThere should be some description of why a perfusion limited model is appropriate.\n\nThe discussion needs to better place the work in context. How will the brain concentration predicted in the model help to aid dosimetry and the potential application to mental health treatment?\n\nEnsure that the MW parameter in the code is correctly specified for each chemical. For example, when PB is dosed is the MW for PB being used to scale that dose initially and then the MW for PI is being used in subsequent conversions?\n\nThe concentration unit in comments in the code is sometimes specified as \"mcmole\" instead of mcmole/L.\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?\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": "81474", "date": "08 Apr 2021", "name": "Quoc Ba Tran", "expertise": [ "Reviewer Expertise Environmental health", "Environmental modeling", "PBPK modeling", "Air pollution" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor issues: Could you please explain why the PBPK model structure consists of seven compartments: the lung, heart, brain, fat, muscle, kidney, and liver?\nThis study simulated the concentration-time courses of psilocin (PI) following a single oral/single intravenous administration of psilocybin (PB) in human/rat tissues (the lung, heart, brain, fat, muscle, kidney, and liver). However, the whole study only gives data/information on the relationship between PB adsorption and PI concentration in plasma, and just a few data on the relationship between PB adsorption and PI concentration in other components of the human body/rat. Therefore, the study should find more solid scientific evidence, that mentions PI concentration will be distributed in those components of the human body/rat if PB is absorbed.\nOne of the main objectives of this study is to quantify brain concentration levels of PI in human brains following PB administration. Therefore, in the Discussion session, please carefully discuss the relationship between PI concentration in brain and PB absorption.\nMinor issues: I have read reference number [18] of this study but have not found the corresponding data, which you acquired for the section “Partition Coefficients\" of Table 1. Please explain how did you acquire the data for the parameters: Fat (PF); Liver (PLI); Muscle (PMU); Lung (PMU); Kidney (PK); and Brain (PMU) in Table 1.\nPart of the information in Table 1 (Blood flow fraction and Tissue Volume fraction) is the basic information that has been published by many studies, so this study may consider incorporating this information into the SI section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-209
https://f1000research.com/articles/9-324/v1	04 May 20	{ "type": "Research Article", "title": "Comparison of WHO and Indonesian growth standards in determining prevalence and determinants of stunting and underweight in children under five: a cross-sectional study from Musi sub-district", "authors": [ "Jeannie Flynn", "Firas Farisi Alkaff", "William Putera Sukmajaya", "Sovia Salamah", "Jeannie Flynn", "William Putera Sukmajaya", "Sovia Salamah" ], "abstract": "Background: Undernutrition among children under five continues to be a critical global public health challenge, especially in developing countries. However, it is believed that Indonesian children are “below” the global standard, thus the WHO standard is not reliable to present the actual prevalence. This study aims to compare the difference between WHO and Indonesian growth standards regarding prevalence of stunting and underweight and its determinants. Methods: This is a cross-sectional study carried out in Musi sub-district, East Nusa Tenggara province in July 2019. East Nusa Tenggara province had the highest prevalence of stunting and underweight in Indonesia. The study population were children under five, and total sampling method was used for this study. Length/height-for-age and weight-for-age were plotted using WHO and national standards. Univariate and multivariate binomial logistic regression were used for statistical analysis. Results: The prevalence of stunting and underweight were higher for the WHO than the national standard (53.9% vs 10.7% and 29.17% vs 17.7%; all p < 0.001). Determinants of stunting were maternal mid-upper arm circumference below 23.5cm and maternal height below 150cm when the WHO standard was used, and no determinant was found when the national standard was used. Determinants of underweight were intrauterine growth restriction, young maternal age during pregnancy, and multiple parities when the WHO standard was used. When the national standard was used, the determinants of underweight were intrauterine growth restriction and maternal education. Conclusions: The WHO standard over-diagnosed stunting and underweight in Musi sub-district. Future studies should be done to re-evaluate the prevalence and determinants of stunting and underweight nationwide using the Indonesian standard.", "keywords": [ "growth chart", "Indonesia", "risk factors", "stunting", "underweight" ], "content": "Introduction\n\nUndernutrition among children under five continues to be a critical global public health challenge, especially in developing countries1. Not only affecting the health of one individual, undernutrition also contributes to many aspects of sustainable development2. There are three indicators to measure nutritional imbalance that lead to undernutrition, which are: stunting (low height for age), underweight (low weight for age), and wasting (low weight for height). Stunting is the result of chronic nutritional deprivation, reflecting the cumulative effects of undernutrition and infection. Underweight is a composite indicator and it includes both acute and chronic undernutrition. Wasting is a symptom of acute undernutrition, usually caused by insufficient food intake or high incidence of infectious disease. High prevalence of those indicators reflects poor nutrition and health status among children under five in the population3.\n\nAccording to the data from 2011, the global incidence of stunting, underweight, and wasting were approximately 164.8 million (25.7%), 100.7 million (15.7%), and 51.5 million (8%) among children under five, respectively. Meanwhile, the global deaths attributed to stunting, underweight, and wasting were approximately 1.017 million (14.7%), 999,000 (14.4%), and 875,000 (12.6%)4. Until 2018, undernutrition rates remained alarming, although the prevalence was declining. Among continents, Asia has the highest prevalence of stunting (55%) and wasting (68%). Based on country income classification, 65% of all stunted and 73% of all wasted children live in lower- and middle-income countries5. However, in the 2018 report, there is no updated data regarding the prevalence of underweight.\n\nThe latest basic health survey in Indonesia in 2018 showed that the prevalence of stunting, underweight, and wasting was 30.8%, 17.7%, and 10.2%, respectively. Among other provinces in Indonesia, East Nusa Tenggara province has the highest prevalence of stunting and underweight, at 42.6% and 29.5%, respectively. Meanwhile, the prevalence of wasting was lower, ranked 8th out of 35 provinces6. According to undernutrition severity classification, the severity of stunting is high and underweight is medium in Indonesia. In East Nusa Tenggara province, the severity of stunting is very high, and the severity of underweight is high7.\n\nThe determinants of child undernutrition are multifaceted and interconnected8. Understanding the determinants of childhood undernutrition is important to improve children’s nutrition by developing the effective and sustained multi-sectorial nutrition programs and interventions over the long term9. Unfortunately, studies evaluating the risk factors of child malnutrition in Indonesia were scarce10. A recent review article showed that determinants of stunting in Indonesia were similar to other countries, including maternal height and education, premature birth and birth length, exclusive breastfeeding, and socioeconomic status11.\n\nHowever, determination of undernutrition always uses the WHO growth standard in Indonesia. It is believed that Indonesian children are “below” the global standard in general, thus the WHO standard is not reliable to present the actual prevalence. Therefore, the Indonesia national growth standard was made using data from National Basic Health Survey12. To this date, no study has been done to scrutinize the difference between these two standards. This study aims to compare the prevalence and determinants of stunting and underweight using WHO and national standards. We use the data from one of the sub-districts in East Nusa Tenggara province because this province had the highest prevalence of stunting and underweight among children under five in Indonesia.\n\n\nMethods\n\nThis study followed the principles of the Declaration of Helsinki and was approved by the Department of Health Timor Tengah Utara district (approval number: DINKES.440/995/XI/2019). This study also complies with STROBE guidelines13,14. All parents gave their written informed consent prior to their children’s inclusion in the study. Information for informed consent was given before the informed consent form was signed. Details that might disclose the identity of the study subjects were omitted from the published data file.\n\nThis study was an observational cross-sectional study conducted in Musi sub-district, one of the sub-districts in East Nusa Tenggara province. Participant recruitment and data collection were conducted in July 2019. Data analysis was conducted in October – December 2019. There were six villages in Musi sub-district. The study population were children aged less than five years old. Total sampling was used for this study. The children and their parents were approached face-to-face by JF during the monthly growth monitoring program in Posyandu (“Pos Pelayanan Terpadu”), a healthcare program by the Indonesian government. Inclusion criteria were children under five who attended the growth monitoring program during the study period, who were born and live with their parents in Musi sub-district, and had both maternal and child health books (Buku Kesehatan Ibu dan Anak / KIA) and health record card (Kartu Menuju Sehat / KMS) published by the Ministry of Health Republic Indonesia. Children with incomplete KIA and KMS were excluded from the determinants analysis.\n\nBoth primary and secondary data was used in this study. Primary data for this study consisted of data obtained through interviews with parents, child anthropometry measurements, and maternal height measurements. The interviews took place in the same location as the Posyandu and were conducted by JF using a predetermined questionnaire. The length of the interview was around five minutes. JF is a female general practitioner who worked in primary healthcare in the sub-district where the study took place. She had worked there for seven months when the study was conducted. Interviews with parents was carried out to obtain information regarding village of origin, parents’ highest education, number of parities, delivery method, and gender and age of their children. Anthropometry measurements of maternal height and child length/height were done by healthcare workers from Oeolo Primary Healthcare. Secondary data from KIA and KMS was used to obtain data regarding birthweight, gestational age, maternal mid-upper arm circumference, and maternal age during pregnancy.\n\nUnderweight and stunting were categorized using WHO child growth standards and Indonesian growth standards for the same sex12,15. Underweight is defined as weight for age below -2 standard deviations (SD), and severe underweight is defined as weight for age below -3 SD. Stunting is defined as length/height for age below -2 SD, and severe stunting is defined as length/height for age below -3 SD. The cut-off level for maternal mid-upper arm circumference was 23.5 cm, for maternal height was 150 cm, and for children’s birthweight was 2500 g. The cut-off level for maternal mid-upper arm circumference was according to the Indonesian national cut-off16, while for maternal height and children’s birthweight, the cut-off was based on a previous study17. Maternal age during pregnancy was categorized to <20 years old, 20–35 years old, and >35 years old. Gestational age and intrauterine growth were categorized based on Lubchenco charts. It categorizes the gestational age to preterm (<37 weeks), term (37–42 weeks), or postterm (>42 weeks) and the intrauterine growth to small for gestational age (SGA) (<10th percentile), appropriate for gestational age (AGA) (10th – 90th percentile), or large for gestational age (LGA) (>90th percentile)18.\n\nAcquired data was analysed using SPSS Statistic for Windows, version 25.0 (IBM Corp., Armonk, N.Y., USA). Data analysis was conducted in two phases. In the first phase, univariate logistic regression was used to identify independent variables that were associated with stunting or underweight. Variables with p < 0.1 were included in the next phase. In second phase, multivariate logistic regression using backward selection was used. Variables with p <0.05 from multivariate analysis were considered as the determinants.\n\n\nResults\n\nThere was a total of 408 children under five in Musi sub-district. Based on WHO standard, the prevalence of stunting and underweight were 53.9% and 29.17%, respectively19,20. Using national standard, the prevalence of stunting and underweight were 10.7% and 17.7%, respectively. There was a significant difference of stunting and underweight between the prevalence from the WHO and national standard (both p <0.001). However, there were only 218 children that fulfilled the criteria to be included for the determinants analysis (Table 1).\n\nChi square test was used.\n\n*p-value between stunted (and severely stunted) and normal.\n\n#p-value between underweight (and severely underweight) and normal.\n\np <0.05 was considered statistically significant.\n\nThe prevalence of stunting and underweight among this study population were 51.4% and 31.7% according to WHO standard and 8.3% and 19.3% according to national standard (Table 1). The number of male and female children was almost equal. More than half of the children were aged between 24 and 59 months old. Majority of the children were born term with a birthweight of more than 2500 g. The education level of both parents was mainly elementary school graduates. Almost half of the mothers had a height of less than 150 cm and more than half of the mothers had a mid-arm circumference of ≤23.5 cm during pregnancy (Table 2).\n\nSGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; MUAC, mid-upper arm circumference.\n\nBased on WHO standard, univariate logistic regression analysis indicated that children with maternal height below 150 cm (OR = 2.844; 95% CI = 1.632 – 4.956) were more likely to be stunted (Table 3). In the multivariate logistic regression analysis, other variables with p-value between 0.05 and 0.1 from the univariate analysis (child’s birthweight, child’s intrauterine growth status, maternal mid-upper arm circumference, and number of parities) were included. Multivariate analysis indicated that children with maternal height below 150 cm (OR = 2.936; 95% CI = 1.672 – 5.154) or maternal mid-upper arm circumference below 23.5 cm (OR = 1.796; 95% CI = 1.008 – 3.105) were more likely to be stunted (Table 4).\n\nSGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; MUAC, mid-upper arm circumference.\n\nSGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; MUAC, mid-upper arm circumference.\n\nBased on national standard, univariate logistic regression analysis indicated that children with birthweight below 2500 g (OR = 2.948; 95% CI = 1.025 – 8.476) or with a father without formal education (OR = 10; 95% CI = 1.094 – 91.441) were more likely to be stunted (Table 3). In multivariate logistic regression analysis, other variables with p-value between 0.05 and 0.1 from the univariate analysis (child’s intrauterine growth status and maternal height) were included. No determinant was found in the multivariate analysis (Table 4).\n\nBased on WHO standard, univariate logistic regression analysis indicated that children with a birthweight below 2500 g (OR = 3.159; 95% CI = 1.507 – 6.622) or intrauterine growth restriction (OR = 3.715; 95% CI = 1.798 – 7.677) were more likely to be underweight. Children with maternal height below 150 cm (OR = 2.098; 95% CI = 1.176 – 3.745) or maternal age under 20 years old during pregnancy (OR = 5.312; 95% CI = 1.989 – 14.186) were also more likely to be underweight (Table 5). In multivariate logistic regression analysis, other variables with p-value between 0.05 and 0.1 from the univariate analysis (paternal education and number of parities) were included. Multivariate analysis indicated that children with intrauterine growth restriction (OR = 3.182; 95% CI = 1.450 – 6.980) were more likely to be underweight. Children with maternal age under 20 years old during pregnancy (OR = 6.252; 95% CI = 1.911 – 20.457) or with mother that had more than four parities (OR = 4.319; 95% CI = 1.189 – 15.689) were also more likely to be underweight (Table 6).\n\nSGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; MUAC, mid-upper arm circumference.\n\nSGA, small for gestational age; AGA, appropriate for gestational age; LGA, large for gestational age; MUAC, mid-upper arm circumference.\n\nBased on national standard, univariate logistic regression analysis indicated that children with birthweight below 2500 g (OR = 3.690; 95% CI = 1.680 – 8.107) or intrauterine growth restriction (OR = 4.825; 95% CI = 2.241 – 10.389) were more likely to be underweight. Children with mother without formal education (OR = 13.95%; CI = 1.207 – 139,959), with height below 150 cm (OR = 2.175; 95% CI = 1.095 – 4.318), or aged under 20 years old during pregnancy (OR = 3.590; 95% CI = 0.011) were also more likely to be underweight (Table 5). In multivariate logistic regression analysis, other variables with p-value between 0.05 and 0.1 from the univariate analysis (paternal education and number of parities) were included. Multivariate analysis indicated that children with intrauterine growth restriction (OR = 4.191; 95% CI = 1.820 – 9.649) were more likely to be underweight. Children with mother without formal education (OR = 27.341; 95% CI =1.281 – 583,318) were also more likely to be underweight (Table 6).\n\n\nDiscussion\n\nIn our study, the prevalence of both stunting and underweight were significantly lower when measured using Indonesian standard compared to when using WHO standard. It has been suggested that overdiagnoses of stunting or underweight are more likely to occur in developing countries21. There are many countries that already proposed their own national growth standard, which are: Korea22, Thailand23, Argentina24, China25, India21, and 18 European countries26. It is argued that the national growth standard of each country is more suitable to reflect the condition in its own population23. However, there were only few published studies that compare the difference between national growth standards and WHO growth standard. A comparison study among Thai children in the first two years of life showed that the prevalence of stunting was higher when using WHO standard in both sexes, but at 24 months the only significant difference was in girls. The prevalence of underweight showed a monotonic increment when using WHO standard, but the Thailand national standard showed a fluctuation23. In Argentina, the prevalence of underweight using WHO standard was 2 times higher than when using their national standard. Meanwhile for stunting, the prevalence when using WHO standard was 1.5 times higher24. In contrary, a comparison study from China showed that the prevalence of stunting and underweight was significantly higher when measured using their national standard25.\n\nThe marked difference in measurements using Indonesian standard and WHO standard probably stems from the difference in methodology during the development of both growth reference standards. The WHO standard was developed using data from five cities in five different countries: United States, Turkey, Norway, Brazil, and India. The children included in the study were healthy children with suitable sociodemographic conditions for growth. Moreover, all participants agreed to follow the feeding recommendation by WHO27. In contrary, the development of Indonesian standard did not have any inclusion and exclusion criteria for study participants. It also did not mention the sociodemographic background of the participants or their feeding habits. The study, however, collected data from all 33 provinces of Indonesia to better reflect the growth of Indonesian children12.\n\nReview article by Beal et al. concluded that the determinants of stunting in Indonesia are maternal height and education, child’s gender, premature birth and birth length, exclusive breastfeeding for six months, living area, and household socio-economic status11. In our study, the determinants of stunting according to WHO standard were maternal height less than 150 cm and maternal upper mid-arm circumference <23.5 cm. In contrast, no determinant was found when Indonesian standard was used. It is because the prevalence of stunting according to Indonesian standard was low. The significant difference in stunting prevalence calculated using Indonesian and WHO standards might be because the WHO standard does not represent local growth appropriately due to population differences in height26, and Indonesian people are generally shorter than the rest of the world.\n\nRegarding underweight, the determinants were also different according to the two different standards. However, there was one common determinant: intrauterine growth restriction. The difference of underweight prevalence between the two standards was not as marked as the difference in stunting prevalence; this may explain that there was still one overlapping determinant. The increased odds of undernutrition in SGA infants are more relevant in low- and middle-income countries28. SGA children are born with lower intrinsic potential for growth due to the persistent effect of growth restriction in utero29,30. SGA is a result of poor maternal nutrition during pregnancy when the child is totally dependent on getting nutrition from the mother through the placenta, hence any nutrition deprivation from the mother will affect the proper growth and development of the fetus31.\n\nThere were several limitations of this study. We did not discern the feeding habits of the participants of this study. Feeding habit could be an important determinant of malnutrition. For example, introduction of complimentary food earlier than four months increased the likelihood of being underweight and stunted32. Data on exclusive breastfeeding and history of immunization cannot be obtained because some of our samples have not yet completed the exclusive breastfeeding and basic immunization period. Data regarding socioeconomic status could not be obtained due to parents’ unstable monthly income. Data regarding the frequency of diarrhea could not be obtained because this was not well documented in primary healthcare medical records. These factors should be accounted for in the ensuing studies. Nevertheless, to our despite all of the limitations, this is the first study that compare the prevalence and determinants of stunting and underweight among Indonesian children under five using Indonesian growth standard and WHO growth standard.\n\n\nConclusion\n\nThe WHO standard was not suitable to diagnose stunting and underweight in Musi sub-district, since the prevalence was significantly higher when using WHO standard compared to when using Indonesian standard. Future studies should be done to re-evaluate the prevalence and determinants of stunting and underweight nationwide using the Indonesian standard. An Indonesian standard for weight-for-height should also be made to re-evaluate the prevalence and determinants of wasting in Indonesia.\n\n\nData availability\n\nFigshare: Growth standard comparison between WHO and Indonesian Growth Chart-Population Data. https://doi.org/10.6084/m9.figshare.12121938.v519\n\nFigshare: Growth standard comparison between WHO and Indonesian Growth Chart-Determinants Data. https://doi.org/10.6084/m9.figshare.12127425.v320\n\nFigshare: STROBE Checklist-Indonesian and WHO Growth Standard Comparison. https://doi.org/10.6084/m9.figshare.12127689.v213\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors thank Dr. Aman Bhakti Pulungan for providing the chart of Indonesian National Growth Standard and to Oeolo primary healthcare workers for their assistance during data collection.\n\n\nReferences\n\nGebre A, Reddy PS, Mulugeta A, et al.: Prevalence of Malnutrition and Associated Factors among Under-Five Children in Pastoral Communities of Afar Regional State, Northeast Ethiopia: A Community-Based Cross-Sectional Study. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63062", "date": "05 May 2020", "name": "Michael Hermanussen", "expertise": [ "Reviewer Expertise child growth", "pediatrics", "endocrinology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aimed to compare Indonesian growth references for height, weight, and BMI with WHO standards. The authors conclude that the WHO standards over-diagnose stunting and underweight in the Musi sub-district.\n\nThis is an important study as it examines whether the globally used WHO growth charts are applicable also for infants and children of the Musi sub-district, Indonesia.\n\nYet, the manuscript needs clarification.\n\nThe authors do not clearly distinguish between the terms “standard” and “reference”: Growth references are statistical summaries of anthropometry, conditioned (usually) on age and sex. References describe how children do grow. Growth standards describe how children should grow. (Garza C, de Onis M. Rationale for developing a new international growth reference. Food Nutr Bull 2004;25(1 Suppl):S5-14.). This is an important distinction. The data presented in this study provide a growth “reference” for the children of the Musi sub-district as they describe how these children grow. They do not describe how these children might grow under different conditions.\n\nThe authors use the terms “stunting”, “wasting”, and “underweight”. These terms refer to body size, they do not describe the nutritional situation. Underweight is defined as: weight for age < –2 standard deviations (SD) of the WHO Child Growth Standards median. Stunting is defined as height for age < –2 SD of the WHO Child Growth Standards median. Wasting is defined as weight for height < –2 SD of the WHO Child Growth Standards median1). The three terms relate to an internationally used “standard”, suggesting that this standard is also applicable to the Indonesian population. But this has been questioned. Healthy Indonesians are shorter and lighter than healthy white US citizens (who grow similar to what the WHO considers their growth standard). The difference in size between Indonesian and US Americans was the rational to develop national Indonesian growth references (Pulungan et al. 2018).\n\nIn addition, the authors use the term “undernutrition”. Undernutrition refers to food: Undernutrition can be defined as \"lack of proper nutrition, caused by not having enough food or not eating enough food containing substances necessary for growth and health\" (https://www.lexico.com/en/definition/undernutrition). Chronic undernutrition has an impact on body size as it results in poor growth, but being short (stunting) is not a synonym of mal- or undernutrition (Scheffler C et al. Stunting is not a synonym of malnutrition. Eur J Clin Nutr. 2019 May 292). Short people may be short for other than nutritional reasons.\n\nIt is not clear whether the authors wanted to study size (length and weight), or the nutritional situation of the children of Musi. They start the manuscript with the words: “Undernutrition among children under five …”. Later it is written: “There are three indicators to measure nutritional imbalance that lead to undernutrition …” suggesting that the manuscript is on nutrition. But where is the data on food? The authors need to provide information on food, or at least on energy deposits in terms of skinfold thickness, arm circumferences etc., or in the case of mal- or undernutrition, show clinical signs of protein or calorie malnutrition, or signs of micronutrient deficiencies. This is not the case.\n\nIt is known to the reviewer that there is international confusion about the terms stunting and malnutrition. Calculating the portion of stunted children refers to the question of how many children are below height for age < –2 SD of the WHO Child Growth Standards median. This does not necessarily mean that this portion of children is also undernourished.\n\nThe manuscript needs major rethinking and re-evaluating of the measurements of height and weight. It is necessary to describe growth of the children of Musi, and to publish these data. The children of Musi are shorter and lighter than suggested by the WHO standard. But this does NOT mean that these children suffer from food shortage. It rather appears that the WHO standard is not applicable for these children, and thus does NOT indicate malnutrition of this child population. This needs to be stressed. The authors should carefully read some of the recent papers discussing the misinterpretation of stunting as a sign of undernutrition (Hermanussen et al. The impact of social identity and social dominance on the regulation of human growth. A viewpoint. Acta Paediatr. 2019 Aug 163).\n\nThe tables show odds ratios. It is much more informative when data are presented as true values. The reader is not so much interested in what proportion of children ranges above or below a certain cut-off, but what are the mean values of height, weight, etc. in this child population.\n\nMinor comments: Correct the term “national standard”. What you mean is: “national reference”. Correct the term “gender”. What you mean is probably “sex”. Table 4 needs to be shortened, there is no need to show empty boxes. The same applies for table 6\n\nDiscussion: The authors write: “SGA is a result of poor maternal nutrition during pregnancy”. This is not quite true. Ample evidence obtained from European countries during periods of war and post-war starvation illustrates that even when pregnant women are severely undernourished, the newborn infants only suffer from minor decreases in body weight (Keys A, Brozek J, Henschel A, Mickelsen O, Longstreet Taylor H. The biology of human starvation. The University of Minnesota Press. Minneapolis. 1950.).\n\nSee various comments in the text.\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?\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": "5518", "date": "27 May 2020", "name": "Firas Farisi Alkaff", "role": "Author Response", "response": "Dear reviewer, Thank you for the comments and suggestions to our manuscript. 1. The authors do not clearly distinguish between the terms “standard” and “reference” Response: We have distinguished the “standard” and “reference” in our revised manuscript. We use the term “reference” for Indonesian growth chart and use the term “standard” for WHO growth chart. 2. The authors use the terms “stunting”, “wasting”, and “underweight”. These terms refer to body size, they do not describe the nutritional situation. Response: We do not refer stunting and underweight as nutritional status in our revised manuscript. 3. It is not clear whether the authors wanted to study size (length and weight), or the nutritional situation of the children of Musi. Response: We have revised the manuscript and limited the aim of our study to body size (length and weight) of the Musi children, not about nutritional status because we do not have the data regarding food, energy deposits, or any clinical sign of undernutrition. 4. It is necessary to describe growth of the children of Musi, and to publish these data. Response: Unfortunately, we do not have the growth record of children in Musi sub-district. The design of our study is cross-sectional, thus we only have one-time height and weight measurement of the Musi children. We have published the measurement data in online repository (https://doi.org/10.6084/m9.figshare.12121938.v5 for population data and https://doi.org/10.6084/m9.figshare.12127425.v3 for determinants data) 5. The authors should carefully read some of the recent papers discussing the misinterpretation of stunting as a sign of undernutrition Response: We have read the corresponding references, and we have made sure to not misinterpret stunting as the sign of undernutrition because the aim of our study is to compare the prevalence of body size using WHO growth standard and Indonesian growth reference. 6. The tables show odds ratios. It is much more informative when data are presented as true values. The reader is not so much interested in what proportion of children ranges above or below a certain cut-off, but what are the mean values of height, weight, etc. in this child population. Response: We would like to give more informative data by presenting the true values of weight and height. However, since the children are not in the same age, we are not sure whether it is appropriate or not. 7. Correct the term “national standard”. What you mean is: “national reference”. Correct the term “gender”. What you mean is probably “sex”. Table 4 needs to be shortened, there is no need to show empty boxes. The same applies for table 6 Response: We have corrected the term “national standard” to “national reference” and term “gender” to “sex”. We also have shortened the table 4 and 6 by omitting the empty boxes. 8. Discussion: The authors write: “SGA is a result of poor maternal nutrition during pregnancy”. This is not quite true. Response: We have removed the discussion regarding the relationship between SGA and poor maternal nutrition. 9. You might better conclude that \"the WHO standard are not suitable for representing child growth in Musi sub-district\". Response: We have edited our conclusion according to your suggestion." } ] }, { "id": "63061", "date": "14 May 2020", "name": "Aroonsri Mongkolchati", "expertise": [ "Reviewer Expertise Maternal and Child 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\nThe abstract was unclear of the result of this study, it should be addressed that after the authors control variable or adjust for confounding factors including mother height and socioeconomic factors. What were the factors have been found to be the determinants factors and this lead to the recommendation and conclusion to be more clear.\nThe statistic was used for data explanation should be more clear such as OR mean Cluded OR, COR, by bivariate whereas Adjusted OR, AOR, used for multivariate analysis.\nThe conclusion should consider one more limitation is that the small sample size of the data particularly on child stunning number on national WHO standard in comparing. As a result, the next research should be more explored.\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": "5519", "date": "27 May 2020", "name": "Firas Farisi Alkaff", "role": "Author Response", "response": "Dear reviewer, Thank you for the comments and suggestions to our manuscript. 1. The abstract was unclear of the result of this study, it should be addressed that after the authors control variable or adjust for confounding factors including mother height and socioeconomic factors. What were the factors have been found to be the determinants factors and this lead to the recommendation and conclusion to be more clear. Response: We have edited the abstract section according to your suggestion. 2. The statistic was used for data explanation should be more clear such as OR mean Cluded OR, COR, by bivariate whereas Adjusted OR, AOR, used for multivariate analysis. Response: We have changed the OR in the bivariate analysis to Crude OR and OR in multivariate analysis to Adjusted OR throughout the manuscript 3. The conclusion should consider one more limitation is that the small sample size of the data particularly on child stunning number on national WHO standard in comparing. As a result, the next research should be more explored Response: We have added the small sample size particularly in the number of stunted children according to national Indonesian reference as one of the limitations in our study" } ] } ]	1	https://f1000research.com/articles/9-324
https://f1000research.com/articles/9-1471/v1	16 Dec 20	{ "type": "Research Article", "title": "Factors influencing non-adherence to opioids in cancer patients: a mixed-methods cross-sectional study", "authors": [ "Rattaphol Seangrung", "Mallika Ahuja", "Koravee Pasutharnchat", "Rungwipha Mahawan", "Mallika Ahuja", "Koravee Pasutharnchat", "Rungwipha Mahawan" ], "abstract": "Background: Strong opioids are mainly utilized to attenuate pain in cancer patients. Adherence to analgesic drugs significantly promotes adequate pain management and improves quality of life. We aimed to identify the factors influencing non-adherence to strong opioids in cancer patients. Methods: A descriptive, cross-sectional, two-phased, mixed methods design was conducted prospectively to evaluate a cohort of 101 cancer patients who are currently prescribed strong opioids from a pain clinic in Thailand between January and March 2018. Participants were asked to complete a questionnaire that included the following sections: general characteristics; the Medication Taking Behavior in Thai (MTB-Thai) for assessing adherence to medications; and factors influencing nonadherence, which were analyzed using multivariate logistic regression. In addition, face-to-face in depth interviews were conducted with patients showing non-adherence to strong opioids (MTB-Thai score ≤21) and analyzed using thematic content analysis. Results: Of 101 cancer pain patients that completed the questionnaire, 39.6% showed non-adherence to strong opioids. Illness understanding (P=0.047) and the use of more than three types of pain medication (P=0.032) were significant factors influencing non-adherence. Qualitative analysis indicated that fear of long-term outcomes, opioid side effects, ineffective pain control, attempts to make the regimen more acceptable, poor understanding, and non-acceptance of disease related to non-adherence. Conclusion: Non-adherence to opioids for cancer patients is a common problem. Awareness of patient factors, medication-related factors, and illness-related factors will provide the knowledge and adequate advice that may enhance adherence to medications.", "keywords": [ "Opioid", "medication non-adherence", "cancer", "pain management" ], "content": "Introduction\n\nCancer is the one of four non-communicable diseases that makes up the majority of global deaths1. In Thailand, an average of 170,000 people were newly diagnosed with cancer in 2018, according to the World Health Organization report2. More than one-third of cancer patients experienced moderate or severe pain3. Improper pain management can be caused by a multitude of factors, including the clinicians’ attitude, patients’ perception, caregiver’s perspective, and the availability or accessibility of analgesic drugs4–9. Significantly, poor adherence to the analgesic regimen can contribute to ineffective cancer pain management10–12. Also, it can lead to a substantial worsening of the disease, death, and increased health care costs12,13.\n\nStrong opioids are the mainstay for treatment of cancer pain. The reported incidence of poor opioid adherence is 50-70% of patients with advanced cancer14. Previous research on the causes of non-adherence has identified various factors, such as illness, drugs, medical personnel, patient characteristics, and socioeconomic factors15. Notably, poor compliance is associated with young age, smoking, fear of drug dependence and side effects, the experience of adverse events, misunderstanding of prescriber instructions, poor beliefs and perceptions, poor family support, and non-acceptance of illness7,16–20. However, no study in Thailand has explored the issue of opioid non-adherence in patient-related factors, which is one of the most significant barriers and is a severe problem that obstructs pain management goals. Furthermore, non-adherence to opioids remains a significant health problem, and more high-quality studies are needed to assess these aspects. A mixed-methods study is required to study the effects of compliance in enhancing the impact of treatment. Therefore, we conducted a questionnaire survey and in-depth interviews to explore factors affecting cancer pain patients’ non-adherence to opioids.\n\n\nMethods\n\nThe present study was a descriptive, cross-sectional, two-phased mixed methods study using both quantitative and qualitative approaches. The study was conducted between January and March 2018\n\nThis study was approved by the Ethics Committee (Chairman Assistant Professor Dr. Chusak Okascharoen) of Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand (09-60-05, 11 January 2018). Participants were informed about the study and provided written informed consent to participate in both the questionnaire and the interviews. All data were confidential.\n\nThe sample size for the study was determined using Taro Yamane sample size formula with 95% confidence level21. The calculation formula of Taro Yamane is presented as: n = N/1 + N (e)2\n\nwhere: n = sample size required, N = number of people in the population.\n\nIn this study cancer pain patients attending a pain clinic at Ramathibodi Hospital, Bangkok, Thailand were the sample population and numbered 134 in the last three months between January and March 2018; therefore, e = allowable error (%)-0.05. A minimum of 101 cancer pain patients who used strong opioids (fentanyl, methadone, morphine) by oral and transdermal routes of administration were required to meet the sample size. Participants were selected using simple random sampling. They were approached during a routine follow up at pain clinic.\n\nThe inclusion criteria were patients aged 18 years or older, diagnosed with cancer pain, strong opioid analgesics for cancer pain prescribed for more than one week for around-the-clock use or as needed, and ability to communicate well in Thai. Exclusion criteria were patients who declined to participate, and who had known or suspected psychotic disease.\n\nAll participants filled out the questionnaire by themselves and participated in the interviews at the hospital. The questionnaire assessed demographic characteristics, pain severity (numerical rating scale in the past week and at the moment) with therapeutic outcomes (pain affect working and social activities, routine daily activities and life), and medication adherence using the Medication Taking Behavior in Thai (MTB-Thai) measure22,23. Other factors associated with non-adherence to strong opioids, including patient factors (knowledge of strong opioid analgesics and patient beliefs about strong opioid analgesics) were assessed using a copyrighted Thai version of the self-report Belief about Medication Questionnaire [Thai-BMQ])24,25. Socioeconomic factors (family and social support), medical personnel factors (satisfy and confident of medical service and staffs), medication-related factors (taste, cost, type of medicines, frequency of taking and side effects) and illness-related factors were also assessed. Both MTB-Thai and Thai-BMQ were used in the study with permission of the originators of the questionnaires. The full questionnaire used in this study was approved by three pain specialists. Content validity was determined by obtaining the item objective congruence (IOC) value for each questionnaire (including general information and pain severity, which influence non-adherence to opioids, MTB-Thai and Thai-BMQ) ranged from 0.80 to 0.92. All were <0.5, indicating good content validity. Cronbach’s alpha coefficient of the questionnaire ranged from 0.702 to 0.788; a score of >0.7 indicates acceptable internal consistency. The questionnaire was not modified after the pilot with 10 patients. A copy of the questionnaire can be found in the Extended data26,27.\n\nOpen-ended interview questions were included at the end of the questionnaire for patients who had a MTB-Thai score ≤21, in order to provide further commentary and suggest other factors that may influence non-adherence to strong opioids. These questions were asked in a face-to-face qualitative in-depth interviews by the second (MA) and the fourth author (RM) that were conducted until data saturation was reached. The interviews were audio-recorded with the patients’ permission, and the interviewers also took field notes.\n\nSPSS for Windows version 18.0 (SPSS Inc., Chicago, IL, USA) was used for quantitative data analysis. Descriptive statistics, such as frequencies, percentages, means, and standard deviations, were used to analyze demographic data. Chi-square test, relative risk, 95% confidence intervals (CI), and p-values were used to measure the association between factors and strong opioid analgesic non-adherence. Multiple logistic regression was performed to identify risk factors for opioid non-adherence and to calculate adjusted risk ratios. P-values less than 0.05 were considered statistically significant.\n\nInterview data (transcribed verbatim from recordings) and the interviewers’ memos were subject to thematic content analysis using ATLAS.ti software version 8.0. Using five stages of data analysis in the framework approach included: 1) familiarization or immersion in the raw data to list key ideas and recurrent themes, 2) identifying a thematic framework (all the key issues, concepts, and themes), 3) indexing or applying the thematic framework or index systematically to all the data in textual form by annotating the transcripts with numerical codes, 4) charting or rearranging the data according to the appropriate part of the thematic framework to which they relate, and forming charts, and 5) mapping and interpretation by using the charts to define concepts, map the range and nature of phenomena.\n\n\nResults\n\nAll 101 participants completed the questionnaire (Figure 1). Most participants were women (N = 52, 51.49%), and the average age was 60.14 years. in total, 40 patients (39.6%) reported non-adherence; the others reported moderate to high adherence. The mean duration of pain treatment was three months (range: 1-11 months). Table 1 shows the demographic data.\n\nMTB-Thai score = Medication taking behavior scale in Thai22.\n\nSD = Standard deviation, IQR = Interquartile range.\n\nStatistically significant differences between the adherence and non-adherence groups were found in the general-overuse dimension of the Thai-BMQ (P = 0.047), illness understanding (P = 0.028), and use of more than three types of pain medication (P = 0.035) (Table 2).\n\nSD = Standard deviation, IQR = Interquartile range.\n\naMTB-Thai score = Medication taking behavior scale in Thai22.\n\nbThai-BMQ = Belief about Medication Questionnaire, Thai version24.\n\ncReversed scale.\n\nP < 0.05 (statistically significant).\n\nThe multivariate analysis showed that two variables had significant associations with opioid non-adherence: illness understanding (P = 0.047) and use of more than three types of pain medication (P = 0.032). The illness understanding and acceptance part of the questionnaire contained five statements. Most participants agreed with either “I understand my illness, and I think I received the best treatment” or “I understand my illness, but I think I could have received better treatment.” Patients who chose the former response were less likely to show non-adherence than those who chose the latter answer (RR = 0.53, 95% CI [0.283–0.993]). Participants prescribed more than three types of analgesics had a 3.04 times higher risk of medication non-adherence than participants prescribed three or fewer types of medication (RR = 3.04, 95% CI [1.099–8.411]) (Table 3).\n\nIQR = Interquartile range, CI = Confidence interval, RR = Risk ratio.\n\nP < 0.05 (statistically significant).\n\n#Results from binary logistic regression analysis (unlike the output from chi-square test in Table 2)\n\naReversed scale.\n\nIn total 10 individual in-depth interviews with patients who had MTB-Thai score ≤21 were conducted, which lasted around 30 to 45 minutes. Five themes related to opioid non-adherence emerged from the data: fear of long-term outcomes, desirable or undesirable opioid side effects, ineffective pain control, attempts to make the regimen more acceptable, and poor understanding and non-acceptance of the disease. Analytic results of the contextual factors associated with non-adherence to opioids in cancer patients are presented in Table 4.\n\nPatient factors\n\nTheme 1: Fear of long-term opioid adverse events\n\nAlmost half of the patients were concerned about opioid addiction. Some chose to be in pain to minimize the chance of addiction. As two patients remarked:\n\n“I could get addicted to the medication, so I don’t want to take morphine syrup more than once a day.”\n\n“I feel uncomfortable taking morphine in front of people. They look at me like I am addicted to drugs.”\n\nMany patients were afraid of liver or kidney damage after long-term opioid use and combined with other pain medications, despite their physicians confirming the safety of their opioid dosage. As some patients mentioned:\n\n“Morphine could reduce my pain but if I take as much as I need, I will suffer from liver or kidney disease in the future.”\n\n“I took lots of medication. I think my liver has had to work too hard, so I wait until I had severe pain, I will take morphine.”\n\n“These drugs can relief the pain, but I wondered if just one or two drugs could control everything. I think that taking four types of drugs every day will damage my health.”\n\nMedication-related factors\n\nTheme 1: Desirable or undesirable opioid side effects\n\nMany patients reported opioid side effects. Some found that the side effects were unbearable and affected their quality of life. Commonly reported severe constipation, upset stomach, and drowsiness. As some patients said:\n\n“I am in pain but I chose to use as low a dose of morphine as I could because it made me constipated.”\n\n“Severe nausea made me afraid to use morphine. Throwing up was much worse than living with this pain.”\n\n“I always had to take a nap after taking morphine. So I could only take it before bedtime otherwise I might sleep all day.”\n\nNotably, some participants found specific side effects beneficial, whereas others found them problematic. As one patient said:\n\n“The drug makes me sleep well at night. I take it every night even if the pain does not bother me much.”\n\nTheme 2: Ineffective pain control\n\nSome patients do not adhere to medical regimens because they find pain medication ineffective. Some participants reported not taking opioids because they did not have the intended effect. As one patient remarked:\n\n“I suffered from pain, but the drug did not make me feel as good as I expected. So there was no reason for taking it.”\n\nSome patients increased their basal opioid doses because their pain had worsened. As one patient remarked:\n\n“I did not know what to do with my pain anymore. I felt so hopeless. I took morphine syrup every hour. The effect was too short-lived, so I tried to take more morphine tablets than the doctor had prescribed, but I ended up feeling sleepier all day.”\n\nTheme 3: Attempts to make the regimen more acceptable\n\nSome patients applied their medication regimen to suit their lifestyle and drug reactions. Some reduced the medication frequency, as they felt uncomfortable taking medicine at midday. Others changed the regimen from around the clock to after meals, because it was easier to remember. Some took all their drugs at bedtime rather than in the morning because of the sedative effect. As some patients said:\n\n“I go to work every day. It’s not easy to bring the drugs with me, so I changed the schedule from a three times daily regimen to a two times daily regimen. I think this work for me.”\n\n“I take the morphine after meals. It is easier to remember. I used to forget to take the pills at 2 pm, which made the pain worse in the evening.”\n\n“I felt sleepy after I took the pills, so I took it all at night before bedtime. This meant that the pain wasn’t well controlled, but that’s better than feeling drowsy all day.”\n\nIllness-related factors\n\nTheme 1: Poor understanding and non-acceptance of the disease\n\nIf doctors can improve patients’ understanding and acceptance of illness, then more collaborative treatment decisions can be made. If patients insist on seeking complete pain relief or a complete cure and are reluctant to adopt modified life goals and activities, it is difficult to set realistic treatment goals. Acceptance-based pain management may be helpful for cancer patients. Many patients reduced or discontinued opioids by themselves when their pain was decreasing. As two patients said:\n\n“I took the pills everyday as advised and they controlled the pain well. I wondered if I could stop taking these drugs, so I tried to stop and the pain came back. Are these drugs helping me with the disease? Or I should try something else?”\n\n“After taking the drug, I feel better, I stopped taking it on some day. I think the disease is getting better.”\n\n\nDiscussion\n\nThe main purpose of this mixed-methods study design was to explore the factors influencing non-adherence to strong opioids in cancer pain patients. Most research has studied the clinical factors associated with adherence to opioids for cancer pain and has not focused the medical non-adherence factor alone. Investigation of non-adherence to opioids in cancer patients is a pro-active useful model for correcting beliefs, attitudes and behavior. This study asked participants about characteristics related to themselves, family and social support, doctor-patient relationships, medication-related factors, and illness-related factors. The incidence of non-adherence of our study was 39.6%, similar to previous study28. Our data indicate that only three out of these five factors were significantly associated with non-adherence: patient factors, medication-related factors, and illness-related factors.\n\nIn the quantitative data, patients’ knowledge of strong opioid analgesics and their beliefs about medication were not significantly related to non-adherence. However, the interviews revealed a concern about adverse effects, side effects, opioid addiction and multiple organ failure from long-term usage, as reported in previous work29–33. This was even though most of the patients from this study had a higher education and good knowledge of strong opioids. They may also be concerns about the adverse effects of opioids. Similarly another study found that some patients who were educated about the side effects of medication showed increased concerns about the risk of addiction34,35.\n\nGenerally, cancer patients require long-term use of strong opioids. Physicians should educate patients during treatment about the prevention of adverse effects. Although patient education is a key component of adherence, more education may make patients anxious and fearful about opioid adverse effects. Patient beliefs and attitudes regarding the effectiveness of the treatment, and lack of motivation, also affects medication adherence36. Healthcare providers should reassure patients by emphasizing the benefits rather than the risks of opioids, and should identify patients’ concerns. Additionally, involvement of patients in the treatment decision-making process may help to reduce fear and facilitate adherence12,37.\n\nA previous study38 found an association between medical adherence and family and social support: adherence was 1.74 times higher in patients from cohesive families and 1.53 times lower in patients from families in conflict. The present findings did not show a correlation between adherence and family support. More than 90% of patients in both the adherence and non-adherence groups confirmed that they received the best care from their families when needed. This may be because Thai people live in large families and therefore find it relatively easy to obtain assistance when needed.\n\nAlmost all patients expressed high satisfaction and confidence in the pain clinic services and staff. Therefore, the doctor-patient relationship was not significantly associated with non-adherence. One previous study39 found a correlation; patients who believed that doctors treat patients as equals, who felt that doctors discuss treatments with patients before making decisions, and who could choose their doctor were more likely to adhere to recommendations.\n\nBoth the quantitative and qualitative data revealed many medication-related reasons for non-adherence, which reflected previous study findings17,40,41. Our quantitative analysis showed that use of more than three types of drugs was associated with a 3 times higher risk of non-adherence than use of less than three types. Furthermore, the qualitative analysis identified three themes related to medication-related non-adherence factors. Similar to a previous study15,40, we found that some patients reduced their opioid dose to avoid unbearable side effects, such as constipation, nausea, vomiting, dyspepsia, or drowsiness. Despite having poor pain control, they could not take the opioid dose prescribed because of the side effects. Most said that their pain was much more bearable than the side effects. Some did not inform their doctor about their pain owing to limited time, being considerate or shame. Moreover, some patients used opioids for the wrong reasons; for example, taking opioids at night to help them sleep even when they had no pain.\n\nIn accordance with previous findings40,41, we found that ineffective pain control was a key reason why some patients refused to take prescribed medication. Some patients increased the prescribed opioid dose for maximal pain relief. Some obtained opioids from many different hospitals to control the pain without discussing this with their doctor. Patients reported many reasons for non-adherence. Some felt that their doctor had insufficient time to listen to their problems. Others were afraid that the doctor would abandon them because they did not use the drugs as prescribed.\n\nOne of the medication-related factors we identified was the attempt to make the drug regimen more acceptable; this has also been reported in previous work40. Patients changed the drug schedule to suit their lifestyle. Some patients reduced the dose frequency as they were not comfortable taking drugs at certain times of the day. Some changed the interval from around the clock to after meals, as it was easier to remember. Some patients took all their medications at bedtime rather than in the morning because of the sedative effects.\n\nPrevious studies of patients with chronic nonmalignant pain indicate that illness acceptance predicts increased psychological, social, and physical functioning42,43 and that acceptance-based pain management may be helpful for cancer patients44. The present quantitative and qualitative data show that patients with poor illness understanding and non-acceptance of the disease show medication non-adherence. Sometimes, patients misunderstood their situation and believed that they could be fully cured and become pain free. Some patients believed that there were better treatment options than the treatment they had received. The results suggest that enhancing the acceptance of pain and cancer may be a clinically relevant management goal.\n\nAlthough many factors were not statistically significant in the quantitative study, such as the patient’s belief about opioid side effects or adverse effects; however, more than three types of pain medication was the critical factor of non-adherence that might raise their concern about long term adverse effects. Also, taking multiple drugs may be a factor that causes patients to adjust their medication as appropriate, which causes inadequate pain relief. From the in-depth interviews, it was clear that concerns about medication side eﬀects, fear of adverse events and poor pain control were barriers for opioid use in cancer pain management.\n\nMainly, the various opioids are very different in bioavailability, metabolism, and response between individual patients. Appropriate opioid use must be selected for each cancer patient, and the dose must be individually titrated. Effective and safe titration of opioids has a significant impact on patient comfort. Obviously, several complex factors affect opioids non-adherence in cancer patients. Therefore, we recommend the following strategies to improve adherence to strong opioid medication for cancer pain.\n\n1. Understanding patients’ reasons for non-adherence to opioids could help doctors to identify how these patients may present clinically, address patients’ concerns about opioids, and encourage doctors to offer patients alternatives to opioid treatment.\n\n2. Reviewing the number of medications because of drug interactions can be managed by reviewing the patient’s medication profile for duplicate or unnecessary medications.\n\n3. Good patient-doctor communication may reduce anxiety, and also improves pain control45. For example, discussing a patient’s concerns about the risk of addiction may help the patient and doctor to set up plans to monitor misuse or identify less risky or more acceptable alternative pain management strategies.\n\nWe only measured non-adherence to strong opioid analgesic medicine. The patients might have been taking other medicines for pain control prescribed simultaneously. As these medicines could have affected pain control, they might have confounded the present results.\n\n\nConclusion\n\nAlmost half of cancer pain patients prescribed opioids showed non-adherence to the medical regimen. Three factors were significantly associated with medication non-adherence: patient factors (fear of long-term outcomes), medication-related factors (use of more than three types of drugs, side effects, ineffective pain control, attempts to make the regimen more acceptable), and illness-related factors (poor illness understanding and non-acceptance of the disease).\n\n\nData availability\n\nThe recordings and transcription of interviews are not openly available in order to conserve the confidential information of participants. All document files were eradicated immediately following data analysis. Themes and quotes from the data analysis are available in Thai. This data can be obtained by application to the Ethical Committee of Faculty of Medicine Ramathibodi Hospital. To apply, please contact the corresponding author at rattaphol_nu@hotmail.com, who will facilitate this process.\n\nFigshare: Data of factors influencing non-adherence to opioids in cancer patients.xls., https://doi.org/10.6084/m9.figshare.13336691.v246.\n\nFigshare: Questionnaire and open ended questions in English.doc., https://doi.org/10.6084/m9.figshare.13336754.v126.\n\nFigshare: Thai version of the questionnaire and open ended questions.docx., https://doi.org/10.6084/m9.figshare.13336766.v227.\n\nFigshare: STROBE checklist for ‘Factors influencing non-adherence to opioids in cancer patients: a mixed-methods cross-sectional study’, https://doi.org/10.6084/m9.figshare.13336778.v147.\n\nFigshare: COREQ checklist for ‘Factors influencing non-adherence to opioids in cancer patients: a mixed-methods cross-sectional study’, https://doi.org/10.6084/m9.figshare.13336793.v148.\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 thank the participants in the study and particularly thank Rojnarin Komonhirun who provided assistance and coordination support. The authors also thank Diane Williams, PhD, from Edanz Group (www.edanzediting.com/ac) for editing the English text of a draft of this manuscript.\n\n\nReferences\n\nWorld Health Organization: Global Health Observatory. Geneva: World Health Organization; 2018; Accessed June 21, 2018. Reference Source\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. CA Cancer J Clin. 2018; 68(6): 394–424. PubMed Abstract \| Publisher Full Text\n\nvan den Beuken-van Everdingen MHJ, de Rijke JM, Kessels AG, et al.: Prevalence of pain in patients with cancer: a systematic review of the past 40 years. Ann Oncol. 2007; 18(9): 1437–49. PubMed Abstract \| Publisher Full Text\n\nJamison RN, Sheehan KA, Scanlan E, et al.: Beliefs and attitudes about opioid prescribing and chronic pain management: survey of primary care providers. J Opioid Manag. 2014; 10(6): 375–82. PubMed Abstract \| Publisher Full Text\n\nPotter VT, Wiseman CE, Dunn SM, et al.: Patient barriers to optimal cancer pain control. Psychooncology. 2003; 12(2): 153–60. PubMed Abstract \| Publisher Full Text\n\nLiang SY, Yates P, Edwards H, et al.: Factors influencing opioid-taking self-efficacy and analgesic adherence in Taiwanese outpatients with cancer. Psychooncology. 2008; 17(11): 1100–07. PubMed Abstract \| Publisher Full Text\n\nGunnarsdottir S, Donovan HS, Serlin RC, et al.: Patient-related barriers to pain management: the Barriers Questionnaire II (BQ-II). Pain. 2002; 99(3): 385–96. PubMed Abstract \| Publisher Full Text\n\nLee BO, Liu Y, Wang YH, et al.: Mediating Effect of Family Caregivers' Hesitancy to Use Analgesics on Homecare Cancer Patients' Analgesic Adherence. J Pain Symptom Manag. 2015; 50(6): 814–21. PubMed Abstract \| Publisher Full Text\n\nReid CM, Gooberman-Hill R, Hanks GW: Opioid analgesics for cancer pain: symptom control for the living or comfort for the dying? A qualitative study to investigate the factors influencing the decision to accept morphine for pain caused by cancer. Ann Oncol. 2008; 19(1): 44–8. PubMed Abstract \| Publisher Full Text\n\nMiaskowski C, Dodd MJ, West C, et al.: Lack of adherence with the analgesic regimen: a significant barrier to effective cancer pain management. J Clin Oncol. 2001; 19(23): 4275–79. PubMed Abstract \| Publisher Full Text\n\nValeberg BT, Miaskowski C, Hanestad BR, et al.: Prevalence rates for and predictors of self-reported adherence of oncology outpatients with analgesic medications. Clin J Pain. 2008; 24(7): 627–36. PubMed Abstract \| Publisher Full Text\n\nOsterberg L, Blaschke T: Adherence to medication. N Engl J Med. 2005; 353(5): 487–97. PubMed Abstract \| Publisher Full Text\n\nCutler RL, FernandezLlimos F, Frommer M, et al.: Economic impact of medication non-adherence by disease groups: a systematic review. BMJ Open. 2018; 8(1): e016982. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbas SQ, Abbas Z: Is opiate compliance a problem in cancer pain? A survey of health-care professionals' views. Int J Palliat Nurs. 2003; 9(2): 56–63. PubMed Abstract \| Publisher Full Text\n\nPound P, Britten N, Morgan M, et al.: Resisting medicines: a synthesis of qualitative studies of medicine taking. Soc Sci Med. 2005; 61(1): 133–55. PubMed Abstract \| Publisher Full Text\n\nJin J, Sklar GE, Min Sen Oh V, et al.: Factors affecting therapeutic compliance: A review from the patient's perspective. Ther Clin Risk Manag. 2008; 4(1): 269–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIngersoll KS, Cohen J: The impact of medication regimen factors on adherence to chronic treatment: a review of literature. J Behav Med. 2008; 31(3): 213–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlhewiti A: Adherence to Long-Term Therapies and Beliefs about Medications. Int J Family Med. 2014; 2014: 479596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKleinsinger F: The Unmet Challenge of Medication Nonadherence. Perm J. 2018; 22: 18–033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen LMT, Rhondali W, De la Cruz M, et al.: Frequency and predictors of patient deviation from prescribed opioids and barriers to opioid pain management in patients with advanced cancer. J Pain Symptom Manage. 2013; 45(3): 506–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamane T: Statistics, An Introductory Analysis. Harper and Row, New York, NY, USA, 2nd edition, 1967. Reference Source\n\nSakthong P, Sonsa-Ardjit N, Sukarnjanaset P, et al.: Development and psychometric testing of the medication taking behavior tool in Thai patients. Int J Clin Pharm. 2016; 38(2): 438–45. PubMed Abstract \| Publisher Full Text\n\nSooksai N, Rittirod T, Pachanasoontorn N, et al.: The Guardian’s Beliefs on Psychotropic Medications Adherence of Autistic Pediatric Patients. Srinagarind Med J. 2019; 34(6): 595–601. Reference Source\n\nLeaungsomnapa Y, Leaungsomnapa S, Promproh S, et al.: Confirmatory factor analysis of beliefs about medicine questionnaire in Thai version. J Prapokklao Hosp Clin Med Educat Center. 2014; 31: 297–310.\n\nSriwarakorn S, Krittiyanunt S, Sakulbumrungsil R: Sensitivity and Specificity of Thai-Version Brief Medication Questionnaire. Journal of Health Research. 2018; 24(3): 129–34. Reference Source\n\nSeangrung R: Questionnaire and open ended questions in English.doc. figshare. Dataset. http://www.doi.org/10.6084/m9.figshare.13336754.v1\n\nSeangrung R: Thai version of the questionnaire and open ended questions.docx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13336766.v2\n\nChou WC, Chen JS, Hung CY, et al.: A nationwide survey of adherence to analgesic drugs among cancer patients in Taiwan: prevalence, determinants, and impact on quality of life. Support Care Cancer. 2019; 27(8): 2857–67. PubMed Abstract \| Publisher Full Text\n\nTzeng JI, Chang CC, Chang HJ: Assessing analgesic regimen adherence with the Morisky Medication Adherence Measure for Taiwanese patients with cancer pain. J Pain Symptom Manage. 2008; 36(2): 157–66. PubMed Abstract \| Publisher Full Text\n\nBerry PE, Ward SE: Barriers to pain management in hospice: a study of family caregivers. Hosp J. 1995; 10(4): 19–33. PubMed Abstract \| Publisher Full Text\n\nCherny NI, Portenoy RK: The management of cancer pain. CA Cancer J Clin. 1994; 44(5): 262–303. PubMed Abstract \| Publisher Full Text\n\nHo RC: Pain in the cancer patient. CA Cancer J Clin. 1994; 44(5): 259–61. PubMed Abstract \| Publisher Full Text\n\nWard SE, Hernandez L: Patient-related barriers to management of cancer pain in Puerto Rico. Pain. 1994; 58(2): 233–8. PubMed Abstract \| Publisher Full Text\n\nDavis MP, Walsh D: Epidemiology of cancer pain and factors influencing poor pain control. Am J Hosp Palliat Med. 2004; 21(2): 137–42. PubMed Abstract \| Publisher Full Text\n\nSun VC, Borneman T, Ferrell B, et al.: Overcoming barriers to cancer pain management: an institutional change model. J Pain Symptom Manage. 2007; 34(4): 359–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorne R, Chapman SC, Parham R, et al.: Understanding patients' adherence-related beliefs about medicines prescribed for long-term conditions: a meta-analytic review of the Necessity-Concerns Framework. PLoS One. 2013; 8(12): e80663. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaynes RB, McDonald HP, Garg AX: Helping patients follow prescribed treatment: clinical applications. JAMA. 2002; 288(22): 2880–3. PubMed Abstract \| Publisher Full Text\n\nDiMatteo MR: Social support and patient adherence to medical treatment: a meta-analysis. Health Psychol. 2004; 23(2): 207–18. PubMed Abstract \| Publisher Full Text\n\nStavropoulou C: Non-adherence to medication and doctor-patient relationship: Evidence from a European survey. Patient Educ Couns. 2011; 83(1): 7–13. PubMed Abstract \| Publisher Full Text\n\nLewis ET, Combs A, Trafton JA: Reasons for under-use of prescribed opioid medications by patients in pain. Pain Med. 2010; 11(6): 861–71. PubMed Abstract \| Publisher Full Text\n\nCrosby FE, Colestro J, Ventura MR, et al.: Survey of pain among veterans in Western New York. Pain Manag Nurs. 2006; 7(1): 12–22. PubMed Abstract \| Publisher Full Text\n\nMcCracken LM, Eccleston C: A prospective study of acceptance of pain and patient functioning with chronic pain. Pain. 2005; 118(1–2): 164–9. PubMed Abstract \| Publisher Full Text\n\nMcCracken LM, Spertus IL, Janeck AS, et al.: Behavioral dimensions of adjustment in persons with chronic pain: pain-related anxiety and acceptance. Pain. 1999; 80(1–2): 283–9. PubMed Abstract \| Publisher Full Text\n\nXu X, Cheng Q, Ou M, et al.: Pain acceptance in cancer patients with chronic pain in Hunan, China: A qualitative study. Int J Nurs Sci. 2019; 6(4): 385–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanja JD: Empathy in the physician's pain practice: benefits, barriers, and recommendations. Pain Med. 2006; 7(3): 265–75. PubMed Abstract \| Publisher Full Text\n\nSeangrung R: Data of factors influencing non-adherence to opioids in cancer patients.xls. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13336691.v2\n\nSeangrung R: STROBE checklist cross-sectional study of factors influencing non-adherence to opioids in cancer patients.docx. figshare. Dataset. http://www.doi.org/10.6084/m9.figshare.13336778.v1\n\nSeangrung R: COREQ Checklist for factors influencing non-adherence to opioids in cancer patients.pdf. figshare. Dataset. http://www.doi.org/10.6084/m9.figshare.13336793.v1" }	[ { "id": "76209", "date": "23 Dec 2020", "name": "Sasikaan Nimmaanrat", "expertise": [ "Reviewer Expertise Pain management including acute", "cancer and chronic non-cancer pain." ], "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 used a mixed method of a survey and an in-depth interview to evaluate factors affecting opioid non-adherence in cancer patients. The questionnaire survey was to identify adherence and non-adherence patients, followed by an in-depth interview in patients with non-adherence until data were saturated. Using the mixed method makes the study more informative and attractive. The flow of the study was clearly presented.\n\nAdequate pain management in cancer patients is mandatory. It is useful to know which factors lead to poor pain control. The authors have found factors which can be classified into patient factors, medication-related factors and illness-related factors. Knowing the contributing factors for inadequate cancer pain management can lead to strategic planning to modify these factors resulting in better pain relief.\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": "6237", "date": "04 Jan 2021", "name": "Rattaphol Seangrung", "role": "Author Response", "response": "Thank you very much." } ] }, { "id": "76208", "date": "11 Jan 2021", "name": "Noraida Mohamed Shah", "expertise": [ "Reviewer Expertise Clinical Pharmacy", "medication adherence" ], "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. It is an interesting read. Non-adherence to opioids for cancer pain is well known in the literature. However, the mixed method employed in this study further explore reasons behind non-adherence specific to the Thai population.\nSuggestions/comments related to the article are as follows:\nEnglish editing is required for this article.\n\nIntroduction: “A mixed-methods study is required to study the effects of compliance in enhancing the impact of treatment” – this sentence is unclear. Was this investigated in this study?\nMethod\nPlease explain simple random sampling used in this study.\n\nPlease state if Medication Taking Behavior in Thai (MTB-Thai) and Belief about Medication Questionnaire (Thai-BMQ) are validated questionnaires.\n\nHow were these questionnaires scored? E.g. How did you classify patients as non-adherent based on MTB-Thai? Similarly, for the other questionnaires.\n\n“item objective congruence (IOC) value for each questionnaire…ranged from 0.80 to 0.92. All were <0.5, indicating good content validity.” - Please check if this statement is correct.\n\nCite references used for values indicating good content validity and acceptable internal consistency as stated in the method section.\nResults\nWould the result be different if number of painkillers is analysed as a continuous variable rather than categorical (< 3 and ≥ 3)? (Table 2).\n\nUnder Table 2, for Patient Factors, Knowledge score (0-10), which questionnaire was used to assess this? Please elaborate under methods section.\n\nWhy only 10 patients interviewed for qualitative analysis? What were the questions asked in the interviews (can explain this under method section: in depth interviews).\n\nWas the patient who had a desirable opioid effect (“The drug makes me sleep well at night. I take it every night even if the pain does not bother me much”) non-adherent to the pain killer? This seems strange as patient would take the medication if it is producing the desired effects.\nDiscussion\nUnder medication-related factors, it was stated that “Some did not inform their doctor about their pain owing to limited time, being considerate or shame”. However, this was not stated under results section. Please clarify.\n\nFor the statement under illness-related factors: “Sometimes, patients misunderstood their situation and believed that they could be fully cured and become pain free. Some patients believed that there were better treatment options than the treatment they had received.” Are these based on your assumption or from the literature?\n\n“however, more than three types of pain medication was the critical factor of non-adherence that might raise their concern about long term adverse effects.” – how did you relate taking > 3 types of pain medication with concern about long term side effects? Was this shown in your study? Similarly, for the next statement “Also, taking multiple drugs may be a factor that causes patients to adjust their medication as appropriate, which causes inadequate pain relief”. How did you relate taking multiple drugs with medication adjustment? Was this seen in your 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6419", "date": "15 Mar 2021", "name": "Rattaphol Seangrung", "role": "Author Response", "response": "Thank you, the reviewer, for your thoughtful suggestions. I have edited the last version under your comments as follows: Introduction: It was written clearly about the sentence of the aims of the study. Method: The sampling method was simple random by a computer-generated random list of the cancer patients who had the follow-up appointment schedule in the study periods. Already state that MTB-Thai and Thai-BMQ are completely validated. If the total score of MTB-Thai 21 indicated medical non-adherence, as follows of reference No. 22. Edited the correct symbol. Added the references that indicate the internal consistency of the questionnaire. Results: Cause of cancer pain is very complicated. In our clinical practice, we prescribed sustained and immediate-release opioids combined with (or without) other adjuvants analgesics in cancer pain treatment, for example, anticonvulsants, antidepressants, Non-steroidal Anti-inflammatory drugs, or acetaminophen. In the section, the patient factors in the part of knowledge score of the questionnaire. A pain specialist created the knowledge assessment of strong opioid analgesics that were completely mentioned in the method section. In the part of face-to-face in-depth interviews by the co-authors. The interviews were audio-recorded, and the interviewers also took field notes. The study was conducted until data saturation was reached that approved by primary and secondary authors. A result of medication-related factors, the patients mentioned using the rescue opioids for sleep well rather than for analgesic effects. Discussion: The authors elaborate on the study results so that it is clear in the description of the second theme (ineffective pain control). Some patients increased their basal opioid doses because their pain had worsened and dared not to call to consult the doctor about ineffective pain control by many reasons. Among the interviews, some patients expressed thoughtfulness. In our study, the patients mentioned as following the results in theme 1: Poor understanding and non-acceptance of the disease. In table 3. Univariate and multivariate logistic regression results. In our study, more than three types of pain medication had significant associations with opioid non-adherence (P = 0.032), which related to the quantitative study that the patients mentioned about adverse effects and multiple organ failure if using medications in the long-term." } ] } ]	1	https://f1000research.com/articles/9-1471
https://f1000research.com/articles/9-323/v1	04 May 20	{ "type": "Brief Report", "title": "Use of routinely collected data in a UK cohort of publicly funded randomised clinical trials", "authors": [ "Andrew J. McKay", "Ashley P. Jones", "Carrol L. Gamble", "Andrew J. Farmer", "Paula R. Williamson", "Andrew J. McKay", "Ashley P. Jones", "Carrol L. Gamble", "Andrew J. Farmer" ], "abstract": "Routinely collected data about health in medical records, registries and hospital activity statistics is now routinely collected in an electronic form. The extent to which such sources of data are now being routinely accessed to deliver efficient clinical trials, is unclear. The aim of this study was to ascertain current practice amongst a United Kingdom (UK) cohort of recently funded and ongoing randomised controlled trials (RCTs) in relation to sources and use of routinely collected outcome data. Recently funded and ongoing RCTs were identified for inclusion by searching the National Institute for Health Research journals library. Trials that have a protocol available were assessed for inclusion and those that use or plan to use routinely collected health data for at least one outcome were included. Routinely collected data sources and outcome information were extracted. A total of 279 studies were identified with 102 eligible for data extraction. An Electronic Health Record (EHR) was the sole source of outcome data for at least one outcome in 46 trials. The most frequent sources are Hospital Episode Statistics (HES) and Office for National Statistics (ONS), with the most common outcome data to be extracted being on mortality, hospital admission, and health service resource use. Our study has found that around half of publicly funded trials in a UK cohort plan to collect outcome data from routinely collected data sources. This is much higher than the figure of 8% found in a cohort of 189 RCTs published since 2000, the majority of were carried out in North America (McCord et al., 2019).", "keywords": [ "Electronic Health Records", "Data linkage", "EHR", "NIHR HTA", "Randomised Clinical Trial", "Randomised Controlled Trial", "RCT", "Registry", "Routinely collected data" ], "content": "Introduction\n\nRoutinely collected data about health in medical records, registries and hospital activity statistics is now routinely collected in an electronic form. Progress in achieving connectivity, data linkage and security now offers the possibility of better use of this data for research purposes. For example, recent evidence shows the utility of long-term follow-up of trial patients through the electronic health record (EHR) (Fitzpatrick et al., 2018). Innovative data-enabled study designs can answer pressing knowledge gaps in research evidence. However, the extent to which such sources of data are now being routinely employed in research to deliver efficient clinical trials, potentially at a wide scale, is unclear.\n\nThe aim of this study was to ascertain current practice amongst a United Kingdom (UK) cohort of recently funded and ongoing randomised controlled trials (RCTs) in relation to sources and use of routinely collected outcome data. We define routinely collected health data to be data collected without specific a priori research questions developed prior to using the data for research.\n\n\nMethods\n\nThe following inclusion criteria were used:\n\n1. Ongoing RCT of any type including feasibility or pilot work, funded by the National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme;\n\n2. use of routinely collected health data for at least one study outcome; and\n\n3. availability of a protocol.\n\nA search of the NIHR Journals Library was undertaken to find protocols registered as of 25/10/2019. The search fields and terms used to select were:\n\n1. Search term: ‘Random’\n\n2. Research type: ‘Primary research’\n\n3. Programme: ‘HTA’\n\n4. Status: ‘Research in progress’\n\nIf the final published report was shown alongside the protocol this was taken to mean that the RCT was not ongoing but the status had not been updated to ‘Published’, and the study was excluded.\n\nIn the absence of a protocol, the study was excluded. For studies with multiple protocol versions, the most recently available version was used.\n\nOne person (AM) extracted the information and categorised each EHR, with a second person (PW) checking classifications and explanations. The information extracted was as follows: Lead Investigator surname, year started, ISRCTN, project title, study type, use of routinely collected health data for at least one study outcome, availability of a protocol, any details of EHR data quality assessment prior to use, EHR name, reasons for sourcing outcome data from EHR, specific outcomes and outcome type where clear data to be used will come from named EHRs.\n\n\nResults\n\nFigure 1 shows the study flow diagram. There were 102 eligible trials available for further study.\n\nTable 1 shows the reasons for collecting trial outcome data from routine sources. The EHR was the sole source of outcome data for at least one outcome in 46 trials (categories 3, 4 and 6 in Table 1). In five of these 46 protocols there was reference to prior feasibility work confirming aspects of the quality of the data to be sufficient for the main trial. Of the 102 trials, 14 (categories 7a-7d in Table 1) planned to assess the feasibility of using the EHR data sources during the trial, although details of the assessment were often lacking. Raw data for Figure 1 and Table 1 and Table 2 are available (see Underlying data, McKay et al. (2020)).\n\nMultiple categories can apply to a single study.\n\nTable 2 shows the sources of outcome data to be used in these 46 studies. The most frequent sources are Hospital Episode Statistics (HES) and Office for National Statistics (ONS), with the most common outcome data to be extracted being on mortality, hospital admission, and health service resource use (see Underlying data, Data Set 5; McKay et al. (2020)). The full list of data sources is given in Extended data, Supplementary Table 1 (McKay et al., 2020).\n\n\nDiscussion\n\nOur study has found that around half of publicly funded trials in a UK cohort plan to collect outcome data from routinely collected data sources. This is much higher than the figure of 8% found in a cohort of 189 RCTs published since 2000, the majority of which were carried out in North America (McCord et al., 2019).\n\nVery few trial teams described any assessments of data quality from EHRs in the protocol. Work is ongoing that should determine whether such information should be reported in the trial publication (Kwakkenbos et al., 2018). An extension to the SPIRIT guidelines for EHR-supported trials is soon to be initiated, and will determine whether this information should be included in the trial protocol. As a minimum, it is recommended that trialists provide evidence in any funding application about the quality of the data from the EHR.\n\n\nData availability\n\nFigshare: Use of routinely collected data in a UK cohort of publicly funded randomised clinical trials. https://doi.org/10.6084/m9.figshare.12185193 (McKay et al., 2020).\n\nThis project contains the following underlying data:\n\nData_Set_1_Details_and_Figure_1_v1.0.csv. (Study identifiers and raw data used for Figure 1.)\n\nData_Set_2_Table_1_v1.0.csv. (Raw data used for Table 1.)\n\nData_set_3_Supp_Table_1_v1.0.csv. (Raw data used for Supplementary Table 1.)\n\nData_set_4_Table_2_v1.0.csv. (Raw data used for Table 2.)\n\nData_set_5_Outcomes_using_EHR_data_v1.0.csv. (Raw data showing details of outcomes using EHR data.)\n\nFigshare: Use of routinely collected data in a UK cohort of publicly funded randomised clinical trials. https://doi.org/10.6084/m9.figshare.12185193 (McKay et al., 2020).\n\nThis project contains the following extended data:\n\nSupplementary Table 1 - EHR sources of outcome data v1.0.pdf. (Supplementary Table 1.)\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\nFitzpatrick T, Perrier L, Shakik S, et al.: Assessment of Long-term Follow-up of Randomized Trial Participants by Linkage to Routinely Collected Data: A Scoping Review and Analysis. JAMA Netw Open. 2018; 1(8): e186019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKwakkenbos L, Juszczak E, Hemkens LG, et al.: Protocol for the development of a CONSORT extension for RCTs using cohorts and routinely collected health data. Res Integr Peer Rev. 2018; 3: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMc Cord KA, Ewald H, Ladanie A, et al.: Current use and costs of electronic health records for clinical trial research: a descriptive study. CMAJ open. 2019; 7(1): E23–E32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKay A, Jones A, Gamble C, et al.: Data sets used and Supplementary Table 1. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12185193.v1" }	[ { "id": "63052", "date": "13 May 2020", "name": "Sharon Love", "expertise": [ "Reviewer Expertise Trial conduct", "particularly monitoring and the use of routinely collected health data." ], "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 review of the protocols of RCTs, currently in progress, funded by NIHR, UK. RCTs were selected from NIHR HTA funding stream list if they claimed to be using routinely collected data for at least one study outcome. The authors found 102 trial protocols matching this criteria and from data extraction that 46 of these were using routinely collected data solely for at least one outcome. The research also found that a handful referenced previous feasibility work confirming the quality of the EHR and also gives a useful table categorising for the 102 trials how they used EHR.\nMajor Comments I have only one major comment and it is the reason for both the ‘partly’ options below. The sample was selected to be using routinely collected data for at least one study outcome. Therefore I think the main result should contain this information. I consider that “in a UK cohort” is not enough of a description of the cohort. The fact that the sample was selected based on using routine data for an outcome is crucial in the interpretation.\nThe main result is that of 102 protocols using routinely collected data for an outcome, 46 were using routinely collected data as their sole source for at least one outcome. 46/102=45%. Around a half of NIHR HTA funded trials that had an uploaded protocol and used routinely collected health data for at least one study outcome, used solely routinely collected data for at least one trial outcome. I think this is an important result.\nMinor comments\nAbstract – last part of the last sentence has a word missing “The majority of which were carried out in North America”.\n\nIf you have space in the text, it would be useful to add the information that 30 were omitted due to not having a protocol.\n\nThe flow chart shows you selected the papers by selecting RCT, those that had a protocol and then those using routinely collected data for at least one outcome. I would be tempted to list the inclusion criteria in the paper in the same order.\n\nThe second inclusion criteria is “use of routinely collected health data”. Elsewhere you use the term EHR. I would be tempted to be consistent.\n\nTable 1: category 10 description appears incomplete.\n\nTable 1: could you add a footnote of the definition of a registry trial?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5523", "date": "01 Jun 2020", "name": "Andrew McKay", "role": "Author Response", "response": "Major comments: Thanks for your important comments. We have made these clearer within the article update to version 2. Major comments part 1: We have now made it clear that the “UK cohort” is “NIHR HTA trials with a protocol” ongoing at the stated data extraction date. Major comments part 2: We have now made this clearer. Minor comments: Thank you for your comments. We have addressed them all within the article update to version 2. In relation to one specific comment, we have chosen to use ‘routinely collected health data (RCHD)’ throughout rather than ‘Electronic Health Record (EHR)’ for consistency." } ] } ]	1	https://f1000research.com/articles/9-323
https://f1000research.com/articles/9-1220/v1	09 Oct 20	{ "type": "Research Article", "title": "Use of social media in the marketing of agricultural products and farmers’ turnover in South-South Nigeria", "authors": [ "Henry Inegbedion", "Emmanuel Inegbedion", "Abiola Asaleye", "Eseosa Obadiaru", "Festus Asamu", "Emmanuel Inegbedion", "Abiola Asaleye", "Eseosa Obadiaru", "Festus Asamu" ], "abstract": "Background: The study investigated the use of social media in the marketing of agricultural products and farmers turnover in South-South Nigeria. The purpose of the study was to determine the extent to which the usage of social media in the marketing of agricultural products in Nigeria can enhance efficiency and farmers’ sales turnover. Methods: It employed the survey research design and data were collected with the help of a structured questionnaire. Research data were analysed using t-test and least square method. Results: The results showed that the use of social media (Facebook, WhatsApp and Instagram) in the marketing of agricultural products enhances efficiency and turnover of farmers through a significant reduction in the cost of marketing agricultural products as well as increased awareness and the attendant increase in demand for agricultural produce. Conclusions: The use of social media (WhatsApp and Instagram) in the marketing of agricultural products significantly influences cost reduction and hence efficiency in marketing as well as enhances turnover of farmers through increased demand for agricultural products. Thus, the adoption of social media in the marketing of agricultural products enhances the efficiency of agricultural marketing and sales turnover of farmers in south-south of Nigeria but only the use of WhatsApp and Instagram predict efficiency and sales turnover of farmers.", "keywords": [ "Agricultural marketing", "efficiency in marketing", "optimal sales turnover", "social media marketing", "demand for agricultural products" ], "content": "Introduction\n\nIn many countries of the world, the agricultural sector is regarded as a stimulant of economic development (Food and Agricultural Statistics, 2004). There is evidence to show consistently that it is not possible for any country to successfully attain industrialized status without first achieving significant success in agricultural performance since the green revolution (investment in food production) is crucial to the industrial revolution (Inegbedion et al., 2020; as well as (Inegbedion et al., 2018). The “agricultural sector performance, particularly through improved productivity, is one of the major ways of reducing poverty in developing countries” (Nebo & Ejionueme, 2017), which is consistent with the key macroeconomic goals of any economy and two of the United Nation’s Sustainable Development Goals: “No Hunger” and “No Poverty”.\n\nOver time, the agricultural sector has suffered neglect in Nigeria that it is largely unattractive to the current generation of youths. It is for this reason that a different type of agricultural marketing deserves to be given due attention to ensure that the earnings potential of agriculture is not undermined. Given the popularity of the use of social media across the different age grades in Nigeria, it is logical to expect that its use in the marketing of agricultural products will make a significant impact on the demand for agricultural products and thus on sales. This explains the rationale for this study.\n\n\nObjectives of the study\n\nThe main objective of this study was to investigate the impact of the adoption of social media in agricultural marketing by farmers in South-South Nigeria on major performance indicators Such as cost of marketing, demand for agricultural products and turnover of farmers.\n\n\nLiterature review\n\nThe traditional marketing of agricultural products consists of determining the farm products, producing in the right quality, storing to ensure that is the products are available, and transporting the products to the places where they are required. It consists mainly of non-verbal communication between the producer and the consumer. The producer (farmer) conceives a product that is required by consumers, produces it in a manner perceived to be consistent with customers` requirement, stores it, and distributes to locations perceived to have adequate demand.\n\nUse of print and electronic media in advertising. Beyond the product-price-storage-distribution strategy, large organisations and government have been known to market their products through advertisements in print and electronic media. The essence of such advertisements is to create awareness of the availability of the products, the locations where they are available as well as the expected benefits of using those products, especially if the products can be substituted for other products.\n\nAdoption of social media for agricultural marketing in South-South Nigeria. Given the shortcomings of the print and electronic media in marketing agricultural products, the need for a more efficient strategy has been long overdue. The rapid growth of the mobile phone use around the world in the last few decades provided a viable marketing alternative for agricultural products in Nigeria and other emerging economies. Mobile phones have contributed significantly to the empowerment of people in developing countries in spreading information networking coverage in remote areas. Consequently, many rural areas are getting great benefit out of its usage in various spheres of endeavour. This has culminated in the improvement of the living standard of poor farmers in developed nations.\n\n\nTheoretical review\n\nAs one of the frequently used theory to underpin Internet usage, the major thrust of TAM is that the users’ beliefs in perceived usefulness and perceived ease of use of the Internet can be used to explain their acceptance of Internet (Chuttur, 2009; Dwivedi et al., 2017). Thus, the extent to which the user believes that technology is useful for the cause and the extent to which he feels that he can easily use technology are critical to his/her willingness to use it. Recent modifications and refinement to TAM have culminated in the extension of its use to enable a better understanding of users` intention to use Internet technology.\n\nThis theory visualizes a model that studies how an agricultural marketing cooperative (AMC), specifically an AMC that is made up of many farmers as members, can adopt a direct selling approach. Under this arrangement, the farmers who are the members of the AMC can sell their products either to the cooperative or in a local market. The model shows that if the farmers decide to sell to the AMC, the decision will induce an anti-competitive effect on the direct selling market, whereas, the option of direct selling has the propensity to create a \"healthy emulation\" among farmers (Agbo et al., 2013). This will then stimulate production and ultimately benefit the cooperative.\n\nThis study adopts the TAM and the theory of agricultural cooperatives as its framework because the ease with which the farmers feel they can utilize social media to market their products and the usefulness of social media marketing to the attainment of their marketing goals are critical to the adoption of social media in the marketing of their products. Furthermore, the cooperative societies to which the farmers belong can play a critical role in the marketing of the farmers’ products.\n\nThe empirical review focuses on cost of marketing agricultural products as well as sales turnover.\n\nBalkrishna & Deshmukh (2017) investigated the “role of social media in agriculture marketing and its scope.” They employed questionnaire and in-depth interviews to collect data. Results indicated that social media is very useful in agricultural marketing.\n\nVassiliadou et al., (2011) investigated “the use of social media among students of Technology Agriculture and their role in promoting agribusiness.” The results showed that social media usage enhances facilitation and flow of knowledge/information, as well as cheap advertisement of products.\n\nMwangi & Wagoki (2016) surveyed leading media groups to investigate “the effect of social media on the performance of advertisement business in the mainstream media in Kenya.” They employed stratified random sampling to select 82 respondents. A questionnaire served as the research instrument. Descriptive and inferential statistics were employed. The findings indicate that the interactivity of social media had a significant positive relationship with the performance of the advertisement. Lashgarara et al., (2011) examined “ICT capabilities in improving the marketing of agricultural productions of Garmsar Township, Iran.” The survey research design was employed with questionnaire serving as the research instrument. The results indicated that ICT capabilities determined 57% variance of agricultural products marketing. Therefore, the following null hypotheses were formulated:\n\nH01: There is no significant relationship between the use of social media in agricultural marketing and cost of marketing agricultural products\n\nH02 : There is no significant relationship between the use of social media (Facebook, WhatsApp and Instagram) in agricultural marketing and farmers’ turnover from agricultural products\n\nOther related studies are “use of information communication technologies among agricultural extension officers in Lesotho” (Akintunde & Oladele, 2019), “adoption of agricultural e-marketing,” (Alavion et al., 2017), as well as the studies of Eze et al. (2019), among others.\n\nGap in the literature. Adoption of social media and other communication technologies in the marketing of agricultural products is not deficient in the empirical literature. White et al. (2014); Balkrishna & Deshmukh (2017) as well as Khou & Suresh (2018), investigated use of social media for agricultural marketing; Akintunde & Oladele (2019) and Alavion et al. (2017) examined determinants of ICT usage in the marketing of agricultural products. Jose & Lokeswari (2018) investigated users and non-users of mobile communication technologies in agricultural marketing; Lashgarara et al. (2011) examined ICT capabilities in improving the marketing of agricultural productions, Ogunniyi & Ojebuyi (2012) investigated “Mobile phone use for agribusiness by farmers in Southwest Nigeria while Mwangi & Wagoki (2016) investigated the effect of social media on the performance of advertisement business in in Kenya. Although a few of the studies investigated the use of social media, none appears to have focused on the implications of the usage of social media in agricultural marketing on marketing efficiency; neither is there adequate empirical evidence on how the use of social media in the marketing of agricultural products can impact on efficiency and sales turnover. Besides, related studies on the research problem in South-South Nigeria are either scant or non-existent. This study sought to fill these gaps.\n\n\nMethods\n\nThe study employed the quantitative research method. Specifically, the conclusive research design consistent with Inegbedion (2018); Inegbedion et al., (2018) and Inegbedion et al., (2016) was employed while the survey method was used in data collection. The study was conducted over July-September, 2019\n\nThe population of the study consisted of 4280 farmers registered in cooperative societies. Of this number, 1620 are from Edo State, 1460 are from Ondo State and 1200 are from Delta state. Yamane’s formula was used to estimate a sample size of 366 (Yamane, 1967) and proportional allocation was used to assign 139, 125 and 102 to Edo, Ondo and Delta States, respectively. Of the 366 respondents that were sampled, 246, representing 67.2% of them voluntarily participated in the study. The participants were randomly selected from farmers’ cooperative societies in the three states (a lottery method was used to achieve randomisation after stratifying the farmers on the basis of crop, poultry and fish farming). Subsequently, the farmers were contacted through any of the social media that they use (Facebook, WhatsApp or Instagram). The major inclusion criterion was membership of farmers’ cooperative while the exclusion criterion was the non-usage of any of the three social media platforms (Facebook, WhatsApp or Instagram). The choice of these states was informed mainly by convenience. Specifically, samples were taken from the current members of the cooperative societies. The sample consisted of the crop, poultry and fish farmers with evidence of usage of social media (Facebook, WhatsApp or Instagram). The sampling frame was requested from the management of the cooperative societies.\n\nAfter the sampling of the respondents, the verbal consent of the management of the cooperative societies was sought. Having obtained the consent of the leaders of the cooperatives, the consents of all the 366 sampled respondents across the three States were sought through the social media. Following the request to participate in the study, through social media 120 of them across the three states declined to participate in the study through their response to the social media message requesting their participation. While a few cited personal reasons for declining, majority of them did not advance any reason, they just refused to respond to the request. Of those approached, 246 gave their consent and thus participated in the study.\n\nBased on the sampling frame, a sample of respondents was selected. Thereafter, the sampled respondents were requested to participate in the study through social media. A survey was constructed and used to examine the use of social media in agricultural marketing and its implication for efficiency and sales turnover through the administration of a questionnaire, which served as the research instrument. The questionnaire contained bio-data questions and 5-point Likert scale questions dealing with social media usage in agricultural marketing and its implication for efficiency and sales turnover in South-South Nigeria. Information was elicited from the respondents via structured questionnaires through the social media channels (Facebook, Instagram and WhatsApp). The questionnaire is available as Extended data (Inegbedion et al., 2020).\n\nA pilot test was conducted on 20 of the sampled respondents. Based on the results obtained from the pilot test, validity and reliability of the instrument were determined. For validity, two approaches were used. First, the instrument was given to experts in management and marketing in the authors’ institution for their expert opinion, this served to fulfil the condition for face validity. Thereafter, content validity index (CVI) was computed. Both scale and item content validity measures were used. The results obtained were 0.66 for scale and 0.67 and 0.67 item CVI of use of social media in marketing agricultural products and cost reduction as well as use of social media in the marketing of agricultural products and farmers’ turnover respectively, thus showing that the instrument was valid since all the values are approximately 0.7 and a value of 0.7 is indicative of a valid instrument (see Table 1).\n\nCronbach’s alpha (α) was used to determine the reliability of the instrument. The computed Cronbach alpha values were 0.70 and 0.75 for use of social medial and cost reduction as well as use of social media and sales turnover respectively while the computed alpha of the entire instrument was 0.84. These values were considered significant since they are approximately 0.7 or more, thus indicating that the items in the instrument are internally consistent. In other words, the instrument is reliable. The foregoing indicates that the instrument is valid and reliable (see Table 2).\n\nCMAP = f (UFB, UWA and UINS) . . . (1)\n\nDAP = f (UFB, UWA and UINS) . . . (2)\n\nIn specific terms, equations 1 and 2 yield\n\nCMAP = β0 + β1 UFB + β2 UWA + UINS+ e . . . (3)\n\nDAP = β0 + β1 UFB + β2 UWA + UINS + e . . .. (4)\n\nWhere\n\nCMAP = Cost of marketing agricultural products;\n\nDAP = Demand for agricultural products;\n\nUFB = usage of Facebook;\n\nUWA = usage of WhatsApp;\n\nUINS = usage of Instagram; and\n\ne. = random error observed along with the variables\n\nResearch data were analysed using the one-sample t-test and least-squares regression. One sample t-test was used to test for significance of the usage of social media constructs in cost reduction and sales turnover while the least-squares method was used to test for the predictive power of the entire constructs (collectively) with respect to cost reduction and sales turnover; besides, the signs of the coefficients of the constructs in the regression model were used to infer the direction of the relationships between usage of social media in agricultural marketing and cost of marketing agricultural products on one hand as well as usage of social media in agricultural marketing and sales turnover on the other hand. Data analysis was conducted with SPSS 20.\n\n\nEthical approval\n\nThe authors sought and got ethical approval to conduct the study from the Landmark University Research Ethical Board. Furthermore, the authors complied with conventional ethical standards in conducting the study, including the request for the consent of the sampled respondents to participate in the study.\n\n\nResults\n\nThe results focus on the impact of social media usage on cost reduction in the marketing of agricultural products and on sales turnover of the farmers. Underlying data are available from Dryad (Inegbedion et al., 2020).\n\nA comparison of the usage of Facebook in agricultural marketing with cost reduction in marketing of agricultural products revealed that respondents who agreed that the usage of Facebook enhances cost reduction had a mean score 3.11. Given the five-point Likert scale, a test value of 3 was used. The computed t- and p-values were 2.04 and 0.046 respectively. This shows that the test was significant at P<0.05. We thus conclude, at the 95% confidence level, that usage of Facebook for marketing agricultural products significantly reduces the cost of marketing (see Table 3).\n\nA comparison of the usage of WhatsApp in agricultural marketing with cost reduction in marketing of agricultural products revealed that respondents who agreed that the usage of Facebook enhances cost reduction had a mean score 3.59. Given the five-point Likert scale, a test value of 3 was used. The computed t- and p-values were 14.58 and 0.001, respectively. This shows that the test was significant at the one per cent level. We thus conclude, at the 99% confidence level, that usage of WhatsApp for marketing agricultural products significantly reduces the cost of marketing (see Table 3).\n\nA comparison of the usage of Instagram in agricultural marketing with cost reduction in marketing of agricultural products revealed that respondents who agreed that the usage of Facebook enhances cost reduction had a mean score 3.09. Given the five-point Likert scale, a test value of 3 was used. The computed t and p values were 1.99 and 0.048, respectively. This shows that the test was significant at the five per cent level. We may thus conclude, at the 95% confidence level, that usage of Instagram for marketing agricultural products significantly reduces the cost of marketing (see Table 3).\n\nA regression model of usage of social media in the marketing of agricultural products and cost of marketing agricultural products revealed that the R2 value was 0.48, thus implying that 48% of the variation in the cost of marketing agricultural products is explained by variation in the usage of social media channels (see Table 4). The ANOVA table shows a calculated F of 10.94 with an associated significant probability of p < 0.001, thus indicating that the F value is significant. The implication is that the overall significance of the model is good (see Table 4). The regression coefficients show that the computed t-values and associated p-values were 3.152 (0.002), 0.337 (0.076), 5.015 (p < 0.001) and 2.570 (0.011) for constant, Facebook usage, WhatsApp usage and Instagram usage, respectively (see Table 4). The implication is that the explanatory variables, consisting of the usage of social media channels (WhatsApp and Instagram) are significant and are thus predictors of cost reduction in the marketing of agricultural products. However, Facebook usage is not significant and is thus not a predictor of cost reduction in the marketing of agricultural products.\n\nA comparison of the usage of Facebook in agricultural marketing with sales turnover of farmers revealed that respondents who agreed that the usage of Facebook enhances farmers’ sales turnover had a mean score 3.214. Given the five-point Likert scale, a test value of 3 was used. The computed t and p values were 5.44 and P <0.001 respectively. This shows that the test was significant at p < 0.05. We thus conclude, at the 95% confidence level, that usage of Facebook for marketing agricultural products significantly enhances the sales turnover of farmers (see Table 5).\n\nA comparison of the usage of WhatsApp in agricultural marketing with sales turnover of agricultural products revealed that respondents who agreed that the usage of Facebook enhances sales turnover had a mean score 3.289. Given the five-point Likert scale, a test value of 3 was used. The computed t and p values were 5.84 and 0.000, respectively. This shows that the test was significant at the one per cent level. We thus conclude, at the 99% confidence level, that usage of WhatsApp for marketing agricultural products significantly optimises the sales turnover of farmers (see Table 5).\n\nA comparison of the usage of Instagram in agricultural marketing with sales turnover of agricultural products revealed that respondents who agreed that the usage of Facebook enhances sales turnover had a mean score 3.115. Given the five-point Likert scale, a test value of 3 was used. The computed t and p values were 4.32 and P < 0.001 respectively. This shows that the test was significant at P < 0.01. We thus conclude, at the 99% confidence level, that usage of Instagram in marketing agricultural products significantly optimises the sales turnover of farmers (see Table 5).\n\nA regression model of usage of social media and turnover of agricultural products revealed that the R2 value was 0.58, thus implying that 58% of the variation in turnover of agricultural products is explained by variation in the usage of social media (see Table 6). The ANOVA table shows a calculated F of 7.21 with an associated significant probability of P < 0.001. The implication is that the overall significance of the model is good (see Table 6). Lastly, the regression coefficients show that the computed t and associated significant probabilities were 12.58 (0.00), 0.816 (0.415), 2.77 (0.006) and 4.614 (p < 0.001) for constant, Facebook usage, WhatsApp usage and Instagram usage, respectively (see Tables 6). The implication is that WhatsApp and Instagram usage are predictors of turnover of agricultural products.\n\nF test was conducted to compare respondents’ perception of the use of social media in the marketing of agricultural products with their demographic variables. A comparison of respondents’ perception of usage of social media in cost reduction and demographic variables had the following computed F and associated significant probabilities 1.12 (0.348), 1.36 (0.25), 0.673 (0.671) and 1.28 (0.28) for age, sex, educational qualification and farm categories, respectively. The implication is that respondents’ perception of the significance of the usage of social media in the reduction of agricultural marketing cost is not influenced by demographic variables (see Table 7).\n\nLastly, a comparison of respondents’ perception of the significance of the usage of social media in enhancing the turnover of agricultural products had the following computed F and associated p-values 0.29 (0.83) 1.26 (0.27), 0.961 (0.51) and 0.734 (0.73) for age, sex, educational qualification and farm categories, respectively. The implication is that respondents’ perception of the significance of the usage of social media in enhancing the turnover of agricultural products is not influenced by demographic variables (see Table 8).\n\n\nDiscussion\n\nResults of the study indicate that the use of Facebook, WhatsApp and Instagram in the marketing of agricultural products has a significant influence on the reduction of the cost of marketing agricultural products. However, while the use of WhatsApp and Instagram were found to be significant predictors of reduction in the cost of marketing agricultural products, the use of Facebook was not found to be significant. The implication of the significance of social media channels (WhatsApp and Instagram) in the reduction of the cost of marketing agricultural products is that the use of social media channels in the marketing of agricultural products enhances efficiency in the marketing of agricultural products. The results are consistent with the findings of Balkrishna & Deshmukh (2017); Mwangi & Wagoki (2016); Ogunniyi & Ojebuyi (2012); White et al., (2014); as well as Vassiliadou et al., (2011). Results of the study further indicate that the use of social media channels (Facebook, WhatsApp and Instagram) in the marketing of agricultural products has a significant influence on the enhancement of the turnover of farmers. However, just like in the previous model, the use of Facebook did not demonstrate a significant predictive power on sales turnover. Thus, only the use of WhatsApp and Instagram have strong predictive powers on sales turnover. The results are consistent with the findings of Mwangi and Wagoki, Khou & Suresh (2018) as well as Ogunniyi & Ojebuyi (2012).\n\nThe first regression model shows that WhatsApp and Instagram usage in the marketing of agricultural products are significant predictors of cost reduction in the marketing of agricultural products while Facebook was not significant. Thus, usage of WhatsApp and Instagram in the marketing of agricultural products has significant implications on the marketing of agricultural products. This tends to suggest that the usage of Facebook by farmers in the marketing of agricultural products is minimal compared to the usage of WhatsApp and Instagram. The coefficient of determination of 0.48 shows that 48% of the variation in advertising cost of farmers is attributable to the usage of social media in advertising. In the same vein, the second regression model shows that the usage of WhatsApp and Instagram are significant predictors of enhanced sales turnover from agricultural products because the two channels of social media help in speedy dissemination of information about the agricultural products to a wide audience of customers, thus leading to increased demand on the short-run. The two regression models indicate that WhatsApp and Instagram are predictors of efficiency of cost of marketing agricultural products and sales turnover from agricultural products.\n\nBased on the research findings, a model of social media marketing of agricultural products, efficiency of agricultural marketing and sales turnover was proposed. The model shows that two social media channels (WhatsApp and Instagram) predict social media impact on the marketing of agricultural products in terms of efficiency and enhanced turnover. The model shows that the use of social media (WhatsApp and Instagram) leads to optimal marketing cost and thus efficient marketing of agricultural products. The optimal cost of marketing agricultural products translates to cost savings and hence optimal turnover in agricultural products. Also, effective use of social media (WhatsApp and Instagram) stimulates demand which also results in optimal sales turnover from agricultural products (see Figure 1).\n\n\nConclusions\n\nGiven the problem definition and findings, the research concludes that: The use of social media (Facebook, WhatsApp and Instagram) in the marketing of agricultural products significantly influences cost reduction and hence efficiency in marketing as well as enhances turnover of farmers through increased demand for agricultural products. Thus, the adoption of social media in the marketing of agricultural products enhances the efficiency of agricultural marketing and sales turnover of farmers in south-south of Nigeria but only the use of WhatsApp and Instagram predicts efficiency of agricultural marketing and sales turnover of farmers.\n\nThis study has made significant contributions to marketing and management knowledge. First, it is among the few studies that have examined the influence of social media marketing on marketing efficiency and farmers’ sales turnover in Nigeria. Secondly, it is among the very few studies to have studied the adoption of social media in agricultural marketing in south-south Nigeria. Thus, a major point of departure of this study from previous studies is its examination of the implications of the usage of social media usage in agricultural marketing on marketing efficiency and sales turnover. It has thus made a concerted effort in reinforcing the interest of farmers in the use of social media in the marketing of agricultural products in south-south Nigeria.\n\nThe study is not without limitations which indicate the need for further studies to minimize the constraints associated with this study. Out of six South-South states in Nigeria, only three were studied. It is uncertain if the three states will adequately represent the six states in the region. The tendency of the three states to inadequately represent the six South-South states is a limitation to the results of this study. Furthermore, the use of only members of the farmers’ cooperative society constitutes a limitation to the results of the study. Farmers who do not belong to the cooperative society may have some dissenting perceptions from those of the members of a cooperative society.\n\nThe significance of the use of social media in the marketing of agricultural products to marketing efficiency and sales turnover implies that policy makers in government and strategic managers of agro-allied firms can embrace the use of social media in the marketing of agricultural products to minimize the cost of marketing and enhance turnover from the sales of agricultural products. Growth in the agricultural sector has implications on a nation’s gross domestic product and by implication, on national development. Given the problem definition and research findings, the following recommendations are suggested. Policymakers in government should be concerned about increasing agricultural production as well as the marketing of agricultural products to enhance earnings from agricultural production. This will attract many unemployed youths to the agricultural sector and thus help to guarantee food security as well as drastically reduce the level of unemployment in the country. Consequently, policymakers in government and other stakeholders like managers of cooperative societies and other farmers’ associations should promote the adoption of social media in agricultural marketing through sensitization of the farmers as well as the outright provision of modern communication gadgets to farmers at subsidized prices.\n\nFuture studies should attempt to minimize limitation associated with cultural bias by including more south-south states in the sample as well as try to include non-members of the farmers’ cooperative society in the sample.\n\n\nData availability\n\nDryad: Use of social media in the marketing of agricultural products and farmers’ turnover in South-South Nigeria. https://doi.org/10.5061/dryad.jwstqjq76 (Inegbedion et al., 2020).\n\nFile ‘Data-Use_of_Social_media_in_Agric_Markt-22’ contains basic demographic information, questionnaire responses and social media use data from each participant.\n\nDryad: Use of social media in the marketing of agricultural products and farmers’ turnover in South-South Nigeria. https://doi.org/10.5061/dryad.jwstqjq76 (Inegbedion et al., 2020).\n\nFile ‘Questionnaire-Use_of_Social_Media_in_Agric_Marketing.docx’ contains a blank copy of the questionnaire used in this study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgement\n\nWe are grateful to the management of Landmark University for providing the necessary funding for this study.\n\n\nReferences\n\nAgbo M, Rousselière D, Salanié J: A Theory of Agricultural Marketing Cooperatives with Direct Selling. Working Papers 1331, Groupe d'Analyse et de Théorie Economique Lyon St-Étienne (GATE Lyon St-Étienne): Université de Lyon.Handle: RePEc:gat:wpaper: 1331. 2013.\n\nAkintunde MAO, Oladele OI: Use of Information Communication Technologies among Agricultural Extension Officers in Lesotho. Journal of Agricultural Extension. 2019; 23(3): 50–65. Publisher Full Text\n\nAlavion SJ, Allahyari MS, Al-Rimawi AS, et al.: Adoption of agricultural e-marketing: Application of the theory of planned behaviour. Journal of International Food Agribusiness Marketing. 2017; 29(1): 1–15. Publisher Full Text\n\nBalkrishna BB, Deshmukh AA: A study on the role of social media in agriculture marketing and its scope. Global Journal of Management and Business Research: E-Marketing. 2017; 17(1): 1–5. Reference Source\n\nChuttur M: Overview of the technology acceptance model: Origins, developments and future direction. All Sprouts Content. 2009; 290. 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Publisher Full Text\n\nInegbedion HE, Obadiaru DE, Bello DV: Factors that influence consumers’ attitude towards internet buying in Nigeria. Journal of Internet Commerce. 2016; 15(4): 353–375. Publisher Full Text\n\nInegbedion H, Inegbedion E, Asaleye A, et al.: Use of social media in the marketing of agricultural products and farmers’ turnover in South-South Nigeria, v2, Dryad, Dataset. 2020. http://www.doi.org/10.5061/dryad.jwstqjq76\n\nJose AM, Lokeswari K: A study on users and non-users of ICT among the farming community. Global Media Journal. 2018; 16(31): 1–5. Reference Source\n\nKhou A, Suresh KR: A Study on the role of social media mobile applications and its impact on agricultural marketing in Puducherry Region. J Manage. 2018; 5(6): 28–35. Publisher Full Text\n\nLashgarara F, Mohammadi R, Najafabadi MO: ICT capabilities in improving the marketing of agricultural productions of Garmsar Township, Iran. Annals of Biological Research. 2011; 2(6): 356–363. Reference Source\n\nNebo GN, Ejionueme N: Adopting Agricultural Marketing Approach for Improving Agricultural Sector Performance in Nigeria. Journal of Business and Management. 2017; 19(4): 4–17. Reference Source\n\nOgunniyi MD, Ojebuyi BR: Mobile Phone Use for Agribusiness by Farmers in Southwest Nigeria. Electronic Journals Service(EJS). 2012; 20(10): 172–187. Reference Source\n\nMwangi MW, Wagoki J: Effect of social media on the performance of advertisement business in the mainstream media in Kenya: A survey of leading media groups in Kenya. International Journal of Economics, Commerce and Management. 2016; 4(4): 159–177. Reference Source\n\nVassiliadou S, Vogiatzi M, Amygdalas T, et al.: The use of social media among students of Technology Agriculture and their role in promoting agribusiness. In: M. Salampasis, A. Matopoulos(eds.): Proceedings of the International Conference on Information and Communication Technologies for Sustainable Agri-production and Environment(HAICTA 2011): Skiathos. 2011. Reference Source\n\nWhite D, Meyers C, Doerfert D, et al.: Exploring agriculturalists' use of social media for agricultural marketing. Journal of Applied Communications. 2014; 98(4): 1–14. Reference Source\n\nYamane T: Statistics, an introductory analysis, Harper and Row. 1967. Reference Source" }	[ { "id": "76594", "date": "16 Feb 2021", "name": "Lauri M Baker", "expertise": [ "Reviewer Expertise Agricultural communication", "social media strategy", "marketing" ], "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 extremely interesting with regard to understanding social media use in agriculture in Nigeria and contributes to the body of knowledge more largely in the area of marketing agricultural products online.\nSince this is the first time I am reviewing for this journal, I wasn’t sure what to expect, but there is a fair amount of copyediting that needs to be done in this manuscript. I have made note of some major issues here in my review, but there are others. Also, perhaps it is acceptable in this journal, but I was trained that in research writing each paragraph needs a minimum of three sentences, and some sentences in this article are shorter than that. Additionally, some of the language used within the article isn’t inclusive. I know many journals now have a stance on this, I am not sure if this journal does or not. The present article uses “he” or he/she instead of they or other neutral pronouns in some places.\nI have the following specific feedback:\nThe end of the second sentence of the manuscript isn’t finished. It ends with “as well as”.\n\nThe first paragraph and first sentence of the literature review has a grammatical error in this section “ensure that is the products are available”. It is unclear exactly what the authors mean because of the sentence structure.\n\nThe first sentence under theoretical framework – theory should be theories.\n\nInternet is chaptalized throughout, but it is no longer considered a proper noun in most style formats.\n\nThe first sentence of the theoretical framework is difficult to understand. I see the authors are trying to convey how they applied this framework, but how that was done is unclear as written.\n\nThe majority of the empirical review section lacks flow and is choppy. It seems the authors have made more of an annotated bibliography instead of synthesizing and apply how each of these studies relates to their present work.\n\nParticipants were described in present tense instead of past tense. This section was also missing commas in the numbers.\n\nSampling procedure was explained well and was adequate, but again the writing in this section should be improved.\n\nI appreciate that the reliability is presented so clearly for each construct, but Tables 1 and 2 are overly complicated. I don’t think you need the 3rd column if you adjust the headings accordingly.\n\n“analysed” is misspelled. It should be analyzed.\n\nThere are places in the results where a capital P is used for significance level. In my training typically a lowercase p is used as in p < .05 and the authors do use this in some cases. I would advocate for the lowercase, but above all consistency.\n\nRounding in the results section is not consistent.\n\nThe use of the Oxford comma in this manuscript is not consistent.\n\nThe conclusions and discussions and implications make sense from the results presented, and I believe the findings provide value in this context. However, the writing in these final sections is difficult to follow and has similar grammatical and flow areas, like the rest of the manuscript.\n\nI would like to see the authors do some serious work in the writing of this manuscript. I hate to reject any article that clearly represents a solid study that can add to the literature in this area, but MAJOR improvements are needed in writing quality, editing, and flow.\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": "6385", "date": "12 Mar 2021", "name": "Henry INEGBEDION", "role": "Author Response", "response": "Comment: but there is a fair amount of copyediting that needs to be done in this manuscript. Response: Significant copyediting has been done Comment: Also, perhaps it is acceptable in this journal, but I was trained that in research writing each paragraph needs a minimum of three sentences, and some sentences in this article are shorter than that. Response: The paragraphs have been adjusted accordingly Comment: Additionally, some of the language used within the article isn’t inclusive. I know many journals now have a stance on this, I am not sure if this journal does or not. The present article uses “he” or he/she instead of they or other neutral pronouns in some places. Response: Efforts have been made to enhance the inclusiveness of the language used Comment: I have the following specific feedback: The end of the second sentence of the manuscript isn’t finished. It ends with “as well as”. Response: This has been corrected Comment: The first paragraph and first sentence of the literature review has a grammatical error in this section “ensure that is the products are available”. It is unclear exactly what the authors mean because of the sentence structure Response: This has been corrected - “ensure that is the products are available” Comment: The first sentence under theoretical framework – theory should be theories. Response: This has been corrected Comment: Internet is chaptalized throughout, but it is no longer considered a proper noun in most style formats. Response: This has now been corrected Comment: The first sentence of the theoretical framework is difficult to understand. I see the authors are trying to convey how they applied this framework, but how that was done is unclear as written. Response: It is actually an attempt to justify the use of the two theories as framework, clarification has been provided. The how has been explained in the theoretical review. Comment: The majority of the empirical review section lacks flow and is choppy. It seems the authors have made more of an annotated bibliography instead of synthesizing and apply how each of these studies relates to their present work Response: Some level of synthesis has been provided to link the empirical literature to the study Comment: Participants were described in present tense instead of past tense. This section was also missing commas in the numbers. Response: The tenses and the missing comas have been corrected Comment: Sampling procedure was explained well and was adequate, but again the writing in this section should be improved. Response: The section has been improved upon Comment: I appreciate that the reliability is presented so clearly for each construct, but Tables 1 and 2 are overly complicated. I don’t think you need the 3rd column if you adjust the headings accordingly. Response: The headings have been adjusted and the third column has been removed Comment: “analysed” is misspelled. It should be analyzed Response: This has been corrected Comment: There are places in the results where a capital P is used for significance level. In my training typically a lowercase p is used as in p < .05 and the authors do use this in some cases. I would advocate for the lowercase, but above all consistency. Response: This has been corrected Comment: Rounding in the results section is not consistent Response: Done Comment: The use of the Oxford comma in this manuscript is not consistent. Response: This has been addressed Comment: The conclusions and discussions and implications make sense from the results presented, and I believe the findings provide value in this context. However, the writing in these final sections is difficult to follow and has similar grammatical and flow areas, like the rest of the manuscript. Response: The grammatical flaws have been significantly addressed Comment: MAJOR improvements are needed in writing quality, editing, and flow Response: The quality of the paper has been enhanced" } ] }, { "id": "73490", "date": "23 Feb 2021", "name": "Philip Olasupo Alege", "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\nAbstract: The Abstract is too long. It should be brief and sharp. The content should include the following: background/motivation; objective of the paper, methodology, data, results and interpretation. It must also include contribution to knowledge. This component of the study should not be more than 250 words.\nIntroduction: This is well written. However, the authors should take another look at line three and check the sentence for consistency. It should be “there are evidences….” You cannot use only one observation to conclude on an issue. Also, there is no need to create a sub-section for “Objective of the Study”\nMAJOR COMMENTS This is mainly on the Structure of the paper. I will suggest the Sections should be numbered for ease of referencing between the different parts of the report. This is also important for the readers. An insight into this is given in what follows:\nLiterature Review\nChannels of advertisement Theoretical Review\nTechnology acceptance model (TAM) Theory of agricultural marketing cooperatives\n\nEmpirical review\nUse of social media in marketing agricultural products and cost of marketing\n\nUse of social media in agricultural marketing and sales turnover\n\nMethods\nStudy Population: Issues of request for consent, validity and Reliability must be discussed here. Model Specification Estimation Technique instead of Statistical Analysis Data Sources instead of Materials and Ethical Matters should be here\n\nEstimation Results and Discussion\nResults Discussion\n\nConclusion\n\nMINOR There are a few typographical errors to be attended to. The authors should check the soft copy.\nGENERAL COMMENTS The paper addresses a very pertinent question in this era of New Normal on the need for the adoption of social media in the marketing of agricultural products. The recommendations are the ways to go. However, if the idea captured in “Figure 1: A model of social media marketing of agricultural products and sales turnover” is a result emanating from this study, then it should be discussed earlier under Section 4.2.\nBesides, the paper is well written and had made a tangible contribution to knowledge.\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": "6384", "date": "12 Mar 2021", "name": "Henry INEGBEDION", "role": "Author Response", "response": "Comment: The Abstract is too long. It should be brief and sharp. The content should include the following: background/motivation; objective of the paper, methodology, data, results and interpretation. It must also include contribution to knowledge. This component of the study should not be more than 250 words. Response: F1000Research provided the template for the structure of abstract. In any case, the abstract has been reduced to below 250 words as suggested Comment: This is well written. However, the authors should take another look at line three and check the sentence for consistency. It should be “there are evidences….” You cannot use only one observation to conclude on an issue. Also, there is no need to create a sub-section for “Objective of the Study” Response: The sentence has been revised for consistency and the section on “objective of the study” has been removed as suggested Comment: This is mainly on the Structure of the paper. I will suggest the Sections should be numbered for ease of referencing between the different parts of the report. This is also important for the readers. An insight into this is given in what follows: Response: The paper has been restructured and the sections of the paper have been numbered as suggested by the reviewer Comment: There are a few typographical errors to be attended to. The authors should check the soft copy. Response: These have been worked on Comment: A model of social media marketing of agricultural products and sales turnover” is a result emanating from this study, then it should be discussed earlier under Section 4.2. Response: This section has been merged with section 4.2 as suggested" } ] } ]	1	https://f1000research.com/articles/9-1220
https://f1000research.com/articles/10-205/v1	11 Mar 21	{ "type": "Opinion Article", "title": "Conducting qualitative research during COVID-19: reflections on methods and challenges when interviewing marginalized refugee women", "authors": [ "Adelaide M. Lusambili", "Sharon Ochieng", "Mary M. Nyikuri", "Constance S. Shumba", "Mary M. Nyikuri", "Constance S. Shumba" ], "abstract": "This reflective opinion article arises from our experience conducting interviews with refugee women attending ante-natal and post-natal services in an urban setting in Kenya in the context of COVID‑19. First, we explain the research context in light of the study objectives. We reflect on the methodological challenges we faced, including researcher’s positionality, and argue that conducting research within the refugee context during the pandemic is unique, therefore research design must reconsider inclusive methodologies tailored to the uniqueness of refugees’ experience in order to obtain useful data. Second, we discuss these challenges in light of our experiences and the implications for addressing the gaps we identified, with particular emphasis on the ways in which professionals working with refugee communities in the context of a pandemic can build trust and obtain meaningful and uncompromised data. Lastly, we provide recommendations for researchers conducting similar research.", "keywords": [ "COVID-19", "refugees", "Qualitative", "Challenges", "ante -natal care", "post -natal care", "Kenya and methodology." ], "content": "Our research context\n\nAlthough there is a growing body of research among refugee women’s access to maternal and child health services in non-pandemic times1, there are limited studies that have focused on informants’ willingness and ability to participate in research2. Over 80% of refugees globally are hosted in low- and middle-income countries, with over half hosted in sub-Saharan Africa region3. However, there are rarely any studies that provide a reflection on the methodological approaches and challenges of working with refugee women during a pandemic in sub-Saharan Africa. Refugee women’s vulnerability is exacerbated in the context of the COVID-19 pandemic, as they face a triple crisis characterized by the impacts of COVID-19, displacement, and gender-based violence4. Pregnant refugee women face barriers in accessing health services with higher rates of adverse pregnancy outcomes5. While it is important to conduct research aimed at understanding how COVID-19 has affected refugee women, to develop evidence-informed pandemic responses and gender responsive maternal and child health interventions, it is plausible that refugee women also experience barriers in participating in research. These barriers may include, but are not limited to, endemic poverty, lack of fluency in the interviewers’ language, fear of authority, mental health issues, and lack of legal status—all of which may affect informants’ ability and willingness to supply the kind of information requested6–10. A gendered reflection of refugee women’s participation in research during the COVID-19 pandemic is therefore important.\n\nIn October 2020, a team of researchers from the department of population health (DPH) at the Medical College at the Aga Khan University conducted research using In-Depth Interviews among refugee women, to investigate the impact of COVID-19 on their access to and utilization of ante-natal and post-natal care services. Our study was approved by Aga Khan University Ethics Review Committee (ERC), National Commission for Science, Technology and Innovation (NACOSTI) and the Nairobi Metropolitan Health Services. The refugee women we interviewed were diverse, with the majority originating from Somalia and a few from Uganda, Tanzania, Ethiopia, South Sudan and Eritrea. Unlike other refugees who live in protected settlements such as Dadaab and Kakuma refugee camps, which are staffed by the United Nations, the refugees we interviewed lived among members of the host communities and did not have legal documentation. As such, they were not able to access free maternity services in public health facilities provided for under the Free Maternity Services Policy5. The refugee women we interviewed were directly linked to the International Organization for Migration (IOM) Wellness Clinic in Eastleigh, where they accessed free health care services, situated in one of Kenya’s urban centres in the capital city. Voluntary informed consent was sought from all the study participants.\n\n\nMethodological challenges and innate bias\n\nRecognizing the importance of a gendered reflection of underrepresented refugee women’s participation in research during the COVID-19 pandemic, we present and discuss herein our unique experiences during the study to investigate the impact of COVID-19 on refugee women’s access to and utilization of ante-natal and post-natal care services. These experiences fall into six main themes: (i) language and trust; (ii) loss in translation; (iii) limited understanding of social research; (iv) childcare challenges; (v) fear, misunderstanding, and insecurity; and (vi) researcher positionality.\n\nThe women were directly recruited through the clinic staff and most of the refugees had passed a screening for their ability to speak the local language, Kiswahili, in which the interviews were to be conducted. However, when we began our interviews, however, we observed that getting the conversations going was very difficult, with many of them indicating that they did not understand Kiswahili. The majority of them struggled to express themselves, and provided only brief and guarded statements. We surmised that the refugees, who were unfamiliar with the researchers, needed to establish trust before delving into their experiences about COVID-19 and how it had impacted their access to health services. This finding is consistent with other authors who have indicated that an extensive time commitment to build trust with marginalized refugee women2.\n\nFirst, when we began to use interpreters fluent in some of the languages spoken by the informants, it soon became evident that a great deal of information was not being translated for us. Some interviewees spoke for 3 minutes in their language, and the interpreter then summarized the information in 1 minute or less. To get the most out of the interpreters, researchers had to watch closely for participants’ facial expressions and body movements. For instance, when participant Y, speaking Somali, tearfully related her experience during delivery, the interpreter nodded her head in disbelief and said, “There is a lot of suffering among refugees and she is sad about what is happening.” But only when we probed why participant Y was so distressed did the interpreter add: “She says she saw a woman giving birth by the road during the lockdown, when hospitals Z and M were closed. The police were harassing everyone, and the woman could not get any help. She heard that the child died, and it was very sad.” This is just one example where valuable information about the effects of the lockdown on pregnant refugees could have been lost in translation, if the interviewer had not probed the interpreter further. This highlights the importance of working with skilled translators, who can directly interpret without overly summarizing or sifting any information.\n\nA number of the participants recruited at the Wellness Clinic did not seem to understand what social research is in general, or what our study aims were in particular. During the interview process, interviewees frequently diverted the discussion to how they could be helped during the COVID-19 epidemic. For instance, when participant X was asked what measures she had put in place for her family to remain COVID-19 safe, she responded, “Any money you can give me, I will use it wisely to put measures in place and feed my four kids and husband. My husband lost his job and we have no income for five months.” Many interviewees took part in our research with the hope of monetary or material assistance, which they commonly inquired or requested during the interview process. It is crucial to communicate clearly about the research purposes and to provide some financial incentives for participants10. We also posit that while research is a first step to understanding the refugee women’s experiences, and could in the long term lead to policy and programmatic changes, however, where possible during the research process, researchers should work with aid agencies that can provide material support or offer referrals for participants in dire circumstances.\n\nThe participants recruited for our study did not have child care and brought children and other family members to interviews. When they came to be interviewed, children who were eligible to wear masks did not have any. For example, participant Q showed up in the interview room with a 2-month-old baby, a 15-month-old girl, a 3-year and a 7-year-old boy. Mothers frequently required to be excused to breast-feed their babies before returning to be interviewed. At the same time the accompanying children and family members were also constantly present throughout the interviews. Study design and planning, especially in pandemic conditions, needs to take into account that refugees have large families, are too poor to buy masks, and bring their dependents to interviews because they cannot afford child care. In hindsight, none of these factors had been taken into account in the planning for this field study. Although the clinic provided masks for the children, this highlights the many risks refugee children may experience by virtue of accompanying their mothers in public without masks. To gather meaningful data and reduce the undue burden on participants, researchers need to consider providing childcare to participants10.\n\nSome women selected for this study felt uncomfortable with the consenting process, and more often they associated it with being deported, as most of them asked, ‘Are you from the government?’ It was also clear that we needed to take time to explain to them what research is, why we were asking for their consent, and why we needed to audio-record the conversations. For instance, although we had spent time explaining the purpose of the study, the importance of voluntary informed consent and that we would record the interview, nevertheless, when participant C saw a researcher turning on an audio-recorder, she asked, ‘Are you recording me on WhatsApp [she immediately stopped talking]?’. At this point, we stopped the interview and explained the process again to her as well as her right to withdraw if she felt uncomfortable with the interview. The interviewee seemed tearful and fearful and we opted not to interview her. This led us to reflect about the extent to which research among refugees could increase their emotional distress and what better ways research among refugees could be conducted. In the case of participant J, she kept looking at the audio-recorder after she had consented and we had started the interview. We noticed that the audio-recorder made her uncomfortable, so we stopped and put the recorder in our bag, but she never spoke again and we ended the interview process. Participant L instantly stopped talking when we started going through the consent form and explained that we will be audio recording. Although she signed the consent form giving us permission to audio-record, she refused to talk. Consenting processes overall took a long time, sometimes requiring health care workers familiar to them to be present to further assure them that the information they gave was confidential, for the purpose of research and not for government monitoring. There might have been a power dynamic resulting in some participants feeling obliged to participate in the study. The use of audio recorders might have compounded the situation. Similarly, other authors have found that refugees may not be comfortable with the use of tape recorders and researchers will need to be content with taking detailed notes instead2. In general, the suitability of the use of audio tapes among this population should be explored in our research context.\n\nAs a community health volunteer explained, the migrants did not have documentation, and most of them were careful in writing, signing or being recorded as they believed this information might be used against them. In addition, most interviewees had not heard about research, and some thought we were officials who had brought them some assistance from the government.\n\nThe issues related to the researchers’ positionality had a tremendous impact on the data collected from participants. In our initial interviews, when we presented ourselves as researchers, participants were guarded about what to say. The second week of data collection, having realised that most of our participants were of Muslim origin, the research team dressed in long clothes and burkas, and spent substantial time at the clinic each day with the aim of immersing with the potential participants and gain acceptance. This approach helped the researchers to yield some useful data from all women, including their perceptions of COVID-19. For example, when participant Q was asked about how COVID-19 had impacted her access to ante-natal services, she retorted, ‘As you know, I am not affected because COVID-19 is not for us (pointing to the researcher wearing a burka) but it is a punishment for Christians and all white people’. As a researcher, we felt that this type of sentiment would only be shared under a sense of trust and that how researchers position themselves as insiders has implications for the richness of the data collected11. For instance, following our failed first attempt at conversations in week one, we obtained rich data in week two when we dressed in attire favoured by refugees and covered our heads. In addition, consenting and audio recording process did not seem to traumatise participants. Although, it seems clear that dressing in burkas and spending substantial amount of time at the clinic finally led to useful data, our shifting positionalities, wearing burkas and being accepted, while it led to useful data, left us with ethical dilemmas as to whether disguising in the form of immersion is an appropriate method to collect data among vulnerable communities who are culturally difficult to engage. Methodologies such as in-depth interviews whereby researchers go to the field to interview vulnerable participants like refugees must be reconsidered. It may be better to engage the local refugee women where possible to collect data among their communities rather than have outsiders do so.\n\n\nConclusions\n\nThe methodological challenges we encountered raise critical issues that must be explored further while conducting research with vulnerable populations such as refugee women. Researchers need to reconsider strategies to engage refugee women and collect useful data in an ethical manner. First, the methodology used in this study calls upon researchers studying refugees or migrant populations who speak a foreign language to consider training individuals in the participants’ own communities to conduct research as well as use refugee interpreters to transcribe such data to avoid meaningful data gaps. This would ensure greater value in the data being collected and could help place participants more at ease. Second, prior to recruiting and interviewing refugee women participants, community sensitization is necessary to build a shared understanding and trust on the purpose and value of research and to avoid perceptions that the research should involve any immediate direct benefit. However, it also appears that small and modest incentives should be considered for refugee women’s participation in research. Third, refugees, and in this specific case refugees of Muslim origin, may have large families and child care can be challenging. Consideration for child care must be in place when interviewing refugee women of reproductive age to ensure quality data collection. Fourth, research about refugees must be conducted in a language in which they are fluent, and if interpreters are needed, they must be people with whom the respondents feel most comfortable and who are competent to translate the interviews without losing valuable information. Fifth, the provision of financial incentives by researchers can improve the participation of under-represented refugee women in research. Finally, researchers need to operate in an ethical manner to balance securing the trust and willingness to participate among respondents with the collection of quality data. To conclude, the need for research among vulnerable populations such as refugees is great, but researchers need to pay close attention to their methodologies to ensure they collect quality data in an ethical manner and in ways that promote inclusion for under-researched refugee women.\n\n\nData availability\n\nNo data is associated with this article", "appendix": "Acknowledgments\n\nWe are grateful to the Wellness Clinic staff in Eastleigh who recruited the study participants and to all the refugee women who protected time to be interviewed.\n\n\nReferences\n\nMengesha ZB, Perz J, Dune T, et al.: Refugee and migrant women's engagement with sexual and reproductive health care in Australia: A socio-ecological analysis of health care professional perspectives. PLoS One. 2017; 12(7): e0181421. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartinez IL, Carter-Pokras O, Brown PB: Addressing the challenges of Latino Health Research: participatory approaches in an emergent Urban Community. J Natl Med Assoc. 2009; 101(9): 908–14. PubMed Abstract \| Publisher Full Text\n\nHernando C, Sabidó M, Casabona J: Facilitators and barriers of participation in a longitudinal research on migrant families in Badalona (Spain): a qualitative approach. Health Soc Care Community. 2018; 26(1): e64–74. PubMed Abstract \| Publisher Full Text\n\nBonevski B, Randell M, Paul C, et al.: Reaching the hard-to-reach: a systematic review of strategies for improving health and medical research with socially disadvantaged groups. BMC Med Res Methodol. 2014; 14: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchein YL, Winje BA, Myhre SL, et al.: A qualitative study of health experiences of Ethiopian asylum seekers in Norway. BMC Health Serv Res. 2019; 19(1): 958. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGabriel P, Kaczorowski J, Berry N: Recruitment of Refugees for Health Research: A Qualitative Study to Add Refugees’ Perspectives. Int J Environ Res Public Health. 2017; 14(2): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLusambili A, Bhanbhro S, Muchanga K: Positionality, Access to the Social Space and Place of Research: Narratives from Research in Low Resource Settings. People Place and Policy. 2020; 14(1): 35–54. Publisher Full Text" }	[ { "id": "93111", "date": "13 Sep 2021", "name": "Sukhjeet Bains", "expertise": [ "Reviewer Expertise Migrant health", "Maternal 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 read this interesting manuscript. Reflecting on methodological challenges in conducting research among vulnerable populations, such as among refugee women, is of critical importance. This is particularly important, as you have described, in the region of Sub-Saharan Africa, where many refugee women are hosted. I commend your commitment to research in this under-explored area.\nOverall, the article was clearly written. I do, however, have a few minor suggestion on how the manuscript could be improved.\nIn Our research context the authors write “A gendered reflection of refugee women’s participation in research during the COVID-19 pandemic is therefore important”. I agree with the authors on the importance of reflection on the gender dimension in research. However, I miss any explanation on what this implies and any examples of how gender may influence researcher-respondent interaction. The importance of gendered reflection is emphasised by the authors in the subheading Methodological challenged and innate bias, and as it seems to be important, it should be elaborated. Although the authors write on page 4, under Researchers’ positionality that the researchers dressed in long clothes and burkas, they do not give information about the gender balance in the research group, whether any man was involved and how it may have impacted the research. If only women were involved (either research team or interpreters for instance?) they should also discuss how that may have impacted their data collection.\n\nI suggest the last sentence on page 4 is rewritten. The authors write: “Although, it seems clear that dressing in burkas and spending substantial amount of time at the clinic finally led to useful data, our shifting positionalities, wearing burkas and being accepted, while it led to useful data, left us with ethical dilemmas as to whether disguising in the form of immersion is an appropriate method to collect data among vulnerable communities who are culturally difficult to engage”. The way it is written now it is unnecessarily long and does not explain what the author means by ‘communities who are culturally difficult to engage’? The authors need to elaborate on this.\nI recommend the authors considers and addresses the concerns described above. I think the article makes some important points about researching on refugee women and I am looking forward to read the article with their findings from the in-depth interviews.\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": "115260", "date": "26 Jan 2022", "name": "Khangelani Moyo", "expertise": [ "Reviewer Expertise migration studies", "refugee governance", "refugee and migrant integration", "migrant transnationalism", "migrant identities", "and social vulnerabilities in the urban periphery." ], "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 paper discusses the challenges encountered by the authors in conducting research on refugee women accessing maternal health services at a clinic in Nairobi, Kenya. According to the authors, the refugees are undocumented and therefore lack the protection of the Kenyan government and also can’t access their entitlements in the form of free healthcare in public hospitals. Amongst other issues, the authors note that they initially struggled with establishing rapport with the participants due to language differences, poverty, unmet expectations, etc.\nThe idea of the article is an important one, especially given the ever-changing research landscape due to the Covid-19 pandemic. Also, as the authors highlight, researchers often neglect some critical aspects that prevent or influence the decisions by participants whether to participate in research studies. However, I have a number of concerns which I discuss below:\n\nThe article places the Covid 19 pandemic at the centre of the analysis, which is commendable and speaks to a developing field since this pandemic is new. However, the experiences described are not unique to the pandemic and may as well have been observed at any other time. Unless there is evidence showing better outcomes without the pandemic then the authors would do well to show how the pandemic is important in relation to how their fieldwork unfolded. In other words, this question arises because it has not been made clear in the paper how the pandemic has material effect on data collection.\n\nRegarding the findings of the paper, the same problem I have noted in point one arises. The authors need to tighten their arguments in order to make a case for their findings. For instance, the language difficulties and trust issues are not unique to the Covid era and can also be experienced whether the interviewees are refugees or not. It would be helpful for the authors to acknowledge this fact and also help the reader with an extended discussion. For example, if they observe that language was an issue – then they should tell the reader, what language did the interviewees speak. Why were the interviews only conducted in Kiswahili, especially when dealing with foreign nationals? The researchers could have sought research assistants who spoke the languages of the refugees rather than adding this as a recommendation at the end. Perhaps the question should be directed to the authors themselves rather than making it a general recommendation because the problems that they experienced could have been avoided if they had done their homework.\n\nThe paper also has some methodological and ethical issues. The authors note that they struggled to connect with the respondents during the first week of data collection and changed their dress code during the second week of fieldwork in order to identify with the respondents. While this may have been a great strategy, it, however raises ethical questions which are not fully explained in the paper. The authors need to discuss their research methods in the paper. For a paper whose main focus is “methodological challenges”, I find the focus on methods particularly thin. There is a need to clearly discuss the research questions, the methods used, the data analysis, the participants from which the data is drawn, and how the ethical issues were handled. For instance, the authors correctly observe that they may have disposed their participants to harm yet there were no measures in place to deal with this. Refugees often escape extreme violence and experience trauma, which implores researchers to engage in responsible and ethical practices during fieldwork. It is not enough for the authors to make recommendations when they should have practiced it in the first place.\n\nSome of the descriptions given amount to “othering” of refugees and the authors need to be careful as certain language may lead to discrimination of an already vulnerable population. For example, the following passage needs attention: At the same time the accompanying children and family members were also constantly present throughout the interviews. Study design and planning, especially in pandemic conditions, needs to take into account that refugees have large families, are too poor to buy masks, and bring their dependents to interviews because they cannot afford child care. In hindsight, none of these factors had been taken into account in the planning for this field study. Although the clinic provided masks for the children, this highlights the many risks refugee children may experience by virtue of accompanying their mothers in public without masks. To gather meaningful data and reduce the undue burden on participants, researchers need to consider providing childcare to participants\"\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? Partly\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-205
https://f1000research.com/articles/9-646/v1	25 Jun 20	{ "type": "Brief Report", "title": "Future scenarios for the SARS-CoV-2 epidemic in Switzerland: an age-structured model", "authors": [ "Janne Estill", "Plamenna Venkova-Marchevska", "Maroussia Roelens", "Erol Orel", "Alexander Temerev", "Antoine Flahault", "Olivia Keiser", "Plamenna Venkova-Marchevska", "Maroussia Roelens", "Erol Orel", "Alexander Temerev", "Antoine Flahault", "Olivia Keiser" ], "abstract": "The recent lifting of COVID-19 related restrictions in Switzerland causes uncertainty about the future of the epidemic. We developed a compartmental model for SARS-CoV-2 transmission in Switzerland and projected the course of the epidemic until the end of year 2020 under various scenarios. The model was age-structured with three categories: children (0-17), adults (18-64) and seniors (65- years). Lifting all restrictions according to the plans disclosed by the Swiss federal authorities by mid-May resulted in a rapid rebound in the epidemic, with the peak expected in July. Measures equivalent to at least 90% reduction in all contacts were able to eradicate the epidemic; a 56% reduction in contacts could keep the intensive care unit occupancy under the critical level and delay the next wave until October. In scenarios where strong contact reductions were only applied in selected age groups, the epidemic could not be suppressed, resulting in an increased risk of a rebound in July, and another stronger wave in September. Future interventions need to cover all age groups to keep the SARS-CoV-2 epidemic under control.", "keywords": [ "COVID-19", "SARS-CoV-2", "mathematical model", "Switzerland" ], "content": "Introduction\n\nSwitzerland has one of the highest incidences of documented SARS-CoV-2 infections per population, with large regional variability1,2. In response to the SARS-CoV-2 pandemic, the Swiss federal government implemented several restrictions, including closures of schools and non-essential shops and services and forbidding gatherings of more than five people3. Many of these restrictions were lifted on May 11, 2020, causing uncertainty about the future of the epidemic. We have developed an age-structured mathematical model to estimate possible scenarios for Switzerland until December 2020, and to identify how different levels of contact reduction between different age groups influence SARS-CoV-2 transmission.\n\n\nMethods\n\nWe used a stochastic compartmental model. The population of Switzerland is divided into three age groups (children (0–17 years), adults (18–64 years) and seniors (≥65 years)) and 11 compartments that represent the epidemiological stage. Most parameters (disease progression; probability of different levels of symptoms) were directly adapted from a model for France4. Relative contact frequencies between children, adults and seniors were retrieved from studies in Belgium, France, Germany and Italy (http://www.socialcontactdata.org/)5,6. Initial conditions (starting date, initial number of exposed individuals), COVID-19 related mortality rates of adults and seniors, overall infectiousness and the relative efficacy of each preventive measure were estimated by calibrating the model results against the daily COVID-19 hospitalizations and deaths2,7. The code and a detailed description of the methodology, including all input parameters and their sources, is available at https://gitlab.com/igh-idmm-public/covid-19/modelling_jestill and is archived with Zenodo8.\n\nBefore running the model for Switzerland, we fitted it to three cantons with epidemics that started at different times (Geneva, Ticino and Bern) to select informative priors for the uncertain parameters. We included the impact of the government-imposed restrictions. We modelled seven scenarios with different assumptions regarding the prevention after 11 May 2020: i) baseline scenario (no further preventive measures introduced); ii) eradication scenario (all contacts reduced by 90%); iii) epidemic control scenario (minimum contact reduction that can keep the number of ICU patients under the critical limit of 1200)9; iv) same contact reduction among adults and seniors as in scenario 3 but without any restriction on children’s contacts; v) same contact reduction in contacts involving adults as in scenario 3, and half of that reduction for other contacts; vi) half of the contact reduction of scenario 3, but 95% contact reduction between seniors and other age groups; and vii) reintroduction of the full lockdown measures as between 20 March–26 April 2020 as soon as the daily new hospitalizations reach 40, until the daily new hospitalizations have remained below 10 for two weeks. We assumed in all scenarios that strong social distancing (restricting all larger gatherings of adults) would continue until 7 June, and “light” social distancing (awareness, hand hygiene) for the rest of the year. Relative reductions in contacts were calculated from this baseline assumption. Contacts are defined as all contact scenarios in which transmission is possible from a single infectious person, regardless of type or duration. We represented future interventions (including contact tracing, intensified screening, and wearing masks) by an overall reduction in either all contacts or contacts between specific age groups. We run the model deterministically until 11 May 2020 and stochastically thereafter, and present the means of 1000 simulations with 95% credible interval (CrI).\n\nWe also conducted several sensitivity analyses to explore how the results depend on some key input assumptions. First, we shortened the latency period so that the serial interval decreased from 7.54 to 4.810. Second, we prolonged the infectiousness by adding a compartment of post-symptomatic infection to all infectious individuals except those who were hospitalized11. Finally, we added a seasonal forcing, which is a potential but currently disputed factor12,13, multiplying the infectiousness by a factor ranging between 0.6 (mid-summer) and 1.0 (mid-winter).\n\n\nResults\n\nEasing of restrictions may lead to a rapid increase in infections from late June, if the population relaxes social distancing and no effective tracing or testing efforts are implemented (baseline scenario, i). In this scenario, 83% of the Swiss population would become infected by the end of the year (Figure 1). By restricting total contacts by 90% (eradication scenario, ii), we estimate a full eradication of the epidemic, resulting in no infectious individuals in Switzerland by 25 July. However, low overall immunity (<5%) leaves the population vulnerable to new outbreaks. A 56% reduction in all contacts would retain the occupancy of ICU beds below the current maximum capacity throughout the year (scenario iii). In this scenario, the effective reproductive number, Re, would stay around 1.4, decreasing to <1 only during summer holidays. In this scenario, a new wave would start in October and only 5.2% (95% CrI 3.4-7.6%) of the population would have been infected by the end of 2020.\n\na) Reproductive number, b) daily COVID-19 related deaths, c) daily intensive care unit (ICU) bed occupancy, and d) cumulative infections in Switzerland from 11 February to 31 December 2020. “Strong reduction” refers to a 56% reduction of contacts (calibrated to prevent ICU overflow with 90% probability), “light reduction” is half of that, and “minimizing contacts” refers to 95% reduction of contacts.\n\nMaintaining a minimum of 56% contact reduction for all age groups is essential. Scenario iv, where this reduction was applied only for contacts among adults and seniors, without restricting contacts involving children, would result in two peaks, in late July and late September, and a substantial ICU overflow and mortality from mid-July until mid-October. In this scenario, 64.9% (95% CrI 63.6-66.2%) of the population would have been infected by the end of 2020. When we reduced contacts involving adults by 56%, and contacts among children and seniors only by 28% (scenario v), we observed no peak in July but a larger peak in autumn, resulting in a similar disease burden (cumulative proportion of infected individuals 54.8%, 95% CrI 54.7-54.9%). A similar pattern in the epidemic, with the next strong peak in October, was also seen in scenario vi when we restricted all contacts by 28%, except those between seniors and other age groups by 95%. In this case the number of deaths was however four times lower and the cumulative proportion of infected higher (63.3%, 95% CrI 63.3-63.6%). All above-mentioned scenarios were sensitive to the parameterisation: for example, in the model calibration for the Canton of Geneva, restricting contacts among adults and seniors only resulted in a stronger peak during summer without the second peak, whereas the bimodal pattern was in turn seen in the two other scenarios.\n\nIn the last modelled scenario (vii), reintroducing and lifting the restrictions based on daily hospitalizations would result in four new lockdowns: 16 June–1 August 2020, 31 August–14 October 2020, 25 October–13 December 2020, and 24 December 2020–13 February 2021. The number of patients in ICU remained below 200 throughout. Overall 5.6% (95% CrI 4.8-6.6%) of the total population would have been infected by 31 December.\n\nThe sensitivity analyses showed that the scenarios were sensitive to the duration of the latency period and infectiousness. Shorter serial interval made it easier to eradicate the epidemic or delay the second wave, whereas assuming post-symptomatic infectiousness meant that stronger contact reductions were needed to control the epidemic. Seasonal forcing also slowed down the epidemic: assuming a strong seasonality factor could delay the next wave to at least September.\n\n\nDiscussion\n\nOur study demonstrates that as long as the virus is present in a community with limited immunity, there is a risk of a rapid rebound of the epidemic if the restrictions are lifted and the people stop following social distancing and other protective behaviour. In the absence of seasonal forcing, the next wave could in theory occur in the summer. Efforts to control the epidemic, such as intensive testing, contact tracing, wearing of masks and hygiene measures, must have sufficient coverage to limit transmission among all age groups. No single intervention is enough. Reducing transmission uniformly may keep the epidemic suppressed only until late autumn. Restricting contacts between seniors and the rest of the population will prevent deaths, but is not sufficient to control the epidemic. A “start-stop” strategy, where the trends in COVID-19 related hospitalizations (or confirmed cases) trigger a new lockdown, is effective but not feasible: this would push the lockdown into the future, with only a few weeks of “normality” in between.\n\nThis study provides a broad range of future scenarios. In reality, the most severe scenarios are very unlikely: we can expect that the public health authorities will react and the people will adapt their behaviour if there is a new increase in cases. Our study has also several limitations. We focused on the average restriction of transmission-supporting contacts, without considering the practical implementation of specific interventions. The baseline contact patterns were based on literature data. They may however not fully reflect transmission patterns as the knowledge on transmission routes is still limited. Some contacts may be easier to track and control than others. Using a compartmental model, we could not differentiate between household and community transmission, or consider the effect of superspreaders.\n\nAn effective response to the COVID-19 pandemic needs several components: the spread of the infection must be kept as low as possible, the vulnerable population groups need additional protection, effective monitoring strategies must be in place, and the society must be ready to reintroduce additional measures if necessary. Only a combined prevention approach that targets all population groups can assure a sufficient control of the epidemic until a vaccine becomes available.\n\n\nData availability\n\nWe used the following data to parameterise our model:\n\nDisease progression parameters (except mortality, infectiousness and relative contact frequency): https://www.medrxiv.org/content/10.1101/2020.04.13.20063933v1 Appendix S1.\n\nRelative contact frequency: www.socialcontactdata.org (online tool https://lwillem.shinyapps.io/socrates_rshiny/, with data from studies “Belgium 2010”, “France 2015”, “Germany 2008” and “Italy 2008”).\n\nCalibration for Switzerland and the Cantons of Bern and Ticino: www.corona-data.ch.\n\nCalibration for the Canton of Geneva: https://www.ge.ch/document/covid-19-situation-epidemiologique-geneve.\n\nModel code available at: https://gitlab.com/igh-idmm-public/covid-19/modelling_jestill/-/tree/master/.\n\nArchived code at time of publication: http://www.doi.org/10.5281/zenodo.3888230\n\nLicense: Creative Commons Attribution 4.0 International.", "appendix": "Acknowledgments\n\nWe thank Rachel Esra, University of Geneva, for editing the paper.\n\n\nReferences\n\nWorldometer: COVID-19 Coronavirus Pandemic. Reference Source\n\nCorona-data.ch: COVID-19 Information for Switzerland. Reference Source\n\nSwiss Confederation: The federal Council. Verordnung 2 über Massnahmen zur Bekämpfung des Coronavirus (COVID-19). Reference Source\n\nDi Domenico L, Pullano G, Sabbatini CE, et al.: Expected impact of lockdown in Île-de-France and possible exit strategies. Reference Source\n\nMossong J, Hens N, Jit M, et al.: Social Contacts and Mixing Patterns Relevant to the Spread of Infectious Diseases. PLoS Med. 2008; 5(3): e74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBéraud G, Kazmercziak S, Beutels P, et al.: The French Connection: The First Large Population-Based Contact Survey in France Relevant for the Spread of Infectious Diseases. PLoS One. 2015; 10(7): e0133203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCOVID-19: Situation épidémiologique à Genève. Reference Source\n\nEstill J, Venkova-Marchevska P, Roelens M, et al.: COVID-19 stochastic age-structured transmission model for Switzerland. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3888230\n\nIcumonitoring: Near-real time monitoring of intensive care occupancy (IES system). Reference Source\n\nNishiura H, Linton NM, Akhmetzhanov AR: Serial Interval of Novel Coronavirus (COVID-19) Infections. Int J Infect Dis. 2020; 93: 284–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang D, Mo G, Yuan X, et al.: Time Kinetics of Viral Clearance and Resolution of Symptoms in Novel Coronavirus Infection. Am J Respir Crit Care Med. 2020; 201(9): 1150–1152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeher RA, Dyrdak R, Druelle V, et al.: Potential Impact of Seasonal Forcing on a SARS-CoV-2 Pandemic. Swiss Med Wkly. 2020; 150: w20224. PubMed Abstract \| Publisher Full Text\n\nJüni P, Rothenbühler M, Bobos P, et al.: Impact of Climate and Public Health Interventions on the COVID-19 Pandemic: A Prospective Cohort Study. CMAJ. 2020. (in press). PubMed Abstract \| Publisher Full Text" }	[ { "id": "68276", "date": "10 Aug 2020", "name": "Andrei R. Akhmetzhanov", "expertise": [ "Reviewer Expertise Infectious disease modeling" ], "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 of Estill et al. reports on possible future scenarios for SARS-CoV-2 epidemic in Switzerland. The results look plausible and may have important implications for a policy-making process. However, I think the study requires a more detailed sensitivity analysis and more flexibility in the proposed scenarios. In my opinion, the adopted values of the parameters (90% of reduction in contacts, 56% reduction, etc.) could be more variable, and additional analysis could help to determine their most efficient values. I also think that the Discussion section could propose a better overview of the problem and give comparison with other studies. Some other remarks of mine are below.\nFigure 1b vs 1c: I would have thought that the deaths counts should be lagged by approx. two weeks compared to ICU bed occupation. However, the black curve in 1b peaks visually at the same time as in 1c. Could the authors check this more carefully and confirm that there is some lag?\n\nFigure 1d vs 1c: What confuses me more is that there is no visually distinct time lag between reported cases and deaths.\n\nFigure 1: what are the Credible intervals for each trajectory?\n\nCompartmental model: it would be nice to have at least a brief description of the adopted model. For example, whether it includes asymptomatic carriers, hospitalized/quarantined people. It would be also good to know if the transition times follow exponential distribution or are described by gamma distribution. The authors could elaborate more than simply saying that all parameters are directly adopted from Ref.4.\n\nI believe that the considered scenarios may represent some minimalistic set, but it would be also nice to have a more detailed sensitivity analysis. For example, the reduction of contacts in Japan was set to 80% during the first wave, and it was proven to be sufficient. The authors of the present study consider the reduction at 90%, which may be more difficult to implement. Hence, I may have a question: how much should we actually reduce the contact rate?\n\nI may have a similar question regarding the seasonal forcing and choice of 7 June for lighting up the social restrictions. What would be a reasonable choice of such date? Is 7 June chosen optimally?\n\nI am a bit unsure if students (in 20s) and workforce subpopulation (in 30s-40s) can be attributed to the same age group. In my opinion, they have a quite distinct contact pattern.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6160", "date": "11 Mar 2021", "name": "Janne Estill", "role": "Author Response", "response": "Dear Dr Akhmetzhanov, Thank you for your constructive comments. Please see our point-to-point response below. Sincerely Janne Estill on behalf of all authors The paper of Estill et al. reports on possible future scenarios for SARS-CoV-2 epidemic in Switzerland. The results look plausible and may have important implications for a policy-making process. However, I think the study requires a more detailed sensitivity analysis and more flexibility in the proposed scenarios. In my opinion, the adopted values of the parameters (90% of reduction in contacts, 56% reduction, etc.) could be more variable, and additional analysis could help to determine their most efficient values. I also think that the Discussion section could propose a better overview of the problem and give comparison with other studies. Some other remarks of mine are below. Authors’ response: Thank you. The contact reduction in scenario iii) was not predefined at 56%: the value was selected after a calibration process as the minimum that allowed to keep the ICU occupancy under the critical limit (1200 beds) throughout the year in at least 90% of the simulations. After rerunning the analysis, we have corrected the value to 54%, which produces more stable results fulfilling the condition. We agree that this was imprecisely expressed and have revised the Methods to clarify that the reduction in itself is a result: “The reduction levels for scenarios ii) and iii) were selected in a manual calibration process, increasing the reduction in increments of 1% until the target condition was met.” The 90% reduction was pre-defined in our original analysis. We have now replaced the fixed 90% threshold also with a target-based scenario, calibrated to reaching a complete eradication (daily cases suppressed persistently to zero) by the beginning of July in at least 90% of the simulations. As a result, we have replaced the 90% overall reduction with a 76% reduction, which was the minimum level to fulfil this condition. Our revised description of Scenario ii) in the Methods (first paragraph) reads now as follows: “ii) eradication scenario (minimum contact reduction to bring the daily new infections to zero by the end of July with 90% probability)” The contact reductions in the remaining analyses were derived from the value 56% (either the same value, or half i.e. 28%), with the exception of the 95% reduction among contacts of seniors in Scenario vi) which was pre-defined (aiming to represent almost-isolation measures). We have decided to keep the same approach, now using the value of 54% instead of 56% (and 27% instead of 54%). Figure 1b vs 1c: I would have thought that the deaths counts should be lagged by approx. two weeks compared to ICU bed occupation. However, the black curve in 1b peaks visually at the same time as in 1c. Could the authors check this more carefully and confirm that there is some lag? Authors’ response: Because of the narrow graph, it is difficult to compare the peak values across panels. For example, in the baseline scenario (black curves), ICU occupancy peaked on 1st August, deaths on 9th August. The difference is 8 days, which is less than two weeks; however, the peak in ICU occupancy does not correspond to the peak in new ICU entries. Figure 1d vs 1c: What confuses me more is that there is no visually distinct time lag between reported cases and deaths. Authors’ response: New infections in the baseline scenario (black curves) peaked on 23th July, which is 17 days before the peak in deaths (9th August). It is understandably difficult to see the peak from the cumulative curve in Fig. 1d. However, we find the cumulative infections more informative than daily infections, because the reported cases do not correspond to true infections (but instead, the cumulative cases can easily be compared with seroprevalence estimates once such become available). We have added a number of new supplementary graphs (including also the daily infections), which will help the interpretability. Figure 1: what are the Credible intervals for each trajectory? Authors’ response: Because of the large number of graphs in each panel, including the credible intervals would probably make the graph unreadable. We have therefore instead included a set of separate supplementary online graphs (see document “covid_model_supplementary_figures.pdf” in the Gitlab repository https://gitlab.com/igh-idmm-public/covid-19/modelling_jestill) with the 95% credible intervals and all 1000 simulations into the appendix. Compartmental model: it would be nice to have at least a brief description of the adopted model. For example, whether it includes asymptomatic carriers, hospitalized/quarantined people. It would be also good to know if the transition times follow exponential distribution or are described by gamma distribution. The authors could elaborate more than simply saying that all parameters are directly adopted from Ref.4. Authors’ response: Our aim was to keep this research paper in a form as a short report. All parameters and a complete description with a graphical representation of the model structure are available from the dedicated Gitlab repository. This is declared at the end of the first paragraph of the Methods. I believe that the considered scenarios may represent some minimalistic set, but it would be also nice to have a more detailed sensitivity analysis. For example, the reduction of contacts in Japan was set to 80% during the first wave, and it was proven to be sufficient. The authors of the present study consider the reduction at 90%, which may be more difficult to implement. Hence, I may have a question: how much should we actually reduce the contact rate? Authors’ response: Please see our response to the first question. The scenario iii) (before 56%, now 54% reduction), and after our revision also scenario ii) (before 90%, now 76% reduction) are set to fulfil given targets. To answer the question, in order to keep the epidemic under control at least 54% reduction throughout the year is needed. In order to completely eradicate the epidemic during the summer, at least 76% reduction (together with a strict control to stop imported infections) would have been needed. We have reformulated the statement in the first paragraph of the Discussion to answer these questions more directly: “Reducing contacts between all people uniformly by at least 54% is needed to control the epidemic until the end of the year, but even in this case the restrictions would need to be tightened during the winter to avoid a rebound in January.” In addition, we have added a short paragraph to the Discussion to put these estimates into context with the true evolvement of the epidemic in the summer and autumn: “The second wave of COVID-19 hit Switzerland severely during October-November 2020. A comparison of the true course of the epidemic and the modelled scenarios shows that Switzerland achieved a relatively high reduction in contact frequencies during the summer and autumn 2020 with measures such as contact tracing, social distancing and mask obligation in public transport. These measures were however not sufficient to control the epidemic. Our findings are however promising in the sense that the overall contact reductions have likely been between 27 and 54% (scenarios iii to vi), giving hope that the new measures introduced by the Swiss Federal Council on 19 and 29 October 2020 (in combination with more stringent measures by many cantons) can, if sustained throughout the winter season, keep the epidemic under control.” I may have a similar question regarding the seasonal forcing and choice of 7 June for lighting up the social restrictions. What would be a reasonable choice of such date? Is 7 June chosen optimally? Authors’ response: The Swiss government announced beforehand 8 June as the date of lifting most restrictions, such as the reopening of many leisure and tourism related destinations and easing the limits of public gatherings and events. We expected therefore that the social contacts would increase at this time. The opening was later changed to be two days earlier, 6 June. We have added the following statement: “(declared beforehand by the authorities as the end date of most restrictions related to leisure activities)” I am a bit unsure if students (in 20s) and workforce subpopulation (in 30s-40s) can be attributed to the same age group. In my opinion, they have a quite distinct contact pattern. Authors’ response: We agree that this is a limitation, particularly in terms of leisure activities where the younger adults can be expected to have more contacts. However we do not believe that the work- and study-related contacts would differ substantially. More than half of the Swiss youth make a vocational education with apprenticeship after compulsory school and enter the workforce, whereas university students (who were affected by all universities switching to online teaching during the lockdown) are more likely to attend jobs where home office is feasible (also de facto mandatory in most cases during the first lockdown). Therefore we have a reason to believe that the differences in contact patterns within the “adult” group are less substantial than the contact patterns compared with children or seniors. We have added the following statement to highlight the limitations: “The division to three age groups cannot catch all heterogeneity in contact patterns between (for example, the more diverse social contacts of students and other young adults versus middle-aged with families; or the differences between healthy seniors slightly above the age of 65 versus more elderly nursing home residents with restricted mobility). However, we believe that the most essential contact differences are covered by the three age groups.”" } ] }, { "id": "74090", "date": "16 Nov 2020", "name": "Subhas Khajanchi", "expertise": [ "Reviewer Expertise Infectious diseases", "Ecological modeling", "Tumor-immune interactions." ], "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 COVID-19 pandemic has already spread throughout the world and the people are aware about the diseases and they are using precautions about the pandemic. But, still COVID-19 is spreading very quickly. Some countries like Spain, Switzerland, Australia, Serbia, China etc. started a second wave of COVID-19.\nTo stop the spread of the diseases a vaccine is needed. But, in absence of the vaccine people must maintain the social distancing. In order to maintain the social distancing people must obey the modeling rule. The introduction needs to be improved by incorporating some recent references on the COVID-19 pandemic. To do so, I suggest some modeling work must be included in the references: Sarkar et al. (20201), Khajanchi and Sarkar (20202) and Samui et al. (20203).\nIn this context an important factor must be include in this study, that is, the impact of the effect of media. How the COVID-19 dynamics have been changed due to incorporation of the media-related awareness like use of face masks, non-pharmaceutical interventions, hand sanitization, etc. The authors must include the manuscript Khajanchi et al. (20204) to study the effect of media.\nIs there any experimental data to validate the mathematical model? The authors at least describe the basic reproduction number R_0 and its impact on COVID-19 pandemic in India. The basic reproduction number R_0 is one of the most crucial quantities in infectious diseases, as R_0 measures how contagious a disease is. For R_0 < 1, the disease is expected to stop spreading, but for R_0 = 1 an infected individual can infect on an average 1 person, that is, the spread of the disease is stable. The disease can spread and become epidemic if R_0 is be greater than 1. In this context the authors can read the manuscript Wang et al. (20025).\nSome references contain errors and inconsistent formatting. It is difficult to give credit to research if even elementary aspects of the work are not error free. This should be corrected with care and love to detail.\nThe manuscript is comprehensive, and I have enjoyed learning about the presented results. I find that the manuscript is written with poor English and the presentation is not good, and I am in principal in favor of indexing, although the following comments should nevertheless be accommodated in one minor revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6161", "date": "11 Mar 2021", "name": "Janne Estill", "role": "Author Response", "response": "Dear Dr Khajanchi, Many thanks for your constructive comments. Please see below our point-to-point response. Sincerely Janne Estill on behalf of all authors The COVID-19 pandemic has already spread throughout the world and the people are aware about the diseases and they are using precautions about the pandemic. But, still COVID-19 is spreading very quickly. Some countries like Spain, Switzerland, Australia, Serbia, China etc. started a second wave of COVID-19. To stop the spread of the diseases a vaccine is needed. But, in absence of the vaccine people must maintain the social distancing. In order to maintain the social distancing people must obey the modeling rule. The introduction needs to be improved by incorporating some recent references on the COVID-19 pandemic. To do so, I suggest some modeling work must be included in the references: Sarkar et al. (20201), Khajanchi and Sarkar (20202) and Samui et al. (20203). Authors’ response: Thank you for pointing this out. We have added a sentence into the Introduction highlighting that until large-scale vaccination, social distancing and other measures need to be kept in place. Since all references suggested by the reviewer refer to India, which is not the focus of this study, we have taken the liberty to instead give references more related to the epidemic in Switzerland and Europe. “A vaccine is likely to be available in the course of the year 2021, but until then, strict social distancing and reduction of contacts together with measures such as testing and tracing are needed to control the epidemic and prevent the health system from a collapse.” In this context an important factor must be include in this study, that is, the impact of the effect of media. How the COVID-19 dynamics have been changed due to incorporation of the media-related awareness like use of face masks, non-pharmaceutical interventions, hand sanitization, etc. The authors must include the manuscript Khajanchi et al. (20204) to study the effect of media. Authors’ response: We agree that the media plays an important role in the network of determinants that influence transmission dynamics. As mentioned in the second paragraph of the Methods, we do not aim to model explicit interventions from the summer onwards, but only use implicit contact reduction levels between children, adults and seniors. We have added the following statement into the Methods (second paragraph): “The future reduction contains both government-ordered measures (such as the mandatory use of masks, new closures of services or locations), as well as indirect influence on behaviour through e.g. efficient communication by the health authorities as well as the mass and social media.“ Is there any experimental data to validate the mathematical model? The authors at least describe the basic reproduction number R_0 and its impact on COVID-19 pandemic in India. The basic reproduction number R_0 is one of the most crucial quantities in infectious diseases, as R_0 measures how contagious a disease is. For R_0 < 1, the disease is expected to stop spreading, but for R_0 = 1 an infected individual can infect on an average 1 person, that is, the spread of the disease is stable. The disease can spread and become epidemic if R_0 is be greater than 1. In this context the authors can read the manuscript Wang et al. (20025). Authors’ response: The model has been calibrated with the observed hospitalization and death data, as explained in the first paragraph of the Methods section. We focus in our paper on the effective reproduction number (Re; see Figure 1a for an estimate of Re over time in all scenarios) instead of the basic reproductive number (R0). These are by definition equivalent in a completely susceptible population, while the Re decreases as the proportion of the population becomes immune. We prefer to use Re instead of R0 since this is used also by e.g. the Swiss COVID-19 Task Force situation report (https://ncs-tf.ch/en/situation-report), allowing a comparison between different modelling studies for Switzerland. We trust that the concept of effective reproduction number is known to the readers and will not require further clarification. Some references contain errors and inconsistent formatting. It is difficult to give credit to research if even elementary aspects of the work are not error free. This should be corrected with care and love to detail. Authors’ response: We have carefully checked all references and found no errors in the links. We trust the editorial office will point out any remaining issues with the references. The manuscript is comprehensive, and I have enjoyed learning about the presented results. I find that the manuscript is written with poor English and the presentation is not good, and I am in principal in favor of indexing, although the following comments should nevertheless be accommodated in one minor revision. Authors’ response: The manuscript has been edited for grammar and style by a native English speaker. We are willing to improve the presentation in our manuscript by further editing, but it is difficult to address this comment without specific details." } ] } ]	1	https://f1000research.com/articles/9-646
https://f1000research.com/articles/10-199/v1	10 Mar 21	{ "type": "Case Report", "title": "Case Report: Pancreatic adenocarcinoma presenting as acute obstructive suppurative pancreatic ductitis", "authors": [ "Taha Sheikh", "Syed Hamza Bin Waqar", "Jordan Burlen", "Toseef Javaid", "Ali Nawras", "Taha Sheikh", "Jordan Burlen", "Toseef Javaid", "Ali Nawras" ], "abstract": "Acute obstructive suppurative pancreatic ductitis (AOSPD) is a rare form of infection primarily arising within the pancreatic duct in the setting of chronic pancreatitis. We present a case of AOSPD precipitated by obstructive adenocarcinoma of pancreatic head in an elderly woman with a past medical history of chronic pancreatitis, alcohol use disorder and, advanced dementia, who developed progressive abdominal pain during her hospital admission for urinary tract infection. Endoscopic retrograde cholangiopancreatography (ERCP) with balloon sweep of pus and stent placement resulted in prompt abdominal pain resolution. Our case highlights a rare presentation of AOSPD as a harbinger of pancreatic malignancy.", "keywords": [ "Pancreatic adenocarcinoma", "AOSPD", "acute suppurative pancreatic ductitis", "malignancy", "pancreas", "chronic pancreatitis", "obstruction", "sphincterotomy" ], "content": "Introduction\n\nAOSPD, first described in 1995, is a rare consequence of chronic pancreatitis characterized by an acute infection of the pancreatic ducts while sparing pancreatic parenchyma. The distinguishing feature is a lack of pseudocyst, abscess, or necrosis formation. Literature remains sparse in the domain of AOSPD, with a recent literature search only mentioning this entity in over 29 cases to date1–12. Pancreatic malignancy is also known to promote AOSPD by obstructing pancreaticobiliary secretion outflow ensuing in subsequent infection2,9. We describe a case of AOSPD precipitated by underlying pancreatic adenocarcinoma.\n\n\nCase presentation\n\nA 71-year-old female with a past medical history of chronic alcohol use, chronic pancreatitis and advanced dementia, presented to our tertiary care setup after a fall with confusion, a day after being discharged from an outpatient facility where she had undergone a colonoscopy. Vitals were remarkable for tachycardia (105 beats per minute), blood pressure 90/70 mmHg, temperature of 100.4F and respiratory rate of 18 per minute. On admission, her blood work-up was significant for an elevated white blood count of 12.4k/µL with urinalysis, positive for nitrites, elevated white blood cells, and bacteria. Blood and urine cultures were sent as part of workup for suspected sepsis, and the patient was started on ceftriaxone (2 grams) to cover for suspected sepsis secondary to urinary tract infection, which was considered as a cause of fall and altered mental status at presentation. Ceftriaxone was continued as urine cultures returned positive with the growth of Klebsiella pneumoniae and Escherichia coli with improvement in mentation 24 hours after admission.\n\nHowever, two days later, the patient developed new onset of progressively worsening, dull epigastric pain with no alleviating or aggravating factors. The pain did not correlate with breathing, position, or food intake. The patient reported that she had for had previous similar abdominal pain episodes six months prior. Magnetic resonance cholangiopancreatography (MRCP) was performed, which showed chronic pancreatitis and a 1 cm calculus in the main pancreatic duct; however, we pursued no intervention. She again had an episode two months prior with elevated white cell count, which responded to a short antibiotic course, but no infection source was found. The patient had no fever or hemodynamic instability. Her labs were significant for AST 156, ALT 182, ALP 949, GGT 2859, Bilirubin 1.1, lipase 68. It was noted that AST, ALT, and ALP were elevated at the previous encounter where she had the first episode of abdominal pain, six months before current admission. Blood cultures at the time of presentation had been negative to date.\n\nDue to epigastric pain, an abdominal ultrasound performed showed a 14mm stone in the pancreatic head with a dilated pancreatic, common hepatic and common bile duct (CBD), measuring 11mm, 9mm, and 11mm respectively (Figure 1 and Figure 2). On day 5, the patient’s hemodynamic parameters worsened with a surge in leukocytosis, and thus, transferal to intensive care with a change in antibiotic regimen to vancomycin and piperacillin-tazobactam was made. Abdominal computed tomography (CT) performed to look for a possible abscess revealed a 3cm obstructive mass in the pancreatic head, suspicious for malignancy with dilation of CBD and pancreatic duct (PD) measuring 16mm and 11mm respectively. CT also demonstrated extensive pancreatic desiccation consistent with the diagnosis of chronic calcific pancreatitis.\n\nEndoscopic retrograde cholangiopancreatography (ERCP) was performed for suspected acute cholangitis secondary to obstruction from pancreatic mass, showing high-grade distal CBD and PD stricture. Sphincterotomy was performed first for the pancreatic duct, then the distal CBD and the stricture in CBD and PD, dilated by using a 4 mm × 4 cm dilating balloon catheter. Upon 4 mm dilation of the PD, a significant amount of pus with debris and stone fragments were extruded. The dilating balloon catheter was removed and a 9–12 mm injecting below retrieval balloon catheter was used to sweep the PD, yielding more pus, debris, and stone fragments. Brushings were obtained from the PD stricture followed by pediatric forceps biopsy of the CBD stricture to rule out malignancy. Subsequently, a 10 Fr × 9 cm biliary stent was placed into the CBD and a 7 Fr × 7 cm stent in the PD with prompt drainage from both stents consistent with the diagnosis of AOSPD (Figure 3 and Figure 4).\n\nThe patient’s sepsis resolved after the pancreatobiliary drainage with prompt resolution of leukocytosis, transaminitis, and total bilirubin levels. Piperacillin-tazobactam was continued for ten days. Biopsy and brushings confirmed hepatobiliary primary invasive adenocarcinoma. Given the grave prognosis, palliative care was proposed. The patient opted for home hospice care.\n\n\nDiscussion\n\nAOSPD is a very rare entity and its mention in literature is sparse. AOSPD presentation overlaps with cholangitis and this mimicry makes misdiagnosis or non-recognition as a separate clinical entity difficult. AOSPD presentation varies from acute to chronic, the most common symptom being abdominal pain1–5,8–12. Less commonly, it may also present as sepsis or septic shock1–4. However, the presence of infection does not always correlate with clinical manifestation. This was evident from cases of asymptomatic presentation, where lab work uncovered the underlying infection incidentally6,7. Pancreatic imaging plays a pivotal role. The main pancreatic duct diameter was significantly higher in AOSPD with Chronic Pancreatitis (CP) than Acute-on-Chronic Pancreatitis patients10. AOSPD as the initial presentation of pancreatic malignancy is reported only once before, besides our case10.\n\nThe presence of pancreatic ductal stones precipitated due to pancreatic secretion stasis also serves as a nidus of infection generation leading to septicemia1–5. Besides enteric pathogens, respiratory pathogens have been increasingly reported in association with AOSPD, especially Klebsiella3,5. In our case, the pancreatic fluid and urinary cultures grew Klebsiella of the same species; however, no bacteremia was present.\n\nThe mainstay of AOSPD diagnosis is through ERCP detection of purulent discharge from the main pancreatic duct, seen in the absence of associated lesions like an abscess or pancreatic pseudocyst. MRCP presumptively diagnosed one case by demonstrating pancreatic duct dilation. However, the diagnosis was still confirmed after ERCP lead to symptom resolution2.\n\nTreatment is through source control via ERCP drainage and appropriate antibiotic administration. Notably, endoscopic naso-pancreatic drainage (ENPD) was additionally performed following ERCP in one reported case11. At this time, there are no established guidelines for the duration of therapy. Broad-spectrum antibiotic with enteric anaerobic coverage (vancomycin with piperacillin-tazobactam, ceftazidime with gentamicin) given for 7–14 days, is the mainstay of therapy1–6.\n\nAOSPD is a rare and potentially fatal diagnosis that requires expeditious recognition and treatment with referral to a facility with ERCP capabilities and expert infectious disease management. Future reviews and studies should work toward universal guidance in management.\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.", "appendix": "References\n\nWeinman DS: Acute suppuration of the pancreatic duct. Gastrointest Endosc. 1995; 41(3): 268–270. PubMed Abstract \| Publisher Full Text\n\nTajima Y, Kuroki T, Susumu S, et al.: Acute suppuration of the pancreatic duct associated with pancreatic ductal obstruction due to pancreas carcinoma. Pancreas. 2006; 33(2): 195–197. PubMed Abstract\n\nDeeb LS, Bajaj J, Bhargava S, et al.: Acute suppuration of the pancreatic duct in a patient with tropical pancreatitis. Case Rep Gastroenterol. 2008; 2(1): 27–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujimori N, Igarashi H, Ito T: Acute obstructive suppurative pancreatic ductitis. Clin Gastroenterol Hepatol. 2011; 9(8): A28. PubMed Abstract \| Publisher Full Text\n\nFujinaga T, Nishida T, Miyazaki M, et al.: Acute suppurative pancreatic ductitis associated with pancreatic duct obstruction. Endoscopy. 2013; 45 Suppl 2 UCTN: E135. PubMed Abstract \| Publisher Full Text\n\nAoki S, Okayama Y, Hayashi K, et al.: A case of purulent pancreatic ductitis successfully treated by endoscopic stenting. Dig Endosc. 2001; 12(4): 341–344. Publisher Full Text\n\nWali E, Koo P, Packer CD: Acute obstructive suppurative pancreatic ductitis in an asymptomatic patient. Case Rep Med. 2015; 2015: 919452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGedam BS, Sadriwala QS, Bansod PY: Chronic pancreatitis with acute obstructive suppurative pancreatic ductitis: a rare case report. J Surg Case Rep. 2017; 2017(2): rjx034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTollivoro T, Palakodeti S, Munroe C: Acute Obstructive Suppurative Pancreatic Ductitis. ACG Case Rep J. 2016; 3(4): e136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKondo H, Naitoh I, Okumura F, et al.: Clinical features of acute obstructive suppurative pancreatic ductitis: A retrospective review of 20 cases. J Gastroenterol Hepatol. 2016; 31(7): 1366–1373. PubMed Abstract \| Publisher Full Text\n\nIwatsuka K, Nakagawara H, Ogawa M, et al.: Spontaneous Development of Acute Obstructive Suppurative Pancreatic Ductitis Associated with Pancreatic Carcinoma: A First Case Report. Intern Med. 2018; 57(9): 1241–1245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShimizuguchi R, Kikuyama M, Kamisawa T, et al.: Acute obstructive suppurative pancreatic ductitis in pancreatic malignancies. Endosc Int Open. 2020; 8(12): E1765–E1768. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "89568", "date": "19 Jul 2021", "name": "Shehzeen Fatima Memon", "expertise": [ "Reviewer Expertise Internal Medicine and Neurology" ], "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 report is well-written and well presented with a firm approach towards the diagnosis from the beginning to the end. Being a rare case, authors have thoroughly covered the appropriate investigations, diagnostic measures and treatments that need to be carried out for pancreatic ductitis presenting in the head of pancreas carcinoma. There are, however, a few grammar mistakes that need to be addressed by the authors.\nThe case given is of an elderly woman with existing co-morbidities of chronic pancreatitis and alcohol use, presenting to the setup with a fall and the diagnosis of UTI was established. Later on, her new abdominal pain episode and a previous history of a similar episode led to a MRCP that showed a stone in main pancreatic duct. Her liver function tests were elevated after the second episode as well as after the first one six months back. Abdominal U/S now showed a stone at the pancreatic head with dilation of surrounding CBD and pancreatic duct. Leukocytosis helped establish the diagnosis of duct inflammation while ERCP revealed a stricture due to obstruction with the balloon sweep showed pus and debris further strengthening the grounds for the presumptive diagnosis. An appropriate therapy of Pipercillin-tazobactam was started as Klebsiella was found in the fluid meanwhile pus drainage was done via balloon catheter and a stent was placed to relieve obstruction. The case has been extensively covered for physicians to benefit from.\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": "89297", "date": "23 Jul 2021", "name": "Andreas Minh Luu", "expertise": [ "Reviewer Expertise Pancreatic surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear authors,\nThank you very much for the opportunity to review this nicely written manuscript. I would suggest minor revisions prior to acceptance of the manuscript.\nFirst of all, what was the primary underlying cause of the main pancreatic duct dilation? Was it the pancreatic stone, the tumor or even both?\nPlease add a CT-scan sequence showing the pancreatic head cancer.\nWas the stone removed completely?\nI am looking forward for your revision.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-199
https://f1000research.com/articles/10-198/v1	10 Mar 21	{ "type": "Research Article", "title": "Characteristics, utilisation and influence of viewpoint articles from the Structured Operational Research and Training Initiative (SORT IT) – 2009-2020", "authors": [ "Mohammed Khogali", "Katie Tayler-Smith", "Anthony D. Harries", "Rony Zachariah", "Ajay Kumar", "Hayk Davtyan", "Srinath Satyanarayana", "Olga Denisiuk", "Johan van Griensven", "Anthony Reid", "Saw Saw", "Selma Dar Berger", "Veerle Hermans", "Abraham Aseffa", "John C. Reeder", "Katie Tayler-Smith", "Anthony D. Harries", "Rony Zachariah", "Ajay Kumar", "Hayk Davtyan", "Srinath Satyanarayana", "Olga Denisiuk", "Johan van Griensven", "Anthony Reid", "Saw Saw", "Selma Dar Berger", "Veerle Hermans", "Abraham Aseffa", "John C. Reeder" ], "abstract": "Background: The Structured Operational Research and Training Initiative (SORT IT) teaches the practical skills of conducting and publishing operational research (OR) to influence health policy and/or practice. In addition to original research articles, viewpoint articles are also produced and published as secondary outputs of SORT IT courses. We assessed the characteristics, use and influence of viewpoint articles derived from all SORT IT courses. Methods: This was a cross-sectional study involving all published viewpoint articles derived from the SORT IT courses held from August 2009 - March 2020. Characteristics of these papers were sourced from the papers themselves and from SORT-IT members involved in writing the papers. Data on use were sourced from the metrics provided on the online publishing platforms and from Google Scholar. Influence on policy and practice was self-assessed by the authors of the papers and was performed only for papers deemed to be ‘calls for action’. Results: A total of 41 viewpoint papers were published. Of these, 15 (37%) were ‘calls for action’. In total, 31 (76%) were published in open-access journals and the remaining 10 in delayed access journals. In 12 (29%) of the papers, first authors were from low and middle-income countries (LMICs). Female authors (54%) were included in 22, but only four (10%) and two (5%) of first and last authors respectively, were female. Only seven (17%) papers had available data regarding online views and downloads. The median citation score for the papers was four (IQR 1-9). Of the 15 ‘call for action’ papers, six influenced OR capacity building, two influenced policy and practice, and three influenced both OR capacity building within SORT IT and policy and practice. Conclusion: Viewpoint articles generated during SORT IT courses appear to complement original OR studies and are valued contributors to the dissemination of OR practices in LMICs.", "keywords": [ "viewpoints", "utilization", "SORT IT", "policy and practice" ], "content": "Background\n\nIn 2009, the International Union Against Tuberculosis and Lung Disease (The Union) and Médecins Sans Frontières (MSF) began an operational research (OR) training course. In 2012, the Special Programme for Research and Training in Tropical Diseases (TDR), hosted at the World Health Organisation (WHO), adopted this model and formed a partnership-based programme known as the Structured Operational Research and Training Initiative (SORT IT). SORT IT seeks to strengthen health systems, improve programme performance, and enhance public health through OR. To this effect, SORT IT enrols participants from low and middle-income countries (LMIC) and teaches them the practical skills of conducting and publishing OR in peer reviewed scientific journals with the goal of influencing policy and/or practice1. Details of the structure and organisation of the SORT IT programme have been described elsewhere1.\n\nIn addition to capacity building, each SORT IT course brings together a rich diversity of people (faculty and participants) from different backgrounds, expertise, experience and countries. This richness often gives rise to vibrant discussions, new ideas and innovative reflections on health-related issues of global interest. Various aspects of OR capacity building to improve public health are also discussed and critically reflected upon. This dynamic - which characterises the modus-operandi of SORT IT – has resulted in the conception and publication of over 40 “viewpoint” articles (classified by journals as viewpoints, editorials, and perspective articles). These viewpoint papers are secondary outputs of SORT IT courses and present critical analysis of a health or policy issue of interest2 and/or challenge the norms of practice (“business as usual”). Many of these papers include a ‘call for action’ to improve public health or advocate for effective approaches to OR capacity building.\n\nSince the inception of SORT IT, we have closely monitored and evaluated the characteristics and outputs of the OR courses, together with the influence that original research papers have had on policy and practice3–9. However, we have yet to carry out any formal assessments of viewpoint articles – in particular examining who are the voices behind the papers, to what extent the papers have been used by others and whether/how they might have influenced policy and or practice.\n\nThis study aimed to assess the characteristics, utilisation and influence of published viewpoint articles derived from all SORT IT courses conducted between August 2009 and March 2020. Specific objectives were to report on i) the characteristics of publications, including LMIC contributions to authorship, collaborative partnerships (all High Income Countries - HIC vs HIC-LMIC); ii) access type and use of published evidence (views, downloads and citations); and iii) how ‘call for action’ papers influenced the implementation of OR capacity building within the SORT IT partnership, and policy and/or practice at national and international levels.\n\n\nMethods\n\nThis was a cross-sectional study involving all published viewpoint articles derived from SORT IT courses conducted between August 2009 and March 2020. Given that journals, publishers and academic communities do not always use standardised terminology for perspective and opinion pieces, we have decided to use the term ‘viewpoint article’ in this study. It includes any publication outside the categories of original research or reviews that discusses a relevant topic from the perspective of the author(s).\n\nBetween August 2009 and March 2020, there were 86 SORT IT courses conducted in Asia, Africa, Europe, South Pacific, and Latin America. A full description of the SORT IT courses, including criteria for selection of participants, programme structure and milestones, mentorship and facilitators has been described previously1. In brief, a regular SORT-IT course consists of three one-week modules implemented over a period of 10 to 12 months. During Modules 1 and 2, participants develop research protocols and electronic data capture and analysis tools. Module 3 focuses on writing a manuscript for scientific publication10. Specific milestones must be achieved if participants are to proceed from one module to the next. A participant is considered to have successfully completed the course if they complete all milestones, including submission of a final manuscript to a peer-reviewed journal. Up until March 2020, almost all modules were residential, during which participants and facilitators would gather every day for five-six days. Diverse discussions related to global health issues and OR capacity building took place during the courses and these served as the driving force behind the viewpoint papers that originated from these SORT IT courses.\n\nIn June 2020, viewpoint articles were obtained from a dedicated database (see underlying data13) that contains all SORT IT viewpoint article publications. Information on the characteristics of the viewpoint papers were sourced from the papers themselves and through consultation with authors. Variables included type of paper (Call for action – papers requesting a change in policy or practice; Descriptive – papers describing an OR or public health related issue), number of authors, nationality, gender and institutional affiliation of the authors, and whether they were involved with work in LMIC.\n\nData pertaining to journal access (open, delayed or subscription-based access) were obtained from each journal’s website; data on a paper’s views and downloads were retrieved from metrics provided by the online publishing platform indexing the article; citation counts were ascertained by examining and cross-checking the citations listed (for each given paper) on the online publishing platform and also in Google Scholar. Final citation counts for each paper were obtained by counting the citations common to both lists, together with any citations found only on the online publishing platform and/or only in Google Scholar.\n\nInfluence on policy and practice was only assessed for papers deemed to be ‘calls for action’. Each paper was examined by a committee of its senior authors in June 2020, allowing time for more recent call for action papers to potentially influence policy and practice (papers having been published between 2010 and 2019). A discussion took place about whether international / national guidelines were modified or changes in policy and/or practice were observed at international or national level as a result of the publication.\n\nData related to the study objectives were collected and analysed during June 2020.\n\nData were entered into Microsoft Excel 2016. Frequencies and proportions were used to summarise categorical variables, and medians and interquartile ranges were used to report continuous variables.\n\n\nResults\n\nAs a result of the SORT-IT courses held between August 2009 and March 2020, a total of 41 viewpoint papers were published; their characteristics are shown in Table 1. Just over a third of the papers constituted ‘calls for action’ (15, 37%). Fewer than a third of first authors were from an LMIC (12, 29%); however, the majority of first authors were involved in work in LMIC (40, 98%) and over two thirds of the papers included authors from LMICs (29, 71%). Only 54% of papers included female authors, and only 10% and 5% of first and last authors, respectively, were female.\n\nMost of the papers included NGO (35, 85%) and academic (38, 93%) author affiliations; 19 (46%) included a ministry of health affiliation and 16 (39%) included a WHO affiliation (some authors had more than one affiliation and these were included in the analysis). There was HIC-LMIC institutional collaboration in over two thirds of the papers (30, 73%).\n\nOf the 41 viewpoint papers, 31 (76%) were published in open access journals and the remaining 10 (24%) in delayed access journals. Online data for article views and downloads were only available for seven (17%) of the papers published in the following journals: BMC Research Notes (2), Frontiers of Public Health (1), Pan American Journal of Public Health (1), Global Health Action (1), F1000 Research (1) and BMJ Global Health (1). The paper with the highest number of views and PDF downloads (3322 and 1089) was a ‘call for action’ paper published in 2019 ( see underlying data13). An overview of citation counts for all papers is shown in Table 2. The median citation score for the call for action papers was four (IQR 2-11) and thus was no different to the median citation score for the descriptive papers (4, IQR 1-8) (data not shown in Table).\n\nInfluence on OR capacity building within the SORT IT partnership and on policy and practice was assessed for the 15 papers that constituted a ‘call for action’. Table 3 provides an overview of these papers and the reported influence. Of these, 11 (73%) resulted in some form of change: six papers influenced the evolution of OR capacity building within the SORT IT partnership, two influenced policy and practice at the national or international level, and three influenced both OR capacity building within SORT IT and policy and practice.\n\nBox 1 provides examples of how some of the ‘call for action’ papers shaped the philosophy and implementation of OR capacity building within the SORT IT partnership; Box 2 provides examples of how policy and practice at a national and international level was influenced.\n\nExample 1\n\nTitle of paper: The published research paper: is it an important indicator of successful OR at programme level?\n\nThe call for action: A call for the published paper to be used as a yardstick for monitoring and measuring the outputs of operational research (OR) in disease control programmes in low to middle income countries (LMICs).\n\nHow this call for action was taken up within the SORT-IT partnership\n\n• Helped the SORT IT partnership to develop the 80-80-80-80 targets for its SORT-IT courses wherein the third target was that 80% or more of submitted papers must be published within 18 months of submission.\n\n• Convinced donors that publications could be considered an independent indicator of OR capacity building programme.\n\nExample 2\n\nTitle of paper: Is OR delivering the goods? The journey to success in low-income countries\n\nThe call for action: This paper emphasised the intricate relation between OR and policy and practice, highlighted why OR findings might fail to affect policy and practice, and provided solutions and a checklist to ensure OR achieves its main goal of meeting health needs and improving health outcomes. This was a ‘call to action’ to say that if the criteria proposed for measuring research to policy and practice are adopted, this is an opportunity to better ensure that OR successfully impacts policy and practice, and to provide an objective way of monitoring the latter.\n\nHow this call for action was taken up within the SORT-IT partnership\n\n• Led Médecins Sans Frontières (MSF) to look beyond publications on ways of monitoring research impact. A software programme to monitor research impact for all MSF research was developed by MSF-UK.\n\n• Led the Union to look beyond publications and to routinely track and report the impact of research publications from SORT IT courses.\n\n• Helped SORT IT to include a target wherein at least 80% of research papers are tracked 18 months after submission to assess their on impact on policy and practice change.\n\nExample 3\n\nTitle of paper: Applying the International Committee of Medical Journal Editors (ICMJE) authorship criteria to operational research in low-income countries: the need to engage programme managers and policy makers\n\nThe call for action: This paper discussed the dissonance between applying the ICJME authorship criteria and the OR goal of translating research findings into policy and practice. The authors called for recognising the contributions of programme managers and policy makers to the OR process and suggested that this recognition be included in the application of the ICMJE guidelines for permitted authorship.\n\nHow this call for action was taken up within the SORT-IT partnership\n\n• The SORT IT partnership embraced the philosophy of early engagement with programme managers and decision makers and their inclusion in study authorship\n\n• In National SORT IT courses (Myanmar, Zimbabwe, Sierra Leone, Liberia), this was taken one step further and national programme staff were invited to participate and provide input during the SORT IT plenaries so that their views of the potential for policy and practice could be included.\n\n• The SORT IT partnership modified its reporting framework to donors and other stakeholders as follows: a) in its quarterly reporting framework, it reported on how many national programme officers and/or policy makers were listed as co-authors on papers (linked to a target of 65%); and b) in its annual reporting logical framework it reported on how many national government staff were included as co-authors on papers. This was assessed annually against a specified indicator.\n\n• The spin-off from the latter is that good relevant research has a good chance of influencing policy and practice – the achievement being 65 – 70%.\n\nExample 1\n\nTitle of paper: Language in tuberculosis services: can we change to patient-centred terminology and stop the paradigm of blaming patients?\n\nThe call for action: An appeal to the global tuberculosis (TB) community – particularly the global Stop TB Partnership - to lead discussions on changing the paradigm of detrimental language in TB services.\n\nHow this call for action was taken up outside the SORT-IT partnership\n\n• Led the STOP TB partnership to convene a meeting to revise the Monitoring and Evaluation framework and lexicon of language in TB.\n\n• Made a major contribution to the crafting of the WHO Guidelines on revised definitions of TB patients and treatment outcomes: “WHO: Definitions and Reporting Framework for Tuberculosis” – 2013 revision (updated December 2014).\n\n• The Union conference and Union journals (IJTLD and PHA) refer to the paper and state that non-judgemental language be used for abstracts, presentations and in all manuscripts (conference submissions and author guidelines).\n\nExample 2\n\nTitle of paper: Building the capacity of public health programmes to become data rich, information rich and action rich\n\nThe call for action: This paper presented the complementary adage of using both operational research and data-to-policy training in a decision-analysis framework, for evidence-informed decision making in public health. It called for Ministers of Health (MoH) to demand that operational research (OR) and decision analysis are used to make their Ministries ‘data-rich, information-rich and action-rich’. In parallel, advocacy and educational efforts were called for, to support MoH to understand the importance of this research evidence and ensure that it is collected and used effectively to improve their health services and health systems.\n\nHow this ‘call for action’ was taken up outside the SORT-IT partnership\n\n• A salient example of how this call for action was taken up is from the National TB programme in Zimbabwe (Takarinda KC, Harries AD, Mutasa-Apollo T, et al. Trend analysis of tuberculosis case notifications with scale-up of antiretroviral therapy and roll-out of isoniazid preventive therapy (IPT) in Zimbabwe, 2000–2018. BMJ Open 2020; 10: e034721).\n\n• The “data rich” was routine aggregate national data on TB case notification, numbers (and proportions) of people living with HIV (PLHIV) in antiretroviral treatment (ART) care and numbers (and proportions) of these ever commenced on IPT.\n\n• The “information rich” was the demonstration a strong association between national ART coverage in PLHIV rising from <1% to 88% between 2004 and 2018, national IPT coverage in PLHIV rising from <1% to 33% between 2012 and 2018 and TB case notification rates dropping 66% from 510 to 173 cases per 100,000 during this whole time period.\n\n• The “action rich” was the impetus to Zimbabwe of i) continuing to expand the HIV test and treat strategy to reach the UNAIDS 90% ART coverage target and the more recent 95% ART coverage target and ii) expanding TB preventive therapy but going for the 12-week course of weekly rifapentine and isoniazid rather than six months of daily isoniazid.\n\nExample 3\n\nTitle of paper: Neglected tropical diseases and the sustainable development goals (SDGs): an urgent call for action from the front line\n\nThe call for action: The international community has pledged through the SDGs to eliminate neglected tropical diseases (NTDs) by 2030. This paper was an urgent call for a change in mind-set to garner support and hasten progress towards achieving this fast-approaching target. The authors advocated for an empowering approach aimed at propelling political momentum, milestones and targets for accountability, new science in drug development and increased funding particularly from G20 countries.\n\nHow this call for action was taken up outside the SORT-IT partnership\n\n• WHO-NTD department has taken on board several of the recommendations which have been integrated in the NTD roadmap.\n\n• The paper and its call were highlighted on the \"World Neglected Tropical Diseases Day\" https://www.itg.be/E/Article/30-january--the-first-ever-world-ntd-day\n\n\nDiscussion\n\nThis is the first time that viewpoint papers derived from SORT IT courses have been formally assessed. Just over a third were ‘calls for action’; the rest were descriptive in nature. LMIC authorship and HIC-LMIC collaboration on these papers was high, but LMIC-first authorship and female representation was low. Three fifths of the ‘call for action’ papers contributed to the evolution of OR capacity building within the SORT-IT partnership.\n\nThe study strengths were that i) all discussion articles produced by the SORT IT partnership over the last 10 years were included and ii) we used universally acceptable parameters to assess access to, and utilisation of the viewpoint articles. The main limitations of this study were that i) online data on views and downloads of articles were only available for 17% of the papers, ii) citation counts might have been underreported without access to subscription-based databases like Web of Science and Scopus. That said, Google Scholar has been shown to be one of the most comprehensive sources for citations across different subject areas11, so we believe that the effect of this limitation is minimal; and iii) the influence on policy and practice was only assessed through self-assessment by the authors of the paper; there was no external objective validation of the results and as such the methodology is open to bias. This is an important limitation of this aspect of the perspective paper.\n\nThis study highlights several interesting findings. First, although more than two thirds of viewpoint papers had LMIC authorship, the proportion of lead and last authors from LMICs was relatively low. Since these papers deal with issues specific to LMICs, having first and last authors from LMICs with knowledge and experience of these issues should be encouraged. Although course participants (predominantly from LMICs) may have an abundance of knowledge and experience to share in discussions and debates, most are not trained in how to craft these ideas and reflections into a paper. Furthermore, these viewpoint papers are not a primary output expected of the SORT IT courses; they are a ‘secondary output’. Thus, the writing process is often led by experienced faculty members (who often enrich or generate ideas from discussions with course participants and formulate these into thoughts), many of whom are from HICs. Fortunately, these faculty members have extensive experience of working in LMICs and are aware of their contexts. However, there is clearly room to do better, to find ways to encourage more LMIC-lead authors – participants and facilitators. Going forward, efforts should be made to increase the number of LMIC lead-authors on viewpoint papers that are generated from SORT IT courses. Many of the individuals who have been trained in SORT IT courses go on to perform OR in the future4,5,8, and those who have co-authored a viewpoint paper, could be encouraged to lead on a viewpoint paper back in their home country as they become more experienced in scientific writing.\n\nSecond, female representation on the papers was low. This may be because there were fewer female mentors and mentees as compared to males on the SORT-IT courses3,6, particularly experienced faculty members. This is corroborated by findings from a previous evaluation of SORT IT courses showing that, of those participants enrolled on SORT IT courses between 2009 and 2014 who went on to become facilitators on subsequent courses, only 37% were female6. As the authors of this evaluation pointed out, socio-cultural factors may also explain this observation - anecdotal evidence suggests that female facilitators experience difficulties in leaving home and travelling to distant course locations. One way to boost female involvement in paper writing is through the SORT IT alumni network; the latter provides an opportunity to actively encourage female SORT IT alumni to become OR fellows and to become drivers of OR.\n\nThird, most of the journals in which viewpoints were published were open access which we recognise as being particularly important if we want to optimise readership in LMICs and ultimately influence policy and practice. Zachariah et al.6, in their evaluation of 20 SORT IT courses conducted between 2009 and 2014, showed that article views/downloads for immediate open access articles were double those of closed access journals. We were unable to assess this correlation, as views and downloads of the viewpoint papers were only available for just under a fifth of the papers. Journals could clearly do a better job of presenting altmetric data of views, downloads, and citations of papers available on their websites. Such metrics are important and should be shared publicly as standard practice on journal websites. In our study, citation counts were used as proxy for utilisation. Establishing the relative value of a single citation count, however, is difficult in the absence of some sort of reference measure. A software tool that many online publishing platforms currently utilise (and which was available for two thirds of our papers) is Dimensions - a linked research data platform12. Dimensions provides various metrics, one of which allows the citation performance of an article relative to similarly aged articles in its subject area to be compared. We cannot vouch for the validity of such a tool (this would need to be further explored), but metrics like this appear to be complimentary to citation counts and may provide a more nuanced overview of the utilisation of papers.\n\nFourth, about three quarters of our viewpoints had an influence within or outside SORT IT and this compares favourably with the 55–66% change in policy and practice reported from previous SORT IT evaluations7,9. However, if we focus on changes in policy and practice outside the SORT IT partnership, this occurred in only a third of our ‘call for action’ papers - in other words, they had less influence in the wider realm than other (original research) SORT IT publications. This is probably because the papers in question are opinion pieces. One opinion paper may not be enough (policy and practice is influenced by all sorts of different inputs) and may not wield the same weight as an original research study when it comes to changing national or global thinking.\n\nFinally, reflecting on the types of viewpoints that are being generated during SORT IT courses (one third being calls for action; two thirds being descriptive) should make us consider the added value of these types of papers. Our ‘call for action’ papers are meant to catalyse change and given the rich diversity and experience of people attending SORT IT courses this initiative should be championed. In addition to monitoring the OR outputs of our SORT IT courses, assessing viewpoint papers appears to be useful and encourages us to systematically monitor whether people we train in SORT IT go on to write viewpoint and perspective papers as they reflect a higher level of scientific thinking and critical reflection. Viewpoints contribute to challenging “more of the same” in health care and to “do things differently”.\n\nIn conclusion, viewpoint articles generated during SORT IT courses appear to complement original OR studies and are valuable contributors to the dissemination of OR practices in LMICs.\n\n\nData availability\n\nDRYAD: Characteristics, utilization and influence of viewpoint articles from the Structured Operational Research and Training Initiative (SORT IT) – 2009–2020. https://doi.org/10.5061/dryad.fj6q573sk13\n\nThis study contains the following underlying data:\n\nSORT IT_publications_viewpoint_articles_2010-2020.xlsx (SORT IT viewpoint article publications)\n\nViewpoint_data.xlsx (article-based metrics of SORT IT viewpoint articles)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nRamsay A, Harries AD, Zachariah R, et al.: The Structured Operational Research and Training Initiative for public health programmes. Public Health Action. 2014; 4(2): 79–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPugh EN Jr: Introducing the viewpoint. J Gen Physiol. 2012; 140(6): 579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZachariah R, Guillerm N, Berger S, et al.: Research to policy and practice change: is capacity building in operational research delivering the goods? Trop Med Int Health. 2014; 19(9): 1068–1075. PubMed Abstract \| Publisher Full Text\n\nGuillerm N, Tayler-Smith K, Berger SD, et al.: What happens after participants complete a Union-MSF structured operational research training course? Public Health Action. 2014; 4(2): 89–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuillerm N, Tayler-Smith K, Dar Berger S, et al.: Research output after participants complete a Structured Operational Research and Training (SORT IT) course. Public Health Action. 2015; 5(4): 266–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZachariah R, Rust S, Berger SD, et al.: Building Global Capacity for Conducting Operational Research Using the SORT IT Model: Where and Who? PLoS One. 2016; 11(8): e0160837. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar AM, Shewade HD, Tripathy JP, et al.: Does research through Structured Operational Research and Training (SORT IT) courses impact policy and practice? Public Health Action. 2016; 6(1): 44–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuillerm N, Dar Berger S, Bissell K, et al.: Sustained research capacity after completing a Structured Operational Research and Training (SORT IT) course. Public Health Action. 2016; 6(3): 207–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTripathy JP, Kumar AM, Guillerm N, et al.: Does the Structured Operational Research and Training Initiative (SORT IT) continue to influence health policy and/or practice? Glob Health Action. 2018; 11(1): 1500762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZachariah R, Ford N, Maher D, et al.: Is operational research delivering the goods? The journey to success in low-income countries. Lancet Infect Dis. 2012; 12(5): 415–21. PubMed Abstract \| Publisher Full Text\n\nMartín-Martín A, Thelwall M, Orduña-Malea E, et al.: Google Scholar, Microsoft Academic, Scopus, Dimensions, Web of Science, and OpenCitations' COCI: a multidisciplinary comparison of coverage via citations. ArXiv. 2020; abs/2004.14329. Publisher Full Text\n\nHook DW, Porter SJ, Herzog C: Dimensions: Building Context for Search and Evaluation. Frontiers in Research. Front Res Metr Anal. 2018; 3: 23. Publisher Full Text\n\nTayler-Smith K: Characteristics, utilization and influence of viewpoint articles from the Structured Operational Research and Training Initiative (SORT IT) – 2009-2020. Dryad, Dataset. 2020. http://www.doi.org/10.5061/dryad.fj6q573sk" }	[ { "id": "81281", "date": "06 Apr 2021", "name": "Desalegn Woldeyohannes", "expertise": [ "Reviewer Expertise Life Science", "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 paper is well written, concise, and clear with the study strengths and limitations clearly outlined. It is also interesting to read how some of the \"view point\"/\"call for action’\" papers shaped the operational research capacity building initiative as well as influence health policy and practice as presented in Box 1 and 2, respectively.\nFew comment/clarifications:\nBackground section: Inconsistent use of the conjunctions such as, \"and or\" (page 3, paragraph three last line) vs. \"and/or\" on the same page, paragraph 4 second from last line.\n\nMethodology section: Inconsistency of using \"international/national\" vs. \"international or national\", page 4, first up to third lines; and again unnecessary space tap appeared between the two words.\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": "87937", "date": "21 Jun 2021", "name": "Prajjwal Pyakurel", "expertise": [ "Reviewer Expertise Non-Communicable 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\nTitle:\nIn the title, utilisation and the influences may carry the same meaning in minutely analyzed. So I think we can avoid overcrowding of words,\n\nBackground:\n\nIn line number 9, what could be the indicators for improving program performance (in terms of ...??)?\n\nIn the subheading, SORT IT course and application we should also mention the follow up period after submission in peer reviewed journal.\n\nMonitored and evaluated the characteristics and outputs of the OR courses. Can we add few examples?\n\nResults:\nInfluence on OR capacity building and on policy and/or practice.\n\nI am not clear about the use of the terminology in some form of change. I think we should be specific.\n\nDiscussion:\nAlthough course participants (predominantly from LMICs) may have an abundance of knowledge and experience to share in discussions and debates, most are not trained in how to craft these ideas and reflections into a paper.\n\nComments: I also believe that besides training English language skills is crucial in writing the paper and non-native English speaker finds it difficult in writing the paper compared to native English Speaker and these things also should be highlighted.\n\nI believe a virtual platform could be another way besides SORT IT alumni network to involve female participants which can be added in the discussion.\n\nConclusion:\nI think we should also add policy and practice implications required further in depth assessment with robust methodology in the conclusion part.\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/10-198
https://f1000research.com/articles/10-5/v1	06 Jan 21	{ "type": "Research Article", "title": "Examining implicit beliefs in a replication attempt of a time-reversed priming task", "authors": [ "Marilyn Schlitz", "Arnaud Delorme", "Arnaud Delorme" ], "abstract": "Background: Psi research is a controversial area of science that examines telepathy, clairvoyance, precognition, and psychokinesis (mind over matter). Central to the debate over the existence of psi is of whether independent investigators can replicate reportedly successful psi experiments. One important variable involves the beliefs of experimenters and participants. A preregistered experiment is presented that sought to replicate and extend previously published parapsychology experiments suggestive of precognition by examining implicit beliefs. Methods: On each trial of the standard (non-psi) priming task, a pleasant or unpleasant word (the \"prime\") is briefly shown on computer screen, followed immediately by a pleasant or unpleasant picture. Trials on which the image and the priming word have different valences are termed “Incongruent”; trials on which the picture and the priming word share a common valence are termed “Congruent”. Participants in such experiments typically respond more slowly on Incongruent trials than on Congruent trials. In this \"time-reversed\" psi version of the experiment, the presumed cause-effect sequence is reversed so that the prime is not flashed until after the participant has already recorded his or her judgment. The experimental hypothesis remains the same: response times will be longer on trials with Incongruent prime/picture pairs than on trials with Congruent prime/picture pairs. Additionally, the study assesses expectations of success on the psi task of 32 experimenters—each testing 12 participants—using self-report questionnaires and the Implicit Association Task (IAT). Results: A significant correlation was found between the Implicit Association Test (IAT) effect and the participants’ reported beliefs in psi, with the effect in the direction opposite to the hypothesized correlation. Conclusions: This study offers an innovative approach to the role of beliefs in psi in a precognition study and speaks to the challenges of replication in controversial science.", "keywords": [ "priming", "Implicit Association Test" ], "content": "Introduction\n\nPsi research involves the study of extended human capacities, including telepathy, clairvoyance, precognition, and psychokinesis (mind over matter). It is an area of controversial research that began in the late 1800’s and continues today. Although proponents can point to an extensive body of evidence that supports the existence of psi, most academic psychologists do not believe that psi effects are likely to exist (Wagner & Monnet, 1979). Central to the debate is the issue of replication. Can independent investigators replicate reportedly successful psi experiments? And if so, under what conditions?\n\nOne important line of research involves the beliefs and expectations about psi. Various studies have shown participants' beliefs toward psi is predictive of successful psi performance. In a set of classic studies, Schmeidler & McConnell (1958) examined what they referred to as a “sheep-goat effect.” In these studies, “sheep” (believers) scored higher on average on psi tests than the “goats” (disbelievers). Several meta-analyses of the literature support the initial findings, noting a significant effect over 49 studies that suggest a “belief-moderated communications anomaly” over more than a 70-year span (Lawrence, 1993; Storm et al., 2012).\n\nPalmer (1972) identified four dimensions that were measured in these studies. These include two impersonal criteria that assessed belief in psi and belief that psi could be demonstrated in the experiment. The other two dimensions include personal beliefs about whether participants believe they have psi abilities and how well the participants expected to demonstrate psi in the study. In these dimensions there is a distinction between beliefs about psi and expectancies about future performance.\n\nThe current study is the third in a series that focused on the correlation of belief and outcome on a psi task (Schlitz et al., 2021). In particular, the focus in the three studies was to replicate an experiment published by Daryl Bem (2011). Using a variety of protocols, Bem’s nine experiments tested for possible retroactive influences of well-established psychological effects (e.g. priming) by “time-reversing” the stimulus and response: on each trial, a participant’s response was recorded before the purportedly causal stimulus was presented. Bem reported statistically significant results in eight of the nine experiments, with a statistically significant mean effect size (d) of 0.22 (Stouffer’s z = 6.66, p = 1.34 × 10-11).\n\nOn each trial of the standard (non-psi) priming task, a pleasant or unpleasant word (the \"prime\") is briefly shown on computer screen, followed immediately by a pleasant or unpleasant picture drawn from the standard International Affective Picture System (IAPS) (Lang & Greenwald, 1993). Trials on which the image and the priming word have different valences (one pleasant and one unpleasant) are termed “Incongruent trials”; trials on which the picture and the priming word share a common valence (both pleasant or both unpleasant) are termed “Congruent trials”. Participants in such experiments typically respond more slowly on Incongruent trials than on Congruent trials.\n\nIn Bem’s \"time-reversed\" psi version of the experiment, the presumed cause-effect sequence is reversed so that the prime is not flashed until after the participant has already recorded his or her judgment of the picture's valence. The experimental hypothesis remains the same: response times will be longer on trials with Incongruent prime/picture pairs than on trials with Congruent prime/picture pairs. Both of Bem's time-reversed priming experiments were successful (Bem, 2011), and a follow-up meta-analysis of 15 such precognitive priming experiments confirmed the hypothesis with an effect size (d) of 0.11, p = .003 (Bem et al., 2015).\n\nIn the time-reversed experiment, two potential primes are pre-designated for each picture, one pleasant and one unpleasant. Immediately after the participant records his or her judgment of the picture as pleasant or unpleasant on a trial, the computer randomly selected one of the two words to serve as the priming word and flashes it briefly on the screen. This procedure thus provides a genuine sampling-with-replacement or an \"open deck\" procedure for determining whether a trial will be congruent or incongruent. Thus the probability of its being congruent or incongruent remains constant at .5 across all trials. As a result, there is no (non-psi) way for a participant to anticipate the kind of trial currently appearing on the screen.\n\nThe two experiments reported by Schlitz et al. (2021) involved three levels of participants: (1) professors and other investigators who recruited student experimenters and were invited to serve as participants themselves; (2) student experimenters who received standardized training in the experimental procedure; and (3) participants who engaged in the psi task. Investigators who conducted the experiment in a university setting obtained their own IRB approvals and were offered the option of co-authorship on the final report. All experiments were pre-registered with the Koestler Parapsychology Unit. All participants were selected based on their interest in the study, but not on their beliefs in psi. As planned, the first 32 experimenters who submitted complete data sets were used in the analysis. (The two other experimenters did not return all the necessary data sets.)\n\nThe study reported here was designed to address a limitation of the first two studies in that expectancies and beliefs were evaluated using self-report questionnaires. Some studies show that when it comes to delicate social and psychological questions, including race, spirituality and core beliefs, people’s introspections are often not aligned with their instinctive responses. The current study addressed this problem by hypothesizing that implicit association tests provide a \"window\" into unconscious beliefs. In a first of its kind protocol, a 12-minute test was added for each experimenter and participant to assess the role of their implicit beliefs and expectancies and to see how these factors affect psi performance. The goal was to identify the extent to which people’s introspection of their belief in psi might not reflect their true core belief. This was tested using an implicit task involving word association and reaction time to assess experimenters’ and participants’ implicit belief in psi.\n\nThe Implicit Association Test (IAT), developed by Greenwald et al. (1998), was adapted to this study by using quick behavioral responses (such as “psi is good”, “psi is bad”) to evaluate implicit belief. The IAT is an assessment that was developed to determine the strength of a person's subconscious association between mental concepts in memory. It is typically used to assess implicit stereotypes that may be held by study participants, such as unconsciously associating stereotypically ethnic names with words consistent with ethnic stereotypes. Participants were instructed to choose if this concept applied to them or not. Their reaction time was used to assess their unconscious belief.\n\n\nMethods\n\nThis study employed a fast-thinking protocol using retrocausal priming. 32 experimenters were recruited through online networks and forums. Once they expressed interest in participating, they were informed that they would need a computer and were responsible for recruiting 12 participants each. When they completed the data collection and returned the data to the researchers, they received a $100 Visa Gift Card and a book on psi research. When they enrolled in the study, they were sent a link to complete the Implicit Association Task (IAT) themselves. They were informed that the experiment takes about 15 minutes to complete. Once they recruited 16 people from their friends and family, they would send a link to the participants for the IAT and psi task. They were contacted several times during the data collection period to check in and confirm the number of participants that had completed the experiment with them. The data were collected during a period from August, 2018 through February, 2019.\n\nBoth experimenters and participants were assessed for their baseline belief in psi phenomena using 5 simple questions to assess belief in psi (see Extended data: Annex 1 in file Documentation_release (Schlitz & Delorme, 2020)). Experimenter and participant implicit belief in paranormal phenomena was assessed using an Implicit-association Test (IAT) 12-minute protocol. Each experimental session consisted of 40 trials. In each trial an image was randomly selected and displayed to the participant, followed by a randomly selected incongruent or congruent priming word.\n\nThis study was approved by the Institute of Noetic Sciences (Protocol DELA_2015). All participants and experimenters signed an informed consent before participating in the study. This form was approved by the IRB. The study analysis was preregistered with the Koestler Parapsychology Registry (KPU Registry 1051).\n\nThe IAT procedure involved a series of seven tasks (Nosek et al., 2007). In the first task, participants were asked to categorize words in two categories – pro psi words: “psychic,\" “paranormal” and “metaphysical “– versus skeptic words: “skeptic”, “materialist”,” nihilist”. Each word appeared in the middle of the screen, and the participants were asked to press the A button on the computer for the psi category and the L button for the skeptic category (with two different hands). On the second task, participants completed a similar sorting procedure with the two categories: good (A key) – words good, great, correct – versus bad (L key) – words bad, awful, wrong. On the third and fourth task (two tasks are created to give participants a break between tasks), individuals were asked to complete a combined task that included both the categories and attributes from the first two tasks. In our case, we linked categories of psi and good (A key) versus skeptic and bad (L key). The fifth task was the same as the first task with the button position for two categories psi (L key) and skeptic (A key) inverted. The sixth and seventh tasks were the same as the third and fourth task with the opposite association, skeptic and good (A key) versus psi and bad (L key). The number of trials in each task was 6, 6, 24, 24, 6, 24, and 24.\n\nOnly the data from tasks 3, 4, 6 and 7 were analyzed, as the other sessions are just for training and making sure experimenters and individuals make the correct associations. All experiments were conducted online. Experimenters sent links to perform the studies to their participants. If experimenters collected data on more than 20 participants, participants above 20 were ignore. Experimenters were compensated $100.\n\nAs in Bem (2011), four statistical tests were performed using two response time data transformation (1/RT and log(RT)) combined with two outlier cutoff criteria (exclude trials with response times >1500 ms and > 2500 ms). These will be referred to as iRT1500 (inverse RT with cutoff at 1500 ms), iRT2500 (inverse RT with cutoff at 2500 ms), lRT1500 (log RT with cutoff at 1500 ms), lRT2500 (log RT with cutoff at 2500 ms).\n\nHypothesis 1 states that the response time will be shorter for trials with congruent words than for trials with incongruent words. Hypotheses 2 – 4 examine the effect of participant IAT effect, experimenter IAT effect, and both participant and experimenter IAT effect, respectively. It was the goal to follow the statistical methodology of KPU registry ID number 1051 as closely as possible, and any deviations from this methodology were on account of model assumption violations that, if ignored, would have rendered the results invalid and untrustworthy. All analyses were conducted using the statistical programming language R v 3.6.3 (R Core Team, 2020). Data visualization was completed using the R package ggplot2 (Wickham, 2016).\n\nThe main data file included participant and experimenter unique ID numbers, the reaction times for 40 trials and the responses (congruent/incongruent, correct/incorrect, photo) in character string format for 40 trials (Underlying data). Since the photo name is not relevant for analysis, for the 40 response columns, the character strings were split on the colon and only the first item retained. This removed the photo information from the response columns, leaving only trial information (e.g. “Ipn1” or “XCpp0”). The data were converted from wide to long format using the R package dplyr (Wickham et al., 2020) and tidyr (Wickham & Henry, 2020) such that the resulting data frame consisted of 5 columns (Participant ID, Experimenter ID, Trial Number, Reaction Time, and Response) and 15360 rows (384 participants × 40 trials each). A new variable Type was defined based on whether the Response character string had a “C” for congruent or “I” for incongruent. Specifically, Type was a binary variable such that “C” denoted congruent trails and “I” denoted incongruent trials. A new variable Correct was defined based on whether the Response character string had a “0” or “X” vs. a “1”. Specifically, if the Response character string had either an “X” or a “0”, Correct was set to “0” (Incorrect). Otherwise, Correct was set to “1” (Correct). All string manipulations were performed using the R package stringr (Wickham, 2019).\n\nParticipants with judging errors on 25% or more trials (i.e. 10 or more trials) were removed. Subsequently, trials with errors in judging the image (Correct = 0) and with response times less than 250 ms (Reaction Time < 250) were excluded. Two new variables were defined, the inverse reaction time (1/RT) and the log reaction time (log(RT)). Then, two new data sets were formed, one such that only reaction times less than or equal to 1500 ms were retained and a second such that only reaction times less than or equal to 2500 ms were retained. For primary analyses, means of inverse reaction time and log reaction time were taken by participant and trial type (i.e. participant specific means were calculated for congruent and incongruent trials), yielding a data set of congruent and incongruent means for 247 participants for the 2500 ms cutoff and 355 participants for the 1500 ms cutoff.\n\nData sets of raw observations and mean reaction times were explored through summary statistics and graphically using boxplots. The mean, median, and standard deviation of mean reaction time, participant IAT Effect, and experimenter IAT effect by trial type were tabulated for data sets of mean reaction times. Boxplots for both data sets (> 2500 ms and >1500 ms cutoff) were generated for mean reaction times, mean inverse reaction times, and mean logged reaction times. Individual participant-specific means were color-coded by participant IAT effect and size denoted experimenter IAT effect. The mean, median, and standard deviation of all observations by trial type were tabulated, and boxplots were generated for the observed reaction times, inverse reaction times, and logged reaction times and color-coded by individual participant. Effect sizes were calculated using the R packages effsize (Torchiano, 2020), effectsize (Ben-Shachar et al., 2020), and WRS2 (Mair & Wilcox, 2020). For paired t-tests, Cohen’s d was calculated with 95% confidence intervals and supplemented by the explanatory measure of effect size from the Yuen’s test on trimmed means for dependent samples. For ANOVAs, Cohen’s f was calculated. For bootstrapped mixed effects models, standardized effect sizes were not possible to calculate due to heteroscedasticity, so unstandardized effect sizes in the form of regression coefficients are presented in the model output with bootstrap confidence intervals.\n\nPrimary analysis. The percentage of participants who had faster average reaction times for congruent trials compared to incongruent trials was calculated for data sets using both the 1500 ms and 2500 ms cutoffs and exact binomial tests were performed to test the alternative hypothesis that the true probability of obtaining a faster congruent trial time is greater than 0.5.\n\nPaired t-tests and bootstrapped paired t-tests were performed to examine differences between congruent and incongruent participant-specific mean (1) inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs, yielding four sets of tests. The difference of congruent and incongruent mean transformed reaction times were examined for adherence to the normality assumption using the Shapiro-Wilk and Kolmogorov-Smirnov tests as well as graphically with histograms and QQ-plots. Subsequently paired t-tests were performed. Bootstrapped paired t-tests were performed using B = 2000 bootstrap samples and the function boot.t.test(), which implements the test outlined in Ch. 16 of Efron & Tibshirani (1993), in the R package MKinfer (Kohl, 2019).\n\nSecondary analysis. (1) Inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to fit linear mixed effects models with type of trial as a factor variable and participant ID as a random effect. Subsequently, ANOVA tables could be generated. However, the models deviated greatly from model assumptions of residual normality and homogeneity of variance. Box-cox transformations were successful in bringing the residuals to normality but resulted in a diamond-shaped residuals vs. fitted plotted that are most commonly seen when the response variable is bounded or truncated. In this case, truncation at 250 ms and the cutoff generated this outcome, still a violation of homogeneity of variance. As a result, the original linear mixed effects model, fitted using the R package lme4 (Bates et al., 2015), was bootstrapped using B = 2000 in the R package lmeresampler (Loy & Steele, 2020). Given the deviations from model assumptions, cases were resampled at both the participant and observation levels. 95% normal and percentile confidence intervals were generated using the R package boot (Canty & Ripley, 2019).\n\nPrimary analysis. Participant-specific mean (1) inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to perform ANCOVAs with experimenter IAT effect as a covariate and type of trial as a factor variable. ANCOVA assumptions were examined through (1) QQ-plots, Shapiro-Wilk tests, and Kolmogorov-Smirnov tests of studentized residuals (normality), (2) the Levene test of variances by type of trial (variance homogeneity), (3) ANOVA with interaction (homogeneity of regression slopes), (4) scatterplots of the data by type of trial (linearity). The normality assumption was violated for all models and as a result, Box-Cox transformations were used to transform the dependent variable such that the normality assumption was not violated. Box-Cox transformations were generated using the R package car (Fox & Weisberg, 2019). Estimated marginal means were calculated using the emmeans package (Lenth, 2019).\n\nSecondary analysis. (1) Inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to fit linear mixed effects models with experimenter IAT effect as a covariate, type of trial as a factor variable, and participant ID as a random effect. Subsequently, ANOVA tables could be utilized to generate ANCOVA outputs. However, the models deviated greatly from model assumptions of residual normality and homogeneity of variance. Homogeneity of variance was somewhat alleviated by weighting observations by participant IAT effect, but normality was still violated. Box-cox transformations were successful in bringing the residuals to normality but resulted in a diamond-shaped residuals vs. fitted plotted that is most commonly seen when the response variable is bounded or truncated. In this case, truncation at 250 ms and the cutoff generated this outcome, still a violation of homogeneity of variance. As a result, generalized linear mixed models (GLMMs) (specifically using the Gamma family), generalized additive mixed models (GAMMs) (using both the Gaussian and Gamma families), and nonparametric linear mixed effects models were attempted without improvement (GLMMs and GAMMs did not resolve violations of assumptions and non-parametric models failed to produce a fit). As a result, the original linear mixed effects model, fitted using the R package lme4 (Bates et al., 2015), was bootstrapped using B = 2000 in the R package lmeresampler (Loy & Steele, 2020). Given the deviations from model assumptions, cases were resampled at both the participant and observation levels. 95% normal and percentile confidence intervals were generated using the R package boot (Canty & Ripley, 2019). Interactions were included in the model.\n\nPrimary analysis. Participant-specific mean (1) inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to perform ANCOVAs with participant IAT effect as a covariate and type of trial as a factor variable. ANCOVA assumptions were examined through (1) QQ-plots, Shapiro-Wilk tests, and Kolmogorov-Smirnov tests of studentized residuals (normality), (2) the Levene test of variances by type of trial (variance homogeneity), (3) ANOVA with interaction (homogeneity of regression slopes), (4) scatterplots of the data by type of trial (linearity). The normality assumption was violated for all models and as a result, Box-Cox transformations were used to transform the dependent variable such that the normality assumption was not violated. Box-Cox transformations were generated using the R package car (Fox & Weisberg, 2019). Estimated marginal means were calculated using the emmeans package (Lenth, 2019).\n\nSecondary analysis. (1) Inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to fit linear mixed effects models with participant IAT effect as a covariate, type of trial as a factor variable, and participant ID as a random effect. Subsequently, ANOVA tables could be utilized to generate ANCOVA outputs. For reasons stated in the previous section, the original linear mixed effects model, fitted using the R package lme4 (Bates et al., 2015), was bootstrapped using B = 2000 in the R package lmeresampler (Loy & Steele, 2020). Given the deviations from model assumptions, cases were resampled at both the participant and observation levels. 95% normal and percentile confidence intervals were generated using the R package boot (Canty & Ripley, 2019). Interactions were included in the model.\n\nPrimary analysis. Participant-specific mean (1) inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to perform ANCOVAs with participant IAT effect and experimenter IAT effect as covariates and type of trial as a factor variable. ANCOVA assumptions were examined through (1) QQ-plots, Shapiro-Wilk tests, and Kolmogorov-Smirnov tests of studentized residuals (normality), (2) the Levene test of variances by type of trial (variance homogeneity), (3) ANOVA with interaction (homogeneity of regression slopes), and (4) scatterplots of the data by type of trial (linearity). The normality assumption was violated for all models and as a result, Box-Cox transformations were used to transform the dependent variable such that the normality assumption was not violated. Box-Cox transformations were generated using the R package car (Fox & Weisberg, 2019). Estimated marginal means were calculated using the emmeans package (Lenth, 2019).\n\nSecondary analysis. (1) Inverse and (2) log reaction times for the (a) 1500 and (b) 2500 cutoffs were used to fit linear mixed effects models with participant IAT and experimenter IAT effect as a covariates, type of trial as a factor variable, and participant ID as a random effect. Subsequently, ANOVA tables could be utilized to generate ANCOVA outputs. For reasons stated in the previous sections, the original linear mixed effects model, fitted using the R package lme4 (Bates et al., 2015), was bootstrapped using B = 2000 in the R package lmeresampler (Loy & Steele, 2020). Given the deviations from model assumptions, cases were resampled at both the participant and observation levels. 95% normal and percentile confidence intervals were generated using the R package boot (Canty & Ripley, 2019). Interactions were included in the model.\n\n\nResults\n\nResults are presented in Table 1–Table 4.\n\n26 participants with judging errors on 25% or more trials were removed, resulting in 14320 observations from 40 trials on 358 participants. 828 additional observations were removed because the reaction time was less than 250 ms and/or the response was incorrect (judging error occurred), yielding 13492 observations on 358 participants. The data set excluding response times >1500 ms was composed of 4449 observations on 308 participants. The data set excluding response times >2500 ms was composed of 10757 observations on 357 participants.\n\nThe mean, median, and standard deviation of mean reaction time, participant IAT Effect, and experimenter IAT effect by trial type are provided in Table 1. Boxplots for both data sets (> 2500 ms and >1500 ms cutoff) showing mean reaction times, mean inverse reaction times, and mean logged reaction times are presented. The mean, median, and standard deviation of all observations by trial type are tabulated in Table 1, and boxplots for the observed reaction times, inverse reaction times, and logged reaction times and color-coded by individual participant are included. Summary statistics and boxplots are comparable between congruent and incongruent trials. There do not appear to be patterns that can be explained by participant IAT effect, experimenter IAT effect, or participant ID.\n\nUsing the 1500 cutoff, 107 out of 246 participants (43.3%) had lower mean reaction times for congruent trials, supporting the null hypothesis that the true probability of a lower mean reaction time for congruent trials is 0.5 (p = 0.9848; 95% CI = (0.38, 1.00)). Using the 2500 cutoff, 160 out of 355 participants (45.1%) had lower mean reaction times for congruent trials, supporting the null hypothesis that the true probability of a lower mean reaction time for congruent trials is 0.5 (p = 0.9721; 95% CI = (0.41, 1.0)). Differences between mean transformed reaction times for congruent and incongruent trials were not normally distributed. There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials using paired t-tests and bootstrapped paired t-tests (p > 0.05) or using two-sample t-test and bootstrapped two-sample t-tests (p > 0.05). There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials using bootstrapped linear mixed effects models. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for Type = Incongruent encompassed 0, indicating that the difference in trial types was not significant at level 0.05.\n\nExperimenter IAT effect was significant only using the > 2500 ms cutoff. Across cutoffs and transformations, the mean transformed difference between congruent and incongruent trials was not significantly different at level 0.05 (Table 2). There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials using bootstrapped linear mixed effects models. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for Type = Incongruent encompassed 0, indicating that the difference in trial types was not significant at level 0.05. There was also no evidence to support an experimenter IAT effect. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for experimenter IAT effect encompassed 0, indicating that the difference in trial types was not significant at level 0.05.\n\nAcross cutoffs and transformations, the mean transformed difference between congruent and incongruent trials was not significantly different at level 0.05 and there was no effect due to participant IAT. There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials using bootstrapped linear mixed effects models. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for Type = Incongruent encompassed 0, indicating that the difference in trial types was not significant at level 0.05. There was also no evidence to support a participant IAT effect. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for participant IAT effect encompassed 0, indicating that the difference in trial types was not significant at level 0.05.\n\nAcross cutoffs and transformations, the mean transformed difference between congruent and incongruent trials was not significantly different at level 0.05, but there was an effect due to the interaction of participant and experimenter IAT effect and the type of trial in inverse and log transformed data with 1500 ms cutoff (p < 0.05), and effects due to the interaction of participant and experimenter IAT effect in inverse and log transformed data with 2500 cutoff (p < 0.05). There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials using bootstrapped linear mixed effects models. Across cutoffs and transformations, both the normal and percentile 95% bootstrap confidence intervals for Type = Incongruent encompassed 0, indicating that the difference in trial types was not significant at level 0.05. There was evidence in the 2500 cutoff data of both experimenter and participant-experimenter interaction IAT effects at level 0.05 (Table 4)\n\n\nDiscussion\n\nWhile the raw data by trial type were positively skewed, the transformation of the truncated reaction times caused the data to be more skewed than if the raw data had been examined. Furthermore, it is important to note that normality and symmetry enter into assumptions differently depending on the tests performed or models fitted. For a paired t-test for example, the difference in pairs is assumed to be normally distributed, whereas in a linear model (and ANOVA, ANCOVA), the residuals are assumed to be normally distributed. In both cases, the assumption does not pertain to the raw data. It also does appear that truncation at 1500 and 2500 ms combined with transformation induced some skew, and it was this truncation that likely prevented parametric analysis for the secondary analysis of Hypotheses I-IV.\n\nThere is no functional difference between the reaction times in congruent and incongruent trials, though means do appear somewhat more differentiated. Boxplots for congruent and incongruent trials overlap entirely. It is worth noting the in Hypothesis I we work with one-tailed hypothesis tests and in Hypotheses II-IV we work with two-tailed hypothesis tests. As a result, care should be taken when interpreting significant effects based on Hypotheses II-IV. It is also worth noting that in general, response times were shorter for trials with incongruent words compared to congruent words, so looking at the other tail for hypothesis testing generally returned low p-values.\n\nA random effect for participant was added to models in the secondary analyses of Hypotheses I – IV. This addition renders the models mixed effects models. ANCOVA tables can still be generated and estimated marginal means examined. The random effect for participant models the similarity in the reaction times for trials performed by the same participant. When multiple observations are taken on subjects, variation between observations within a subject different from variation in observations between subjects. Random effects allow us to model this variation appropriately. Without an adjustment like a random effect, the assumption that all observations are independent is clearly false. In other words, we have committed pseudoreplication (Lazic, 2010; Magezi, 2015).\n\nLinear mixed effects models were fitted by bootstrapping cases rather than residuals because bootstrapping cases is less sensitive to model assumptions (Efron & Tibshirani, 1993), which were violated in all of these models. Cases were bootstrapped at both levels of the model and with 2000 bootstrap samples this provided a robust interpretation of model estimates. Use of the bootstrap means that we must look to the bootstrap confidence intervals rather than an ANCOVA table to form conclusions, but in order to adhere to transformation and cutoff requirements, a bootstrap approach was needed. Fitting GLMMs on the original, untransformed and untruncated data set would provide a complement to bootstrapped results and provide the satisfaction of ANCOVA table production. An interaction was fitted in all of the mixed effects models of hypotheses II – IV to be sure the ANCOVA homogeneity of regression slopes assumption was not violated.\n\nThe first hypothesis stated that response times would be shorter for trials with congruent words compared to trials with incongruent words. This hypothesis was not supported at level 0.05, and in fact response times were shorter for trials with incongruent words compared to trials with congruent words. The second hypothesis stated that response time effects will be greater for experimenters with positive implicit belief about the experimental outcome. This hypothesis was only supported at the 2500 cutoff for the primary analysis and only at the 2500 cutoff for the log transformed data for the secondary analysis. The third hypothesis, that response time effects will be greater for participants with positive expectations about the experimental outcome was not supported. The fourth hypothesis involved response time effects and interaction between experimenters and participants belief in psi. For the primary hypothesis, at the 1500 cutoff, there was a significant (p < 0.05) effect due to the interaction between participant IAT effect, experimenter IAT effect and type of trial. At the 2500 cutoff, there was a significant (p < 0.05) experimenter IAT effect and a significant participant IAT and experimenter IAT effect. The secondary hypothesis was not supported at the 1500 cutoff. At the 2500 cutoff, there were significant effects due to experimenter IAT and the interaction between participant and experimenter IAT effect.\n\n\nConclusions\n\nThis study illustrates the development of a progressive research program that has explored the role of belief in psi replication. None of the hypotheses were verified except for Hypothesis IV of an interaction between participant-experimenter interaction and IAT effects. This study made use of an innovative approach to testing a sheep/goat effect by introducing the IAT as a way of assessing unconscious biases. Contrary to the initial prediction, the central hypothesis shows a significant trend in the direction of slower response times in relation to coherent targets in the priming paradigm.\n\nThese three studies in the series aim to build upon previous research by exploring whether the observations about beliefs in psi may play a role in the replication of anomalous results under controlled conditions. A limitation of the first two experiments (Schlitz et al., 2021) is that expectancies and beliefs were evaluated using self-report questionnaires; this study sought to address this weakness. The implicit association test originally developed by Greenwald et al. (1998) has shown that overt responses of participants do not necessarily reflect their unconscious beliefs. Results of the current study support the utility of this approach.\n\nOverall, this research must be seen as a failure to replicate the original Bem findings. Of course, interpretation is left to the reader. One may consider two possible ways of accounting for the results in this study and the previous two in the series. It is possible that the interpretations and meta-cultural dimensions of the experimental exchanges were unexpected variables. It is also possible that a more subtle, unanticipated and uncontrolled factor may have disrupted the production of an overall effect on the main pre-registered hypothesis. For example, the study took place in diverse settings with no consistent environment, set, or setting across sub-experiments. The background and experiences of the experimenters were uncontrolled, with the exception of the interventions. Future studies might aim to select participants and experimenters that have shown talent at performing this task.\n\nIt may also be argued that previous results by Bem and others represented chance findings or undetected subtle artifacts. In this approach, it could be said that the results reported here accurately reflect the absence of a psi effect. The magnitude of the previous findings casts this interpretation in doubt. Having said this, the methodology employed in the current study was more ambitious in scope and sophisticated in terms of the use of preregistration than Bem’s original studies.\n\nStudies in both psychology and sociology show that people tend to interpret ambiguous evidence in alignment with their prior beliefs (see, e.g. Roe, 1999). As such, it is predicted (though not pre-registered) that proponents of psi will tend to favor the first interpretation of the data and skeptics the latter. However, the inconsistent nature of our findings does not allow for a firm acceptance or rejection of either interpretation, and the issue will only be resolved by further research. The controversy generated by research into the possible existence of psi abilities reflects the theoretical and practical importance of the questions raised by such potential abilities, and we believe this justifies the additional work needed to help resolve the type of inconsistent results reported here.\n\n\nData availability\n\nOpen Science Framework: Experimenter effect, https://doi.org/10.17605/OSF.IO/XQRC5 (Schlitz & Delorme, 2020) (registered on 28th October 2020, https://osf.io/6qxwy).\n\nThis project contains the following underlying data in folder ‘experiment 3’ (information about these data files is included in Documentation_release.pdf documentation available on OSF):\n\n- data.txt\n\n- data_additional.txt\n\n- expertimenter_additional_info.txt\n\n- expertimenter_info.txt\n\n- participants_additional_info.txt\n\n- participants_info.txt\n\nOpen Science Framework: Experimenter effect, https://doi.org/10.17605/OSF.IO/XQRC5 (Schlitz & Delorme, 2020) (registered on 28th October 2020, https://osf.io/6qxwy).\n\nThis project contains the following extended data:\n\n- Documentation_release.pdf: contains information pertaining to the protocol for the present study (experiment 3), data file descriptions, and Annex 1 (questions about belief in psi).\n\n- Guide to the Original RPriming Data Files.pdf: contains target pictures and primes for the present study (experiment 3).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBates D, Maechler M, Bolker B, et al.: Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw. 2015; 67(1): 1–48. Publisher Full Text\n\nBem DJ: Feeling the future: experimental evidence for anomalous retroactive influences on cognition and affect. J Pers Soc Psychol. 2011; 100(3): 407. PubMed Abstract \| Publisher Full Text\n\nBem DJ, Tressoldi P, Rabeyron T, et al.: Feeling the future: A meta-analysis of 90 experiments on the anomalous anticipation of random future events [version 2; referees: 2 approved]. F1000Res. 2015; 4: 1188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBen-Shachar, Makowski, Lüdecke: Compute and interpret indices of effect size. CRAN, 2020. Reference Source\n\nCanty A, Ripley B: boot: Bootstrap R (S-Plus) Functions. R package version 1.3-23. 2019.\n\nEfron B, Tibshirani RJ: An Introduction to the Bootstrap. Chapman and Hall/CRC. 1993. Reference Source\n\nFox J, Weisberg S: An R Companion to Applied Regression, Third Edition. Thousand Oaks, CA: Sage. URL 2019. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLenth R: emmeans: Estimated Marginal Means, aka Least-Squares Means. R package version 1.4.3.01. 2019. Reference Source\n\nLoy A, Steele S: lmeresampler: Bootstrap Methods for Nesting Linear Mixed Effects Models. R package version 0.1.1. 2020. Reference Source\n\nMagezi DA: Linear mixed-effects models for within-participant psychology experiments: an introductory tutorial and free, graphical user interface (LMMgui). Front Psychol. 2015; 6: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMair P, Wilcox RR: Robust Statistical Methods in R Using the WRS2 Package. Behav Res Methods. Forthcoming. 2020; 52(2): 464–488. PubMed Abstract \| Publisher Full Text\n\nNosek BA, Greenwald AG, Banaji MR: The Implicit Association Test at age 7: A methodological and conceptual review. Automatic processes in social thinking and behavior. 2007; 4: 265–292. Reference Source\n\nPalmer J: ESP scoring as predicted from four definitions of the sheep-goat variable. Research in parapsychology. 1972; 37–39.\n\nR Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna Austria, URL 2020. Reference Source\n\nRoe CA: Critical thinking and belief in the paranormal: A re-evaluation. Br J Psychol. 1999; 90(Pt 1): 85–98. PubMed Abstract \| Publisher Full Text\n\nSchlitz M, Delorme A: Experimenter effect. 2020. http://www.doi.org/10.17605/OSF.IO/XQRC5\n\nSchlitz M, Bem D, Marcusson-Clavertz D, et al.: Examining Experimenter and Participant Beliefs and Expectations:Preregistered Replications of a Time-Reversed Priming Task. Journal of the Society of Scientific Exploration. 2021; 35(1): 69–94. Publisher Full Text\n\nSchmeidler GR, McConnell RA: ESP and personality characteristics. 1958.\n\nStorm L, Tressoldi PE, Di Risio L: Meta-analysis of ESP studies, 1987-2010: Assessing the success of the forced-choice design in parapsychology. J Parapsychol. 2012; 76(2): 243–273. Reference Source\n\nTorchiano M: effsize: Efficient Effect Size Computation. R package version 0.8.0. 2020. Reference Source\n\nWagner MW, Monnet M: Attitudes of college professors toward extra-sensory perception. Zetetic Scholar. 1979; 5: 7– 17.\n\nWickham H: ggplot2: Elegant graphics for data analysis. Springer-Verlag New York, 2016; 2016.\n\nWickham H: stringr: Simple, consistent wrappers for common string operations. R package version 1.4.0. 2019. Reference Source\n\nWickham H, Henry L: tidyr: Tidy messy data. R package version 1.0.2. 2020. Reference Source\n\nWickham H, Fran?ois R, Henry L, et al.: dplyr: A grammar of data manipulation. R package version 0.8.5. 2020. Reference Source" }	[ { "id": "76816", "date": "21 Jan 2021", "name": "Chris A. Roe", "expertise": [ "Reviewer Expertise Parapsychology", "Transpersonal Psychology", "Anomalous Experience" ], "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 reports on a multi-researcher experiment that represents an extension of the Bem ‘feeling the future’ paradigm. The premise of Bem’s work is that one can take a standard protocol from an established area of perceptual or cognitive psychology and reverse the elements such that success at the task is possible only if the participant is able to access information that only exists in the future; in other words, predicted condition differences constitute a test for precognition. This controversial claim has unsurprisingly provoked a vociferous reaction, but also a large number of replication attempts. Although a recent meta-analysis of these replications suggests an overall significant effect, it is clear that there is wide variation in outcome across labs. Schlitz and Delorme’s paper represents a systematic attempt to explore factors that might contribute to that variation. This focus is framed in terms of ‘experimenter effects’, but it is important to be clear that these are not peculiar to parapsychological research. For example, Rosenthal (1994, p. 176)1 refers to 464 studies of interpersonal expectancy effects with an overall d of .63 (r = .30) that demonstrates the effect in a variety of contexts, including studies of reaction time, interpretation of inkblots, animal learning, person perception and skill learning. Harris and Rosenthal (1985)2 provide a meta-analysis of 135 studies that focus on 16 behaviours hypothesised to mediate the effect, including warm interpersonal climate, experimenter expectancy, focused attention, and feedback. Notwithstanding this, Schlitz and Delorme’s exploration in the context of claimed precognition effects is important. It is not clear, however, why priming was chosen as the method to focus on – was this especially susceptible to more polarised outcomes in the meta-analyses? Was there more scope with this method for experimenter effects to gain purchase? Belief is clearly an important factor that can drive experimenter effects, but there are other factors linked especially to rapport and social comfort, and it would be useful to adopt a more multivariate approach to interpersonal dynamics in future studies. Having said that, the use of an implicit association test was a clever way to avoid social desirability confounds when looking at belief and expectation per se. It would have been helpful to know what other instructions were given to participant-experimenters regarding the conditions under which trials should be run; for example, whether participants were monitored during their trial, whether they were required to use a quiet space in which they would not be disturbed, whether minimum requirements for internet bandwidth and stability were in place (it is not stated that the experiment takes place online, but is implied by the description that the authors ”send a link to the participants for the IAT and psi task). Were the criteria used in data cleaning specified in the pre-registration? It would be important to say so – it’s a bit too open ended to simply state “It was the goal to follow the statistical methodology of KPU registry ID number 1051 as closely as possible”. The analysis strategy seems exhaustive and transparent. Having said that, I found the report of outcomes rather opaque with a number of references to outcomes supporting the null hypothesis rather than the conventional formulation that they failed to support the experimental hypothesis, and in places, the null was treated as the experimental (“There was no evidence to support the null hypothesis that reaction times are lower for congruent compared to incongruent trials”, bottom p 9). This elucidation could be usefully simplified. Reporting of H2-H4 seemed similarly unfocused. Given the analysis strategy, I expected IAT analyses to be reported in terms of ANOVA outcomes, with one factor being incongruent/congruent condition and a second factor being belief group using something like a median split; in which case, the belief effect would show up in the interaction. The discussion gets rather bogged down in technical issues regarding the distributions of raw data, and lacks a clear statement concerning how the findings relate to the issue we began with, namely accounting for variations in outcomes across Bem replications in terms of belief and motivation factors. As a consequence, the citations in this section relate only to statistical matters, at the expense of, say, relating current findings to previous successful and unsuccessful replication attempts that have used the priming task (e.g. Rabeyron 2014).3 It is proper to acknowledge that overall the experiment has failed to replicate Bem’s original finding. It would be interesting to speculate on design features that might have contributed to that; it is notable that many failures of Bem’s suite of studies have used on-line presentation in which participants’ attention is not directly monitored, and if the current study was similarly designed then this could have been a contributory factor. The opening paragraphs of the conclusions are more like summaries of design features than conclusions per se.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6352", "date": "12 Feb 2021", "name": "Marilyn Schlitz", "role": "Author Response", "response": "As author I have read this review and will modify the paper to address the concerns raised." }, { "c_id": "6402", "date": "10 Mar 2021", "name": "Marilyn Schlitz", "role": "Author Response", "response": "R1 The authors appreciate the thoughtful review and have addressed the feedback throughout the paper. These changes are noted below. We have expanded the narrative to include the topic of experimenter effects in psychology and have included references to Rosenthal and Harris and Rosental in order to better position this study within the mainstream psychology approach to expectancy effects and to reference existing meta-analyses. Notwithstanding this, Schlitz and Delorme’s exploration in the context of claimed precognition effects is important. It is not clear, however, why priming was chosen as the method to focus on – was this especially susceptible to more polarised outcomes in the meta-analyses? Was there more scope with this method for experimenter effects to gain purchase? We make note of the fact that a multivariate approach to interpersonal dynamics would be a useful next step in future studies. We appreciate that the reviewer makes not of the innovative nature of the implicit association test to study social desirability confounds in looking at belief and expectations. We elaborated on the instructions that were given to the participants, including that the trials should be conducted in a quiet space and that the task took place online. All analyses, including data cleaning specifications, were included in the preplanned analyses. Further, we simplified the statistical discussion. The ANOVA analysis was not included as it had not be pre-registered. The narrative regarding the findings and how they relate to Bem’s previous research have been expanded. Likewise, we explicitly noted that the replication was unsuccessful and speculate in the Conclusion on how design features of this study, including the fact that it was conducted online, have been added." } ] }, { "id": "76817", "date": "11 Feb 2021", "name": "Jeffery Martin", "expertise": [ "Reviewer Expertise psychology", "cognitive science", "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\nOne of the benefits of this platform is the ability to review previous reviews, and thus being able to reduce duplicate effort regarding observations. In this case, that includes a comprehensive review written by Chris Roe of the University of Northampton. I recommend starting with Roe's review prior to reading this one, as I occasionally refer to his comments.\nTo Roe's observations, I would add that this is a straightforward, dense and primarily technically focused paper. It continues work by this and other teams that importantly and meaningfully furthers the work being done in this domain.\nStyle-wise, the paper could probably use a bit more narrative padding and context for the average academic reader. Given that, relatively few psychology researchers are familiar with the debates around the IAT and its use, or in many cases even familiar with it at all, the paper would benefit from providing some additional context for it. It would also be helpful to have the hypotheses more clearly stated early in the paper, and to have a bit more contextual analysis and narrative at both the hypothesis and field levels, as Roe notes.\n\nThese style differences aside, the paper is sound from a research and technical perspective, and comprehensive. Some authors prefer a more terse and technical style that demands more of their professional readers, and I do not feel they should be penalized for this. Everything a scholar needs to both evaluate and design a reproduction of the research is present (excepting things like the actual IAT software), though the paper's terse nature can make this take some digging to piece together.\nAuthors of papers involving the IAT are wise to go overboard detailing their analytical procedures. In this, these authors do not disappoint. Typically, data analysis is kept at a much more cursory, but familiar level (i.e.: ANOVA outcomes, etc. - as Roe mentions). It seems as though the authors may have meant to include a bit more of this. For example, there's a section in the paper that seems to imply that they meant to include boxplots for both datasets. Given that they are mentioned several times, this would be a helpful addition.\nFor a paper dealing with IAT data, it is as or more important to demonstrate that the highly specific technical aspects of the data analysis were understood and properly implemented. These authors achieve this. As Roe also notes, many of the citations in the paper deal with methodology and data analytics. I think this is important because for the average psychology researcher to truly understand the nuances of IAT data, some of these will most likely need to be chased down and absorbed.\nThe authors clearly make use of both standard and advanced statistics, but primarily report on the latter. The type and degree of analysis here is a plus for those who are able to understand it, but I am concerned that it will be a minus for those who can't. The latter could be left without an understanding of the nuances provided in the reported results. In some sense, that is just part of the divide currently present in the field between those skilled in the traditional statistics it has long depended upon, and other, often younger, scholars who also have strong expertise in the more recent advances in data analytics and what they can reveal.\nOne of the benefits of the analysis from someone like Delorme, whose skills are well known to and highly regarded on both sides of this divide, is that he likely carefully thought through what he felt would best answer the hypotheses given the available data rather than just including the standard statistical fare that might actually tell less of the story. When this is the case, and other more traditional statistics are not as present, I think it is helpful for the authors to provide some detail around why they arrived at that decision.\nMost of this feedback involves differences in style, and my other comments do not rise to the level of rejection. Therefore, I accept this paper, though I encourage the authors to reflect upon these comments and make changes based on them that they feel might benefit the reader.\nOverall I find this to be a very helpful article for those interested in this field and a strong and novel contribution.\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": "6351", "date": "12 Feb 2021", "name": "Marilyn Schlitz", "role": "Author Response", "response": "We have reviewed this review and will be making modifications based on the suggestions." }, { "c_id": "6403", "date": "10 Mar 2021", "name": "Marilyn Schlitz", "role": "Author Response", "response": "Thank you for this thoughtful review. We have revised the paper based on the suggestions. These include the following: The authors have read and responded to Roe’s comments. To address style concerns, we provided more narrative and context regarding the contributions of the study to understanding of experimenter effects and the debate and use of IAT. Further, we moved the hypotheses up so that they might be more easily identified. By providing more narrative and reducing unnecessary details regarding the analyses, we have sought to reduce the paper’s “terse nature.” We elaborated on the critiques of IAT and addressed these in terms of how we handled them in this study. We did not include ANOVA outcomes as they were not part of our preregistered analyses. Given that the reviewer feels we have done an adequate job of detailing our analytical procedures, we have removed references to boxplots." } ] } ]	1	https://f1000research.com/articles/10-5
https://f1000research.com/articles/10-197/v1	10 Mar 21	{ "type": "Study Protocol", "title": "Clinical trial of a rehabilitation device based on electrical stimulation for patients with obstructive sleep apnoea (OSA): a study protocol", "authors": [ "William D. Moscoso-Barrera", "Luis Mauricio Agudelo‑Otalora", "Angélica María Moreno-Giraldo", "Javier Burguete", "Secundino Fernández", "Elena Urrestarazu Bolumburu", "Diego Fernando Gómez", "María Alejandra Pérez-Perdomo", "José Miguel Paipa", "María Angélica Bazurto", "Natalia Gelvez", "Carolina Bermúdez", "Luis F. Giraldo-Cadavid", "William D. Moscoso-Barrera", "Luis Mauricio Agudelo‑Otalora", "Angélica María Moreno-Giraldo", "Javier Burguete", "Secundino Fernández", "Elena Urrestarazu Bolumburu", "Diego Fernando Gómez", "María Alejandra Pérez-Perdomo", "José Miguel Paipa", "María Angélica Bazurto", "Natalia Gelvez", "Carolina Bermúdez" ], "abstract": "Obstructive sleep apnoea-hypopnoea syndrome (OSA) is a respiratory disorder characterised by repetitive obstruction of the upper airway, leading to several interruptions during sleep. It is currently one of the main public health problems worldwide and one of the main cardiovascular risk factors in developed and intermediate developing countries, whose populations are increasing in rates of obesity and age. One of the common treatments for OSA is a continuous positive airway pressure (CPAP) device, which pumps air through a hose, reaches a mask that the patient has over his or her nose and travels the airway, keeping the upper airway open during sleep and avoiding episodes of airway collapse. The problem is that CPAP is not accepted by some patients due to a lack of adaptation, so alternative treatments may be needed. For some years, there have been explorations of treatments related to electrical stimulation of the muscles of the upper airway as therapy to reduce the number of episodes of apnoea (measured through the apnoea–hypopnoea index) during the night, strengthening these muscles through stimulation. This is the protocol of the first clinical study of a rehabilitation device for home use that not only provides functional stimulation of the upper-airway dilator muscles but also provides sensory stimulation. This device works by strengthening the dilating muscles of the upper respiratory tract and improving the sensory capacity of the laryngo-pharyngeal tract and is based on existing publications on the effectiveness of functional and somatosensory neurostimulation through neuroplasticity in the recovery of neurological deficits. Trial registration: Clinicaltrials.gov NCT04607343 (29/10/2020)", "keywords": [ "Rehabilitation device", "Electrical stimulation", "Obstructive Sleep Apnea", "Genioglossus muscle", "Laryngopharyngeal sensory test." ], "content": "Introduction\n\nSleep apnoea is a significant cardiovascular risk factor. Its consequences include systemic high blood pressure, pulmonary hypertension, myocardial infarction and cerebrovascular disease [(Caples et al., 2005), (Rangel-León et al., 2015)]. It also produces metabolic alterations related to the control of glycemia and lipids (Caples et al., 2005). Additionally, it is linked to obesity, which creates a vicious cycle: obesity causes sleep apnoea, and sleep apnoea worsens obesity by altering the response to hormonal mediators of satiety (Romero-Corral et al., 2010).\n\nDespite representing a pathology with multiple complications, there are limited treatments for obstructive sleep apnoea-hypopnoea syndrome (OSA). The adherent use of continuous positive airway pressure (CPAP) has been shown to have beneficial effects on OSA outcomes, including avoiding further deterioration in the airway (Maurer et al., 2012); however, the adherence rate for this device is low (even less than 40% in some cases), forcing researchers to look for alternatives (Kezirian et al., 2010). Surgical procedures related to the treatment of OSA have variable results, with high mortality rates (Maurer et al., 2012) and effectiveness ranging from 20% to 60% (Maurer et al., 2012).\n\nRecent research was investigated electrical stimulation of the upper airway in animals and humans with OSA. Animal studies have consisted of inserting needle electrodes orally into the genioglossus muscle of awake dogs or stimulating the hypoglossal nerve in anaesthetised and non-anaesthetised felines, checking the response of the airway muscles in stimulations with frequencies up to 50 Hertz (Hz) (Kezirian et al., 2010). Similarly, rats were stimulated on the upper laryngeal nerve with frequencies between 30 Hz and 40 Hz for 50 seconds, causing swallowing reflexes, which shows the effects of the action on the muscles (Tsuji et al., 2015).\n\nHuman studies for the management of OSA began with the intraoral electrical stimulation of the upper airways carried out by Guilleminault in the 1970s (Guilleminault et al., 1978). These early interventions did not detail the muscle groups on which electrostimulation was exerted, nor did they have diagnostic methods for determining whether the stimulation was taking place on dilating muscles or airway constrictors. Any of these reasons could explain a simultaneous stimulation of dilating and constrictor muscles of the airway, which could be responsible for the failure of such interventions.\n\nAround ten years later, research focused specifically on the main dilating muscle of the airway, the genioglossus, showing that genioglossus stimulation reduced the apnoea–hypopnoea index (AHI) (Schwartz et al., 1993) and daytime sleepiness. However, the successful stimulation of upper airway muscles and relief from upper airway obstruction still has problems, such causing the patient to wake during sleep and the need to implant electrodes invasively on the hypoglossal nerve (Yoo & Durand, 2005).\n\nMore recently, a device has been developed that seeks to stimulate the hypoglossal nerve by electric current (Maurer et al., 2012). This device performs functions similar to a pacemaker, generating an electrical stimulus synchronously with breathing (Kezirian et al., 2010; Maurer et al., 2012). Several studies have shown that electrical stimulation in the hypoglossal nerve through this device improves the airspace of the oropharynx and hypopharynx, thus decreasing obstructive episodes during sleep by up to 68% (Kezirian et al., 2010; Lorenzi-Filho et al., 2017; Maurer et al., 2012). However, the implantation of this device is done in an invasive way and is highly irreversible.\n\nFurthermore, there is a new therapeutic horizon that until now has not been explored in OSA: the use of sensory recovery interventions based on the concepts of neuroplasticity that are the basis of neurological rehabilitation (Gillick & Zirpel, 2012; Howlett et al., 2015; Page et al., 2015; Robbins et al., 2006). In fact, there is already preliminary information on the effectiveness of such interventions in the recovery of the sensitivity of patients with oropharyngeal dysphagia (Gallas et al., 2010; Pauloski et al., 2013; Rofes et al., 2013).\n\nThe evidence provided by previous research on the effectiveness of electrostimulation in OSA supports the potential of such interventions for the treatment of this entity, as long as stimulation is applied on the dilating muscles of the airway and stimulation of the constrictor muscles is avoided. Likewise, there is evidence that supports the potential benefit of electrostimulation in the recovery of sensory alterations of the laryngopharyngeal tract, which may have a role in the OSA pathogenesis. However, there are a number of problems with this treatment that have yet to be solved, including the development of a non-invasive, effective device; the alteration in sleep architecture by micro-arousals, or awakenings caused by the electric current; and the lack of a device for that a patient can used with minimal level training, on an outpatient basis.\n\n\nMethods\n\nHow effective a new electrostimulation device is in dilating the upper airway, improving AHI, desaturation indices and quality of life in patients with obstructive sleep apnea\n\nTo evaluate in a first clinical trial a rehabilitation device for patients with OSA based on oropharyngeal electrical stimulation, which strengthens the dilating muscles of the upper airway, improves mechanical laryngopharyngeal sensitivity and improves OSA.\n\nPerform experimental electrotherapy, nasoendoscopy, polygraphy and polysomnography tests with the device in order to calculate thresholds of functional and sensory intensities on the dilating muscles of the upper airway in patients with OSA. These tests also include the detection of signals that measure breathing events during sleep in patients with OSA in order to use them in the control mechanisms of the electrostimulation device.\n\nAssess the effects and safety of the device in a group of five healthy volunteers for preliminary results.\n\nEvaluate in a small group of patients with OSA the device capacity to stimulate the upper-airway dilating muscles and to reduce the AHI and oxygen desaturation indices when the electrostimulation device is used over a medium-term period (eight weeks) with morning and evening therapy sessions.\n\nA feasibility clinical study of a medical device (Abdel-Aleem, 2009; CDHR et al., 2013) will be performed in patients prospectively recruited at the Sleep Medicine Service of Fundacion Neumologica Colombiana a tertiary care referral university medical centre specialized in respiratory and sleep medicine, located at Bogota. Patients will initially be evaluated by the Pulmonary and Sleep Medicine Service of Fundacion Neumologica Colombiana to confirm the diagnosis of OSA, for which the presence of symptoms of the disease plus a basal overnight polysomnogram with an AHI greater than 5 will be required.\n\nIn total, 12 patients with OSA will be recruited from those referred for full overnight polysomnography: four with mild, four with moderate and four with severe OSA. Patients will be recruited prospectively and consecutively after explaining the study and signing the informed consent. They will be evaluated by a pulmonary and sleep medicine doctor to confirm the diagnosis of OSA. For diagnosing OSA it will be required the presence of symptoms of the disease plus a baseline polysomnogram with an apnea-hypopnea index (AHI) greater than 5/h. Their general medical condition will also be evaluated and their admission to the protocol defined in accordance with the inclusion / exclusion criteria. Afterwards, the patients will undergo a clinical evaluation by a speech language pathologist trained in myofunctional therapy for OSA (see Figure 1). The study will also include a group of 5 healthy volunteers. The standard clinical evaluation of all patients and healthy volunteers will include the application of a standardized questionnaire assessing for the comorbidities of the Charlson Comorbidity Index (Charlson et al., 1994). Candidates for participating as healthy volunteers will also be screened with the STOP-BANG questionnaire (Baldini et al., 2017; Chung et al., 2016). A healthy volunteer will be define by the absence of signs and symptoms of acute conditions and comorbidities, and a STOP-BANG score < 3 (Baldini et al., 2017; Chung et al., 2016).\n\nThe classifications of the subjects in each category of OSA and as healthy controls will be done by pulmonary and sleep medicine physicians after the standard clinical evaluation. If it is considered that the subject applies as a control, they will be included in the study after signing the informed consent The definition of mild, moderate or severe OSA will depend on the presence of symptoms plus an AHI (measured during a full overnight polysomnography) between 5 and 15 in the case of mild OSA, >15 to <30 in the case of moderate OSA and ≥ 30 in the case of severe OSA.\n\nSample size calculation. Considering that this will be the first and pilot study of the electrostimulation device, there are no previous studies to make precise sample size calculations. Therefore, we considered it more appropriate to calculate the sample size using the effect size, that is, how large is the difference in the AHI before and after the intervention in terms of the standard deviation (SD) of the AHI (Cohen, 1988; Machin et al., 2009; Rosenthal, 1996). This method avoids the requirement of knowing the precise values for the means and SDs of the variables under study. For this pilot study, in which the priority is assessing feasibility, we considered that the AHI difference before and after the intervention should correspond to a large effect size to be considered significant. Future studies looking for smaller differences will use the results of this pilot study for sample size calculations. Effect size larger than 0.8 are considered large (Cohen, 1988; Rosenthal, 1996).\n\nTherefore, the sample size was calculated looking for an effect size of 1.0 (a decrease in the AHI after the intervention of 1.0 SD of the AHI, which could correspond to about 15 apnoea-hypopnoea events per hour (Butler et al., 2019)), assuming a significance of 95%, a power of 80% for comparing repeated measures (Machin et al., 2009). With such assumptions, we calculated a minimum sample size of 10 subjects to detect statistically significant differences in the repeated measures comparison of pre-and post-intervention AHI (Machin et al., 2009), and two subjects were added to compensate for the potential loss to follow-up, for a total of 12 subjects.\n\nInclusion criteria for patients. Patients over 18 years of age with confirmed OSA (four with mild OSA, four with moderate OSA and four with severe OSA) who sign their informed consent to participate in the study.\n\nInclusion criteria for controls:\n\nSubjects over 18 years of age\n\nExclusion criteria for patients and controls:\n\n1. Pregnancy\n\n2. Basal polysomnography that does not meet validity criteria to be interpreted.\n\n3. Anticoagulation (although not a contraindication for laryngopharyngeal sensory endoscopic testing, anticoagulation is an exclusion criterion for this study to maintain the lowest level of risk).\n\n4. Haemorrhagic diathesis (to avoid risk of severe epistaxis during nasal endoscopy).\n\n5. Glasgow scale less than 15 (to avoid confusion with sensory or motor laryngopharyngeal involvement due to neurological disease that compromises the state of consciousness).\n\n6. Basal oxygen saturation by awake pulse oximetry below 88%.\n\n7. Patients with more than 5% of the total apnoea events being of central origin measured during a full overnight polysomnography(to avoid including patients with central sleep apnoea in whom laryngopharyngeal electrostimulation would have no effect).\n\n8. Inflammatory or infectious lesions on the face or neck\n\n9. Skin hypersensitivity\n\n10. Anaesthetic areas, burns, bruises or recent wounds in the area of electrical stimulation\n\n11. Cardiac pacemakers or other telemetry-controlled devices,\n\n12. History of maxillofacial or pharyngeal surgery.\n\n13. Active cancer\n\n14. Tumours of the laryngopharyngeal tract.\n\n15. Significant mental and/or behavioural conditions or inability of the patient to cooperate during the examination/intervention.\n\n16. Epilepsy\n\n17. Central nervous system (CNS) surgery in the last three months (to avoid confusion with muscle or sensory laryngopharyngeal compromise).\n\n18. Brain trauma in the last three months (to avoid confusion with muscle or sensory laryngopharyngeal compromise).\n\n19. Neurological sequelae of any cause compromising the head and neck muscles (to avoid confusion with muscle or sensory laryngopharyngeal compromise).\n\n20. Underlying neuromuscular diseases affecting the head and neck muscles (to avoid confusion with muscle or sensory laryngopharyngeal involvement due to neuromuscular disease).\n\n21. Chronic use of systemic corticosteroids at doses greater than or equal to 20 mg daily of prednisone or equivalent (to avoid confusion with corticosteroid myopathy that compromises the laryngopharyngeal region).\n\n22. Bone prostheses or osteosynthesis (with polarized currents there is danger of chemical burn and bone resorption)\n\n23. Acute febrile processes\n\n24. Chronic decompensated diseases\n\n25. Diseases in terminal states\n\n26. Refusal to participate in the study.\n\nAdditional exclusion criteria for controls: The following exclusion criteria will apply to controls in order to ensure that they do not have significant comorbidities:\n\n1. Previous diagnosis of OSA\n\n2. STOP-BANG score >3 (Baldini et al., 2017; Chung et al., 2016)\n\n3. Age > 60\n\n4. Body mass index > 30\n\n5. Any comorbidity of the Charlson Comorbidity index (Charlson et al., 1994) determined by the physician evaluating the selection criteria.\n\nAnthropometric variables, including weight in kilograms, size in metres, body mass index in kilograms/squared meters (BMI), and the neck's circumference in centimetres, will be measured. In addition, the Epworth Sleepiness Scale (Johns, 1991), the Sleep Apnoea Quality of Life Index (SAQLI) (Coman et al., 2016) including the CPAP tolerance domain, will be applied for OSA patients and the STOP-BANG questionnaire for controls (Baldini et al., 2017; Chung et al., 2016). A full list of variables is provided in Table 1.\n\nCandidates to be enrolled (patients and controls) will receive detailed information about the study and will sign their informed consent to participate in it.\n\nElectrostimulation. Experimental tests with transcutaneous electrical stimulation at different intensities and at the points of the submandibular region previously explored by this and other research groups (Rangel-León et al., 2015) will be conducted first of all on the five healthy volunteer subjects. For the electrostimulation, the experimental device will be used. The stimulation with the electrode will be carried out on the upper airway dilator muscles, starting with the extralaryngeal muscles and then stimulating facial muscles. Electrostimulation will be performed in specific facial and extralaryngeal muscles, using a current type Biphasic Symmetric TENS, with a phase duration of 100 microseconds (µs), a frequency of 40 Hertz (Hz), an intensity of about 12 milliamps (mA) and a constant voltage, during a time of 15 minutes. The current intensity will be titrated to the tolerance of the patient or until obtaining visible muscle contraction. During stimulation, the region corresponding to the carotid sinus and the stellate ganglion will be avoided. During these tests, the neuromuscular functional and sensory thresholds of the upper airway, the most effective points of electrostimulation and the tolerance of intervention will be determined. The determination of the functional threshold will be made by clinical evaluation of the subject during stimulation (determining the current intensity required to induce contraction of the stimulated muscle).\n\nElectrostimulation begins with electrodes placed on the skin in the submentonean and submandibular triangles (Figure 2), making sure not to place electrodes on the carotid sinus. The goal of this stimulation will be topographical closeness of the genioglossus, geniohyoid, mylohyoid, platysma and digastric muscles. For this purpose, symmetrical biphasic transcutaneous electrical nerve stimulation type (TENS-type) currents will be used, with a phase duration of 100 microseconds (µs), a frequency of 40 Hz and an intensity between 9–12 milliamps (mA), until the patient cannot tolerate electrostimulation or until visible muscle contraction (Rangel-León et al., 2015).\n\nThe electrodes for electrostimlation will be placed in the red area.\n\nThe functional threshold corresponds to the smallest current intensity capable of obtaining contraction of the airway's dilating muscles. The presence of contraction of the stimulated muscles will be determined by asking the patient about the sensation they feel, as well as by external and endoscopic inspection. These thresholds will be measured using the experimental device, which provides real-time information of the current characteristics (including intensity, voltage and frequency). The sensory threshold corresponds to 75% of the functional threshold’s current intensity (Robbins et al., 2006; Rofes et al., 2013). The degree of opening of the upper airway obtained on these thresholds will be determined by conventional video-flexible-nasolaryngoscopy measuring the upper airway diameters using software designed ad hoc and previously validated.\n\nAfter characterizing the electrostimulation thresholds in healthy volunteers (functional and sensory thresholds), as well as their desired and secondary effects, the 12 subjects with OSA will undergo electrostimulation at the same area of the neck using similar current characteristics and titrating the current intensity to determine the functional and sensory thresholds for each patient. The neuromuscular thresholds of the upper airway and the most effective points of electrostimulation will be determined during these tests. Again, the determination of the functional threshold will be made by clinical evaluation of the subject during stimulation, and the degree of opening of the upper airway based on these thresholds will then be evaluated by conventional video-flexible-nasolaryngoscopy.\n\nWe will conduct an analysis of the correlation of the signals obtained in experimental tests, elaborate a map of more effective points for electrical stimulation and determine the intensities required for each patient. We will assess the possibility of using the same standard point map for all patients.\n\nAfterwards, the 12 OSA patients will receive an eight-week therapeutic intervention, using a protocol similar to that used by our group (Rangel-León et al., 2015). The effects of the intervention will be evaluated looking for improvement of the AHI, the desaturation indices, the Epworth Sleepiness Scale (ESS) (Johns, 1991) and the Sleep Apnoea Quality of Life Index SAQLI. (Coman et al., 2016). The improvement of the AHI and desaturation indices will be determined by comparing full overnight polysomnograms conducted before and after the intervention and looking at the changes in such indices. Polysomnograms will include standard electroencephalography, electrooculography, mandible and leg electromyography, nasal and oral airflow, and electrocardiography; respiratory effort will be measured by diaphragmatic excursion, and oxygen saturation will be measured by pulse oximetry. Polysomnograms will be performed, read and interpreted in the sleep laboratory of the Colombian Pneumological Foundation by certified personnel blinded to the patient intervention. The person in charge of reading and interpreting the polysomnograms will be blinded to the patient’s intervention. Sleep stages and respiratory events (including apnoea-hypopnoea and desaturation events) will be scored according to the rules of the American Academy of Sleep Medicine (AASM) Manual for Scoring of Sleep and Associated Events (Berry et al., 2012). The clinical diagnosis of OSA will be defined by an apnoea-hypopnea index (AHI) > 5 events/hour of sleep, with symptoms of sleep fragmentation and daytime somnolence according to the American Academy of Sleep Medicine. The severity of OSA will be determined according to the AHI as follows: mild, 5–14.9; moderate, 15–30; and severe > 30 events/hour (Takegami et al., 2009).\n\nPatients will undergo a conventional video-flexible-nasolaryngoscopy in which the degree of obstruction and the laryngopharyngeal sensitivity during wakefulness will be determined in accordance with the protocol previously validated by our research group (Giraldo-Cadavid et al., 2017; Giraldo-Cadavid et al., 2017). Briefly, the upper airway mucosa is stimulated at precise points (aryepiglottic folds, lateral pharyngeal walls and soft palate) using air-pulses of different intensities, the sensory thresholds correspond to the lower air-pulse intensity perceived by the patient (Giraldo-Cadavid et al., 2017; Giraldo-Cadavid et al., 2017). This test is performed using a thin flexible endoscope with a working channel of 1.2 mm. The endoscopist introduces the endoscope through the patient’s nasal cavity. It is lubricated with water-soluble gel to decrease discomfort. The endoscopist does not use anesthetics because the test only produces mild to moderate discomfort, it is infrequently associated with pain, and anesthetics might alter the laryngopharyngeal tract reflexes and the measurements of their thresholds.\n\nThe sensory evaluation consists of administering pulses of air at different intensities to the patient to stimulate and determine the reflex thresholds of the laryngeal adductor (LART), cough (CRT), gag reflexes (GRT) and psychophysical sensitivity thresholds.\n\nEffectiveness assessment will also include video-flexible-nasolaryngoscopies with measurement of laryngopharyngeal sensitivity as previously detailed, and Drug-induced sleep endoscopy (DISE) each performed before the intervention and a second of each test performed after the intervention. During DISE the level and degree of upper airway obstruction will be determined at the soft palate, tonsils, tongue base and the epiglottis using software designed ad hoc for measuring the airway diameter and perimeter and previously validated.\n\nThe image processing software is made in Python and determines the airway obstruction degree as follows:\n\nThe system takes the video made during DISEs, divides it into image format frames for edition. Subsequently, the rater selects the frames with the airway region where he wants to make the measurements while there are not OSA events, usually in the initial phase of sedation (in light sleep), he also selects frames with the same airway regions to make the measurements during the OSA events, usually in more profound stages of sleep. In each frame, the rater delimits the areas and diameters to be measured in the image using the computer's mouse. The software processes the images to calculate the areas' and diameters' size in pixels and displays their corresponding values in a graph. To calculate the percentage of obstruction the computer divides the measures of the areas and diameters during OSA events by the corresponding measures taken at the beginning of the DISE (during the lightest stage of sleep) when there are not obstructive sleep apnoeas.\n\nThe pre-intervention tests, including clinical evaluation (history, anthropometric measures and physical exam), basal polysomnogram, SAQLI, ESS, STOP-BANG, and DISE will be performed a maximum of one month before the start of the intervention, and the post-intervention tests will be performed a maximum of one month after the intervention.\n\nDuring the study, patients will continue with the therapy ordered by their treating physician for the management of OSA, including CPAP and lifestyle changes.\n\nAny adverse event will be notified to the institutional review board and competent authorities in the expected time according to its severity: less than 24 hours in case of serious adverse events.\n\nThe effectiveness assessment will be based on the improvement of the following outcomes:\n\nAHI during sleep.\n\nAHI during rapid eye movements (REM) sleep.\n\nAHI during non-REM sleep.\n\nDesaturation index: number of desaturations per hour during sleep.\n\nT90: percentage of sleep time spent under 90% oxygen saturation.\n\nSpO2 nadir: lowest oxygen saturation measured by pulse oximetry.\n\nMean SpO2: mean oxygen saturation measured by pulse oximetry.\n\nNumber of patients who lower one or more categories in the severity of OSA (moving from severe to moderate, severe to mild or moderate to mild OSA or normalising the AHI).\n\nNumber of patients who climb one or more categories in the severity of OSA (moving from mild to moderate, mild to severe or moderate to severe OSA).\n\nImprovement in the quality of life as measured by SAQLI.\n\nImprovement in daytime sleepiness as measured by the ESS.\n\nQualitative variables will be described with absolute and relative frequencies, and comparisons between before and after the intervention will be made by the McNemar test.\n\nQuantitative variables will be evaluated for assumptions of normality by the Shapiro-Wilk test. If they have normal distribution, they will be described with means and standard deviations; otherwise, they will be described with medians and interquartile ranges. Comparisons of before and after the intervention will be made by student t-tests for paired measurements if the distribution is normal or by the Wilcoxon signed range tests otherwise.\n\nStatistical analysis will be performed using Stata version 16.1 (StataCorp, Lakeway Drive, USA).\n\n\nEthics\n\nThe type of electric current that will be used in this study is safe in its application with a low probability of causing burns or adverse events in deep tissues. Electrostimulation is an invention widely used today for pain management for cancer, precordial pain, herpes, vertebral pain and immediate postoperative pain (Rodríguez, 2004). However, because new and experimental equipment not yet approved by regulatory bodies will be used, there is a higher than minimum risk present. This protocol was approved by the ethics committee (IRB) of Fundación Neumológica Colombiana with act number 234 of March 2, 2018 any modification in this protocol will be submitted to the IRB for approval before its implementation.\n\nThe study will be explained to each patient verbally and in a written document that summarises the objectives and characteristics of the study. Informed written consent will be obtained from patients who agree to enter the study.\n\nThe database with patient information will be anonymised to preserve patient confidentiality. Insurance will be taken out to cover the risks of side effects in patients.\n\nThis protocol was registered with ClinicalTrials.gov on 29th October 2020 (NCT04607343).\n\n\nPlan for dissemination of study results\n\nThe study results will be summarized and presented in at least one international and one national medical conference. Afterwards a final report in the form of a scientific original article will be submitted to an international peer review journal.\n\n\nStudy status\n\nNot yet recruiting.\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: Clinical trial of a rehabilitation device based on electrical stimulation for patients with obstructive sleep apnoea (OSA): a study protocol. https://doi.org/10.17605/OSF.IO/AU8F4 (Giraldo-Cadavid et al., 2021)", "appendix": "References\n\nAbdel-Aleem S: Medical Device Regulations, Combination Product, Study Committees, and FDA‐Sponsor Meetings. In: Abdel-Aleem S, editor. Design, execution, and management of medical device clinical trials. Hoboken, New Jersey, USA: John Wiley & Sons, Inc. 2009; 129–94. Publisher Full Text\n\nBaldini M, Chiapella MN, Fernández MA: [STOP-BANG, a useful and easy tool for the screening of obstructive sleep apnea]. Medicina (B Aires). 2017; 77(3): 191–195. 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Reference Source\n\nComan AC, Borzan C, Vesa CS, et al.: Obstructive sleep apnea syndrome and the quality of life. Clujul medical. 2016; 89(3): 390–395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallas S, Marie JP, Leroi AM, et al.: Sensory transcutaneous electrical stimulation improves post-stroke dysphagic patients. Dysphagia. 2010; 25(4): 291–7. PubMed Abstract \| Publisher Full Text\n\nGiraldo-Cadavid LF, Bastidas AR, Padilla-Ortiz DM, et al.: Accuracy and reliability of the sensory test performed using the laryngopharyngeal endoscopic esthesiometer and rangefinder in patients with suspected obstructive sleep apnoea hypopnoea: protocol for a prospective double-blinded, randomised, exploratory study. BMJ Open. 2017; 7(8): e015235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiraldo-Cadavid LF, Burguete J, Rueda F, et al.: Reliability of a laryngo-pharyngeal esthesiometer and a method for measuring laryngo-pharyngeal mechano-sensitivity in a prospectively recruited cohort of patients. Eur Arch Otorhinolaryngol. 2017; 274(7): 2861–2870. PubMed Abstract \| Publisher Full Text\n\nGiraldo-Cadavid LF, Moscoso-Barrera WD, Agudelo-Otalora LM, et al.: Clinical trial of a rehabilitation device based on electrical stimulation for patients with obstructive sleep apnoea (OSA). 2021. http://www.doi.org/10.17605/OSF.IO/AU8F4\n\nGillick BT, Zirpel L: Neuroplasticity: an appreciation from synapse to system. Arch Phys Med Rehabil. 2012; 93(10): 1846–55. PubMed Abstract \| Publisher Full Text\n\nGuilleminault C, Hill MW, Simmons FB, et al.: Obstructive sleep apnea: electromyographic and fiberoptic studies. Exp Neurol. 1978; 62(1): 48–67. PubMed Abstract \| Publisher Full Text\n\nHowlett OA, Lannin NA, Ada L, et al.: Functional Electrical Stimulation Improves Activity After Stroke: A Systematic Review With Meta-Analysis. Arch Phys Med Rehabil. 2015; 96(5): 934–943. PubMed Abstract \| Publisher Full Text\n\nJohns MW: A new method for measuring daytime sleepiness: the Epworth sleepiness scale. Sleep. 1991; 14(6): 540–545. PubMed Abstract \| Publisher Full Text\n\nKezirian EJ, Boudewyns A, Eisele DW, et al.: Electrical stimulation of the hypoglossal nerve in the treatment of obstructive sleep apnea. Sleep Med Rev. 2010; 14(5): 299–305. PubMed Abstract \| Publisher Full Text\n\nLorenzi-Filho G, Almeida FR, Strollo PJ: Treating OSA: Current and emerging therapies beyond CPAP. Respirology. 2017; 22(8): 1500–1507. PubMed Abstract \| Publisher Full Text\n\nMachin D, Campbell MJ, Tan SB, et al.: Sample Size Tables for Clinical Studies. West Sussex, UK, Wiley-Blackwell. 2009. Reference Source\n\nMaurer JT, Van De Heyning P, Lin HS, et al.: Operative technique of upper airway stimulation: an implantable treatment of obstructive sleep apnea. Oper Tech Otolayngol Head Neck Surg. 2012; 23(3): 227–233. Publisher Full Text\n\nPage SJ, Cunningham DA, Plow E, et al.: It takes two: noninvasive brain stimulation combined with neurorehabilitation. Arch Phys Med Rehabil. 2015; 96(4 Suppl): S89–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPauloski BR, Logemann JA, Rademaker AW, et al.: Effects of enhanced bolus flavors on oropharyngeal swallow in patients treated for head and neck cancer. Head Neck. 2013; 35(8): 1124–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRangel-León YJ, Rengifo-Varona ML, Gálvis-Gómez AM, et al.: Rehabilitacion de musculos orofaringeos con ejercicios y electroterapia para el sindrome de apnea-hipoapnea obstructiva del sueno. Rehabilitacion. 2015; 49(1): 4–9. Publisher Full Text\n\nRobbins SM, Houghton PE, Woodbury MG, et al.: The therapeutic effect of functional and transcutaneous electric stimulation on improving gait speed in stroke patients: a meta-analysis. Arch Phys Med Rehabil. 2006; 87(6): 853–9. PubMed Abstract \| Publisher Full Text\n\nRodriguez M: Electrotherapy in physical therapy. Second edition: Buenos Aires. Pan-American Publishing: 2004.\n\nRofes L, Arreola V, Lopez I, et al.: Effect of surface sensory and motor electrical stimulation on chronic poststroke oropharyngeal dysfunction. Neurogastroenterol Motil. 2013; 25(11): 888–e701. PubMed Abstract \| Publisher Full Text\n\nRomero-Corral A, Caples SM, Lopez-Jimenez F, et al.: Interactions between obesity and obstructive sleep apnea: implications for treatment. Chest. 2010; 137(3): 711–719. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenthal JA: Qualitative Descriptors of Strength of Association and Effect Size. Journal of Social Service Research. 1996; 21(4): 37–59. Publisher Full Text\n\nSchwartz AR, Thut DC, Russ B, et al.: Effect of electrical stimulation of the hypoglossal nerve on airflow mechanics in the isolated upper airway. Am Rev Respir Dis. 1993; 147(5): 1144–1150. PubMed Abstract \| Publisher Full Text\n\nTakegami M, Hayashino Y, Chin K, et al.: Simple four-variable screening tool for identification of patients with sleep-disordered breathing. Sleep. 2009; 32(7): 939–48. PubMed Abstract \| Free Full Text\n\nTsuji K, Tsujimura T, Magara J, et al.: Changes in the frequency of swallowing during electrical stimulation of superior laryngeal nerve in rats. Brain Res Bull. 2015; 111: 53–61. PubMed Abstract \| Publisher Full Text\n\nYoo PB, Durand DM: Effects of selective hypoglossal nerve stimulation on canine upper airway mechanics. J Appl Physiol. 2005; 99(3): 937–943. PubMed Abstract \| Publisher Full Text" }	[ { "id": "255741", "date": "25 May 2024", "name": "Carlos O'Connor-Reina", "expertise": [ "Reviewer Expertise sleep disordered breathig" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors have presented the protocol of the first clinical study of a rehabilitation device for home use that not only provides functional stimulation of the upper-airway dilator muscles but also provides sensory stimulation. My concerns are the next. Authors considered that awake examination is the same as sleep examination. DISE should be performed in all the patients to evaluate the anatomical findings more suitable to use the device. Authors commented an evaluation from myofunctional therapists they did not mention what is the protocol followed or what are the texts used. conscious anatomical evaluation i.e. Friedman, or Mallanpati is not performed. The degree of opening awake is not the same as sleep. Authors do not consider positional sleep apnea in their evaluation.\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? Not applicable", "responses": [] }, { "id": "86369", "date": "21 Aug 2024", "name": "Joachim T. Maurer", "expertise": [ "Reviewer Expertise Sleep medicine", "functional treatment", "obstructive sleep apnoea" ], "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\nSpecific comments: Page 3, Right column, 3rd paragraph: \"Likewise, there is evidence that supports the potential benefit of electrostimulation in the recovery of sensory alterations of the laryngopharyngeal tract, which may have a role in the OSA pathogenesis\"\n\nPlease add the literature you refer to.\nFigure 1: What does SLP mean? Can the authors please elaborate?\nGeneral comments: - The citations are mainly old or very old. Only very few are as recent as 8-10 years ago. More recent publications regarding functional treatments such as hypoglossal nerve stimulation and myofunctional treatment are missing but need to be integrated.\n\nMajor comment: The protocol does not present an extraordinary approach, and it is unclear why publication and indexing of the protocol is necessary at this stage without any preliminary results. Additionally, there appears to have been no patient recruitment over the past three years. Providing information on any progress or preliminary findings could strengthen the case for its publication and indexing. I suggest that the authors do their study and publish their results where the study protocol is described in the methods section.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-197
https://f1000research.com/articles/10-196/v1	10 Mar 21	{ "type": "Brief Report", "title": "Most Japanese individuals are genetically predisposed to recognize an immunogenic protein fragment shared between COVID-19 and common cold coronaviruses", "authors": [ "Johannes M. Dijkstra", "Aaron P. Frenette", "Brian Dixon", "Aaron P. Frenette", "Brian Dixon" ], "abstract": "In the spring of 2020, we and others hypothesized that T cells in COVID-19 patients may recognize identical protein fragments shared between the coronaviruses of the common cold and COVID-19 and thereby confer cross-virus immune memory. Here, we look at this issue by screening studies that, since that time, have experimentally addressed COVID-19 associated T cell specificities. Currently, the identical T cell epitope shared between COVID-19 and common cold coronaviruses most convincingly identified as immunogenic is the CD8+ T cell epitope VYIGDPAQL if presented by the MHC class I allele HLA-A24:02. The HLA-A24:02 allele is found in the majority of Japanese individuals and several indigenous populations in Asia, Oceania, and the Americas. In combination with histories of common cold infections, HLA-A24:02 may affect their protection from COVID-19.", "keywords": [ "COVID-19", "T cell", "MHC", "HLA", "peptide", "epitope", "Japanese", "VYIGDPAQL", "SPRWYFYYL" ], "content": "Introduction\n\nThe virus causing the COVID-19 pandemic is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Wu et al., 2020; Zhou et al., 2020). SARS-CoV-2 is one of the seven coronaviruses that are known to infect humans, the others being SARS-CoV-1 (causing SARS), Middle East respiratory syndrome coronavirus (MERS-CoV), and the common cold coronaviruses (CCCoVs): human coronavirus OC43 (HCoV-OC43), HCoV-HKU1, HCoV-229E, and HCoV-NL63. These coronaviruses belong to the serological/phylogenetic clades designated as group I (alphacoronaviruses) and group II (betacoronaviruses) (Figure 1). SARS-CoV-1 and MERS-CoV infected relatively few people and therefore should have little effect on global cross-virus immune memory. On the other hand, the CCCoVs cause ~20% of common cold cases, are globally distributed, and all adults have probably been infected with them multiple times in their lives (Hirsch et al., 2013; Mäkelä et al., 1998; Zhou et al., 2013).\n\nViruses closely related to SARS-CoV-2 are found in bats. Bats and civets are the probable sources of SARS-CoV-1, and camels are alternative hosts for MERS-CoV. The first reported outbreaks in people by infections with SARS-CoV-1, MERS-CoV, and SARS-CoV-2 occurred in 2002, 2012, and 2019, respectively, with differences in number of deaths and case fatality ratios among registered cases (percentages indicated in regular font between parentheses) (https://www.who.int/publications/m/item/summary-of-probable-sars-cases-with-onset-of-illness-from-1-november-2002-to-31-july-2003; https://www.who.int/health-topics/middle-east-respiratory-syndrome-coronavirus-mers; https://coronavirus.jhu.edu/map.html). SARS-CoV-2 infections and deaths are not always registered and based on data from New York it was estimated that the true fatality rates may be ~1.4% (Italic font) (Yang et al., 2021).\n\nThe immune defense against viruses includes both innate and adaptive immune responses. Cell types that specialize in adaptive immunity (immune memory) are B cells, CD4+ T cells, and CD8+ T cells. B cells can secrete antibodies, but there is probably little or no protective cross-virus anti-SARS-CoV-2 humoral immunity deriving from infections by CCCoVs (e.g., Amanat et al., 2020; Shrock et al., 2020). CD8+ T cells recognize peptides presented by major histocompatibility complex class I (MHC-I) cells and can kill the presenting cell; the peptides bound by MHC-I are approximately 8-13 amino acids (aa) length, and mostly are 9 aa (“9-mers”) (Rammensee et al., 1995; Schellens et al., 2015). CD4+ T cells recognize peptides if presented by MHC class II (MHC-II) molecules and help regulate immune responses; the peptides bound to MHC-II typically are 12-25 aa (Rammensee et al., 1995), although the part binding within the MHC-II groove is only 9 aa as commonly found for MHC-I (Stern & Wiley, 1994). In the case of MHC-I alleles, available computational software provides a powerful in silico tool in predicting which peptides are presented. For a broader discussion on T cell functions in COVID-19 patients, including whether T cell responses are always beneficial or could also be detrimental, we refer to other studies (Altmann & Boyton, 2020; Bacher et al., 2020b; Jarjour et al., 2020).\n\nIn the spring of 2020, Nguyen et al. (2020) and Dijkstra & Hashimoto (2020) reported on possible MHC-I binding epitopes shared between CCCoVs and SARS-CoV-2 based on in silico analyses and speculated on cross-virus T cell immune memory. Whereas Nguyen et al. (2020) made a comprehensive analysis of possible MHC epitopes of SARS-CoV-2, Dijkstra & Hashimoto (2020) concentrated on identical 9-mers shared between SARS-CoV-2 and at least one of the CCCoVs. Probably for that reason, Dijkstra & Hashimoto (2020) were more accurate in identifying such 9-mers, as they found >230 whereas Nguyen et al. (2020) only listed 144. These identical 9-mers, or even 8-mers, were mostly found in several well-conserved nonstructural proteins that are expressed as part of the ORF1ab polyprotein, while they are absent or nearly absent in the structural proteins S, M, N, and E (Dijkstra & Hashimoto, 2020; Lee et al., 2020; Nguyen et al., 2020). Since then, it has been shown indeed that a recent history of CCCoV infections reduces the severity of COVID-19 infections (Sagar et al., 2021), and a considerable number of studies have investigated SARS-CoV-2 T cell epitopes (summarized in this article). The present study screened the recent literature to investigate—in line with the hypothesis of our earlier report (Dijkstra & Hashimoto, 2020)—which of the identical peptides shared between SARS-CoV-2 and any of the CCCoVs have been confirmed experimentally to bind to MHC molecules and/or to stimulate T cells. Arguably, the only one of such peptides convincingly reported as immunogenic by independent research groups is the helicase-derived peptide VYIGDPAQL which binds MHC-I allele HLA-A24:02 and then can stimulate CD8+ T cells. In some populations, such as the Japanese, HLA-A24:02 is found in >50% of the individuals and the allele may affect their resistance against COVID-19.\n\n\nMethods\n\nAs we did before (Dijkstra & Hashimoto, 2020), proteins encoded by a reported genomic sequence for SARS-CoV-2 (GenBank MN908947; Wu et al., 2020) were compared with those for HCoV-OC43 (NC_005147; Vijgen et al., 2005), HCoV-HKU1 (NC_006577; Woo et al., 2005), HCoV-229E (NC_002645; Thiel et al., 2001), and HCoV-NL63 (NC_005831; van der Hoek et al., 2004) by performing BLAST homology searches at the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and by making multiple sequence alignments using CLUSTALW software (https://www.genome.jp/tools-bin/clustalw); continuous stretches of 9 aa acids identical between SARS-CoV-2 and one of the other viruses were identified manually. A complete list of the detected 9-mers, minus a very few that we had missed at that time, are shown in Dijkstra & Hashimoto (2020). Furthermore, from published reports found by Google and PubMed searches, sequences of SARS-CoV-2 peptides reported to activate T cells were compared with the above listed CCCoV proteomes using tblastn (align) function at the NCBI database, in search for identical sequences in the case of 8-mers (which we did not find) or stretches of ≥9 consecutive identical aa in the case of 9-mers or larger peptides. The peptide sequences collected by either method were screened against the Immune Epitope Database (IEDB; http://www.iedb.org/; Dhanda et al., 2019) for reports in the human species context, and, unless these database reports represented the same studies that also appeared in article form, their IEDB information was added to Table 2 (available here). The collected peptide sequences were also analyzed by ANN 4.0 software at IEDB Analysis Resource (http://tools.immuneepitope.org/mhci/) for prediction of their affinity to a set of representative human MHC-I alleles, which were chosen because of their global abundancy, their relevance for the presented data, or as representatives of MHC-I supertypes (Lund et al., 2004). Identical 9-mers for which no MHC binding was found or predicted, for which no labeling or activation of T cells was reported, and which were not part of larger immunogenic T cell epitopes, were not included in Table 2.\n\nThe HLA allele frequencies as shown in Table 3 are based on data as summarized in the Allele Frequency Net Database http://www.allelefrequencies.net/ (settings: HLA > HLA classical allele freq search) (Gonzalez-Galarza et al., 2020).\n\n\nResults and discussion\n\nThe first two studies that experimentally investigated possible CCCoV-induced T-cell memory against SARS-CoV-2 were Grifoni et al. (2020) and Braun et al. (2020) (Leslie, 2020). Braun et al. (2020) tested CD4+ T cell activation using S (spike) protein derived peptide pools, whereas Grifoni et al. (2020) investigated both CD4+ T and CD8+ T cell activations using peptide pools derived from various SARS-CoV-2 proteins. Both groups found SARS-CoV-2 peptide pools to activate T cells from healthy donors (HD), proposedly representing memory T cells primed by similar peptides during earlier CCCoV infections. Notably, T cells of HD were also activated if their peripheral blood mononuclear cells (PBMC) were incubated with pools of peptides derived from not very well conserved SARS-CoV-2 proteins such as S (Braun et al., 2020; Grifoni et al., 2020), although—depending on the virus isolates—SARS-CoV-2 S protein does not share identical 9-mers with any of the CCCoVs but may share two identical 8-mer sequences (Dijkstra & Hashimoto, 2020). Braun et al. (2020) assumed that even a <50% identity between the corresponding SARS-CoV-2 and CCCoV peptides might explain the assumed cross-virus CD4+ T cell memory. Similarly, the authors of the Grifoni et al. (2020) study—answering our question on how S-derived peptides could activate cross-virus CD4+ and CD8+ T cell memory despite not sharing identical 9-mers or (presumably) immunogenic identical 8-mers—explained that CD8+ T cell activation in their type of in vitro assay could be found for a substantial part of any peptides sharing only ≥70% identity and that for CD4+ T cell activation the requirements for peptide similarity were even lower (see comments section below the Grifoni et al., 2020 article at https://www.cell.com/cell/fulltext/S0092-8674(20)30610-3). Although it has been well established that T cells can be promiscuous in recognizing pMHC (MHC + peptide) complexes, the extents to which this is relevant for in vivo T cell memory and does or does not tend to involve peptides with very similar sequences are being debated (e.g., Grant et al., 2016; Petrova et al., 2012). As explained by Peng et al. (2020), in vitro observations that suggested the existence of CCCoV-primed anti-SARS-CoV-2 “cross-reactive” T cell memory might alternatively be caused by the activation of naive T cells or of T cells primed by not-so-similar peptides of non-related pathogens. Possibilities for explaining in vitro observations suggesting cross-virus T cell memory in the case of non-identical peptides are summarized in Figure 2. Naturally, the proportions of false negative and false positive outcomes depend on the sensitivity of the assay. The best chance for in vitro results truly representing stimulation of the same T cells (TCR-identical T cells) activated during CCCoV and subsequent SARS-CoV-2 infections—hence representing functional memory—is if the MHC-presented peptides are identical. Hence, the present article predominantly focuses on such identical peptides.\n\nEven if the CCCoVs do possess a very similar sequence, the in vitro activation does not need to be indicative for peptide-A being an epitope for in vivo cross-virus T cell memory. In the model, peptide-A is either used directly or as part of pMHC complexes, and the T cells are stimulated after their isolation or as part of PBMC.\n\nTable 1 summarizes the Grifoni et al. (2020) and Braun et al. (2020) studies, as well as later studies that experimentally investigated potential SARS-CoV-2 T cell epitopes. Section A of the table summarizes studies that only investigated peptide pools and/or intact proteins, and section B summarizes the studies that (also) investigated individual peptides. The table explains whether indications for CD4+ and/or CD8+ T cell activation were observed, whether MHC association was addressed and/or MHC binding tested, and whether indications for SARS-CoV-2 and CCCoV cross-virus T cell memory were investigated and observed. Importantly, the consensus was that anti-SARS-CoV-2 T cell memory in individuals that had only been infected with CCCoVs was weak and only detectable in a subset of individuals (Sette & Crotty, 2020). It should be noted that none of the studies listed in Table 1 had specifically selected individuals with a known recent history of CCCoV infection, although presumably in such group a stronger T cell response against SARS-CoV-2 antigens would be found (Sagar et al., 2021).\n\n(a) In most of the listed studies experimental evidence was obtained for the existence of SARS-CoV-2-specific CD4+ and/or CD8+ T cells in COVID-19 convalescent donors\n\n(b) In many of the listed studies experimental evidence was obtained suggesting that CCCoV infections induced, or could induce, anti-SARS-CoV-2 T cell memory. Naturally, no samples were used of healthy donors without CCCoV infection history, and for this table, as done in the majority of the listed studies, all positive reactions in healthy donors that indicated SARS-CoV-2-specific T cell activation were interpreted as indications for possible cross-virus T cell memory. In the Habel et al. (2020) study, for T cells from healthy donors activations of similar extent were found for SARS-CoV-2 peptides and peptides from other pathogens for which the donors did not have an infection history.\n\n(c) Some of the listed studies determined the association (a) of T cell responses with MHC alleles or found binding (b) of peptides to MHC alleles\n\n(d) This column lists the proteins or peptides that were investigated. In most cases, though not all, there had been a preselection of peptides based on software predictions for MHC binding. In addition, positive findings for identical 9-mers shared between SARS-CoV-2 and at least one of the CCCoVs are summarized, with VYI and SPR peptides highlighted in bold.\n\n(e) The 3-letter names for peptides here only refer to the 9-mers \"Not specified\" indicates that it was not determined whether reacting cells were CD4+ or CD8+ T cells.\n\nA question mark is added if we are uncertain about what the authors did.\n\nn.d. = not determined\n\nData, and also the nomenclature, were retrieved from the Allele Frequency Net Database.\n\nOnly populations with HLA-A24:02 frequencies ≥0.29 and Japanese Ainu are listed.\n\nTable 2 lists the subset of the 9-mers that are identical between SARS-CoV-2 and at least one of the CCCoVs (Dijkstra & Hashimoto, 2020) and additionally were predicted to bind representative MHC-I alleles or experimentally found to bind MHC-I, be part of MHC-II binding peptides, or to activate T cells. It also lists >9-mers for which evidence of T cell activation was reported. The experimental results summarized within Table 2 were collected from the articles listed in Table 1, from articles on SARS-CoV-1, and from reports uniquely deposited to the Immune Epitope Database and Analysis Resource (IEDB; http://www.iedb.org/; Dhanda et al., 2019). The 9-mer peptides listed in Table 2 are referred to in Table 1 by using the letter code of their N-terminal three amino acids.\n\nThe only identical T cell epitope repeatedly found to be immunogenic by independent research groups was peptide “VYI” (VYIGDPAQL) (highlighted in bold in Table 1; details in Table 2). The VYI peptide is shared between SARS-CoV-2, HCoV-HKU1, and HCoV-OC43 viruses and part of a larger identical stretch AKHYVYIGDPAQLPAPR in their helicase protein (aka nonstructural protein 13 or NSP13). Prachar et al. (2020) showed that the VYI peptide bound to HLA-A24:02, although not very stable. To our knowledge, all studies that investigated the VYI peptide—three in total—found it to stimulate T cells in an HLA-A24:02 context (Ferretti et al., 2020; Kared et al., 2020; Nelde et al., 2021). Ferretti et al. (2020) investigated the entire SARS-CoV-2 proteome by a “T-scan” assay measuring activation of CD8+ memory T cells from COVID-19 convalescent donors after incubation with HEK293 cells engineered to express a single HLA allele and one of a set of overlapping 61 aa stretches. By this method, Ferretti et al. (2020) detected only three SARS-CoV-2 stretches with “dominant” epitopes that activated CD8+ T cells from multiple HLA-A24:02+ COVID-19 convalescent donors; one of these three stretches encompassed the VYI peptide and stimulated two of five investigated samples. Involvement of the VYI peptide was confirmed by activation of the HLA-A24:02+ T cells upon coculturing with HLA-A24:02+ target cells pulsed with VYI peptide. If for the T-scan screen 61 aa stretches of CCCoV proteomes were used instead, Ferretti et al. (2020) appear to have found a weak but noticeable response by HLA-A24:02+ memory CD8+ T cells in the cases of HCoV-HKU1 and HCoV-OC43 (our interpretation of their Figure 5A). Kared et al. (2020) performed a binding assay, testing 94 peptides from across the SARS-CoV-2 proteome—predicted by software to bind HLA-A24:02— using pHLA-A24:02 tetramers for labeling of CD8+ T cells from five HLA-A24:02+ COVID-19 convalescent donors. They found positive reactions for only eight of the 94 peptides. One of these eight peptides was the VIY peptide, which detectably labeled T cells of only one of the five donors. Regarding possible CCCoV-induced anti-SARS-CoV-2 T cell memory, Kared et al. (2020) mentioned “Notably, SARS-CoV-2 specific CD8+ T cells were not detected in any of the healthy donors recruited before the official SARSCoV-2 pandemic;” their number of HD controls, however, was low, and for their HLA-A24:02-matched experiments seems to have been between one and four. Nelde et al. (2021) tested ten predicted HLA-A24:02 SARS-CoV-2 epitopes and found the VYI peptide to be one of the three “dominant T cell epitopes” as it elicited activation of CD8+ T cells from seven of ten HLA-A24:02+ COVID-19 convalescent donors upon incubation with their PBMC. On the other hand, for PBMC of 17 healthy HLA-A24:02+ donors a stimulation of CD8+ T cells could not be observed. To summarize these three studies, the VYI peptide is among the most immunogenic SARS-CoV-2 T cell epitopes in HLA-A24:02+ individuals, although there is no evidence yet that this was primed by previous CCCoV infections. Presumably, because of the latter, none of the three studies mentioned that VYI peptide is shared between SARS-CoV-2, HCoV-HKU1, and HCoV-OC43 (Ferretti et al., 2020; Kared et al., 2020; Nelde et al., 2021). We assume that not finding anti-VYI T cells in HLA-A24:02+ HD was only a matter of assay sensitivity, because CCCoV-induced T cell memory is expected as the VYI peptide is embedded in a longer identical AKHYVYIGDPAQLPAPR stretch shared between SARS-CoV-2, HCoV-HKU1, and HCoV-OC43; thus, the MHC-I pathway processing of the peptide is expected to be similar in each viral background, and software predicts that the immunoproteasome efficiently generates the VYI peptide from all three viruses (https://imed.med.ucm.es/Tools/pcps/; Gomez-Perosanz et al., 2020). An additional reason for assuming the involvement of CCCoV-induced T cell memory is that the helicase is not one of the more abundant (structural) viral proteins (Davidson et al., 2020) whereas nevertheless VYI is consistently found as one of the dominant T cell epitopes. Future experiments selectively investigating HLA-A24:02 donors with a recent HCoV-HKU1 or HCoV-OC43 infection will probably be more sensitive in detecting anti-VYI CD8+ T cells primed by CCCoV infection.\n\nHLA-A23:01 belongs to the same “supertype” as HLA-A24:02 (Lund et al., 2004) meaning that they tend to bind similar peptides, and in the IEDB database it is reported that HLA-A23:01 also binds VYI peptide (IEDB Reference:1000425). It has not been described yet whether VYI peptide can stimulate T cells in an HLA-A23:01 context.\n\nAlthough not identical throughout the sequence, as an exception, Table 2 also lists the 9-mer SPRWYFYYL (“SPR”) for SARS-CoV-2 and its matching LPRWYFYYL (“LPR”) for HCoV-HKU1 and HCoV-OC43. Several independent studies (Table 1; SPR indicated in bold) suggest that SPR is highly immunogenic and involved in cross-virus immune memory (summarized in Table 2). The only amino acid difference between the SPR and LPR peptides is at the P1 position, which is not necessarily important for peptide conformation in pMHC-I complexes or for binding T cell receptors (TCRs) (e.g., Sewell, 2012). Thus, it seems plausible that a fraction of the T cells primed by pMHC/LPRWYFYYL may also recognize pMHC/SPRWYFYYL. It was convincingly shown that SPR peptide binds HLA-B07:02 alleles and in that context can stimulate CD8+ T cells from COVID-19 convalescent donors (Ferretti et al., 2020; Kared et al., 2020; Peng et al., 2020; Schulien et al., 2021; Snyder et al., 2020). CD8+ T cells from HLA-B07:02+ HD could also be stimulated by SPR, suggesting cross-virus CD8+ T cell memory induced by the LPR peptide of HCoV-HKU1 or HCoV-OC43 (Schulien et al., 2021), which is consistent with in vitro results showing that CD8+ memory T cells from HLA-B07:02+ COVID-19 convalescent donors were activated by HLA-matched cells expressing 61 aa fragments of HCoV-HKU1 or HCoV-OC43 encompassing the LPR peptide (Ferretti et al., 2020). However, SARS-CoV-2 peptides encompassing or even only partially overlapping the SPR peptide also induced responses—remarkably strong in some cases—of CD4+ T cells from COVID-19 convalescent donors and, to a lesser extent, from HD (also summarized in Table 2; Le Bert et al., 2020; Nelde et al., 2021; Peng et al., 2020; Tarke et al., 2020). The combined results suggest an overlap of highly immunogenic MHC-I and MHC-II epitopes, and—although such overlap is not impossible—some caution for the possibility that SPR peptide might (additionally) cause non-MHC-restricted immune stimulation seems to be warranted. Except for HLA-B07:02, SPR peptide was also found to bind the MHC-I alleles HLA-B51:01, -53:01, and -54:01 (IEDB database Reference:1000425).\n\nOther than VYI, we did not find any other identical peptides shared between SARS-CoV-2 and CCCoVs for which current publications convincingly indicate a high immunogenicity. However, the 9-mers FVDGVPFVV (“FVD”) and LPYPDPSRI (“LPY”) could be promising, although they were only reported as immunogenic CD8+ T cell epitopes in single publications. Snyder et al. (2020) appear to have identified FVD peptide as an immunodominant SARS-CoV-2 T cell epitope in the HLA-A02:01 context, although their descriptions of this matter could be more detailed. Tarke et al. (2020) found that CD8+ T cells from a few HLA-B51:01+ COVID-19 convalescent donors could be stimulated by several peptides that encompassed the LPY 9-mer peptide sequence, namely peptide DYVYLPYPDPSRI (a 13-mer shared between SARS-CoV-2 and HCoV-HKU1), its shorter versions VYLPYPDPSRI (an 11-mer) or YLPYPDPSRI (a 10-mer), or peptide LPYPDPSRIL (a 10-mer); software predicts that the encompassed LPYPDPSRI (a 9-mer) is the best HLA-B51:01 binder and that of the other lengths only the 10-mers YLPYPDPSRI and LPYPDPSRIL are expected to bind this allele (Table 2), suggesting that some processing of the longer peptides may explain the combined results. Future analysis of the immunogenicity of the LPY 9-mer peptide would be interesting.\n\nIn addition to the above, Mateus et al. (2020) and Tarke et al. (2020) found some other SARS-CoV-2 peptides that encompassed ≥9-mers shared with CCCoVs and stimulated CD4+ T cells (Table 2), but whether those identical stretches formed the immunogenic epitope has not been determined yet.\n\nFor speculation on the global implications of the ability of different MHC-I alleles to bind conserved peptides, their global distributions must be appreciated. The online Allele Frequency Net Database (AFND; http://allelefrequencies.net/; Gonzalez-Galarza et al., 2020) comprises information of MHC-I allele frequencies in different populations, and Figure 3 includes their visual summaries of global distributions of the alleles HLA-A24:02 (binder of VYI peptide), HLA-B07:02 (binder of SPR peptide), HLA-A02:01 (binder of FVD peptide), and HLA-B51:01 (predicted binder of LPY peptide). Table 3 lists populations with the highest frequencies of HLA-A24:02 according to AFND. For all studies summarized in Table 3 the allele frequencies and for a subset also the percentage of individuals carrying the allele had been determined (note that if randomly distributed, an allele frequency of >0.29 would correspond to a prevalence of >50%). Data for populations in Japan and for Japanese in the USA are highlighted in shades of gray (Table 3). In Japanese populations the HLA-A24:02 allele frequencies are ≥0.33 except for in the Ainu which are a racial minority indigenous to Hokkaido in Northern Japan. Ikeda et al. (2015; the “Japan pop 16” study in Table 3) investigated 18604 individuals from “all parts of Japan” and the authors concluded that the HLA-A24:02 allele is distributed in ~60% of the Japanese population. Other populations in which HLA-A24:02 is present in >50% of the investigated members are various indigenous populations in Asia, Oceania, and the Americas (Table 3). Those non-Japanese populations are not further discussed in this study as it is harder to collect their data relevant to COVID-19.\n\nCircles refer to individual studies with the color indicating the detected allele frequency following the color bar. See http://www.allelefrequencies.net/ for more detailed information on those studies. Permission to reproduce this image was obtained from AFND.\n\nHLA-B07:02 is common in Northern Europe (Figure 3) and is carried by approximately a third of the Irish population (reports at AFND). HLA-A02:01 is common in Europe, and, for example, found in 49% of the Polish population (report at AFND). HLA-B51:01 is common in Southern Europe and the Middle East (Figure 3) and was found at an allele frequency of 0.19 in Saudi Arabia (report at AFND).\n\nA year into the pandemic, the accumulated number of COVID-19 deaths per million inhabitants in Japan is 45, which is >30 times fewer than in countries such as Italy (1465), the UK (1559), and the USA (1362) (on February 1st, 2021, according to https://www.worldometers.info/coronavirus/). At least for the initial wave of the disease in the first half of 2020 the apparent low prevalence of COVID-19 was supported by finding specific antibodies in only ~0.1% of citizens of Tokyo in June 2020 (governmental report https://www.mhlw.go.jp/content/000648706.pdf) and minimal or even negative excess mortality rates until at least July 2020 (Yorifuji et al., 2021). These low numbers are quite surprising since the stringency of behavioral regulations to protect against COVID-19 have been less severe in Japan than in most Western countries (https://www.bsg.ox.ac.uk/research/research-projects/coronavirus-government-response-tracker; Hale et al., 2020). The surprise about the relatively low incidence of COVID-19 in Japan was captured well in the title of a BBC article on July 2020 “Coronavirus: Japan's mysteriously low virus death rate” (https://www.bbc.com/news/world-asia-53188847). Most of the difference with Western countries can probably be explained by voluntary behaviors such as the willingness of the Japanese to wear masks (despite absence of obligation) and by better individual health status such as a relatively low prevalence of obesity. As a note, however, the Japanese are not per se better protected against respiratory viruses, as mortalities per capita resulting from the 2009 influenza pandemic were higher than in European countries (Simonsen et al., 2013).\n\nIn Japan, as in other countries, CCCoV infections are poorly monitored, but available data indicate that from the winter 2014–2015 until November 2019 (we were not able to find more recent data) the most common CCCoV species in Japan has been HCoV-OC43. Especially during the winters of 2014–2015 and 2018–2019, this virus species appears to have been prevalent (Komabayashi et al., 2020; Kubota-Koketsu et al., 2020). Thus, many Japanese individuals probably received a relatively recent immune boost with the VYI peptide.\n\nCurrently, in the winter 2020–2021, there has been a surge in the number of COVID-19 cases and deaths in Japan (https://www.worldometers.info/coronavirus/; https://www.aljazeera.com/news/2021/1/4/japan-weighs-state-of-emergency-amid-severe-covid-19-surge), and it is unclear whether the factors that during the first half of 2020 protected the Japanese better than the populations of many other countries are still in place. Possibly, the warm winter season of 2019–2020 (Japan Meteorological Agency reports https://www.data.jma.go.jp/obd/stats/data/en/smp/index.html), and the associated early hay fever season (https://global.weathernews.com/news/13178/; http://kafun.taiki.go.jp/), may have helped to protect the Japanese population during the first half of 2020. In Japan, largely because of aging monoculture coniferous forests, depending on the definition approximately half of the population may be considered to suffer from pollen-induced allergic rhinitis, which has been called a national affliction (e.g., Minami et al., 2019; Nakamura et al., 2019; Yamada et al., 2014). Although speculative, the associated inflammation of the respiratory tract might non-specifically elevate both innate and specific (possibly anti-VYI T cells) immunity against COVID-19. Although each of these individual biological factors probably is not very protective against COVID-19 (see also the below paragraph), at the population scale a combination of such factors might significantly impact the virus reproduction number (R).\n\nIn short, (i) most Japanese individuals possess the MHC-I allele HLA-A24:02 that presents a highly immunogenic SARS-CoV-2 T cell epitope VYI, (ii) in recent years many of them have been exposed to this epitope by HCoV-OC43 infection, and (iii) at the time of the first COVID-19 wave many of them had an elevated immune status of their respiratory tracts because of pollen allergy.\n\nMHC polymorphism is believed to be driven by differences in immune responses conferred by the different alleles, but actual evidence for this to cause differences in disease resistance is close to absent (Kelly & Trowsdale, 2017; Yamaguchi & Dijkstra, 2019). Presumably, this is caused by each set of MHC alleles having enough choice within a pathogen proteome for presenting some peptides efficiently to the immune system. Theoretically, the MHC allelic effect on differences in disease resistance should become larger if the choice of possible immunogenic epitopes becomes more limited. Hence, the effect of MHC polymorphism on immune memory induced against a related virus should be larger than on immune memory against the same virus. However, several studies have investigated the association of MHC polymorphism with differences in COVID-19 resistance, and—arguably—no convincing associations have yet been presented (Iturrieta-Zuazo et al., 2020; Littera et al., 2020; Lorente et al., 2021; Wang et al., 2020). From these combined within population studies, however, it probably follows that HLA-A24:02 does not necessarily have a detectable positive or negative impact on the two commonly analyzed COVID-19 parameters, which are the frequency of contracting COVID-19 and the severity of the disease. As far as we know, there has not been a thorough investigation yet of a possible association between MHC polymorphism and the virus titers in the upper respiratory tract. The severity of COVID-19 is predominantly determined by whether the virus infects the lower respiratory tract (Cyranoski, 2020), which, curiously, has been described as largely disconnected from the level of virus replication in the upper respiratory tract (He et al., 2020; Lavezzo et al., 2020). He et al. (2020), for example, stated “There was no obvious difference in viral loads across sex, age groups and disease severity.” Therefore, if cross-virus memory T cells would not be sufficient to help block an infection but instead help to expedite the end of upper respiratory tract infections, the HLA-A24:02 allele might give protection at the population level by limiting spread without having an impact on the COVID-19 parameters typically studied in the HLA-association studies (contraction and severity at the level of individuals). We speculate that HLA-24:02-restricted anti-VYI CD8+ T cell immune memory—especially if recently boosted by HCoV-OC43 or HCoV-HKU1 infections or nonspecific immune stimulations—can reduce the total number of virus particles secreted by COVID-19 patients and so the replication number (R) of the virus at the population level.\n\n\nConcluding remarks\n\nThe only CD8+ T cell epitope shared between CCCoVs and SARS-CoV-2 for which the immunogenicity was convincingly proven is the VYIGDPAQL peptide if presented by HLA-A24:02. This MHC-I allele is found in the majority of the Japanese population and may help explain their surprising resistance to the virus. More studies on T cells activated during CCCoV infections and on possible associations of MHC alleles with COVID-19 parameters are necessary. Considering that for the S protein there may not be meaningful CCCoV-induced anti-SARS-CoV-2 cross-virus immune memory (depending on how one interprets the various publications)—related to the weak conservative pressure on this protein—it is feasible that SARS-CoV-2 will mutate its S proteins and escape the immune protection induced by the current generation of S-only vaccines. Potentiating such vaccines by adding immunogenic peptides from better conserved parts of the proteome, for example VYI peptide in the case of HLA-A24:02+ individuals, may be a viable option to help prevent such immune escape.\n\n\nData availability\n\nSevere acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, Accession number MN908947: https://www.ncbi.nlm.nih.gov/nuccore/MN908947\n\nHuman coronavirus OC43, complete genome, Accession number NC_005147.1: https://www.ncbi.nlm.nih.gov/nuccore/NC_005147.1\n\nHuman coronavirus HKU1, complete genome, Accession number NC_006577: https://www.ncbi.nlm.nih.gov/nuccore/NC_006577\n\nHuman coronavirus 229E, complete genome, Accession number NC_002645: https://www.ncbi.nlm.nih.gov/nuccore/NC_002645\n\nHuman Coronavirus NL63, complete genome, Accession number NC_005831: https://www.ncbi.nlm.nih.gov/nuccore/NC_005831", "appendix": "References\n\nAltmann DM, Boyton RJ: SARS-CoV-2 T cell immunity: Specificity, function, durability, and role in protection. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou W, Wang W, Wang H, et al.: First infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood. BMC Infect Dis. 2013; 13: 433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–273. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "81416", "date": "26 Apr 2021", "name": "Brian M. Baker", "expertise": [ "Reviewer Expertise T cell immunology", "T cell recognition", "antigen presentation", "immune crossreactivity." ], "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\nDijkstra and colleagues describe a meta analysis of potential cross-reactivity between T-cell epitopes of common coronaviruses and SARS-CoV2. After a comprehensive review, they identify only one possible HLA-A2402 presented cross-reactive epitope. As a meta analysis there is no new experimental data provided to review, but the the analysis of prior data is comprehensive and rigorous. The work will be valuable in responding to the ongoing CoV2 pandemic, understanding immunity (and potential pre-existing immunity), and in the planning of next generation vaccines.\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": "81412", "date": "26 Apr 2021", "name": "Andrea J. Sant", "expertise": [ "Reviewer Expertise CD4 T cells and MHC restricted antigen presentation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the process of definitive epitope discovery for SARS-Cov-2 and the relevance of particular peptide epitopes because of the prevalence of particular HLA restriction elements in particular populations, and the potential for cross-reactive memory to be recalled from immunity established from endemic seasonal coronavirus infections. Thus, the impact of definitive epitope discovery is high in these situations.\nWhile the conclusions of the study and the major strategies offered are valuable, some trimming of the manuscript could make this more accessible to the reader. I recommend that the authors select the most essential features of past studies to illustrate key points that are necessary to present to the reader.\nI also recommend that the peptide be named by its full spanning amino acids (first and last) for clarity rather than “VYI”.\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? I 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": "81415", "date": "05 May 2021", "name": "Anna Gil", "expertise": [ "Reviewer Expertise T cell immunology in humans", "T cell receptor repertoire", "CD8+T cell cross-reactivity" ], "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 summarizes a very important report on possible MHC-I binding epitopes shared between common cold coronaviruses and SARS-CoV-2. It presents a detailed and thorough analysis of existing literature in a single setting. Based on the reports focused on T cell response restricted to HLA-A24:02, the authors rightly suggest that the future vaccine design should include immunogenic peptides from more conserved parts of the SARS-CoV2 proteome.\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? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-196
https://f1000research.com/articles/9-1316/v1	12 Nov 20	{ "type": "Systematic Review", "title": "Global prevalence and pathogenesis of headache in COVID-19: A systematic review and meta-analysis", "authors": [ "Endang Mutiawati", "Syahrul Syahrul", "Marhami Fahriani", "Jonny Karunia Fajar", "Sukamto S. Mamada", "Helnida Anggun Maliga", "Nur Samsu", "Muhammad Ilmawan", "Yeni Purnamasari", "Annisa Ayu Asmiragani", "Ichsan Ichsan", "Talha Bin Emran", "Ali A. Rabaan", "Sri Masyeni", "Firzan Nainu", "Harapan Harapan", "Syahrul Syahrul", "Marhami Fahriani", "Jonny Karunia Fajar", "Sukamto S. Mamada", "Helnida Anggun Maliga", "Nur Samsu", "Muhammad Ilmawan", "Yeni Purnamasari", "Annisa Ayu Asmiragani", "Ichsan Ichsan", "Talha Bin Emran", "Ali A. Rabaan", "Sri Masyeni", "Firzan Nainu", "Harapan Harapan" ], "abstract": "Background: This study was conducted to determine the prevalence of headache in coronavirus disease 2019 (COVID-19) and to assess its association as a predictor for COVID-19. This study also aimed to discuss the possible pathogenesis of headache in COVID-19. Methods: Available articles from PubMed, Scopus, and Web of Science were searched as of September 2nd, 2020. Data on characteristics of the study, headache and COVID-19 were extracted following the PRISMA guidelines. Biases were assessed using the Newcastle-Ottawa scale. The cumulative prevalence of headache was calculated for the general population (i.e. adults and children). The pooled odd ratio (OR) with 95% confidence intervals (95%CI) was calculated using the Z test to assess the association between headache and the presence of COVID-19 cases. Results: We included 104,751 COVID-19 cases from 78 eligible studies to calculate the global prevalence of headache in COVID-19 and 17 studies were included to calculate the association of headache and COVID-19. The cumulative prevalence of headache in COVID-19 was 25.2% (26,464 out of 104,751 cases). Headache was found to be more prevalent, approximately by two-fold, in COVID-19 patients than in non-COVID-19 patients with symptoms of other respiratory viral infections, OR: 1.73; 95% CI: 1.94, 2.5 with p=0.04. Conclusion: Headache is common among COVID-19 patients and seems to be more common in COVID-19 patients compared to those with the non-COVID-19 viral infection. No definitive mechanisms on how headache emerges in COVID-19 patients but several possible hypotheses have been proposed. However, extensive studies are warranted to elucidate the mechanisms. PROSPERO registration: CRD42020210332 (28/09/2020)", "keywords": [ "COVID-19", "SARS-CoV-2", "headache", "severity", "predictor" ], "content": "Introduction\n\nThe current coronavirus disease 2019 (COVID-19) pandemic has caused a global crisis for both the health and economic sectors. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a member of the Coronavirinae family and Betacoronavirus subfamily together with severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle Eastern respiratory syndrome coronavirus (MERS-CoV)1. The virus is primarily transmitted from person-to-person through droplets from symptomatic and pre-symptomatic patients, and is is likely to also be transmitted by asymptomatic individuals2–6. Currently, no effective vaccines or pharmaceutical agents against SARS-CoV-2 are available but some progressions have been made to produce vaccines and drugs against the disease7–10.\n\nAlthough up to 20.3% of hospitalized patients require admission to the intensive care unit (ICU)11 with complications such as hypoxemia, acute respiratory distress syndrome (ARDS), arrhythmia, shock, acute cardiac injury, and acute kidney injury12–14, most SARS-CoV-2 infections are asymptomatic or have mild symptoms1,15,16. The common clinical symptoms of COVID-19 include fever, dry cough, dyspnea, chest pain, fatigue and myalgia1,12,17,18. In some cases, other neurological manifestations such as headache, dizziness, seizure, taste and smell impairment were also reported12,18–21. Headache is one of the symptoms that is also reported in various viral infections such as dengue and chikungunya that are common in the tropical regions22,23 and therefore may not be specific for COVID-19. In addition, the prevalence of headache in COVID-19 patients varies across studies19,20,24. A study found that the prevalence of headache was 17.4% (94/540) in Hubei province, the epicenter of the outbreak, and 14.1% (111/788) among patients outside the epicenter21. Another study in European countries found that the headache was reported in more than 40% of 417 COVID-19 patients19. Furthermore, the association of headache with the presence of COVID-19 is unknown. This systematic review was undertaken to provide robust evidence on the prevalence of headache in COVID-19 patients globally and its association with COVID-19 cases. Information described in this study might help clinicians to decide whether headache could be used as one of the basic symptoms to be included in diagnosing SARS-CoV-2 infection, especially those in the front line with limited resources.\n\n\nMethods\n\nThis systematic review was conducted as recommended by the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines25. The protocol of this study was registered with PROSPERO, an international database of prospectively registered systematic reviews at the University of York, on 28th September 2020 (CRD42020210332).\n\nArticles reporting headache as a symptom of COVID-19 cases were included. COVID-19 cases should be diagnosed with RT-PCR test using either nasopharyngeal and oropharyngeal swab, bronchoalveolar lavage or cerebrospinal fluid (CSF). All cross-sectional and cohort studies that included COVID-19 cases randomly selected from the population were considered eligible while case reports and case series including all editorials, reviews, and commentaries were excluded. Case-control studies with pre-allocated number of patients with headache and non-headache were excluded. Studies that were conducted in specific populations only such as in pregnancy, children, cancer patients and other groups were excluded. Only articles written in English were included in this study.\n\nTo identify potential articles for analysis, systematic searches were conducted using three bibliographical databases (PubMed, Scopus, and Web of Science as of September 2nd, 2020). The search criteria were as follows. Pubmed ([Title] \"SARS-CoV-2\" OR \"COVID-19\" OR \"Wuhan coronavirus\" OR \"Wuhan virus\" OR \"novel coronavirus\" OR \"nCoV\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus disease 2019 virus\" OR \"2019-nCoV\" OR \"2019 novel coronavirus\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus\" OR \"coronaviruses\" OR \"SARS 2\" OR \"2019-nCoV acute respiratory disease\" OR \"novel coronavirus pneumonia\"OR \"COVID\") AND ([All] “Headache”). Scopus ([Title] \"SARS-CoV-2\" OR \"COVID-19\" OR \"Wuhan coronavirus\" OR \"Wuhan virus\" OR \"novel coronavirus\" OR \"nCoV\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus disease 2019 virus\" OR \"2019-nCoV\" OR \"2019 novel coronavirus\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus\" OR \"coronaviruses\" OR \"SARS 2\" OR \"2019-nCoV acute respiratory disease\" OR \"novel coronavirus pneumonia\"OR \"COVID\") AND ([All] “Headache”). Web of Science ([Title] \"SARS-CoV-2\" OR \"COVID-19\" OR \"Wuhan coronavirus\" OR \"Wuhan virus\" OR \"novel coronavirus\" OR \"nCoV\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus disease 2019 virus\" OR \"2019-nCoV\" OR \"2019 novel coronavirus\" OR \"severe acute respiratory syndrome coronavirus 2\" OR \"coronavirus\" OR \"coronaviruses\" OR \"SARS 2\" OR \"2019-nCoV acute respiratory disease\" OR \"novel coronavirus pneumonia\"OR \"COVID\") AND ([All] “Headache”). English as language limitation was imposed in the searches. Only peer-reviewed articles were included. Data were extracted both from the articles and the supplementary materials. Reference lists from the eligible articles were retrieved for further relevant studies.\n\nAll titles and abstracts of identified articles were imported into the EndNote X9 (Thompson Reuters, Philadelphia, PA, USA) and duplicate records between databases were removed. Retrieved articles were initially screened based on title and abstract to identify possible eligible studies. The full texts of potentially eligible articles were then reviewed. The screening and review processes were conducted by two authors (MF and JKF). After reviewing the full texts, the eligibility of each study was decided.\n\nThe following data were extracted from eligible articles: study characteristics (author, title, journal, study site and study design), headache characteristics (number of patients with headache, type of headache, localization, and severity), COVID-19 characteristics (number of patients with COVID-19, severity, and outcome).\n\nThis study received no external funding.\n\nThe primary outcomes of this systematic review were: a) the prevalence of headache in COVID-19 cases; and b) the association between headache and COVID-19 cases compared to other viral infections.\n\nThe cumulative prevalence rate of headache was calculated for COVID-19 cases in the general population. The prevalence was calculated as the number of COVID-19 cases with headache divided by the total number of COVID-19 cases with and without headache, expressed as a percentage (%). Pooled odd ratios (OR) and 95% confidence intervals (95% CI) were calculated to assess the association of headache and COVID-19 compared to non-COVID-19 cases.\n\nTo reduce sample selection bias, a critical assessment was specifically conducted in terms of setting of study and diagnosis of COVID-19. The quality of eligible studies was assessed using critical appraisals based on the Newcastle-Ottawa scale (NOS)26.\n\nThe association between headache and the presence of COVID-19 was assessed by the calculation of a pooled OR and 95%CI using the Z test (p<0.05 was considered statistically significant). Prior to analysis, gathered data from studies were evaluated for heterogeneity and potential publication bias. Heterogeneity among studies was assessed using the Q test. Initial analysis found that the data had heterogeneity (p<0.10) and therefore a random effect model was employed. Egger’s test and a funnel plot were used to assess the reporting or publication bias (p<0.05 was considered having potential for publication bias). The data were analyzed using Review Manager version 5.327. The cumulative pooled OR and 95%CI was presented in a forest plot.\n\n\nResults\n\nThe literature searches yielded 732 articles, of which 229 were excluded as duplicates between databases. Following a screening process of the titles and abstracts of the remaining 503 articles, an additional 253 articles were excluded due to irrelevant studies leaving 250 articles (Figure 1). The full texts of the remaining 250 articles were retrieved and screened for eligibility. This process excluded additional 49 articles that were not eligible as they did not fulfill the inclusion criteria. A full assessment was conducted on 201 articles.\n\nTo calculate the prevalence of headache in COVID-19, full-text assessment resulted in the exclusion of 123 articles for the following reasons: case control studies (n=2), case report studies (n=69), case series (n=29), duplicated dataset (n=1), and conducted in specific population only (n=23). The targeted population studies were conducted among healthcare workers28–31, diabetic patients32, pediatrics33, pregnant women34, cancer patients35, children and young adults36–40, patient undergone surgery41, critical patients42, women undergone delivery43, patients with mild-moderate COVID-1944–46, patients with gastrointestinal symptoms47, patients with severe headache only48, and patients with anosmia only49. In total, 78 studies were included to calculate the prevalence of headache in COVID-19 and all studies were published in 2020. The studies were conducted in Brazil50, China13,51–96, Egypt97, France98–101, Germany102, India103,104, Italy105–111, Japan112,113, Jordan114, Somalia115, South Korea116,117, Spain118,119, Turkey120,121, and the US122–126. Two studies were cross-sectional119,122, five were prospective cohort studies65,90,98,110,121 and the remaining 71 studies were retrospective studies.\n\nTo calculate the association between headache and COVID-19, the full-text assessment yielded 16 eligible studies. The rest of the references had been excluded for these reasons: (a) the studies were case reports or case-series (n=98); (b) the full-text did not include data of outcome of interest (n=84); and (c) low quality of study (n=3) (Figure 1). The included studies were conducted in a wide ranges of regions: Australia127, Belgium29, Brazil50, China70, France99, Hongkong128, Israel129, Italy130, Germany131, Netherlands31, Turkey120, and the US28,105,122,132,133. Out of the studies, ten were case-control28,29,31,99,105,120,128,130,132, four were cross-sectional50,70,122,131,133, and two were prospective cohort studies127,129.\n\nOur systematic review included 78 studies consisting of 104,751 COVID-19 patients and headache was reported in 26,464 patients with a cumulative prevalence of 25.26%. The list of the studies and the prevalence of headache of each study is presented in Table 1. One study which included 51 patients described the specific location of headache: 1.96% (1/51) was a temporal headache, 35.29% (18/51) was a frontal headache, 23.52% (12/51) was a retro-orbital headache, and 39.21% (20/51) was a diffuse headache99. Another study which involved 46 patients reported that 40 (86%) had tension-type pain and 6 (14%) had migraine-like headache107. Data from 18 studies indicated that 72.17% (236/327) of headaches were reported in mild-moderate COVID-19 cases51,53,55–58,62,63,67,76,88,92–97,117. The prevalence of headache in severe COVID-19 cases from 15 studies was 27.83% (86/309)53,55,57,58,62,63,73,76,88,92,93,95–97,117.\n\nA total of 16 studies, consisting of 5,407 COVID-19 cases in adults and 94,818 adults with non-COVID-19 infections (mostly COVID-19-like respiratory viral infections), were analyzed to determine the association between headache and COVID-19. Of these studies, an association between headache and the occurrence of COVID-19 was observed in 9 studies28,29,31,105,122,127,129,130,132 while seven studies reported no association50,70,99,120,131,133,134 (Table 2). Our cumulative calculation revealed that headache was found to be 1.7-fold more prevalent in patients with COVID-19 compared to those with non-COVID-19 respiratory viral infections, OR: 1.73; 95% CI: 1.94, 2.51 with p=0.04. The correlation between headache and COVID-19 is presented in Figure 2.\n\n\nDiscussion\n\nAs a non-specific symptom, headache might present not only in COVID-19 cases but also in other viral diseases, therefore, headache might not raise suspicion of SARS-CoV-2 infection22,23. However, a study described that headache is one of the main neurological symptoms of coronavirus infection including SARS-CoV-2135. The global prevalence of headache in our systematic review is more than 25% out of 104,751 COVID-19 cases. This result was almost double that of the previously reported prevalence from studies in China early in the pandemic that ranged from 6.5–13.1%53,88. This suggests that headache is prevalent in SARS-CoV-2 infection and therefore could potentially be used as one of the indicators to diagnose COVID-19 cases.\n\nOur study also highlights that headache was significantly more prevalent in COVID-19 patients, 2.2-fold, than suspected non-COVID-19 viral infection. A study found that only around 11% of MERS patients reported they suffered from headaches136. A study in COVID-19 patients with pre-existing primary headache disorders revealed that the headache during COVID-19 had an unusual presentation with 42% (44/104) reporting a recent onset of headaches, 49% (51/104) had a change in headache pattern, and 39% (39/104) reported the worst headache they had ever had119. These results suggest that new onset headache and changes of headache pattern should be carefully explored as this might be able to differentiate patients with COVID-19 from those without.\n\nWe explored the available literature to broaden our knowledge of the pathogenesis of headache in COVID-19. In general, three main primary headaches are observed in COVID-19 patients i.e. migraine, cluster headache and tension-type headache137–140. No fixed mechanisms have been reported on how these headaches emerge in COVID-19 patients. However, it has been proposed that the activation of trigeminal nerve ending in the periphery followed by the sensitization of various sites in the brain is one of the main pathomechanism of headache in these patients137,138.\n\nA headache attack is initiated by the release of several vasoactive neuropeptides such as glutamate, calcitonin gene-related peptide (CGRP), substance P and pituitary adenylate cyclase-activating polypeptide (PACAP), from nociceptive sensory fibers (especially nociceptive C-fibers and Aδ-fibers) innervating blood vessels located in the meninges and other cranial structures leading to vasodilation, degranulation of mast cells and plasma protein extravasation in those vascular structures141–143. The release of those peptides from the fibers could be due to either electrical, chemical or mechanical induction emerged from three branches of the trigeminal nerve i.e. ophthalmic, maxillary and mandibular branches143. However, because of its wider area of innervation in the meninges and cranial blood vessels, ophthalmic branch seems to play more of a role in stimulating nociceptive processes in meningeal structures than the other two branches143,144.\n\nNext, any physiological events, such as vasodilation, that have occurred in the meningeal and large cerebral blood vessels will become a stimulus which is sent to the trigeminal ganglion (TG) where other nociceptive information from other afferent trigeminal branches are also converging141. Although cerebral and meningeal vasodilation is not the sole cause of headache145, most studies have agreed on the critical role of blood vessel dilation in the emergence of headaches.\n\nFrom the TG, the stimulus is projected to an area in the brainstem called the trigeminocervical complex (TCC) via first-order neurons143. These transmissions are then projected to the diencephalon structures, including the thalamus and hypothalamus, via the second-order neurons143,146. The third-order neurons are subsequently responsible for transmitting the information from diencephalic systems to various cortical areas associated with motoric, somatosensory, auditory, retrosplenial and visual functions141,143, leading to the manifestation of headache pain and other related symptoms (Figure 3).\n\nNociceptive information coming from peripheral networks is transmitted to trigeminal ganglion acting as central hub between peripheral and central nervous systems. Next, this information is sent to TCC located in the brainstem, transmitted to diencephalon structures and terminated in various areas in cortex. The transmission in this pathway is linked to the pivotal involvement of neurotransmitters (e.g. glutamate, GABA and serotonin) and nociceptive neuropeptides (e.g. CGRP, substance P and PACAP) released from nerve fibers synapses, particularly nociceptive C-fibers and Aδ-fibers. The receptors of these signaling molecules are identified in both peripheral blood vessel, trigeminal ganglion and central structures, such as in cerebrospinal fluid and TCC142,147.\n\nDuring these transmission processes, the release of neuropeptides, especially CGRP, is limited only in the meninges and in the central terminals of trigeminal afferents147. When the transmission reaches TCC structures, CGRP and substance P may act to induce the release of glutamate and reduce gamma aminobutyric acid (GABA) production147,148. It has been proposed that during a headache attack, the level of glutamate in the TCC increases, while GABA release is decreased148. This condition could result in the increase of nociceptive neurons excitability149. Moreover, low level of serotonin in trigeminal nerve might also be involved in migraine pathophysiology as the release of this neurotransmitter has been linked to the inhibition of CGRP in trigeminal nerves150,151.\n\nSeveral mechanisms have been postulated to explain how trigeminovascular system is activated in COVID-19. Firstly, direct invasion of the virus may activate the peripheral trigeminal system137. This theory is hypothesized according to a study, confirming that trigeminal ganglia possess an angiotensinergic activity152. Thus, the viral attack would hypothetically disturb the activity of the renin-angiotensin-aldosterone system (RAAS), which may increase the level of CGRP153. Although the invasion of SARS-CoV-2 into the olfactory nerve ending seems to be the main route154–156, the action of the virus on the trigeminal nerve must not be overlooked, as suggested by Perlman et al. (1989). They demonstrated that the trigeminal nerve, in addition to the olfactory nerve, was a route used by neurotropic murine coronavirus to invade the central nervous system (CNS)157. The hypothesis of trigeminal nerve attack by SARS-CoV-2 is also supported by the fact that olfactory mucosa is innervated by the trigeminal nerve158,159 suggesting the invasion of the olfactory mucosa by SARS-CoV-2 may also induce trigeminal nerve injury.\n\nFollowing entry into the trigeminal nerve, SARS-CoV-2 may hijack the transneuronal transport system to direct the virus to enter the nucleus via a retrograde axonal transport mechanism. This transport occurs by the involvement of cytoskeletal motor proteins called dynein supported by cofactor dynactin that function to move substances, such as endosomes and vesicles, including hijacking viruses, on microtubule towards the cell body160. Once the virus gains access to the nucleus in the cell body through the microtubule-organizing center (MTOC), viral replication is initiated161. Finally, viral progenies may spread to other areas of the body, including the CNS, via anterograde axonal transport assisted by the kinesin motor protein family160.\n\nA study suggested a transneuronal transport system used by coronavirus after investigating the neuroinvasiveness of HCoV-OC43 in mice162. Furthermore, the movement of SARS-CoV-2 via retrograde axonal transport is also hypothesized as it has been reported that the envelope protein of SARS-CoV could subvert dynein function either directly or indirectly163. The role of dynein in the retrograde axonal movement of several viruses, such as herpesviruses, West Nile virus, rabies, and influenza virus, upon their penetration in the neuronal plasma membrane has also been reported164–169.\n\nSecondly, SARS-CoV-2 may also invade the trigeminal nerve by indirect mechanisms. Cytokine storm and vasculopathy mechanisms are also proposed to explain the activation of trigeminal nerve upon SARS-CoV-2 infection137. Cytokine storm has attracted substantial interest from researchers and clinicians as this unwanted condition is strongly suggested to be related to the increased mortality in SARS-CoV-2-infected patients170,171. It is hypothesized that the headache suffered by COVID-19 patients at the later stage of this infection is induced by the cytokine storm139,172,173. This notion has been supported by the fact that proinflammatory cytokines, such as IL-1, IL-6 and TNF-α, have been linked to the activation of the trigeminovascular system, which is responsible for the emergence and development of headache through the modulation of CGRP174–177.\n\nMoreover, the presence of angiotensin-converting enzyme 2 (ACE2) receptor on the endothelial cells makes the blood vessels vulnerable to invasion by SARS-CoV-2178,179. It is known that ACE2 is associated with several protective mechanisms within the body, such as vasodilation180 and antinociception181. ACE2 also diminishes excessive free radical production which prevents oxidative stress182. The utilization of this receptor by the virus may decrease its activities, leading to the disturbance of vascular function. The perivascular trigeminal nerve may in turn be affected resulting in the COVID-19-related headache137. More studies are required to improve our understanding on the role of the ACE2 receptor in headache pathophysiology.\n\nAnother hypothesis by which SARS-CoV-2 could induce headache is offered by Abboud et al. (2020). They proposed that gas exchange disturbance in alveolar tissues triggered by the viruses would induce hypoxia, which in turn leads to ischemia170,183. Ischemia itself has been known to have a strong relation with headache incidents184 that could be induced by exaggerating the production of free radicals.\n\nIn regards to the headache characteristics presented by COVID-19 patients compared to the headache induced by other viral infections, no apparent differences could be observed. Headache in COVID-19 could be worsened by physical or head movement, felt in either the entire head (holocranial) or unilaterally (hemicranial) and and the pain is typically pressing or tightening139. It is hypothesized that headaches occurring in COVID-19 patients might be the result of the same mechanisms as observed in influenza A and influenza B infections, which could be related to the activity of cytokines173,185–187. A recent report on dengue-related headache suggested that the headache could be pulsating and either affect the entire brain, only frontal, or orbital area, which may resemble primary headaches reported in COVID-19 patients139,172,188. Therefore, although we found that headache is more frequent in COVID-19 patients than those of non-COVID-19 patients, diagnosis of COVID-19 should not be based on the presence of a headache.\n\nIn conclusion, headache is a common symptom in COVID-19 cases. Some mechanisms have been proposed as to the mechanism for headaches in COVID-19 such as the activation of the trigeminovascular system by either direct action of the virus or indirect mechanisms induced by cytokine storm, vasculopathy, or ischemia induced by gas exchange disturbance in COVID-19 patients. Extensive efforts must be carried out to provide definitive answers about COVID-19-related headaches. Detailed investigations on the mechanisms by which SARS-CoV-2 attacks the CNS and thus generates headaches are important to improve our understanding on the pathophysiology of COVID-19, and therefore influences possible pharmacological intervention decisions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Global prevalence and pathogenesis of headache in COVID-19: A systematic review and meta-analysis’, https://doi.org/10.6084/m9.figshare.13166783.v1189\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nAuthors would like to thank HT Editorial Services in assisting during the study and writing processes.\n\n\nReferences\n\nHarapan H, Itoh N, Yufika A, et al.: Coronavirus disease 2019 (COVID-19): A literature review. J Infect Public Health. 2020; 13(5): 667–73. 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PubMed Abstract \| Publisher Full Text\n\nVillalón CM, VanDenBrink AM: The Role of 5-Hydroxytryptamine in the Pathophysiology of Migraine and its Relevance to the Design of Novel Treatments. Mini Rev Med Chem. 2017; 17(11): 928–38. PubMed Abstract \| Publisher Full Text\n\nImboden H, Patil J, Nussberger J, et al.: Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia. Regul Pept. 2009; 154(1–3): 23–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPortaluppi F, Vergnani L, Margutti A, et al.: Modulatory effect of the renin-angiotensin system on the plasma levels of calcitonin gene-related peptide in normal man. J Clin Endocrinol Metab. 1993; 77(3): 816–20. PubMed Abstract \| Publisher Full Text\n\nAgyeman AA, Chin KL, Landersdorfer CB, et al.: Smell and Taste Dysfunction in Patients With COVID-19: A Systematic Review and Meta-analysis. Mayo Clin Proc. 2020; 95(8): 1621–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEliezer M, Hautefort C, Hamel AL, et al.: Sudden and Complete Olfactory Loss of Function as a Possible Symptom of COVID-19. JAMA Otolaryngol Head Neck Surg. 2020; 146(7): 674–5. PubMed Abstract \| Publisher Full Text\n\nXydakis MS, Dehgani-Mobaraki P, Holbrook EH, et al.: Smell and taste dysfunction in patients with COVID-19. Lancet Infect Dis. 2020; 20(9): 1015–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerlman S, Jacobsen G, Afifi A: Spread of a neurotropic murine coronavirus into the CNS via the trigeminal and olfactory nerves. Virology. 1989; 170(2): 556–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrand G: Olfactory/trigeminal interactions in nasal chemoreception. Neurosci Biobehav Rev. 2006; 30(7): 908–17. PubMed Abstract \| Publisher Full Text\n\nSchaefer ML, Böttger B, Silver WL, et al.: Trigeminal collaterals in the nasal epithelium and olfactory bulb: a potential route for direct modulation of olfactory information by trigeminal stimuli. J Comp Neurol. 2002; 444(3): 221–6. PubMed Abstract \| Publisher Full Text\n\nMiranda-Saksena M, Denes CE, Diefenbach RJ, et al.: Infection and Transport of Herpes Simplex Virus Type 1 in Neurons: Role of the Cytoskeleton. Viruses. 2018; 10(2): 92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerino-Gracia J, García-Mayoral MF, Rodríguez-Crespo I: The association of viral proteins with host cell dynein components during virus infection. FEBS J. 2011; 278(17): 2997–3011. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSt-Jean JR, Jacomy H, Desforges M, et al.: Human respiratory coronavirus OC43: genetic stability and neuroinvasion. J Virol. 2004; 78(16): 8824–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlvarez E, DeDiego ML, Nieto-Torres JL, et al.: The envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated. Virology. 2010; 402(2): 281–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBearer EL, Breakefield XO, Schuback D, et al.: Retrograde axonal transport of herpes simplex virus: evidence for a single mechanism and a role for tegument. Proc Natl Acad Sci U S A. 2000; 97(14): 8146–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntinone SE, Smith GA: Retrograde axon transport of herpes simplex virus and pseudorabies virus: a live-cell comparative analysis. J Virol. 2010; 84(3): 1504–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBohmwald K, Gálvez NMS, Ríos M, et al.: Neurologic Alterations Due to Respiratory Virus Infections. Front Cell Neurosci. 2018; 12: 386. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrigoryan S, Kinchington PR, Yang IH, et al.: Retrograde axonal transport of VZV: kinetic studies in hESC-derived neurons. J Neurovirol. 2012; 18(6): 462–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunsperger EA, Roehrig JT: Temporal analyses of the neuropathogenesis of a West Nile virus infection in mice. J Neurovirol. 2006; 12(2): 129–39. PubMed Abstract \| Publisher Full Text\n\nUgolini G: Rabies virus as a transneuronal tracer of neuronal connections. Adv Virus Res. 2011; 79: 165–202. PubMed Abstract \| Publisher Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehta P, McAuley DF, Brown M, et al.: COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020; 395(10229): 1033–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelvis R: Headaches During COVID-19: My Clinical Case and Review of the Literature. Headache. 2020; 60(7): 1422–1426. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh J, Ali A: Headache as the Presenting Symptom in 2 Patients With COVID-19 and a History of Migraine: 2 Case Reports. Headache. 2020; 60(8): 1773–1776. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfroz S, Arakaki R, Iwasa T, et al.: CGRP Induces Differential Regulation of Cytokines from Satellite Glial Cells in Trigeminal Ganglia and Orofacial Nociception. Int J Mol Sci. 2019; 20(3): 711. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKristiansen KA, Edvinsson L: Neurogenic inflammation: a study of rat trigeminal ganglion. J Headache Pain. 2010; 11(6): 485–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeeb L, Hellen P, Hoffmann J, et al.: Methylprednisolone blocks interleukin 1 beta induced calcitonin gene related peptide release in trigeminal ganglia cells. J Headache Pain. 2016; 17(1): 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdvinsson L, Haanes KA, Warfvinge K: Does inflammation have a role in migraine? Nat Rev Neurol. 2019; 15(8): 483–90. PubMed Abstract \| Publisher Full Text\n\nVarga Z, Flammer AJ, Steiger P, et al.: Endothelial cell infection and endotheliitis in COVID-19. Lancet. 2020; 395(10234): 1417–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHikmet F, Méar L, Edvinsson Å, , et al.: The protein expression profile of ACE2 in human tissues. Mol Syst Biol. 2020; 16(7): e9610. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Moraes PL, Kangussu LM, Castro CH, et al.: Vasodilator Effect of Angiotensin-(1-7) on Vascular Coronary Bed of Rats: Role of Mas, ACE and ACE2. Protein Pept Lett. 2017; 24(9): 869–75. PubMed Abstract \| Publisher Full Text\n\nForte BL, Slosky LM, Zhang H, et al.: Angiotensin-(1-7)/Mas receptor as an antinociceptive agent in cancer-induced bone pain. Pain. 2016; 157(12): 2709–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRabelo LA, Alenina N, Bader M: ACE2-angiotensin-(1-7)-Mas axis and oxidative stress in cardiovascular disease. Hypertens Res. 2011; 34(2): 154–60. PubMed Abstract \| Publisher Full Text\n\nAbboud H, Abboud FZ, Kharbouch H, et al.: COVID-19 and SARS-Cov-2 Infection: Pathophysiology and Clinical Effects on the Nervous System. World Neurosurg. 2020; 140: 49–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlesen J, Friberg L, Olsen TS, et al.: Ischaemia-induced (symptomatic) migraine attacks may be more frequent than migraine-induced ischaemic insults. Brain. 1993; 116(Pt 1): 187–202. PubMed Abstract \| Publisher Full Text\n\nEccles R: Understanding the symptoms of the common cold and influenza. Lancet Infect Dis. 2005; 5(11): 718–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNicholson KG: Clinical features of influenza. Semin Respir Infect. 1992; 7(1): 26–37. PubMed Abstract\n\nSmith RS: The cytokine theory of headache. Med Hypotheses. 1992; 39(2): 168–74. PubMed Abstract \| Publisher Full Text\n\nde Abreu LV, Oliveira CB, Bordini CA, et al.: New Daily Persistent Headache Following Dengue Fever: Report of Three Cases and an Epidemiological Study. Headache. 2020; 60(1): 265–8. PubMed Abstract \| Publisher Full Text\n\nHarapan H: Global prevalence and pathogenesis of headache in COVID-19: A systematic review and meta-analysis. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13166783.v1" }	[ { "id": "74699", "date": "04 Dec 2020", "name": "Erni J. Nelwan", "expertise": [ "Reviewer Expertise infectious 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\nAuthors seem not considered potential important bias while choosing headaches to be analysed. While among patients with fever (mainly), headache is in the same pathophysiology response due to prostaglandin release. Not to include other symptoms, so it is not merely a headache as a symptom.\nHow about the existence of co-morbidity such as Hypertension (uncontrolled hypertension)? It is not clear how to justify data with such condition concomitantly.\nIn the results section, what does this statement mean?\n\"Headache was found to be more prevalent, approximately by two-fold, in COVID-19 patients than in non-COVID-19 patients with symptoms of other respiratory viral infections, OR: 1.73; 95% CI: 1.94, 2.5 with p=0.04.\"\nWhat kind of non-covid-19 patients with symptoms of other respiratory viral infections? This is a confusing statement.\nAs a clinician taking care of Covid-19 patients, the importance of this article is low.\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? No\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6388", "date": "10 Mar 2021", "name": "Endang Mutiawati", "role": "Author Response", "response": "Dear Dr Erni J. Nelwan We agreed that our study have limitation since we did not able to analysed the data based on existence of COVID-19 co-morbidity. This is mainly because the available of data. The data was scarce. We have added this in our manuscript: \"In this analysis we did not analyze the prevalence of headache based on COVID-19 severity, the existence of COVID-19 co-morbidity (such as diabetes and hypertension) and based on demographic characteristics such as gender due to scarcity of the available data. Therefore, whenever enough data are available, such sub-analyses are critical to be conducted.\" We have provided a clear explanation about the non-COVID-19 in Method section as requestioned. Not-COVID-19 in our study refers to other respiratory viral infections such as rhinovirus, influenza, parainfluenza and respiratory syncytial virus. We added: \"non-COVID-19 cases (other respiratory viral infections such as rhinovirus, influenza, parainfluenza and respiratory syncytial virus)\" in text. We also also agreed that the pathogenesis of headache is complex including the production of prostaglandin while fever. Therefore in our study we compared the occurrence of headache in COVID-19 and other non-COVID-19 respiratory infection as control. This to reduce the effect of fever since in both COVID-19 and non-COVID-19 cases the fever are present. Due to this complexity we did not claim any diagnostic for direct clinical practice." } ] }, { "id": "79512", "date": "15 Feb 2021", "name": "Morteza Arab-Zozani", "expertise": [ "Reviewer Expertise Health Policy and Management", "an expert in systematic review and meta-analysis methodology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for considering a great area of interest related to COVID-19. This study aimed to investigate the prevalence and pathogenesis of headache in COVID-19 patients, globally. The methodology is completely rigorous and the results are interesting. I have a few minor comments to the authors as follow:\nIn the study selection and/or quality appraisal stages are the authors faced with discrepancies? Please state how resolved these potential discrepancies.\n\nWas it not possible to analyze the subgroups? For example, based on age groups or gender.\n\nPlease add some details about the NOS checklist and also add the result of the quality appraisal to the manuscript.\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": "6389", "date": "10 Mar 2021", "name": "Endang Mutiawati", "role": "Author Response", "response": "Dear Dr Morteza Arab-Zozani, Thank you for your comments. 1. We have provide the approach how to solve in case there was discrepancies between two authors during evaluation of the studies. 2. We were unable to do sub-group analysis based on age and gender due to available of data. We have added this as one of our limitation: We have added this in our manuscript: \"In this analysis we did not analyze the prevalence of headache based on COVID-19 severity, the existence of COVID-19 co-morbidity (such as diabetes and hypertension) and based on demographic characteristics such as gender due to scarcity of the available data. Therefore, whenever enough data are available, such sub-analyses are critical to be conducted.\" 3. We have provide more detail information of Newcastle-Ottawa scale (NOS) - What items that were assessed and how to score as well how the studies classified. We also have added the NOS scores briefly to all studies included in the study. Thank you" } ] } ]	1	https://f1000research.com/articles/9-1316
https://f1000research.com/articles/10-194/v1	09 Mar 21	{ "type": "Software Tool Article", "title": "Meta-Signer: Metagenomic Signature Identifier based onrank aggregation of features", "authors": [ "Derek Reiman", "Ahmed Metwally", "Jun Sun", "Yang Dai", "Derek Reiman", "Ahmed Metwally", "Jun Sun" ], "abstract": "The advance of metagenomic studies provides the opportunity to identify microbial taxa that are associated with human diseases. Multiple methods exist for the association analysis. However, the results could be inconsistent, presenting challenges in interpreting the host-microbiome interactions. To address this issue, we develop Meta-Signer, a novel Metagenomic Signature Identifier tool based on rank aggregation of features identified from multiple machine learning models including Random Forest, Support Vector Machines, Logistic Regression, and Multi-Layer Perceptron Neural Networks. Meta-Signer generates ranked taxa lists by training individual machine learning models over multiple training partitions and aggregates the ranked lists into a single list by an optimization procedure to represent the most informative and robust microbial features. A User will receive speedy assessment on the predictive performance of each ma-chine learning model using different numbers of the ranked features and determine the final models to be used for evaluation on external datasets. Meta-Signer is user-friendly and customizable, allowing users to explore their datasets quickly and efficiently.", "keywords": [ "Metagenome-wide Association Study", "Feature Extraction", "Machine Learning", "Rank Aggregation" ], "content": "Introduction\n\nRecent metagenomic studies of the gut microbiome have linked dysbiosis to many human diseases1–3. The identification of microbial taxa associated with human disease has been one of the important efforts in metagenomics data analysis4. Various metagenomic studies use parametric or non-parametric statistical tests to detect differentially abundant individual taxa between disease and control groups5–9. These types of methods can potentially miss taxa with weak associations but together can present strong statistical association.\n\nIn order to capture group association, several methods are proposed by exploring related taxa on a phylogenetic taxonomic tree10–14.\n\nThese elaborated statistical methods enhanced the detection of the microbial group association. However, they may still fail to detect complex multivariate nonlinear associations.\n\nAlternative approaches of using machine learning (ML) models have been advocated for the prediction of the host phenotype15. This is motivated by the findings that a microbial signature for the host phenotype may be complex, involving simultaneous over- and under-representations of multiple microbial taxa potentially interacting with each other. Classical ML models, such as Random Forest (RF), Logistic Regression and Support Vector Machines (SVMs), and deep neural networks (DNNs) have been applied to host phenotype prediction using microbial abundance features16–22.\n\nWhile the ML approaches demonstrated promising results on host phenotype prediction23,24, it is a challenging task for users to determine what is the best ML model and how many features are needed in order to achieve robust prediction, especially on external validation datasets. In addition, each ML algorithm may generate different feature importance rankings12,22,25, complicating the decision on a consistent and informative signature for the host phenotype of interest.\n\nIn this work, we introduce a novel tool, Meta-Signer, a Metagenomic Signature Identifier based on rank aggregation of informative taxa learned from individual ML models. Meta-Signer uses RF, SVM, penalized Logistic Regression, and multiple-layer perceptron neural network (MLPNN) models to evaluate importance of each microbial taxon and generates a ranked list of features (i.e., taxa) per model. It aggregates all the ranked lists using a procedure \"RankAggreg\" based on the cross-entropy method or the genetic algorithm26. Finally, Meta-Signer reports the top ranking features specified by the user and generates the ML models using these features. Meta-Signer’s workflow is shown in Figure 1.\n\nLarge rounded rectangles represent different modules of the workflow. Microbial abundance is preprocessed and filtered, and then used to train ML models. Features are ranked for each model and an overall aggregated feature ranking is constructed. Meta-Signer generates portable, user-friendly HTML files for visualization as well as ML models trained on a subset of high ranking features. SVM, support Vector Machine; MLPNN, multiple-layer perceptron neural network; ML, machine learning.\n\nMeta-Signer is user-friendly and easy to run. It provides a readable summaries as HTML outputs as well as final trained ML models. Meta-Signer is distributed as Python tool and available at https://github.com/YDaiLab/Meta-Signer.\n\n\nMethods\n\nThe inputs to Meta-Signer are (1) a tab separated file of taxa abundance values where each row represents a taxon and each column represents a sample, and (2) a line separated list of response values where each row represents the phenotypic response of a sample. The first column in the abundance table should be the taxonomic identification of the taxon. An additional required file is (3) the run configuration file with user specified parameters. A final optional file is (4) the model parameters for the neural network architectures in JSON format. If this file is not found, Meta-Signer will tune the parameters and save them for later use.\n\nMeta-Signer includes three classic ML models (RF, Linear SVM, Logistic Regression), as well as an MLPNN model. RFs are decision tree learning models trained in an ensemble fashion, taking the average of the ensemble to give a robust decision tree27. While growing each tree, a decision is made at each node by selecting the feature from a random subset of features that best splits the data into two subsets based on the Gini impurity of each subset. Given a set of data points with k classes, let pi be the proportion of samples of class i for i ∈ {1...k}. The Gini impurity of the set is calculated as\n\n\n\nOur method implements the RF model using the scikit-learn Python library. Once trained, features are then extracted by evaluating the mean decrease impurity. For each node, the importance of the feature node being split on the decision tree is calculated as the decrease in Gini impurity before and after the split. This value is then weighted by the proportion of total samples that were split upon that node. A feature’s importance is then calculated by averaging the weighted importance values of nodes that split using that feature across all trees in the ensemble.\n\nSVMs are supervised ML models that learn the best hyperplane to separate two classes of data28. In case of linear SVMs, a set of weights (w), and an intercept (b) will be learned. The class of the sample xi can then be determined as\n\n\n\nSince the weights can be used to rank the importance of features, we used the linear SVMs in Meta-Signer for feature extraction. Once trained, features can be ranked using the absolute value of the learned weight parameters. SVM models are tuned with a grid search using the scikit-learn Python library.\n\nLogistic Regression fits a logistic function to estimate the probability of binary classification; however, it can be extended to multi-class scenarios29. The model predicts the probability of a sample xi being class 1 as\n\n\n\nwhere β are the weight parameters which are learned. We apply shrinkage to reduce the total number of model parameters in the final model using L1 regularization in order to penalize the absolute value of the weights, eliminating a portion of the features to create a sparse model. Given a set of samples xi (i = 1,...,n) where each sample has m features and a binary class label yi, the model minimizes the cost\n\n\n\nwhere the weight parameters are penalized with the regularization parameter λ. Logistic regression models are trained using the scikit-learn Python library. Once trained, the β values are used to rank features based on their absolute value. Neural networks are consisted of multiple layers of nodes that are fully connected with edges constituting weights30. The values of a hidden layer are a linear combination of the values from the previous layer which is passed through a non-linear activation function. More explicitly, the values of a hidden layer hl is calculated as\n\n\n\nwhere hl−1 are the values from the previous hidden layer, Wl are the weights connecting hl−1 to hl, bl is a bias value, and 𝜓 is a non-linear activation function. Meta-Signer uses the Rectified Linear Unit activation function for hidden layers and the softmax activation function on the output layer. The entire network is trained using a single loss function\n\n\n\nHere ac is the predicted softmax probability of a sample’s true class c. The second term performs L2 regularization on the network weights and is penalized by λ. Networks are trained using early stopping with a validation subset of 10% of the training data.\n\nBefore training, Meta-Signer checks for a file containing network hyper-parameters for MLPNN models. If it does not find this file, Meta-Signer will use the first partition of the cross-validation to empirically determine the hyper-parameters. This is done using another cross-validation on the training set of the first partition. In addition, using the configuration file, the user can set custom parameters and even disable any of the learning models if they do not wish to incorporate it into their results. In Meta-Signer, RF, SVM, and Logistic Regression are trained using the scikit-learn python package and MLPNN models are trained using Tensorflow.\n\nFor each ML model in each cross-validated partition, Meta-Signer extracts the feature scores and uses the scores to construct a ranked feature list. RF features were scored using a method called mean decrease impurity. For each node, the importance of the feature being split upon is calculated as the decrease in Gini impurity from before and after the split. This value is then weighted by the proportion of total samples that were split upon that node. A feature’s importance is then calculated by averaging the weighted importance values of nodes that split using that feature across all trees in the ensemble. Features in Logistic Regression and SVM models were scored based on the magnitude of their coefficients in the decision functions.\n\nThe extraction of features from DNN models is a challenging task in general. We use a procedure developed in 31 to evaluate features in MLPNNs. Briefly, the MLPNN features were evaluated by calculating the cumulative weight across all layers by taking the running product of all the weight matrices in the learned networks. The product results in a matrix that has a column for each class and a row for each feature, and the value at a given index is that feature’s cumulative impact for that class. We then consider a feature’s importance as the maximum impact across classes to create a single ranked list.\n\nFor each partition of the cross-validation, we generate a single ranked list for each of ML models. Once the entirety of the cross-validated training is complete, the entire set of all ranked lists across all models is aggregated into a single top-k ranked list by minimizing the distance between the set of ranked lists and the top-k list, where k is specified by the user in the configuration file. More specifically, given a set of ranked lists {ℓ1,...,ℓm}, the top-k ranked list, θ^, is determined as,\n\n\n\nHere, L is the state space of top-k rankings, wi is a weight associated with ℓi, and d(θ,ℓi) is the distance between a proposed top-k ranked list, θ, and ℓi.\n\nThe aggregation is performed using the R package RankAggreg26. This package uses either a genetic algorithm or cross-entropy based approach with Markov Chain Monte Carlo sampling to find the top-k features that minimize the sum of the distances between each of the input sets and the generated top-k set. The distance used is the Spearman’s Correlation. Each input ranked list is weighted in the aggregation by the area under the receiver operating curve (AUC). After the model predictions are evaluated and the features are ranked into a single list, Meta-Signer provides a summary of the results in a portable HTML file. The file contains a description of the run and evaluation metrics for the different models in the form of boxplots. It also provides the distribution of the feature importance scores for each ML model. Lastly, it provides a list of the top-k taxa selected from the original taxa, the proportion of individual ranking sets that each taxon was present in the top-k, the rank and p-value under a PERMANOVA test32, and the class in which the taxon was found to be predictive for. All images are encoded into the file, allowing the HTML file to be moved without considering the location of the images.\n\nMeta-Signer’s general workflow proceeds as described above and as shown in Figure 1. Meta-Signer was designed for Python 3.7 and requires the following Python packages: numpy, pandas, scipy, scikit-learn, scikit-bio, Tensorflow, matplotlib, seaborn. In addition, a working version of R version 3.0 or higher is required. Code and instructions for configuration and running can be found at https://github.com/YDaiLab/Meta-Signer.\n\n\nUse cases\n\nIn this section we will demonstrate how to run Meta-Signer on a dataset of patients with inflammatory bowel disease (IBD) which is provided in the GitHub repository. This dataset came from the Prospective Registry in IBD Study at MGH (PRISM)33, which enrolled patients with a diagnosis of either Crohn’s disease (CD) or ulcerative colitis (UC). The dataset includes 68 samples with CD, 53 samples with UC, and 34 healthy samples.\n\nIn addition, Meta-Signer includes another IBD dataset for external testing. This dataset consists of two independent cohorts from the Netherlands34. The first cohort consists of 22 healthy subjects who participated in the general population study LifeLines-DEEP in the northern Netherlands. The second cohort consists of subjects with IBD from the Department of Gastroenterology and Hepatology, University Medical Center Groningen, Netherlands and includes 20 samples with CD and 23 samples with UC. Together, both the PRISM dataset and the external IBD dataset included 201 microbial features. Datasets were evaluated using all three classes as well as in a binary case by combining CD and UC samples.\n\nTo use Meta-Signer and the data provided, the user needs to download Meta-Signer from the GitHub repository using the following command:\n\ngit clone https://github.com/YDaiLab/Meta-Signer.git\n\ncd Meta-Signer\n\nThe user must make sure that all the Python and R dependencies are met before running Meta-Signer (see Operation). The user can either install these manually, or use the provided Meta-Signer Conda environment file provided using the following command:\n\nconda env create -f meta-signer.yml\n\nsource activate meta-signer\n\nBefore running Meta-Signer, the user should open the file config.py in the src directory. This file can be used to change the run parameters of Meta-Signer and turn off and on different ML methods. An example is shown in Figure 2.\n\nUsing these parameters, we will run 10 iterations of 10-fold cross-validation on the data found in the PRISM_3 directory under the data folder. Any taxon not found in at least 10% of samples will be removed as well as any taxon with less than 0.001 mean abundance. We will use the genetic algorithm method for rank aggregation to generate a candidate list with a maximum of 50 features. Descriptions for each parameter can be found on Meta-Signer’s GitHub page as well as in the configuration file. The user can then generate the aggregated ranked list using the following command:\n\npython generate_feature_ranking.py\n\nThis will perform the cross-validation evaluation and feature ranking, storing all the results in the results folder under a directory named after the dataset. This directory will contain internal cross-validation results as well as an HTML file that allows the user to see how different ML methods perform on the dataset. Performance of the 10 iterations of 10-fold cross-validation on the PRISM dataset is shown in Table 1. We include an analysis of both binary class (IBD vs Healthy) and tertiary class (IBD vs CD vs UC) scenarios.\n\nStandard deviation is shown in parentheses. RF, Random Forest; SVM, Support Vector Machine; MLPNN, multiple-layer perceptron neural network; AUC, area under the receiver operating curve; MCC, Matthews correlation coefficient; F1, F1 score.\n\nThe HTML output displays these results in the form of boxplots. An example for the PRISM dataset is shown in Figure 3. In addition, Meta-Signer will evaluate the different ML methods’ training performance using increasing numbers of ranked features. Since these models will eventually overfit, the saturation of performance can help guide the user on how many features are needed to train the final model on. For the PRISM dataset, we see this saturation around 30 features, as shown in Figure 4\n\nButtons on the top allow users to cycle through different metrics. RF, Random Forest; SVM, Support Vector Machine; MLPNN, multiple-layer perceptron neural network; AUC, area under the receiver operating curve; MCC, Matthews correlation coefficient; F1, F1 score.\n\nRF, Random Forest; SVM, Support Vector Machine; MLPNN, multiple-layer perceptron neural network; AUC, area under the receiver operating curve.\n\nUsing the point of saturation on the AUC curve as a guide, the user can then select the number of features and train final models using the following command:\n\npython generate_final_models.py PRISM_3 30\n\nHere PRISM_3 points to the result directory labeled PRISM_3 that Meta-Signer has just generated and 30 represents the number of features the user would like to have in the final models. In addition, the user can provide an external dataset to evaluate,\n\npython generate_final_models.py\n\nPRISM_3 30 -e PRISM_3_external\n\nNote that PRISM_3_external must be a directory in the data directory with it’s own abundance.tsv and labels.txt files. This command will generate a directory that will contain the final trained ML models as well as another HTML file showing the ranked lists for each model as well as the aggregated ranked list as shown in Figure 5.\n\nCD, Crohn’s disease; UC, ulcerative colitis.\n\nIn addition, if an external dataset is available for evaluation, the HTML file will display a table of metrics for both evaluation on the training set and evaluation on the external dataset. An example is shown in Figure 6.\n\nRF, Random Forest; SVM, Support Vector Machine; MLPNN, multiple-layer perceptron neural network; AUC, area under the receiver operating curve; MCC, ; F1, .\n\n\nDiscussion\n\nWe have developed Meta-Signer, a user-friendly tool for the extraction of robust microbial taxa that are predictive to host phenotype from multiple ML models. By training different types of ML models, Meta-Signer exploits the similarities in the ranked lists of taxa learned by individual ML models to create a single aggregated set of informative microbial taxa for host phenotype prediction. We have shown that Meta-Signer can provide more informative features when compared to similar methods in both binary and multi-class scenarios.\n\nTo evaluate Meta-Signer, we benchmark against other a previously published methods Biosigner35 and a non-parametric PERMANOVA test32. Biosigner is a generic ML driven feature selection method for omics data and available in R. It uses trained RF, SVM and Partial-Least Squared Discriminant Analysis models to selectively eliminate features, resulting in a single set of remaining features. The non-parametric PERMANOVA test was included as a baseline method of feature ranking for comparison. Results for the benchmarking can be found in the Extended data document on the Meta-Signer GitHub page.\n\n\nConclusions\n\nIn conclusion, Meta-Signer is a user-friendly tool to identify a robust set of highly informative microbial taxa that are predictive of human disease status from a metagenomic dataset.\n\n\nData availability\n\nOriginal data and information for both the PRISM and external IBD datasets can be obtained from Dataset 4 of the original study http://doi.org/10.1038/s41564-018-0306-433. The data for the PRISM and external IBD datsets as formatted for use of Meta-Signer can be found at Zenodo: derekreiman/Meta-Signer: Original Release. http://doi.org/10.5281/zenodo.407740336. This project contains microbial abundance and sample class data formatted for Meta-Signer as TSV, TXT and JSON files within the folder ’data’.\n\n1. abundance.tsv - tab separated file for microbial abundance data\n\n2. labels.txt - text file of class labels\n\n3. model_parameters.json - JSON file containing neural network hyper-parameters\n\nZenodo: derekreiman/Meta-Signer: Original Release. http://doi.org/10.5281/zenodo.407740336. This project contains the following extended data:\n\nExtendedData.pdf (Data for the benchmarking of Meta-Signer against Biosigner and PERMANOVA ranked features)\n\nData are under the GPL-3 license.\n\n\nSoftware availability\n\nSource code available from: https://github.com/YDaiLab/Meta-Signer\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.407740336\n\nLicense: GPL-3.0", "appendix": "Acknowledgements\n\nWe gratefully acknowledge the support of NVIDIA Corporation with the donation of the Titan Xp GPU used for this research.\n\n\nReferences\n\nMarchesi JR, Adams DH, Fava F, et al.: The gut microbiota and host health: a new clinical frontier. Gut. 2016; 65(2): 330–339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Jia H: Metagenome-wide association studies: fine-mining the microbiome. Nat Rev Microbiol. 2016; 14(8): 508–22. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDitzler G, Polikar R, Rosen G, et al.: Multi-Layer and Recursive Neural Networks for Metagenomic Classification. IEEE Trans Nanobioscience. 2015; 14(6): 608–616. PubMed Abstract \| Publisher Full Text\n\nPasolli E, Truong DT, Malik F, et al.: Machine Learning Meta-analysis of Large Metagenomic Datasets: Tools and Biological Insights. PLoS Comput Biol. 2016; 12(7): e1004977. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFioravanti D, Giarratano Y, Maggio V, et al.: Phylogenetic convolutional neural networks in metagenomics. BMC Bioinformatics. 2018; 19(Suppl 2): 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReiman D, Metwally A, Dai Y: Using convolutional neural networks to explore the microbiome. Annu Int Conf IEEE Eng Med Biol Soc. 2017; 2017: 4269–4272. PubMed Abstract \| Publisher Full Text\n\nOudah M, Henschel A: Taxonomy-aware feature engineering for microbiome classification. BMC Bioinformatics. 2018; 19(1): 227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMetwally AA, Yu PS, Reiman D, et al.: Utilizing longitudinal microbiome taxonomic profiles to predict food allergy via Long Short-Term Memory networks. PLoS Comput Biol. 2019; 15(2): e1006693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReiman D, Metwally AA, Sun J, et al.: PopPhy-CNN: A Phylogenetic Tree Embedded Architecture for Convolutional Neural Networks to Predict Host Phenotype From Metagenomic Data. IEEE J Biomed Health Inform. 2020; 24(10): 2993–3001. PubMed Abstract \| Publisher Full Text\n\nLaPierre N, Ju CJT, Zhou G, et al.: MetaPheno: A critical evaluation of deep learning and machine learning in metagenome-based disease prediction. Methods. 2019; 166: 74–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou YH, Gallins P: A Review and Tutorial of Machine Learning Methods for Microbiome Host Trait Prediction. Front Genet. 2019; 10: 579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Q, Abel H, Wells A, et al.: Selection of models for the analysis of risk-factor trees: leveraging biological knowledge to mine large sets of risk factors with application to microbiome data. Bioinformatics. 2015; 31(10): 1607–1613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPihur V, Datta S, Datta S: RankAggreg, an R package for weighted rank aggregation. BMC Bioinformatics. 2009; 10: 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo TK: Random decision forests. Proceedings of the Third International Conference on Document Analysis and Recognition (Volume 1) -Volume 1. 1995. Publisher Full Text\n\nCortes C, Vapnik V: Support-vector networks. Mach Learn. 1995; 20(3): 273–297. Publisher Full Text\n\nHastie T, Tibshirani R, Friedman J, et al.: The elements of statistical learning: data mining, inference, and prediction. New York: Springer. 2009. Reference Source\n\nAggarwal CC: Neural networks in deep learning. Springer. 2018. Reference Source\n\nDanaee P, Ghaeini R, Hendrix DA: A deep learning approach for cancer detection and relevant gene identification. Pac Symp Biocomput. 2017; 22: 219–229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson MJ: Permutational multivariate analysis of variance (permanova). In: N. Balakrishnan et al., editors, In Wiley StatsRef: Statistics Reference Online. Wiley, 2017; 1–15. Publisher Full Text\n\nFranzosa EA, Sirota-Madi A, Avila-Pacheco J, et al.: Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nat Microbiol. 2019; 4(2): 293–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTigchelaar EF, Zhernakova A, Dekens JAM, et al.: Cohort profile: LifeLines DEEP, a prospective, general population cohort study in the northern Netherlands: study design and baseline characteristics. BMJ Open. 2015; 5(8): e006772. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRinaudo P, Boudah S, Junot C, et al.: biosigner: A New Method for the Discovery of Significant Molecular Signatures from Omics Data. Front Mol Biosci. 2016; 3: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReiman D, DaiLab Y: derekreiman/meta-signer: Original release. 2020. http://www.doi.org/10.5281/zenodo.4077403" }	[ { "id": "86264", "date": "29 Jun 2021", "name": "Braden T Tierney", "expertise": [ "Reviewer Expertise microbiology", "metagenomics", "bioinformatics", "data science" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a machine-learning software tool designed for use on compositional, specifically microbiome, data. This is a worthy endeavor -- the microbiome field is in need of well-designed statistical methods. I am specifically enthusiastic about using multiple machine learning approaches on the same dataset.\n\nHowever, I do not believe this software is ready for wide use. The lofty goal of comparing multiple machine learning tools sets an extremely high bar for usability, especially to those in the microbiome field who are not machine learning experts. Its complexity, while impressive and indicative of substantial effort, requires that the authors spend a fair amount of time attempting to make its specific use-cases and pitfalls clear to users.\n\nAdditionally, I believe the manuscript itself needs substantially more detail in order to inform users as to what is going on under the hood. If I had to choose, I would like to see less time spent on the theory underlying each method and more on the justification and interpretation of the model input/output. As a result, I would recommend a major revisions to the tool and manuscript before its widespread adoptions.\nMajor Comments:\nSoftware structure\nI was unable to locate any evidence of a unit testing suite -- for a tool this complex, this is essential. If the authors do not have experience with professional-level Pythonic development, I highly recommend that they take some time and look into the fundamentals of unit-testing and writing object-oriented code.\n\nUsers should have a clear statement in the README of when the software is appropriate and when it is not\n\nThe example in the extended data of the manuscript should ideally be featured in the documentation (referenced in the README) so users have a clear use-case right on hand.\n\nWhat about data on runtime or recommended sample sizes for different algorithms? What about memory requirements? A deep network has very different requirements than logistic regression -- this should be made clear in the documentation. You really do not want to accidentally encourage improper use of these algorithms -- eg using deep learning on a tiny dataset will yield overfitting.\n\nOn this note, I am not entirely sure deep learning should even be enabled by default. The sample sizes required for this to be meaningful are orders of magnitude larger than most microbiome studies currently available (certainly the ones used in the IBD example). Moreover, the power of deep learning when used appropriately comes from its customizability -- number of layers, window sizes, etc. I am not clear that it is an algorithm optimized for a “one click solution.” I would rather see it replaced with, perhaps, a gradient boosted machine or something similar.\n\nParameters -- The authors are aggregating vastly dissimilar machine learning techniques. Again, this is a great sentiment, but each of them has so many different drawbacks and potential strategies that the authors need to clarify and justify their default choices in the GitHub repository. The config.py file has only a small subset of the options. Perhaps they could argue that Meta-Signer is a “first pass” tool, and that it could later be used to select an optimal method for refinement?\n\nPlease provide clear resources for the users to interpret your output (e.g. a model and an AUC is not enough). What do you recommend they do with the information they now have?\n\nThe manuscript leaves a series of very basic questions on the table.\n\nWho is the target audience for this? How much of a machine learning background should they have? This perhaps sounds pedantic, but it is important. A machine learning expert would likely fit their own models to have better control over the data. A novice would likely not be able to use the software without more detailed documentation or -- worse -- they would use it and not fully understand what is going on technically.\n\nWhy is this specifically targeted towards microbiome data? Why not other compositional data? Or can you use count data instead?\n\nWhy were these particular modeling strategies included? Why not others?\n\nCan you discuss why you are distinct from other tools -- like BioSigner -- and why someone would use yours over that? Again, I would really like to see the results from this comparison actually discussed in depth. It is not sufficient to just have a pdf on the GitHub. Please discuss why you saw the differences you did.\n\nCan you include other potential demographic variables or confounders? We now know the microbiome is wildly confounded by environmental variables -- any modeling approach that does not explicitly discuss these should be treated with suspicion. See https://pubmed.ncbi.nlm.nih.gov/33149306/1 or https://www.nature.com/articles/nature157662. Simply predicting a Y with a series of X’s is not likely to be reproducible at scale.\n\nIt is critical that the discussion has a clear mention of the benefits and drawbacks of this approach? Can you discuss overfitting at least? An AUC of 1 on your training dataset is likely too good to be true (and this was seen to be the case in the validation cohort, Figure 6).\nIn summary, I do believe there is value to this tool -- but the authors need to exert more effort to clarify its utility and, most importantly, ensure it can be used to uncover meaningful biology.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [] }, { "id": "87185", "date": "27 Jul 2021", "name": "Boyang JI", "expertise": [ "Reviewer Expertise microbiome", "omics integration", "mathematical modeling", "human disease", "systems biology", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article “Meta-Signer: Metagenomic Signature Identifier based onrank aggregation of features”, Reiman et.al. developed a pipeline for identifying microbial features linked to human health/disease using multiple machine learning methods with rank aggregation of feature lists. Four different methods for classifications including random forest, support vector machine, logistic regression and multiple-layer perceptron neural network were applied. Also authors applied the pipeline with a dataset including healthy controls and IBD patients.\nOverall, the pipeline is well-designed, and the paper is well written. Also this software represents an alternative way identify microbial signatures linked to disease. However, I found that there are some points that could be analyzed and discussed more fully.\nMajor points:\nIn this workflow, the input abundance data were pre-processed, filtering and normalized by log transformation. More details about the pre-processing and filtering need to be included in the manuscript. Also lot of evidences (Weiss et al. Microbiome (2017) 5:27 DOI 10.1186/s40168-017-0237-y; Pereira et al. BMCGenomics (2018) 19:274 DOI 10.1186/s12864-018-4637-6; McKnight et al. Methods Ecol Evol. (2018) 1–12 DOI 10.1111/2041-210X.13115) had shown that the methods of normalization had significant influences in the analysis of metagenome abundance data. Therefore, the use of log transformation of relative abundance may be not a good choice here. Authors can add more choices for normalization or just provide the direct functions for importing normalized abundance data.\n\nAlthough authors provide the visualization of the relationship between number of features and the model performances (e.g. AUC) in the script of “generate_feature_ranking.py“, I still think authors can provide a completely automatic way for machine learning parameter optimization and the selection of best number of features.\nMinor point:\nSince the configuration file is a python file, I strongly suggest authors to change current “config.py” to other readable names.\n\nSome spelling errors in the title and main text\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/10-194
https://f1000research.com/articles/8-256/v1	05 Mar 19	{ "type": "Systematic Review", "title": "Application of genus Cassia in the treatment of Constipation: A systematic review", "authors": [ "Muhammad Shahzad Aslam" ], "abstract": "Purpose: Role of genus cassia in the treatment of Constipation Methods: Methodological analysis, systematic review, and meta-analysis of identified studies using RevMan Result and Discussion: Cassia fistula was partially effected in treating constipation however there is a need for improvement in the protocol of studies to reduce biases. These results were only limited to one species so it cannot be generalized among all species of Cassia. Conclusion: Cassia fistula is partially effective in reducing the pain and consistency of stool during constipation among children.", "keywords": [ "Cassia", "Cassia fistula", "Constipation", "Pediatric gastroenterology" ], "content": "Introduction\n\nConstipation is a clinical disorder attributed to ineffectual colonic impulsion and/or increased resistance to the proliferation of colonic matters1. Approximately 20% of the world population suffers from chronic constipation2. It is one of the most common pediatric problems3. It was found to be the second most stated disorder in the field of pediatric gastroenterology. Treatment costs for children with constipation will be around three times higher than children without constipation in the United States4. African American children, particularly girls, are greatly affected by constipation, which has been associated with poor hygiene conditions5.\n\nCommonly, constipation is treated by Cisapride in children6, other treatments include polyethylene glycol 3350 and lactulose, however polyethylene glycol 3350 has been found to be more effective7. Supplemented and non-supplemented yogurt helps in reducing abdominal pain and to enhance defecation frequency8. It has been observed that different species of Cassia act an effective as a laxative such as Cassia fistula, Cassia alata, and Cassia augustifolia9–11. The genus Cassia is well known in alternative medicine as hepatoprotective12, laxative, and in the treatment of ringworm infection13, skin diseases14 and leprosy15. It has many pharmacological properties including acting as a hypolipidemic agent16, anti-microbial17, anti-fungal18, and anti-cancer agent19. Genus Cassia contains a number of bioactive compounds such as anthraquinone20, tannin21, coumarins22, triterpene, volatile oil23, phenolic glycoside24, flavonoids25 from different parts of the plant. Different species of Cassia possess laxative properties due to various anthraquinone derivative such as aloe-emodin, rhein, chrysophanol and chrysoobtusin. In this review, we systematically assessed the laxative potential of different species of Cassia.\n\n\nMethods\n\nA systematic literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Using the keywords (Senna AND Laxatives AND Clinical trial) (Cassia AND Laxatives AND Clinical trial) (Cassia AND Clinical trial) (Senna AND Clinical trial) and publication range from 01 January 1960 till 31 December 2018 for identification of the records. Table 1 shows the search strategy for PubMed Central. During screening of the records only full-length open access articles were considered. Abstract only or closed access articles were excluded11. Only articles involving children aged between 2–15 were included. All review articles, in-vivo studies and those >10 years from the search data were excluded. A preliminary search of the PubMed, CNKI, Scopus, Web of Science, Google Scholar and PsycINFO databases and digital archive such as PubMed Central, yielded 2207 papers published in English from the last ten years. Duplicate and irrelevant articles were removed (n=2203). One article was further removed during screening due to closed access (n=3). One publication was removed because the article did not meet the eligibility criteria (n=2). A PRISMA Flow Diagram is given in Figure 1.\n\nIdentification of articles were performed at level 1 using the search strategy as mentioned in Table 1. Duplicate articles, irrelevant articles such as polyherbal formulation, review articles, or any article other than Cassia or Senna were removed at level 2. Only four4 articles were identified as being relevant. One record was excluded due to not being a full text article. Abstracts were being reviewed for the following inclusion and exclusion criteria at level 4 and one article was removed for not meeting the eligibility criteria i.e. Randomized, clinical trial on Constipation, full-length open access articles, Pediatric Functional Constipation (age range: 2–15 years).\n\nTypes of studies. The author has selected studies of randomized open label, prospective, controlled, parallel-group clinical trial for meta-analysis. Baseline characteristics of randomized trials of studies included on pediatric functional constipation are presented in Table 4. Characteristics of the studies included are mentioned in Table 5.\n\nTypes of participants. The author included studies involving patients (aged 2–15 years) with Functional constipation. The diagnosis of Functional constipation was according to according to the Rome III criteria26. Inclusion and exclusion criteria were based on Study design, participants, intervention, outcome (SPIO) criteria and indicated in Table 2.\n\nTypes of interventions. Included studies were focused on the role Cassia in the treatment of Functional constipation. Unfortunately, there were only two studies identified.\n\nTypes of outcomes. Eligible studies included consisted of the following outcomes: improvement in the episodes of fecal incontinence per week, improvement in the episodes of retentive posturing per week, improvement in the average of severity of pain of defecation (by VAS), improvement in defecation frequency per week, patient’s drug compliance and improvement in the average of consistency of stool defecated (by VAS).\n\nMethodological quality assessment was made on the basis of following criteria. 1) Aims and Hypothesis clearly defined, adequate sample representation, patient care quality, ethical approval protocol, outcomes assessment, validity and reliability of outcome measure, attempt to blind researcher, follow-up, appropriate statistical analysis and missing data reported. Ten item defined evaluation of methodological quality (MQ) is presented in Figure 2. Risk of Bias were assessed using Cochrane collaboration's tool on the basis of the following criteria such as selection bias, performance bias, attrition bias, reporting bias and miscellaneous. Cochrane Collaboration’s tool for assessing the risk of bias was used and the results are presented in Table 627.\n\nThe following data were extracted according to study characteristics (e.g., first author, year of publication, search dates, and number of included studies), patient characteristics (functional constipated children, aged between 2–15), sample size, study type (e.g., Randomized open label, prospective, controlled, parallel-group clinical trial study), randomization methods (e.g., \"systematic randomization and simple randomization”) and outcome measures/variables (e.g., improvement in defecation frequency per week, improvement in the episodes of fecal incontinence per week, improvement in the episodes of retentive posturing per week, improvement in the average of severity of pain of defecation (by VAS), improvement in the average of consistency of stool defecated (by VAS) and patient’s drug compliance). Data extraction was performed by Muhammad Shazad Aslam. Transcripts were analysed, coded and data was extracted using the demo version of qualitative data analysis software Atlas.ti 8.0 . Table 3 represent all the data that was extracted. All the meta-data are available as Dataset 1.\n\nMeta-analysis was conducted using the Review Manager (RevMan) 5.3 software28. The summary measures were reported as odds ratios (ORs) or as a standard mean difference (SMD) with 95% confidence intervals (CI). The presence of heterogeneity among trials was assessed using the Chi-square test, and the extent of the inconsistency was measured by I2 statistics. Output file from RevMan is available as Underlying data29.\n\n\nResults and discussion\n\nBoth of the selected studies were not blinded during intervention and outcome assessment that will result in performance bias and detection bias respectively. These biases occur where the investigators know about the participant's treatment group. Performance bias can also refer to the fact that participants can change their responses or behaviour if they know which group they are allocated in. Blinding of outcome assessment may decrease the risk of the investigator or participant being aware of the treatment that a patient is receiving. If the participants and the caregivers are aware of the intervention and outcome that may affect the behavior of the participants, these behavioral changes may affect the performance of the treatment. Clinical trials on adults were also excluded, such as a randomized clinical trial of a phytotherapeutic compound containing Pimpinella anisum, Foeniculum vulgare, Sambucus nigra, and Cassia augustifolia for chronic constipation9. Results of both included studies were non-significant when comparing their baseline characteristics of pediatric functional constipation as presented in Table 5. During analysis of study characteristics, it was found that both of studies demonstrated Cassia fistula is helping to treat constipation among the children as shown in Table 3, but there is a risk of bias according to Cochrane Collaboration’s tool (Table 6). Moreover, both studies found were from one country (Iran). During a methodological assessment, many flaws were identified such as inadequate patient care, attempt to blind the researcher and missing data (Figure 2). During meta-analysis, the comparison was made before and after treatment among different variables such as defaecation, fecal incontinence, retentive posturing, the severity of pain, and consistency of stool. All the variable (before and after treatment) were found to be symmetrical when plotted on a funnel plot as shown in Figure 4, Figure 6, Figure 8, Figure 10, Figure 12, and Figure 14 respectively. The overall effect for some variables is statistically insignificant (P=0.11, P=0.49, P=0.24) such as fecal incontinence, retentive posturing, and acceptance, tolerance respectively. High heterogeneity was found in two variables i.e severity of pain (90%) and consistency of stool (77%). All the forest plot of defaecation, fecal incontinence, retentive posturing, severity of pain, consistency of stool and acceptance and tolerance are represented in Figure 3, Figure 5, Figure 7, Figure 9, Figure 11 and Figure 13 respectively.\n\n\nConclusion\n\nAfter evaluation of results, it was found that Cassia fistula was not completely effective. It was partly effective in reducing the pain and consistency of stool during constipation. However, these results cannot be generalized among all population. A well designed, expert validated protocol is required in the future. There is a need to develop an instrument that will be free from bias. Moreover, the results cannot be generalized among all species of Cassia as the studies available are only for one species. There is a need to isolate identified bioactive compounds from different species of Cassia and evaluate the effect of different factors such as duration of constipation, defecation, incontinence or retentive posturing under clinical trial.\n\n\nDeclarations\n\nUnderlying data. Open Science Framework: Application of genus Cassia in the treatment of Constipation: A systematic review. https://doi.org/10.17605/OSF.IO/PKR4N29\n\nThis project contains the following underlying data:\n\n- Cassia Senna for Constipation.rm5 (study RevMan file)\n\n- Quotation Manager.xlsx (study characteristics of citations included in this study)\n\nOpen Science Framework: PRISMA diagram and flowchart for the study “Application of genus Cassia in the treatment of constipation: A systematic review”. https://doi.org/10.17605/OSF.IO/PKR4N29\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\nThis research was supported by Xiamen University Malaysia.\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\nBharucha AE: Constipation. Best Pract Res Clin Gastroenterol. 2007; 21(4): 709–731. PubMed Abstract \| Publisher Full Text\n\nCamilleri M, Ford AC, Mawe GM, et al.: Chronic constipation. Nat Rev Dis Primers. 2017; 3: 17095. PubMed Abstract \| Publisher Full Text\n\nLe Luyer B, Ménard M: [Constipation in children]. Rev Prat. 1998; 48(4): 376–381. PubMed Abstract\n\nLiem O, Harman J, Benninga M, et al.: Health utilization and cost impact of childhood constipation in the United States. J Pediatr. 2009; 154(2): 258–262. PubMed Abstract \| Publisher Full Text\n\nUc A, Hyman PE, Walker LS: Functional gastrointestinal disorders in African American children in primary care. J Pediatr Gastroenterol Nutr. 2006; 42(3): 270–274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNurko S, Garcia-Aranda JA, Worona LB, et al.: Cisapride for the treatment of constipation in children: A double-blind study. J Pediatr. 2000; 136(1): 35–40. PubMed Abstract \| Publisher Full Text\n\nGremse DA, Hixon J, Crutchfield A: Comparison of polyethylene glycol 3350 and lactulose for treatment of chronic constipation in children. Clin Pediatr (Phila). 2002; 41(4): 225–229. PubMed Abstract \| Publisher Full Text\n\nGuerra PV, Lima LN, Souza TC, et al.: Pediatric functional constipation treatment with Bifidobacterium-containing yogurt: a crossover, double-blind, controlled trial. World J Gastroenterol. 2011; 17(34): 3916–3921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPicon PD, Picon RV, Costa AF, et al.: Randomized clinical trial of a phytotherapic compound containing Pimpinella anisum, Foeniculum vulgare, Sambucus nigra, and Cassia augustifolia for chronic constipation. BMC Complement Altern Med. 2010; 10: 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMozaffarpur SA, Naseri M, Esmaeilidooki MR, et al.: The effect of cassia fistula emulsion on pediatric functional constipation in comparison with mineral oil: a randomized, clinical trial. Daru. 2012; 20(1): 83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThamlikitkul V, Bunyapraphatsara N, Dechatiwongse T, et al.: Randomized controlled trial of Cassia alata Linn. for constipation. J Med Assoc Thai. 1990; 73(4): 217–222. PubMed Abstract\n\nJafri MA, Jalis Subhani M, Javed K, et al.: Hepatoprotective activity of leaves of Cassia occidentalis against paracetamol and ethyl alcohol intoxication in rats. J Ethnopharmacol. 1999; 66(3): 355–361. PubMed Abstract \| Publisher Full Text\n\nAbubacker MN, Ramanathan R, Senthil Kumar T: In vitro antifungal activity of cassia alata linn. Flower extract. Indian Journal of Natural Products and Resources. 2008; 7(1): 6–9. Reference Source\n\nBenjamin TV, Lamikanra A: Investigation of Cassia alata, a Plant Used in Nigeria in the Treatment of Skin Diseases. Q J Crude Drug Res. 1981; 19(2–3): 93–96. Publisher Full Text\n\nSingh S, Singh SK, Yadav A: A Review on Cassia species: Pharmacological, Traditional and Medicinal Aspects in Various Countries. American Journal of Phytomedicine and Clinical Therapeutics. 2013; [cited 2019 Jan 11]; 3: 291–312. Reference Source\n\nPatil UK, Saraf S, Dixit VK: Hypolipidemic activity of seeds of Cassia tora Linn. J Ethnopharmacol. 2004; 90(2–3): 249–252. PubMed Abstract \| Publisher Full Text\n\nGaddam SA, Kotakadi VS, Sai Gopal DV, et al.: Efficient and robust biofabrication of silver nanoparticles by cassia alata leaf extract and their antimicrobial activity. J Nanostruct Chem. 2014; 4(1): 82. Publisher Full Text\n\nSumathy V, Zakaria Z, Jothy SL, et al.: In vitro and in vivo antifungal activity of Cassia surattensis flower against Aspergillus niger . Microb Pathog. 2014; 77: 7–12. PubMed Abstract \| Publisher Full Text\n\nYuenyongsawad S, Bunluepuech K, Wattanapiromsakul C, et al.: Anti-cancer activity of compounds from Cassia garrettiana heartwood. Songklanakarin J Sci Technol. 2014; 36(2): 189–194, [cited 2019 Jan 11] Reference Source\n\nJung HA, Ali MY, Jung HJ, et al.: Inhibitory activities of major anthraquinones and other constituents from Cassia obtusifolia against β-secretase and cholinesterases. J Ethnopharmacol. 2016; 191: 152–160. PubMed Abstract \| Publisher Full Text\n\nSivakumar V, Ilanhtiraiyan S, Ilayaraja K, et al.: Influence of ultrasound on Avaram bark (Cassia auriculata) tannin extraction and tanning. Chem Eng Res Des. 2014; 92(10): 1827–1833. Publisher Full Text\n\nNgoc TM, Nhiem NX, Khoi NM, et al.: A new coumarin and cytotoxic activities of constituents from Cinnamomum cassia. Nat Prod Commun. 2014; 9(4): 487–488. PubMed Abstract\n\nJeyaratnam N, Nour AH, Kanthasamy R, et al.: Essential oil from Cinnamomum cassia bark through hydrodistillation and advanced microwave assisted hydrodistillation. Ind Crops Prod. 2016; 92: 57–66. Publisher Full Text\n\nZeng J, Xue Y, Lai Y, et al.: A new phenolic glycoside from the barks of Cinnamomum cassia. Molecules. 2014; 19(11): 17727–17734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuximon-Ramma A, Bahorun T, Soobrattee MA, et al.: Antioxidant activities of phenolic, proanthocyanidin, and flavonoid components in extracts of Cassia fistula. J Agric Food Chem. 2002; 50(18): 5042–5047. PubMed Abstract \| Publisher Full Text\n\nFabricant DS, Farnsworth NR: The value of plants used in traditional medicine for drug discovery. Environ Health Perspect. 2001; 109 Suppl 1: 69–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCollaboration C: Cochrane handbook for systematic reviews of interventions. 2008. Reference Source\n\nCollaboration C: Review manager (RevMan)[computer program]. 2011.\n\nAslam MS: Application of genus Cassia in the treatment of Constipation: A systematic review. 2019. https://www.doi.org/10.17605/OSF.IO/PKR4N" }	[ { "id": "47237", "date": "23 May 2019", "name": "Massimo Bellini", "expertise": [ "Reviewer Expertise Gastroenterology: Functional Digestive 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\nI’m really puzzled about the usefulness of a review which takes into consideration only two papers and altogether only 132 patients. Constipation, as the author himself states, is a very frequent condition, so the conclusions are inevitably flawed by this bias. The author should address this issue in the conclusions\nHence, I have significant reservations.\nMoreover, here enclosed you'll find a copy of the paper. The authors can find some suggestions, as tracked changes and comments to the original manuscript, which could improve the quality of the manuscript.\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": "4663", "date": "23 May 2019", "name": "Muhammad Shahzad Aslam", "role": "Reviewer Response", "response": "Dear,I have read the report. The selected studies were according to the criteria given inside the paper. So, there were only two selected studies for the systematic review. The comments you have mentioned are appreciable. Thanks" }, { "c_id": "6404", "date": "08 Mar 2021", "name": "Muhammad Shahzad Aslam", "role": "Author Response", "response": "Please see inclusion criteria (Only articles involving children aged between 2–15 were included. Only articles involving children aged between 2–15 were included.)" } ] }, { "id": "50310", "date": "28 Jun 2019", "name": "Farhad Shokraneh", "expertise": [ "Reviewer Expertise Evidence synthesis", "Systematic review", "Overview", "Scoping review", "Rapid review", "Randomized controlled trials", "Open data", "Open access", "Open source", "Medical journalism", "Peer-review", "Open source", "Outcomes", "Interventions", "Cochrane" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author investigates the effect of Cassia fistula on three outcomes based on two included studies. It seems a valuable research that could potentially direct the pharmaceutical companies toward a new agent to treat the constipation.\n\nTitle The title should be “Cassia fistula for treatment of children and adolescents with constipation: A systematic review of randomized controlled trials”.\n\nAuthorship The systematic review requires a process that involves at least two people for screening and data extraction. I suggest the author to find another systematic reviewer to be involved in this work.\n\nAbstract The abstract is very brief and sentences are incomplete. Abstract should be re-written following the example from similar systematic reviews and following PRISMA for Abstract.\n\nMinor edits\n“act an effective as a laxative” should be “act as effective as laxatives”.\n\n“search data” should be “search date”.\nMethods\nThe standard search filter for finding RCTs as reported in Cochrane Handbook should be used.\n\nIt is advised in Cochrane Handbook searching Embase, MEDLINE/PubMed, and CENTRAL for systematic reviews of RCTs and this review reports searching only MEDLINE/PubMed.\n\nExcluding closed access papers without trying to order them from library or contacting the authors introduces full-text or open access bias.\n\nThe structure of the method should change and start with 1: Eligibility Criteria 2: Databases and search strategy 3: Screening process 4: Data extraction process 5: Assessment of risk of bias process 6: Data analysis plan. Currently, the first section of the methods reports info on search and eligibility criteria while there are specific subheadings for these sections.\n\nThe rationale behind excluding studies older than 10 years from search date is not clear.\n\nThe reported number of search results 2207 is not reasonable. A proper search should not find more than 100-200 results for this topic.\n\nThe search date has not been reported.\n\nFollowing a published Cochrane review of RCTs in terms of structure and all the items in PRISMA Checklist for full systematic reviews is recommended.\n\nConstipation is missing from search strategy and MeSH terms in the table.\n\nThe numbers in PRISMA flow diagram does not match the numbers reported in the text and it does not seem to be correct. Following one of the existing published systematic reviews of RCTs from Systematic Reviews journal or Cochrane Database of Systematic Reviews is recommended.\n\nThe quality of included studies is being assessed using Cochrane Risk of Bias tool. The role of MQ and using Atlas.ti is not clear in this review. These two are not usually used for SR of RCTs.\n\nStudying the meta-analysis section in the Cochrane Handbook which is freely available online could help revising the statistical analysis.\n\nTable 2 should report PICOS not SPIO.\n\nThe time point for the outcome is not clear.\n\nThe outcome measure has not been described properly.\n\nThe complications and adverse effects have not been reported in the review.\nResults\nThe sample size reported in the Data Extraction table does not match the numbers in meta-analysis. For example the total for experimental and control group in Review Manager file is 109 for Esmaeilidooki 2016 and 71 for Mozaffarpur 2012 while in the table is 51 and 81 respectively.\n\nThe analysis for before treatment is not required. All before treatments can be deleted.\n\nFunnel plots only work with about 10-20 studies. All funnel plots can be deleted.\n\nRisk of bias based on Cochrane tool is not clear. It should be low risk, unclear risk and high risk. The current description of Yes/No does not provide enough information.\n\nIn meta-analysis, “one randomized always analysed” while the numbers in Table 4 are not the numbers in meta-analysis in Review Manager.\n\nThe control groups in two studies are different so I am not sure if combining the data from two studies in a meta-analysis is a right when there is such heterogeneity in PICOS components.\n\nSD is greater than mean is some outcomes which is an indicator of skewed data or error in reporting. Contacting the trials authors might make some of the points clear.\nDiscussion and Conclusion Current data and status of the review may not be appropriate for discussion or making a conclusion. The current evidence is limited and more rigorous studies will be required to make any conclusion or recommendations.\n\nI understand the author’s passion for conducting a systematic review and so I request the author to work with an expert supervisor in the field before revising the work.\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? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "6405", "date": "08 Mar 2021", "name": "Muhammad Shahzad Aslam", "role": "Author Response", "response": "WHO defines 'Adolescents' as individuals in the 10-19 years. So title cannot changed" } ] } ]	1	https://f1000research.com/articles/8-256
https://f1000research.com/articles/10-51/v1	28 Jan 21	{ "type": "Brief Report", "title": "Genome-wide screening of upstream transcription factors using an expression library", "authors": [ "Naoya Yahagi", "Yoshinori Takeuchi", "Yoshinori Takeuchi" ], "abstract": "The identification of upstream transcription factors regulating the expression of a gene is generally not an easy process. To facilitate this task, we constructed an expression cDNA library named Transcription Factor Expression Library (TFEL), which is composed of nearly all the transcription factors in the mouse genome. Genome-wide screening using this library (TFEL scan method) enables us to easily identify transcription factors controlling any given promoter or enhancer of interest in a chromosomal context-dependent manner. Thus, TFEL scan method is a powerful approach to explore transcriptional regulatory networks.", "keywords": [ "Transcription", "Transcription factor", "Expression cloning" ], "content": "Abbreviations\n\nTFEL, transcription factor expression library; VEGF, vascular endothelial growth factor; Fasn, fatty acid synthase; SREBP, sterol regulatory element-binding protein; HIF, hypoxia-inducible factor; LXR, liver X receptor; KLF, Kruppel-like factor.\n\n\nIntroduction\n\nTranscriptional regulation is widely involved in many biological processes of relevance such as cellular development, tissue differentiation, reprogramming and apoptosis as well as nutrient metabolism; in many cases, such processes involve complicated regulatory networks1. Generally, downstream targets of a transcription factor can be identified in a convincing way through transcriptome analyses after overexpression or knockdown of the transcription factor, or through investigating the binding interaction between the transcription factor and genomic DNA using chromatin immunoprecipitation (ChIP)-sequencing method. However, no assured methods of identifying upstream transcription factors regulating a specific promoter or enhancer exists, since the relatively poor amount of transcription factors expressed in protein pools or cDNA libraries makes purification difficult, while binding factor prediction using computer analysis based on binding motif databases suffers from high false-positive and false-negative rates in general2. Therefore, we constructed a comprehensive expression library of transcription factors in the mouse genome, the Transcription Factor Expression Library (TFEL), and developed a new method of identifying upstream transcription factors through an expression cloning technique using the TFEL (TFEL scan method). Using this TFEL scan method, we can easily identify the specific transcription factor or transcription factor complex trans-acting against any given cis-element of interest.\n\n\nMethods\n\nThe mouse transcription factor DNABook™ was purchased from DNAFORM, Tokyo, Japan3. Each spot on the sheets of DNABook was punched, and plasmid DNA was eluted in 10μl distilled water. Competent DH5α E. coli was transformed with 1μl of the eluted DNA solution, and plasmids were cloned in a standard manner.\n\nAmong 1,588 clones in the DNABook, 1,350 clones were on pFLCI vector, 204 clones on modified BluescriptI(+), 23 clones on pFLCIII, and 11 clones were on pFLCII.\n\nThe clones on pFLCI were digested by restriction enzymes EcoRI and BamHI, clones on pFLCII by XhoI and BamHI, pFLCIII by Bsu36I and BamHI, and clones on modified BluescriptI(+) were cut by SacI and XhoI on a 37 ˚C heat block. The restricted DNA fragments were separated on agarose gel (Cat. # 5091, Agarose Basic TAKARA) by electrophoresis, and verified if they are correspondent with the predicted band pattern calculated from the database (GenBank / EMBL / DDBJ) sequence. Unfortunately, information about which clones are included in the DNABook cannot be made public due to a confidentiality agreement. For more details, please contact the corresponding author via email (nyahagi-tky@umin.ac.jp)\n\nTFEL was constructed by transferring all the DNABook clones into pcDNA3.1 expression vector (Invitrogen). To facilitate this process, we first modified pcDNA3.1 vector and inserted the PCR-amplified kanamycin-resistant gene from pENTR4 vector (Invitrogen) into the original pcDNA3.1 at the SmaI-SalI sites by replacement of neomycin-resistant gene to make the vector resistant to kanamycin. By this modification, we could ligate an insert DNA fragment to the pcDNA3.1 vector directly without agarose gel isolation of the insert from vector backbone fragment and undigested plasmid after restriction enzyme digestion of DNABook clones, which are ampicillin-resistant.\n\nBecause DNABook clones were on as many as 23 versions of various vectors (pFLCI: 12, pFLCII: 3, pFLCIII: 6, and modified BluescriptI(+): 2), multiple strategies based on the calculation of restriction sites were used to construct TFEL clones.\n\nFor pFLCI clones, the combination of EcoRI and BamHI digestion was first tried to cut insert fragments out of vectors when inserts were not predicted to be cut by the two restriction enzymes, and excised fragments were inserted into pcDNA3.1(-) vector at EcoRI and BamHI sites. For pFLCI clones whose inserts were cut by EcoRI or BamHI, SfiI digestion and ligation to pcDNA3.1(-)-SfiI (XhoI-EcoRI part of pcDNA3.1(-) multiple cloning site was replaced with synthesized SfiI sequence) was next attempted. For clones to which neither strategy was applicable, either EcoRI-ApaI (into pcDNA3.1(+)), EcoRI-KpnI (into pcDNA3.1(-)), or SacI-BamHI (into pcDNA3.1(+)-SacI-mod whose SacI site is modified to be unique by introducing mutation to the other SacI site outside of the multiple cloning site) strategy was tried. For the other 73 clones to which these strategies were not applicable, SfiI digested multiple fragments were inserted into pcDNA3.1(-)-SfiI at one time. When another restriction enzyme such as ApaI, KpnI, NotI, or ScaI could be used to cut the vector backbone without cutting the insert, this treatment was done at the same time to reduce the possibility of a vector fragment being cloned into pcDNA3.1.\n\nFor modified BluescriptI(+) clones, the SfiI (into pcDNA3.1(-)-SfiI) or XhoI-SacI (into pcDNA3.1(-)-SacI-mod) strategy was used. For 47 plasmids on ZA vector whose orientation of the insert is opposite to that of others, corresponding “(+)” version of pcDNA3.1 vectors were used. 7 clones on ZX vector had corrupted 5’-side SfiI and XhoI sites, therefore KpnI-SfiI, KpnI-BglII, or BssHII strategy was used instead.\n\nFor pFLCII clones, the XhoI-BamHI strategy was used when applicable. Others were excised with SacI and cloned into SacI site of pcDNA3.1(-)-SacI-mod, after which the inserted direction was checked, and clones constructed in the correct direction was selected.\n\npFLCIII clones were ligated using I-CeuI and PI-SceI.\n\nThe mouse Vegfa-luc plasmid (-1210 to +246) was generated from a PCR-amplified fragment inserted into pGL2-basic vector (Cat. #E1641, Promega) at SmaI site. The PCR primers used were 5’-AAGATGAACCGTAAGCCTAGGCT-3’ and 5’-AACCGTTGGCACGATTTAAGA-3’ and amplification was performed according to a 3-step method. The rat Fasn-luc plasmid (-397 to +28) was prepared as described previously4. The mouse Srebf1c-luc plasmids (-2.2k to +40 and -249 to -144) were constructed as described elsewhere5,6.\n\nTo screen transcription factors regulating a target promoter, TFEL clones were co-transfected with the specific promoter-luc plasmid into HEK293 cells using SuperFect Transfection Reagent (Cat. #301305, QIAGEN). The screenings were performed by co-transfecting 10–20 clones with each luciferase plasmid per one well. Transfection was performed using 0.5μg of plasmid DNA pooled equally from 10 or 20 TFEL clones per well. The luciferase activity in transfectants was measured on a luminometer (BERTHOLD) with a standard assay kit (Cat. #E1483, Promega). First screen was performed with 10 or 20-clone pools per well, and significantly shifted pools were further tested with one clone at a time.\n\n\nData analysis\n\nAll the data from a luminometer were output as Excel sheets, and directly input to graphs. All the graphs were drawn in Microsoft Excel 2016 (16.0.5083.1000).\n\n\nResults\n\nTo construct the TFEL expression plasmid library, we used clones from the RIKEN FANTOM libraries (Figure 1A). The clone subset consisting of genome-wide transcription factors is available from DNAFORM as Mouse Transcription factor DNABook™, and is comprised of 1,588 nonredundant genome-wide mouse transcription factor genes7. We retrieved all the clones in the DNABook using Escherichia coli transformation. After checking the band patterns using several restriction enzymes and sequencing if necessary, each excised insert DNA fragment was transferred to a pcDNA3.1 expression vector. In this manner, we constructed a pcDNA3.1 expression library composed of 1,588 clones of genome-wide and nonredundant mouse transcription factors.\n\n(A) Construction of Transcription Factor Expression Library (TFEL). The original clones were derived from the RIKEN FANTOM1/2/3 libraries, and 1,588 clones that are predicted to be transcription factor genes by RIKEN TFdb are distributed as Mouse Transcription factor DNABook™ from DNAFORM Inc. TFEL consists of the DNABook clones transferred into pcDNA3.1 expression vectors through recombination using several restriction enzymes optimized to each clone. (B) Workflow of identifying upstream transcription factors using TFEL. A luciferase plasmid with a specific promoter of interest is co-transfected with the TFEL clone(s) into mammalian cells and the luciferase reporter activities are measured.\n\nNext, we evaluated the validity of this expression cloning method (TFEL scan method) using the TFEL (Figure 1B8). Figure 2A–C8 shows three cases in which the upstream transcription factors influencing well -known promoters were examined: A, vascular endothelial growth factor (gene name: Vegfa), B, fatty acid synthase (gene name: Fasn), and C, sterol regulatory element-binding protein (SREBP)-1c (gene name: Srebf1c). In these cases, well-known determinant transcription factors (HIF-1α and -2α for Vegfa-luc, SREBP-1a and -2 for Fasn-luc, and LXRα for Srebf1c-luc) were identified as expected. These screenings were performed by co-transfecting 10–20 clones with each luciferase plasmid per one well.\n\n(A–C) Representative cases of the analysis of upstream transcription factors using Transcription Factor Expression Library (TFEL) against specific promoters: (A) vascular endothelial growth factor (gene name: Vegfa), (B) fatty acid synthase (gene name: Fasn), and (C) sterol regulatory element-binding protein-1c (gene name: Srebf1c). Transfection was performed using 0.5μg of plasmid DNA pooled equally from 10 or 20 TFEL clones per well. (D) Representative case identifying the combinatorial interactions on a specific promoter. In this assay, we screened for transcription factor(s) that suppress LXR activity on the SREBP-1c promoter (-249 to -144). To activate LXR and to improve the sensitivity of the assay, we added an LXR ligand T0901317 to the culture media. The first screening was performed using a 20-clone pool assay, and individual clones were examined in the second screening.\n\nThese results demonstrated that the identification of upstream transcription factors using this expression screening method works well.\n\nWe further attempted to identify unknown partner(s) interacting with a specific transcription factor on a specific gene promoter. To this end, we screened interacting partner(s) for LXR on the SREBP-1c promoter6. As shown in Figure 2D13, KLF4 was picked up as a candidate from the first screening performed by co-transfecting 20 clones with an SREBP-1c promoter luciferase plasmid in the presence of an LXR ligand T0901317. Further screening of the clones one by one revealed that KLF4 and KLF15 interacted with and suppressed LXR on the SREBP-1c promoter (data not shown, see ref 6). Thus, the TFEL scan method was proved to be useful for searching the interacting partner against a specific transcription factor of interest in a DNA-sequence-dependent manner.\n\n\nDiscussion\n\nIn the present study, we clearly demonstrated that our new method of TFEL scan is a powerful approach to explore transcriptional regulatory networks by identifying transcription factor(s) involved in the regulation.\n\nFirst, we showed that in three cases (Vegfa-luc, Fasn-luc and Srebf1c-luc), we could easily identify well-known determinant transcription factors (HIF-1α and -2α for Vegfa-luc, SREBP-1a and -2 for Fasn-luc, and LXRα for Srebf1c-luc) as expected (Figure 2A–C8). In addition, we recently succeeded in identifying the binding transcription factor for an important SNP at the diabetes-associated TCF7L2 gene locus through our TFEL scan method9. It is suggested that TCF7L2 is the single largest effect of a common SNP on type 2 diabetes risk in European populations10.\n\nThus, we demonstrated that the TFEL scan method works very well to identify the regulatory transcription factor in a simple model.\n\nNext, we additionally succeeded in identifying transcription factors forming a complex such as LXR and KLF4 (Figure 2D8). Therefore, the TFEL scan method was proved to be similarly effective even in these more complex cases.\n\nIt is well known that the transcriptional output of a gene is due to the joint activity of many transcription factors, the binding and activation of which are highly interdependent1. This cooperation is often mediated by direct physical contact between two or more transcription factors, forming homodimers, heterodimers, or larger transcriptional complexes11. In fact, it has been estimated that approximately 75% of all metazoan transcription factors heterodimerize with other factors12. Thus, the importance of transcription factor combinations has been highlighted more and more, and the mapping of the combinatorial interactions among transcription factors has been attempted using a mammalian two-hybrid system11. Our successful results identifying KLFs as interacting partners of LXR on Srebf1c promoter clearly showed that the expression cloning method using the TFEL (TFEL scan method) may also be useful for exploring interacting partner(s) among transcription factors on a specific DNA fragment of interest. In contrast, binding site prediction based on binding motif databases suffers from high false-positive and false-negative rates in general13. In particular, the sequence-based approach has no power to predict transcription factor complexes formed by protein-protein interaction. Conversely, the search for protein-protein interactions among transcription factors alone, for example, using a mammalian two-hybrid system, may not be sufficient to elucidate the regulatory complexes in certain situations including the present case of LXR-KLFs complex, because complexes like LXR-KLFs are also dependent on DNA binding and therefore locus-specific. This relatively weak interaction supported by the DNA backbone enables gene-specific regulations and may possibly give more diversity to transcriptional networks. Thus, our strategy of screening for the transcription factor complex in a chromosomal context-dependent manner using the TFEL scan method can be a very effective and powerful approach for exploring sophisticated transcriptional networks in detail, because this screening system is performed under more physiological conditions than traditional methods.\n\nOur initial approach using a mixed pool assay handling 20 clones at a time failed to detect KLF15 in the first screening described above. Interference among the co-transfected transcription factors in the pools was thought to have affected the results and veiled the KLF15-LXR interaction, suggesting that a single clone assay is preferable, especially for screening for combinatorial interactions. In relation to this point, interactions with endogenous transcription factors should also affect the results to varying degrees, and screenings in different cell lines should provide more information.\n\nOur TFEL library is based on the Mouse Transcription factor DNABook™, and a small portion of transcription factors are missing, as they previously reported7. For example, among the 17 KLF family members, 7 KLFs (KLF7, 9, 10, 11, 12, 14 and 17) are missing. We are planning to fix this problem and to complete the construction of a comprehensive expression library in the near future.\n\nIn summary, the present study clearly demonstrates that our expression cloning method using the TFEL (TFEL scan method) enables us to efficiently identify regulatory transcription factors as well as to elucidate combinatorial interactions among transcription factors in a chromosomal context-dependent manner. Thus, the TFEL scan method is a powerful approach to explore transcriptional regulatory networks.\n\n\nData availability\n\nFigshare: Underlying data of Figure 2. Representative cases of the analysis of upstream transcription factors using TFEL against specific promoters. https://doi.org/10.6084/m9.figshare.132373198\n\nThis project contains the following underlying data:\n\n- Fig2A_090109_BxVEGF1.3k.xls\n\nRepresentative cases of the analysis of upstream transcription factors using TFEL against specific promoters: (A) vascular endothelial growth factor (gene name: Vegfa).\n\n- Fig2B_081218_AxFAS0.4k.xls\n\nRepresentative cases of the analysis of upstream transcription factors using TFEL against specific promoters: (B) fatty acid synthase (gene name: Fasn).\n\n- Fig2C_101026_B(10)xSREBP1c2.2k.xls\n\nRepresentative cases of the analysis of upstream transcription factors using TFEL against specific promoters: (C) sterol regulatory element-binding protein-1c (gene name: Srebf1c).\n\n- Fig2D_081219_AxSREBP1c-T_1st.xls\n\nRepresentative case identifying the combinatorial interactions on a specific promoter. In this assay, we screened for transcription factor(s) that suppress LXR activity on the SREBP-1c promoter (-249 to -144). 1st screening data.\n\n- Fig2D_081225_SREBP1c-T_2nd_A28.xls\n\nRepresentative case identifying the combinatorial interactions on a specific promoter. In this assay, we screened for transcription factor(s) that suppress LXR activity on the SREBP-1c promoter (-249 to -144). 2nd screening data.\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 Yuji Teraoka, Nobue Suzuki, and Kazuko Shibahara for skilled technical assistance.\n\n\nReferences\n\nLambert SA, Jolma A, Campitelli LF, et al.: The Human Transcription Factors. Cell. 2018; 172(4): 650–665. PubMed Abstract \| Publisher Full Text\n\nWasserman WW, Sandelin A: Applied bioinformatics for the identification of regulatory elements. Nat Rev Genet. 2004; 5(4); 276–287. PubMed Abstract \| Publisher Full Text\n\nCarninci P, Kasukawa T, Katayama S, et al.: The transcriptional landscape of the mammalian genome. Science. 2005; 309(5740): 1559–1563. PubMed Abstract \| Publisher Full Text\n\nAmemiya-Kudo M, Shimano H, Hasty AH, et al.: Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes. J Lipid Res. 2002; 43(8): 1220–1235. PubMed Abstract \| Publisher Full Text\n\nTakeuchi Y, Yahagi N, Nakagawa Y, et al.: In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression. Biochem Biophys Res Commun. 2007; 363(2): 329–335. PubMed Abstract \| Publisher Full Text\n\nTakeuchi Y, Yahagi N, Aita Y, et al.: KLF15 enables rapid switching between lipogenesis and gluconeogenesis during fasting. Cell Rep. 2016; 16(9): 2373–2386. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanamori M, Konno H, Osato N, et al.: A genome-wide and nonredundant mouse transcription factor database. Biochem Biophys Res Commun. 2004; 322(3): 787–793. PubMed Abstract \| Publisher Full Text\n\nYahagi N: Underlying data of Figure 2. Representative cases of the analysis of upstream transcription factors using TFEL against specific promoters. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13237319.v1\n\nPiao X, Yahagi N, Takeuchi Y, et al.: A candidate functional SNP rs7074440 in TCF7L2 alters gene expression through C-FOS in hepatocytes. FEBS Lett. 2018; 592(3): 422–433. PubMed Abstract \| Publisher Full Text\n\nZeggini E, Weedon MN, Lindgren CM, et al.: Replication of genome-wide association signals in UK samples reveals risk loci for type 2 diabetes. Science. 2007; 316(5829): 1336–1341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRavasi T, Suzuki H, Cannistraci CV, et al.: An Atlas of Combinatorial Transcriptional Regulation in Mouse and Man. Cell. 2010; 140(5): 744–752. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalhout AJM: Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping. Genome Res. 2006; 16(12): 1445–1454. PubMed Abstract \| Publisher Full Text\n\nKim TM, Park PJ: Advances in analysis of transcriptional regulatory networks. Wiley Interdiscip Rev Syst Biol Med. 2011; 3(1): 21–35. PubMed Abstract \| Publisher Full Text" }	[ { "id": "78980", "date": "09 Feb 2021", "name": "Kazuhiko Igarashi", "expertise": [ "Reviewer Expertise gene regulation by transcription factors", "their functions in cell differentiation and 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\nThis is a very important resource paper for the fields of gene regulation, metabolism, differentiation and stress response. Overall, the manuscript is well written and the results are clearly presented. There are some issues readers may like to know better.\nHuman genome is estimated to encode for 1639 transcription factors (Lambert SA et al, Cell 172, 650-, 20181). How much of the mouse transcription factors in the expression library is overlapping with the human transcription factors. A brief discussion will be helpful.\n\nMany of the transcription factors characterised thus far are responsive to stresses or ligands. The results in this manuscript show that such transcription factors can be identified by overexpression. A brief discussion is needed.\n\nHow will this expression library be shared?\n\nThe description in the text of experiment in Fig. 2D may be improved. Did authors try to identify factors that restrict/inhibit LXR? Why was this important for the regulation of Srebf1c promoter?\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": "6406", "date": "08 Mar 2021", "name": "Naoya Yahagi", "role": "Author Response", "response": "1. Human genome is estimated to encode for 1639 transcription factors (Lambert SA et al, Cell 172, 650-, 20181). How much of the mouse transcription factors in the expression library is overlapping with the human transcription factors. A brief discussion will be helpful. We thank the reviewer for the valuable advice. According to this suggestion, we checked the overlapping among the transcription factor databases such as RIKEN TFdb (the original database that the TFEL is based on; Kanamori M et al, Biochem Biophys Res Commun. 2004;322(3):787-793 (ref.7)), Human TF (Lambert SA et al, Cell, 2018;172(4):650-665.), and Animal TFDB 3.0 (Hu H et al, Nucleic Acids Research, 2019;47(D1):D33-D38.), using a gene symbol converter available at http://biodb.jp/. The results were as follows: the overlap between RIKEN TFdb and Human TF was 889 genes, app 65% of Human TF and 53% of RIKEN TFdb, and the overlap between RIKEN TFdb and Animal TFDB 3.0 was 958 genes, app 59% of Animal TFDB 3.0 and 57% of RIKEN TFdb. The differences were a little larger than expected and even when we compared the same Animal TFDB human and mouse databases, the overlap was limited to 1,340 genes, which were app. 80% of the 1665 human TF genes in the database. Therefore, app. 300 TF genes (20%) seemed to be different between human and mouse. Thanks to the reviewer’s comment, we could better understand the differences among TF databases, but because this content is beyond the scope of this article, we will keep it within the Comments. Also, we realized during these series of gene symbol checking that KLF 9, 10, 11 had been included in the TFEL library, and corrected the description in the discussion. 2. Many of the transcription factors characterized thus far are responsive to stresses or ligands. The results in this manuscript show that such transcription factors can be identified by overexpression. A brief discussion is needed. We thank the reviewer for the valuable comment. According to the reviewer’s advice, we added a new paragraph of discussion to the revised manuscript like this: Many of the transcription factors characterized thus far are responsive to stresses or ligands. The results of the case of LXR shown here demonstrate that such transcription factors can also be identified by overexpression. However, at the same time, we have experienced that some transcription factors activated in a ligand-dependent manner, for example, glucocorticoid receptor (GR), cannot be identified by mere overexpression without ligands (data not shown). Therefore, it may depend on transcription factors whether they can be detected without ligands/additional activators or not. 3. How will this expression library be shared? Thank you for the comment. We are planning to talk with DNAFORM about how to share this expression library in the near future. 4. The description in the text of experiment in Fig. 2D may be improved. Did authors try to identify factors that restrict/inhibit LXR? Why was this important for the regulation of Srebf1c promoter? Thank you for the question. Actually, there is a long story behind this. To make the long story short, we had found an important element shown to exert a suppressive effect on the SREBP-1c promoter in a fasting state (Takeuchi Y, et al, Cell Rep, 2016;16(9):2373-2386 (ref. 6)). According to the reviewer’s advice, we added this explanation to the revised manuscript." } ] }, { "id": "79133", "date": "19 Feb 2021", "name": "Harukazu Suzuki", "expertise": [ "Reviewer Expertise Regulation of gene expression", "TF-mediated DNA demethylation", "Cell reprogramming" ], "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 a method to identify transcription factors (TFs) regulating target gene(s). The expression constructs that authors has made are highly useful for scientific community who are interested in TF-mediated gene regulation. The manuscript is written well, but there are several issues to be addressed.\nIdentification of upstream TF(s) should be objective. Test cases shown in Figure 2A, B and C should be statistically evaluated. HIF-1a,2a and SREBP-1a,-2 may be evident, however I am not sure whether LXRa-mediated luciferase activity is significantly high or not.\n\nIn figure 2D, authors selected 4 pools. I think this selection should also be objective by using statistical evaluation. If authors statistically selected pools for further one by one test, the pool with KLF4 has been selected?\n\nOne of the most important value in this work must be TF expression plasmids that authors have systematically constructed. I think this resource should be available to scientific community.\n\nTFs regulate downstream genes by associating with promoters or enhancers. It is worth if the screen system works for TFs associating with enhancers.\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? 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? Yes", "responses": [ { "c_id": "6407", "date": "08 Mar 2021", "name": "Naoya Yahagi", "role": "Author Response", "response": "1. Identification of upstream TF(s) should be objective. Test cases shown in Figure 2A, B and C should be statistically evaluated. HIF-1a,2a and SREBP-1a,-2 may be evident, however I am not sure whether LXRa-mediated luciferase activity is significantly high or not. We thank the reviewer for the valuable advice. Because the data have been obtained for the screening purpose, each pool has just single value, so robust statistical analyses to show significance are a little difficult to perform. However, as an empirical method, 2SD (standard deviation) criteria is often used. For Figure 2A (Vegfa-luc), only the pools #5 and #29 (black bars; which means that they actually contain positive clones) are above the 2SD line. For Figure 2B (Fasn-luc), the pools #30 and #60 (black bar) are above the 2SD line, but the pool #30 was revealed to contain no positive clones after the 2nd screening of each clone in the pool. For Figure 2C (Srebf1c-luc), the pools #5, #50, #76 (black bar) and #78 are above the 2SD line, but only the pool #76 was revealed to have a positive clone (LXRα) after the 2nd screening. Our actual strategy was to pick up the top 5% (or the bottom 5% for the search of suppressors as shown in Figure D) for the 2nd screening. As a result, both approaches (the 2SD criterion and the top 5% criterion) are proved to work sufficiently as a screening method. Based on this discussion, we added the following paragraph to the revised manuscript: From the statistical point of view, we picked up the top (or the bottom for D) 5% pools for the 2nd screening, and as a result, this criterion was similar to the 2SD (standard deviation) criterion in these cases. 2. In figure 2D, authors selected 4 pools. I think this selection should also be objective by using statistical evaluation. If authors statistically selected pools for further one by one test, the pool with KLF4 has been selected? Thank you for the question. We selected the top or bottom 5% pools for the 2nd screening, and only the pool #28 containing KLF4 was proved to be a true hit after the 2nd screening. 3. One of the most important value in this work must be TF expression plasmids that authors have systematically constructed. I think this resource should be available to scientific community. Thank you for the comment. We are planning to talk with DNAFORM about how to share this expression library in the near future. 4. TFs regulate downstream genes by associating with promoters or enhancers. It is worth if the screen system works for TFs associating with enhancers. We thank the reviewer for the valuable comment. Yes, we think that our method is also effective for enhancers to be matched with TFs, as shown in the case of TCF7L2 gene (Piao X, et al, FEBS Lett, 2018;592(3):422-433 (ref. 9)). According to the reviewer’s advice, we added the following sentence to the revised manuscript: It is noteworthy that the TFEL scan method is also applicable to enhancers as well as promoters as shown in the case of TCF7L2 gene." } ] } ]	1	https://f1000research.com/articles/10-51
https://f1000research.com/articles/10-187/v1	08 Mar 21	{ "type": "Research Article", "title": "Building capacity through open approaches: Lessons from developing undergraduate electrophysiology practicals", "authors": [ "Erin C. McKiernan", "Lucía Medina Gómez", "Lucía Medina Gómez" ], "abstract": "Background: Electrophysiology has a wide range of biomedical research and clinical applications. As such, education in the theoretical basis and hands-on practice of electrophysiological techniques is essential for biomedical students, including at the undergraduate level. However, offering hands-on learning experiences is particularly difficult in environments with limited resources and infrastructure. Methods: In 2017, we began a project to design and incorporate electrophysiology laboratory practicals into our Biomedical Physics undergraduate curriculum at the Universidad Nacional Autónoma de México. We describe some of the challenges we faced, how we maximized resources to overcome some of these challenges, and in particular, how we used open scholarship approaches to build both educational and research capacity. Results: We succeeded in developing a number of experimental and data analysis practicals in electrophysiology, including electrocardiogram, electromyogram, and electrooculogram techniques. The use of open tools, open platforms, and open licenses was key to the success and broader impact of our project. We share examples of our practicals and explain how we use these activities to strengthen interdisciplinary learning, namely the application of concepts in physics to understanding functions of the human body. Conclusions: Open scholarship provides multiple opportunities for universities to build capacity. Our goal is to provide ideas, materials, and strategies for educators working in similar resource-limited environments.", "keywords": [ "biomedical sciences", "electrophysiology", "higher education", "undergraduate education", "open education", "open research", "open scholarship", "open science" ], "content": "Introduction\n\nElectrophysiological techniques, like electromyogram (EMG), electrocardiogram (ECG), and electroencephalogram (EEG) recording, are commonly used in both clinical settings and biomedical research. For example, EMG recordings are used to study neuromuscular disorders1 and spinal cord injury2,3; ECG recordings are used to detect cardiac conduction disorders4 and heart attack5; and EEG recordings are used to study epileptic seizures6,7 and sleep disorders8,9. Considering the importance of these techniques, it is vital that biomedical students receive training in their physiological basis, how to perform recordings, and how to analyze electrophysiological data, starting preferably at the undergraduate level.\n\nAs recently as a decade ago, several factors made doing electrophysiology with groups of students difficult if not prohibitive. Recording equipment was large, not portable, costly, and required expertise to operate. However, in recent years, companies have emerged dedicated to the production of low-cost but high-quality electrophysiology equipment, ideal for use in educational settings. For example, Backyard Brains (BYB; backyardbrains.com) is a company that designs and sells equipment to record action potentials (APs) in insects and plants, EMG and ECG in human subjects, and a variety of other electrophysiology products and accessories, most at prices below $300 U.S. dollars (USD). Many of these devices fit in the palm of your hand and connect to any smartphone, making them highly portable and easy to use. We have entered a new era when electrophysiology can now be easily brought into the classroom. However, many lesson plans and degree programs have yet to catch up.\n\nIn 2014, the Faculty of Science at the Universidad Nacional Autónoma de México (UNAM) – Latin America’s largest public university –launched its first undergraduate degree program in biomedical physics10,11. The overall goal of the program is to provide students with integrative theoretical and practical training in the areas of physics, mathematics, and biomedical sciences, to produce inter-disciplinary professionals that can work in diverse clinical and research environments. Specifically, the objectives of the program include, but are not limited to, educating students in: (1) physics applied to the study of the human body; (2) physics applied to medical diagnosis and therapy; and (3) physical principles underlying the instrumentation and function of the latest biomedical devices11. We believe electrophysiology training is an important part of meeting these educational objectives. However, due to limited resources and infrastructure, none of our core courses previously included laboratory practicals in electrophysiology. The same limitations were also affecting our ability to develop electrophysiology research projects with our students.\n\nIn 2017 and 2019, we received funds through UNAM’s educational innovation grants (PAPIME) program to develop electrophysiology practicals for our biomedical physics students. With a total of nearly $17,000 USD over the last three years, we were able to buy recording equipment, microscopes, computers, instrumentation accessories, and more, and successfully developed electrophysiology practicals which we have released (electrophys.wordpress.com and github.com/emckiernan/electrophys) as Open Educational Resources (OERs)12,13. Here we share examples of some of these practicals, their use in biomedical physics education, and how we integrated them into our curriculum. Furthermore, we describe the techniques and tools we used to make the most of the grant funds in a limited-resource environment, and specifically how open scholarship practices (open data, open education, open hardware, open protocols, open source) helped us broaden our impact and build not just educational but also research capacity. We hope sharing our experience will help other academics working in similar environments.\n\n\nInstitutional context\n\nTo explain some of the motivation behind this project and its potential impact, it is important to first understand the environment in which we work, both within UNAM and the Faculty of Science.\n\nUNAM is the largest public university in Latin America14. As of 2019-2020, over 360,000 students were enrolled at UNAM, including more than 217,000 undergraduates and 30,000 graduate students15. The university has 129 undergraduate and 41 graduate degree programs. Also, in 2019 UNAM served over one million students through its continuing education, including online, programs15.\n\nAs a public insitution, education at UNAM is nearly free, subsidized by federal funds. Students pay an annual registration fee of just 20 Mexican cents (equivalent to ∼0.01 USD). This is combination with UNAM’s prestige and reputation for quality education results in a high demand for entry. Each year, less than 10% of applicants are accepted at the undergraduate level through UNAM’s admissions testing16. In other words, a huge percentage of the eligible student population in Mexico is unable to study at this university that receives the largest share of public funds – an annual budget equivalent to approximately 2 billion USD17,18. One could argue that, more than any other public institution in Mexico, UNAM has a responsibility to give back to the community. On the other hand, while UNAM receives more funds than other public universities in Mexico, it still operates on a relatively limited budget considering its size and the number of services offered by the institution. For comparison, consider the University of California, which has a similar though smaller population of over 285,000 students19 but almost 20 times the budget of UNAM20. So, how can institutions like UNAM maximize the use of public funds, both for their benefit and that of the larger Mexican population?\n\nUNAM comprises 15 faculties, 34 institutes, and various other centers, schools, and units15. There are fundamental differences for academics working in faculties versus institutes, which are important for understanding our work as professors in the Faculty of Science.\n\nIn institutes, the primary focus is research. Laboratory space is assigned to many faculty at the time of hiring and their teaching load is low. According to the UNAM Statute of Academic Personnel, researchers in institutes must teach a minimum of 3 contact hours per week each semester21, equivalent to a 1-1 teaching load at Canadian or U.S. institutions. In contrast, faculties are focused on teaching. Entry-level professors are required to teach a minimum of 9 contact hours per week each semester21, equivalent to a 3-3 teaching load. Unlike at many institutions in North America, there are no standard mechanisms for ‘buying out’ of teaching if a professor receives a grant. In addition, professors are expected to contribute significantly to ‘formation of human resources’ by directing student social service projects and theses, serving on committees, and tutoring. Many professors work almost exclusively with undergraduates, especially for the first few years when their professoriate level does not allow advising graduate students in many degree programs. Despite the heavy teaching and service load, there is still a research expectation. However, professors are not necessarily assigned laboratory space and receive no start-up funds. Availability of laboratory space in the Faculty of Science has become especially problematic as student and academic population growth puts increasing demands on an already overloaded infrastructure.\n\nThese conditions raise a number of questions for professors working in faculties like UNAM’s Faculty of Science: With limited resources and infrastructure, how do I provide high-quality, hands-on educational experiences for my classes?; how do I develop research projects for social service and thesis students?; and how do I build up my own research program and start producing?\n\n\nPAPIME educational grants\n\nA partial answer to some of the above questions comes in the form of internal grants offered by the General Directorate for Academic Personnel Affairs (Dirección General Asuntos del Personal Académico; DGAPA) at UNAM. One of these grant programs – the Support Program for Projects to Innovate and Improve Education (Programa de Apoyo a Proyectos para Innovar y Mejorar la Educación; PAPIME) – focuses on education22. This program has been key for us in building capacity and is a funding mechanism we believe more universities should emulate. The goal of PAPIME is to, “Promote the improvement and development of academic staff by supporting projects that lead to innovation and improvement of the teaching-learning process and benefit students...Teaching innovation projects should revolve around themes that allow creative teaching, with new ways of thinking, to motivate the interest and imagination of students”22.\n\nThe 2020 call for applications23 shows that these grants fund a wide range of projects and diverse products, including but not limited to: (1) teaching materials, like exercises or practicals, case design, tutorials, digital applications, software, and websites; (2) publications, like books or articles in areas such as educational research; (3) innovative educational evaluation systems, strategies, and instruments; (4) organization and participation in academic events, like colloquia and seminars; and (5) training activities, like in-person or online courses and workshops, or fieldwork.\n\nThese grants are typically 1 year in duration, and as of 2020 can be awarded up to $250,000 MXN annually23, or ∼12,500 USD. Interestingly, while not explicitly using the language, PAPIME grants can function to a certain extent as OER grants. Products resulting from PAPIME projects are required to be uploaded to UNAM’s Repository of Educational Innovation (Repositorio de Innovación Educativa, RIE: innovacioneducativa.unam.mx). Digital materials in particular must be uploaded to UNAM’s University Learning Network (Red Universitaria de Aprendizaje, RUA: rua.unam.mx). The stated objective of sharing these materials is to “disseminate and extend coverage for the benefit of the university community and thus optimize the resources invested by UNAM in development of the project”23. In line with this, projects are evaluated on several characteristics related to broadness of impact, including: (1) number of students that will benefit from the project; (2) where students come from, whether inside the academic entity, university, or beyond; (3) number and names of classes that will benefit from the materials; and (4) number of professors that will use the products. Using an open approach can help academics argue broader impact, i.e. a larger population of both students and educators are reached, within and beyond the institution, and materials can be reused, revised, remixed, and redistributed12.\n\n\nElectrophysiology grant\n\nWe were awarded our first PAPIME grant in 2017 and our second in 2019. The idea for the overall project came from what we perceived to be a gap in the education of our Biomedical Physics undergraduates, namely a lack of hands-on training in electrophysiology and related skills. We set out to design electrophysiology laboratory practicals that could be incorporated into our plan of study. Not all these practicals were intended to be 100% novel; resources exist on the basics of EMG24,25 and ECG26,27 recording, for example. BYB has already developed over 60 experiments that can be performed using their equipment and released these on their website (backyardbrains.com/experiments) as OERs under an open license. However, there are a few ways we wanted to expand and extend existing work.\n\nFirst, we wanted all our practicals to be accompanied by more in-depth lesson plans. The BYB tutorials are excellent starting points, but are too simple for our fourth-semester human physiology undergraduates (e.g., their EMG tutorial24 is marked as ‘beginner’ for elementary school students 5th grade and up). On the other hand, many of the resources we found in the scientific literature were too complicated, aimed more at graduate students or working professionals (e.g., 25). In addition, many of these latter resources have a clinical rather than biophysics focus. We saw a need for electrophysiology OERs designed for a more intermediate, undergraduate level that would reinforce material seen in our core courses, including physics as applied to the human body.\n\nSecond, we aimed to develop novel practicals that would combine electrophysiology with other physiological measurements like spirometry, helping our students see how different systems in the body work together. Currently, as in many universities, our human physiology course is taught as a sequence of system-based modules (e.g., nervous system, cardiovascular system, etc.). As Conford28 writes, “One assumption that many modular courses presently reflect is that effective learning proceeds via self-contained chunks of information...Modules, however, by their very structure, tend to fragment knowledge rather than to integrate it” (pg. 243). We see these practicals as a way to recover this integration and connect concepts across modules.\n\nThird, we sought to develop bilingual materials. We have struggled to find quality Spanish-language OERs, especially in biophysics. Language can be a significant barrier to OER reuse and remixing29–32. From an UNESCO report33, “Not only does the English language dominate OER provision, but English-language content tends to be based on Western learning theory. This limits the relevance and accessibility of OER materials in non-English, non-Western settings. There is a risk that language barriers and cultural differences could consign less developed countries to the role of OER consumers rather than contributors to the expansion of knowledge” (pg. 12).\n\nFinally, we wanted to develop a suite of products around each practical and release not just written OERs but also accompanying code, data, images, and videos, all under open licenses. We reasoned this was one important way to increase the impact of the project. For example, educators without the resources to buy recording equipment could at least reuse our data and code to graph and analyze electrophysiology recordings with their students.\n\n\nBuilding capacity\n\nWith our first grant in 2017, we were awarded the equivalent of ∼$10,500 USD. We used the bulk of the funds to purchase electrophysiology recording equipment (Table 1), microscopes, and related accessories like electrodes and dissection tools (Table 2). The remaining funds were used to finance scholarships for two undergraduates to work on the project. With our second grant in 2019, we were awarded ∼$6,300 USD that we used to purchase computer equipment, surface electrodes for recording, instrumentation accessories like Raspberry Pi 3 Model B and Module V2 cameras, Arduino sensor kits, and food and bedding for experimental animals. (Many of these are standard products available from multiple providers, so we did not itemize these in table form). We also gave scholarships to two more undergraduates. While the amounts awarded us may sound sizeable – similar OER grants in the U.S. and Canada often cap at $5,000 USD34–36 – this was still a limited budget considering we were starting from zero in terms of equipment and materials. We had to maximize use of these funds to build capacity.\n\nAll prices in USD. Prices at time of purchase, not including shipping and handling, taxes, etc.\n\nAll electrophysiological recording equipment was obtained from BYB (backyardbrains.com), including devices to record APs in insects (Neuron SpikerBox), and EMGs (Muscle SpikerBox) or ECGs (Heart and Brain SpikerBox) in human subjects (Table 1). We purchased these as bundles, which included the recording device, cables, surface electrodes, conductive gel, and other accessories. The low cost of these bundles (<$250 USD each), compared to conventional electrophysiology equipment, allowed us to purchase multiple devices. With 3 or 4 devices, we could work in groups of 5-10 and pilot practicals with classes of 20–30 students.\n\nWe also purchased DIY kits from BYB to build additional recording devices. This served two purposes. First, students will assemble the devices, learning valuable instrumentation skills in the process, which is one of the core objectives of our Biomedical Physics plan of study. Students will fully document the assembly process with step-by-step protocols, photos, and videos, which will be shared online as OERs. Second, at around half the price of the fully assembled device bundles, DIY kits allowed us to buy more equipment without exceeding our budget. Once assembled, our recording capacity will double, meaning we can work with more students.\n\nAffordability was not the only advantage of the BYB equipment. The small size and portability of the devices meant we did not need a dedicated laboratory space, solving one of our infrastructure issues. We could take these devices into any classroom and record with students using their smartphones. We also allow students to borrow these devices and take them home to work on individual research projects. A few years ago, having students do electrophysiology at home would have been impossible. Now we can offer them this unique experience, which can be a huge motivating factor in their academic development. Since 2017, students and professors in our program have used the equipment in core and elective coursework, social service projects, and thesis research. In other words, purchasing a small amount of equipment has greatly increased our capacity to provide high-quality educational and research opportunities for our undergraduates.\n\nWe purchased several accessories to improve both recording experiences for students and potentially research capacity (Table 2). For example, while the BYB electrodes that come with the Neuron SpikerBox are sufficient for basic AP recording in large insects, they are stainless steel sewing needles with a relatively large tip diameter (0.25–0.6 mm)37, non-insulated, and not ideal for finer recordings in smaller preparations or cells. So, we purchased insulated Tungsten electrodes with a 2–3 µm tip diameter. At ∼$19 USD each, these electrodes are only $9 more than BYB’s, but should provide a substantial improvement in recording capabilities and quality. For less than $200 USD we can buy a packet of 10 Tungstens and upgrade 10 SpikerBoxes.\n\nAll prices in USD. For items purchased in MXN, an exchange rate of 19 pesos to the dollar was used to estimate amounts. Prices at time of purchase, not including shipping and handling, taxes, etc.\n\nWe also purchased manipulators to improve control and precision of electrode placement. Conventional 3-axis manual micromanipulators, like those made by Narishige (usa.narishige-group.com), cost ∼1,000 USD and were out of our price range. However, BYB provides a 3-D printed plastic manipulator with 3 axes of movement in the millimeter range and adjustable electrode angle through 135 degrees for just $99.99. Furthermore, BYB’s open hardware approach means the plans for printing and building the manipulators are available on their website, which will allow us to reduce costs in the future by printing more manipulators at a university facility. In fact, the growing open labware/maker movement is increasingly allowing researchers to 3-D print their own lab equipment, including electrophysiology devices and accessories, for a fraction of the cost38,39.\n\nWe also bought accessories from a local provider (SIET México), including Arduino kits, sensor kits, Raspberry Pi 3 Model B, and Raspberry Pi Cameras Module V2. Arduino kits include an Arduino Uno R3, servo and step motors with drivers, a variety of sensors (infrared, humidity, temperature), and other accessories such as cables, resistors, and LEDs. Sensor kits are designed to be used in conjunction with Arduinos, and include heartbeat, temperature, touch, and sound sensors, as well as buzzers, joysticks, and switches. Similar Arduino and sensor kits can be purchased through Amazon or eBay. Kit components can be used for a variety of electrophysiology-related projects, including instrumentation of simple myoelectric prosthetic prototypes40,41.\n\nElectrophysiology often involves dissection to prepare tissues or cells for recording. Dissection tools, especially those for fine dissection, are costly. Fortunately, companies like VWR provide economic solutions in the form of classroom dissection sets, which include scissors, forceps, scalpels, pins, and more. With tools for up to 20 students and priced at ∼$200 USD or less, the cost comes out to only ∼$10 USD per student. With the remaining funds we had for tools, we bought just two fine dissection kits for use in more advanced, individual student projects.\n\nDissection and fine detail instrumentation also requires visualization and magnification. With this in mind, we purchased several microscopes with different characteristics. The Fisherbrand Illuminated Pocket Microscope weighs just 85 grams, measures 140L × 38W × 22H mm, and has 60–100x magnification. Similarly, the BYB High Power RoachScope weighs 400 grams, measures 142L × 94W × 74H mm, and can be used in combination with any smartphone camera. With digital zoom, it has 5–100x magnification. Both these microscopes are designed for maximum portability, so they can be taken into the classroom. In addition, both cost less than $100 USD each, so we could buy several to work with groups of students. We also purchased three Fisher Science Education Advanced Stereomicroscopes for just under $300 USD each. These microscopes are not very portable, but should give us better optics. To increase the utility of these microscopes, we are planning on 3-D printing a low-cost adapter that will attach to the eyepiece and allow us to mount any smartphone to take high-quality pictures or video. Open plans for such an adapter are available via the NIH 3D Print Exchange42 and pictured in 39.\n\nFinally, we purchased an advanced trinocular microscope (National Optical via Fisher Scientific) with 4x, 10x, 40x, and 100x objectives and a built-in digital camera (Moticam 1080 HDMI & USB) for high-resolution viewing of tissues and cells. The higher cost of this microscope meant we could only buy one. However, connecting the camera to a large computer monitor allows us to carry out demonstrations and have groups of students view samples simultaneously. We have also hosted “open house” events for new students using this microscope.\n\n\nData acquisition and analysis\n\nCommercial software used to record, process, and analyze electrophysiology data is often a significant expense for many laboratories. We did not have the budget to pay for software licenses, but also felt to do so would be incompatible with the open spirit of the project. It was important to us that any software we used be open source, and that any analysis code we created also be open to facilitate reuse. BYB provides the SpikeRecorder application free through their website (backyardbrains.com/products -> Software) and source code via GitHub (github.com/BackyardBrains/Spike-Recorder-IOS). The app can be downloaded and installed on students’ phones in minutes to begin recording. All recordings are saved as .wav audio files, which can then be played back, visualized, and some analysis performed within the same app43. However, for analysis we felt we needed more control and customization, so we wrote code in Python (version 3.7.4) using the following packages: math, Matplotlib44, NumPy45,46, os, pandas47, random, SciPy48, statistics, sys, wave, and wfdb. All our code is available via our GitHub repository at github.com/emckiernan/electrophys and archived on Zenodo at doi.org/10.5281/zenodo.455442049.\n\nPython code was developed inside Jupyter notebooks, which provide an interactive way to document and share code50,51. Our notebooks walk students through the process of opening and graphing recordings, applying filters, and quantifying aspects of electrical activity. The notebooks include exercises for students to perform in or outside of class as data analysis practicals. In other words, we create OERs out of this shared code52–54. As Downes53 writes, use of Jupyter notebooks in this way “changes the conception of an educational resource from something static to something that’s interactive, to something that can be used to create, as well as to consume” (pg. 9). This also helps us meet another core learning objective of the Biomedical Physics plan of study, namely programming skills. In fourth semester, when students start with our electrophysiology practicals in their human physiology course, they also take a programming course which primarily teaches Python. Data analysis practicals are a good way for them to apply new programming skills to biomedically relevant data analysis, and integrate knowledge from these two core courses.\n\n\nWorkflow and related tools\n\nThe development of most of our practicals began as a free-form process. We had a general idea of the type of recording we wanted to perform, and piloted these ideas first with classes of 20–30 undergraduates. Students were encouraged to experiment, for example by trying out different electrode placements and exercises. Subsequently, students wrote individual reports with background information, protocols, results, and conclusions. Students shared their photographs, videos, data, and reports with us via Google Drive. From the resources provided by students, we collected the best examples and used this information to build our master documents for each practical. In addition, four students were given scholarships with PAPIME funding to help us run pilots, gather materials, analyze data, and draft protocols. In this way, students played an active role in OER development, reinforcing Buckland’s idea of “students as content creators”55.\n\nOur master documents were written in LaTeX using the Overleaf platform (overleaf.com). LaTeX presents a variety of advantages over word processing software, including control over document layout and figure placement, excellent equation handling, and automatic reference formatting56. The Overleaf platform in particular provides several benefits. First, basic accounts are free, so there were no additional costs, as would be incurred by using commercial packages like Microsoft Office. Second, we could easily share and collaborate on a master file with integrated commenting functions and version control. Finally, Overleaf provides a rich text viewing option, which is more user-friendly, especially for undergraduates just starting out with LaTeX.\n\nOnce we had final versions of the master documents, these were uploaded to a public repository on GitHub (github.com/emckiernan/electrophys), along with images, data, and code associated with each practical. GitHub provides Git version control57,58, which means OERs can continue to evolve as necessary while preserving the history of resource development59. GitHub also provides collaboration features, which we hope students and educators will use to improve and customize these materials. However, we recognize not everyone uses GitHub, and that only hosting our materials there could represent a barrier to reuse. So, we built a Wordpress website to share materials in a more user-friendly way. This was done by converting our LaTeX documents to html using Pandoc (pandoc.org), and then copying the html to a free Wordpress template (wordpress.org). Minor formatting to improve visual presentation was done by hand. Jupyter notebooks were uploaded by creating public gists (gist.github.com) and then copying these links to the Wordpress site for embedding. Using the free Wordpress services meant we did not incur any costs for website creation or hosting.\n\nOur workflow is visualized in Figure 1. Moving forward, there are ways we could improve this workflow. For example, a more efficient way to set up our website would be to use GitHub Pages (pages.github.com). This would allow for automatic syncing of the website when the repository materials are updated, but requires more in-depth html knowledge to properly format and maintain the site. We would also like to explore open source alternatives to several of the tools we used. Bosman and Kramer outline a potential open science workflow (as well as other workflows ranging from traditional to experimental) that could be useful for researchers and educators60. More information on tools, including some open source alternatives, is available in Table 3.\n\n\nPracticals and course integration\n\nIn fourth semester, students in UNAM’s Biomedical Physics undergraduate degree program take a human physiology course, which is divided into modules: (1) nervous, (2) musculoskeletal, (3) biofluids (4) cardiovascular, (5) respiratory (6) gastrointestinal, and (7) renal systems. At present, this class is only lecture. One of our goals with this project was to design hands-on electrophysiology activities to be integrated throughout the course. We briefly describe some of these practicals, where they fit into the course, and how they reinforce concepts from our plan of study. The format of our written documentation accompanying each practical is modeled after BYB’s experiment manual64, with clear learning objectives for before, during, and after practical completion. All our practicals – finished and under development – are at github.com/emckiernan/electrophys, and select ones at electrophys.wordpress.com. A full list of practicals and links to documents, data, and code is available as extended data via Zenodo doi.org/10.5281/zenodo.454035565.\n\nThe first practical is designed to teach students the basics of EMG recording, carried out at the end of the musculoskeletal system module. The background written information reinforces physiology concepts seen in class, as well as the application of basic physics concepts seen in other coursework. It begins with a description of how muscle-bone-joint complexes function as lever systems. Students are encouraged to think back to the three types of classical lever system and find corresponding examples of these in the human body. This involves visualizing biomechanics and how the relative position of bones, joints, muscles, and loads will affect movement. The written documentation goes on to reinforce concepts such as how muscle structure affects tension development, length-tension relationships, and the energy requirements for muscle contraction. We then describe the basics of EMG recording, comparing the advantages and disadvantages of invasive versus surface recording, and the basic bipolar differential recording configuration. Study questions prompt students to think about where they will need to place electrodes to record from different muscles and what potential limitations they might encounter.\n\nStudents then move on to the experimental phase of the practical where they carry out their own EMG recordings, in groups of 4-6 students depending on class size. Step-by-step instructions on how to perform the recordings are included in the written documentation. However, these are designed to be informative without being too prescriptive and still allowing for exploratory learning. The only requirement is that students record from at least one muscle from each type of lever system, but we do not tell them which muscles to record from or how they should activate these muscles. Students are encouraged to design their own experiments using everyday items available in the classroom or simple exercise aids, like resistance bands or hand grippers, brought from home. Students have performed EMGs from facial muscles while eating, tricep muscles while doing pushups, bicep muscles while arm wrestling or lifting their backpacks, and forearm muscles while performing martial arts movements. Students are also encouraged to explore different types of contraction, including intermittent versus sustained and increasing versus decreasing force.\n\nWe have several other EMG practicals still under development, including experimental ones to measure fatigue in the bicep muscle, dual recordings from antagonistic muscle pairs, simultaneous recording of EMG and force sensor measurements from the forearm, and others. In addition, we are developing EMG data analysis practicals. Students will take the recordings they gathered in the first practical, graph them, and learn about the design and application of band-pass and low-pass filters to process their data. They will learn about different techniques used to smooth data, calculate an envelope, and use thresholds to detect start and stop times of muscle contractions. These practicals will be carried out using Jupyter notebooks running Python, thereby simultaneously strengthening students’ programming skills. All practicals are listed in the extended data (doi.org/10.5281/zenodo.454035565).\n\nIn module 3 of their human physiology course, students learn about the cardiovascular system and carry out a practical to record their ECG before and after exercise. The background written information begins with a description of how the heart performs external mechanical work. Students are encouraged to visualize the heart as a single-chamber pump with inflow and outflow valves, and examine the pressure-volume relationships similar to the way one would with an internal combustion engine66. Students learn about sequential pressure and volume changes in different chambers of the heart during the cardiac cycle, and how to graph this with a pressure-volume loop. The documentation goes on to describe the electrical activity of specialized populations of cells in the heart, including the ionic basis of APs in these cells. Discussing cardiac muscle activity also encourages students to think back to module 2 of the human physiology course when we discussed contraction mechanisms in this muscle type. Finally, we describe the basics of ECG recording, including how the summation of individual potentials leads to the extracellularly recorded events, different recording configurations, and the importance of electrode placement.\n\nStudents then move on to the experimental phase, working in groups of 4–6. Volunteers from each group record their ECGs, while other students help with organizing and exporting the data. Students first record their baseline ECG under resting conditions for at least 1–2 minutes. Then, they disconnect the recording device while leaving the electrodes in place and perform light to moderate exercise for at least 5 minutes. After this, students reconnect the device and record their ECG again for at least 1–2 minutes. Students can choose the type of physical activity they perform. For example, students have done push-ups or burpees, ran laps around the building, or gone up and down stairs outside the classroom. We encourage students to compare how different levels of activity change the ECG signal, and how the signal varies across subjects (e.g., athletes versus non-athletes).\n\nWe are developing additional experimental practicals designed to explore the relationship between heart and respiratory activity, using simultaneous ECG and spirometry, for example. We are also working on ECG data analysis practicals. Students will take the recordings they gathered during the first ECG practical, graph them, and learn how to detect the peaks of the QRS complex to calculate heart rate and quantify how it changes after different levels of exercise. They will also examine techniques for detecting the P and T waves, and calculating intervals important in clinical evaluations. A full list of these practicals is available in the extended data (doi.org/10.5281/zenodo.454035565).\n\nIn module 5 of their human physiology course, students learn about the respiratory system, an important part of which is understanding the mechanics of breathing. How do respiratory muscles expand or contract the thoracic cavity and change pressure gradients? How does the participation of different muscles change when respiration is normal versus forced? And to relate back to the musculoskeletal module, how is respiratory muscle contraction related to electrical activity?\n\nIn this practical, students record EMGs from the rectus abdominis. This muscle is known as an accessory respiratory muscle because it is not activated during normal exhalation, but is activated during forced exhalation when additional effort is needed to reduce the volume of the thoracic cavity beyond that accomplished by simple elastic recoil67. While recording rectus abdominis EMG, students simultaneously use a spirometer (Vernier) to measure the volume of air moved in and out of the lungs. Students are instructed to perform a sequence of normal breaths interspersed with maximal forced inhalations and exhalations.\n\nThe dual recordings allow students to see firsthand that, during normal respiration and forced inhalation, little to no electrical activity is recorded on the EMG because the rectus abdominis is not contracting. However, during forced exhalation, the EMG shows an increase in both the amplitude and frequency of the signal with increased effort and increased volume exhaled. The written materials for the practical are designed to reinforce several physical concepts applied to the study of respiration, including: (1) pressure-volume relationships and Boyle’s Law as applied to the lungs; (2) importance of pressure gradients and Ohm’s Law as applied to airflow; (3) Poiseuille’s Law as applied to measuring airflow through a spirometer, and (4) biomechanics of active lung expansion versus passive elastic recoil. This practical also gives students the opportunity to integrate knowledge from two modules to understand how the musculoskeletal and respiratory systems work together. We are working on developing more practicals that combine electrophysiological recordings with other physiological measurements (e.g., from force, displacement, or gas sensors) to provide similar integrative learning experiences.\n\n\nDiscussion\n\nLess than a decade ago, providing hands-on electrophysiology learning experiences for undergraduates, especially large classes, was not feasible. However, over the last few years, technological advancements have opened up new possibilities for educators. With the introduction of the BYB Neuron SpikerBox in 2011, an easy-to-use, low-cost bioamplifier brought neurophysiology into the classroom37. Since then, the single-channel SpikerBox, and the later two-channel version, have been used to design practicals for undergraduates to record from cricket sensory organs68, grasshopper neurons responding to visual stimuli69, and to study AP conduction velocity in earthworms70. Surveys from these studies indicate that students not only enjoy these hands-on activites, but that they also improve learning outcomes, increasing test scores by as much as 25% on average70. The SpikerBox has even been used as part of a larger program to provide undergraduates the opportunity to teach neuroscience to highschool students71.\n\nIn recent years, BYB has released more complex devices for recording ECG, EMG, and single-channel EEG, which have also been used in undergraduate class settings to improve learning. For example, Catena and Carbonneau (2018) describe using the BYB Muscle SpikerBox Pro to record dual-channel EMG as part of an undergraduate biomechanics course72. Their survey results show that students reported “better motivation\" and higher “personal responsibility for learning\". Test scores for students who had these hands-on learning experiences were also 7% higher compared to students who did not72. Similarly, Judge and colleagues (2020) used BYB equipment to develop ECG and EMG exercises for community college anatomy and physiology courses73. Students who carried out these exercises showed “significant learning gains\"73.\n\nOther groups have also developed and shared plans for low-cost electrophysiological recording devices, and in the process created instrumentation exercises for undergraduates. Matsuzaka and colleagues (2012) describe the development of a low-cost (only $85 USD per unit) amplifier for recording EEGs, EMGs, and other electrophysiological signals with students74. Importantly, the authors mention that potential problems of reproducibility and quality control when building these devices “could be resolved if the optimized circuit layout is freely available” (pg. A124). Crisp and colleagues (2016) provide step-by-step instructions for students to build a simple EMG device using a breadboard amplifier, with few components and an assembly time of just 30 minutes75. Wyttenbach and colleagues (2018) review these and other devices as part of a larger discussion on “reducing the cost of electrophysiology in the teaching laboratory”76.\n\nIt is not just low cost that is important, but moreso the open approaches taken that have increased the impact of many projects like the ones described above. Sharing hardware schematics and building instructions, openly licensing and publicly documenting code, and growing online support communities – characteristics of projects like Arduino, BYB, and Raspberry Pi – have allowed classrooms and laboratories in limited-resource countries to build capacity77.\n\nOne of the most interesting aspects of this project for us has been working at the intersection of open education and open research, and experiencing firsthand how these approaches can build on one another. Open access, open data, open education, and open source have historically different developments, and are often treated as separate areas of advocacy. However, all these areas share common goals: (1) increased access to information, whether in the form of a textbook, an article, a data set, or code; (2) increased participation, whether in education, research, citizen science, or software development; and (3) better outcomes, whether that means better learning outcomes, more reproducible research, or improved software. How can these open approaches learn from each other and work together to further these goals? In particular, in a limited-resource environment, we wondered whether open educational approaches could also help us build research capacity, and whether open research approaches could also help us create OERs. In our opinion, the answer to both of these questions is a resounding ‘yes’.\n\nThe clearest example of this for us was in observing the connections between open education and open source. When we thought about code as not just for research but also an educational resource, it changed how we thought about sharing this product. Previously, we might have simply shared our code as a raw Python file in a GitHub repository, and included some in-line comments and a README file as documentation. However, when we envisioned students or educators reusing our code to learn, we realized it needed more in-depth explanations and exercises. Using Jupyter notebooks, we built up tutorials or lesson plans surrounding the code and transformed these into OERs52–54. Importantly, after completing these resources we not only had quality OERs to use for our classes, but we also had a bank of well-documented analysis tools to use for future research projects. Organizing and documenting our code in this way may help us with lab group onboarding, as any incoming students can go through the tutorials and quickly get up to speed on our data analysis techniques. Furthermore, in the process of elaborating didactic explanations of our analysis, sometimes we realized ways in which our code could be improved. Therefore, the interaction went both ways: building educational capacity through open practices led to building research capacity and vice versa.\n\nWe have visually mapped out some of the potential connections between different open approaches (Figure 2). While this is not an exhaustive map – other open approaches could be included and other connections explored – it is a representation of how these connected for us in this project.\n\nImportantly, one open approach did not automatically lead into the next; there were transformations of the materials and certain conditions that needed to be met at each stage to maintain the flow between each. For example, simply sharing our data would not necessarily allow others to develop new analysis code (Figure 2, upper right arrow). For this to occur, the data need to be well organized, labelled, and documented, with meta-data included. Admittedly, we are still struggling with the best ways to do this to optimize reuse of our data, and believe this is one area where researchers would benefit from more training. Similarly, shared code does not necessarily become an OER (Figure 2, central left-pointing arrow). This requires that the code be well explained, often with a surrounding lesson plan and exercises. Open licensing at each stage was also key, since locking down content at any point would stop the flow. However, licensing is different for code, data, and documents. To select licenses for each product, we used resources like the Creative Commons License Chooser (creativecommons.org/choose) and GitHub’s Choose an Open Source License tool (choosealicense.com).\n\nWe would also like to encourage researchers to expand their ideas of what they consider an OER. When working with students, there is no clear line where education ends and research begins. The research we do with students, especially with undergraduates, is not necessarily to discover new things but rather to teach students how to do research. It is more about the process than the end result, and as such, everything we create during that process – protocols, code, data, notebooks – can potentially be transformed into an OER to train others. We also believe that in thinking of research as a teaching-learning process, with all the documentation and explanations that entails, we may in turn enhance the research itself, improving experimental design and reproducibility.\n\nWe are not the first to think about the potential connections or intersections between different open approaches. For years, libraries have been at the forefront of conceptualizing, creating, and managing all kinds of open content78–80, and thinking about how open practices might connect. The following are all projects led by librarians and information specialists, and/or based in libraries. In 2015, Atenas and Havemann published a book81 arguing that “while Open Data is not always OER, it certainly becomes OER when used within pedagogical contexts” (pg. 22), and presented five case studies where open data were used to teach students programming skills, data literacy, and even promote civic engagement. Elder82,83 and Walz84 have looked at the differences between open access and open education, but in the process also found areas where these overlap and where they can learn from one another. For the last few years, Virginia Tech libraries has been hosting a series called “Connecting the Opens”, where they invite experts to discuss possible connections between open practices (recordings found at VTechWorks vtechworks.lib.vt.edu). Makerspaces, which often combine aspects of open hardware, open source software, and open education, are increasingly being established and run by libraries85–87. We hope to see even more of this intersectional work in coming years, and expect that much of it will arise in libraries.\n\nLibraries are also increasingly both leading and funding open scholarship projects, including the development and implementation of OERs. In a 2016 survey of U.S. universities, 64% responded that it was the library who had originated affordable course content (ACC) or OER initiatives at their institutions88. For those with governing bodies overseeing these initiatives, 89% said that libraries were participating members and half said that libraries led the group. Over half of respondents also indicated that funding for ACC/OER initiatives came from library general operating budgets – more than any other institutional or external funding source.\n\nDespite library support for open initiatives, it seems other institutional policies have not necessarily caught up. Walz and colleagues88 write, “survey responses indicate that current university-wide tenure and promotion policies do not explicitly encourage faculty adoption, adaptation, or creation of ACC/OER” (pg. 5). We believe it was important for the success of this project, as well as our own professional development, that our department recognizes and values participation in PAPIME projects in annual performance, promotion, and tenure reviews, and gives us space on evaluation forms to report on non-traditional digital products, including OERs. We encourage institutions to rethink and reform their evaluation policies to incentivize open scholarship, including OER development and adoption, and to seek guidance from libraries on how best to do this.\n\n\nConclusions\n\nWe succeeded in developing a suite of electrophysiology practicals to give biomedical physics undergraduates hands-on training in this important area of study. Our materials are shared as open educational resources to increase their reach and impact. Importantly, open approaches, such as open data, open hardware, and open source software, were crucial to our project’s success, allowing us to maximize limited resources and build both educational and research capacity. We believe open scholarship has a key role to play in the future of undergraduate education, and hope the examples and strategies we have shared here will benefit other educators working to improve learning experiences in the biomedical sciences and beyond.\n\n\nData availability\n\nSource repository: GitHub. Electrophysiology practicals for undergraduate students. https://github.com/emckiernan/electrophys.\n\nArchived at time of publication -\n\nZenodo: electrophys v1.0.1 http://doi.org/10.5281/zenodo.455442049\n\nOur GitHub repository, archived via Zenodo, contains all the lesson plans for our electrophysiology practicals, raw electrophysiology data, and data analysis code.\n\nLicense depends on resource type. Practicals (documents) are shared under the Creative Commons Attribution 4.0 International (CC BY 4.0) license; code under the MIT License; and data (recordings) under the Creative Commons CC0 1.0 Universal license. For more information, see our license file.\n\nZenodo. Electrophysiology practicals for undergraduates: links to code, data, and lesson plans (Version v1.0). http://doi.org/10.5281/zenodo.454035565\n\nThis extended data includes a pdf document with a full list of our practicals (developed and under development) with links to the resources.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThis paper grew from a talk given by ECM at the Open Education Global 2018 conference. The authors thank the conference organizers and attendees for their inspiration and feedback. The authors also thank participating professors Marco A. Herrera Valdez, Rodrigo A. Martín Salas, and Sergio E. Solis Najera, and contributing students Diego Alberto Barceló Nieves, Rogelio Barrios Rosas, Diana Alejandra Cuevas Salazar, Daniel Gómez Pérez, José Carlos Hernández Herrerías, Carlos Ignacio Herrera Nolasco, Carol Molina Martínez, Juan Carlos Morales Cordova, Jenifer Alejandra Parra Reyes, Noel Isaías Plascencia Díaz, Estíbaliz Margarita Ramírez Vázquez, Ana Daniela del Río Pulido, Angélica Rubio García, Mitsui Myrna Salgado Saito, and in general, UNAM’s Biomedical Physics students. Finally, the authors thank Lorena Barba, Abbey Elder, Abigail Goben, Rajiv Jhangiani, Vahid Masrour, Alejandro Pisanty, Michelle Reed, Nick Shockey, and Anita Walz who contributed ideas, references, and other feedback that shaped this paper.\n\n\nReferences\n\nHogrel JY: Clinical applications of surface electromyography in neuromuscular disorders. Neurophysiol Clin. 2005; 35(2–3): 59–71. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColpitts KN, Seymour KP, Bozer ALH: Development of an Introductory Neuroscience Teaching Experience for Undergraduates with a Low-Cost Neuroscience Summer Academy. J Undergrad Neurosci Educ. 2019; 17(2): A125–A129. PubMed Abstract \| Free Full Text\n\nCatena RD, Carbonneau KJ: Guided Hands-On Activities Can Improve Student Learning in a Lecture-Based Qualitative Biomechanics Course. Anat Sci Educ. 2019; 12(5): 485–493. PubMed Abstract \| Publisher Full Text\n\nJudge JL, Cazares VA, Thompson Z, et al.: Development of low-cost cardiac and skeletal muscle laboratory activities to teach physiology concepts and the scientific method. Adv Physiol Educ. 2020; 44(2): 181–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsuzaka Y, Ichihara T, Abe T, et al.: Bio-amplifier with Driven Shield Inputs to Reduce Electrical Noise and its Application to Laboratory Teaching of Electrophysiology. J Undergrad Neurosci Educ. 2012; 10(2): A118–A124. PubMed Abstract \| Free Full Text\n\nCrisp KM, Lin H, Prosper I: Breadboard Amplifier: Building and Using Simple Electrophysiology Equipment. J Undergrad Neurosci Educ. 2016; 14(2): A124–A131. PubMed Abstract \| Free Full Text\n\nWyttenbach RA, Johnson BR, Hoy RR: Reducing the Cost of Electrophysiology in the Teaching Laboratory. J Undergrad Neurosci Educ. 2018; 16(3): A277–A281. PubMed Abstract \| Free Full Text\n\nYusuf S, Baden T, Prieto-Godino LL: Bridging the Gap: establishing the necessary infrastructure and knowledge for teaching and research in neuroscience in Africa. Metab Brain Dis. 2014; 29(2): 217–220. PubMed Abstract \| Publisher Full Text\n\nvan der Werf T: Open Content Activities in Libraries: Same Direction, Different Trajectories — Findings from the 2018 OCLC Global Council Survey. 2020; Accessed July 2020. Publisher Full Text\n\nWest Q: Librarians in the pursuit of open practices. In R.S. Jhangiani and R. 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Publisher Full Text" }	[ { "id": "81109", "date": "25 Mar 2021", "name": "Javiera Atenas", "expertise": [ "Reviewer Expertise Open Education", "Open Data", "Curriculum Design" ], "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 a solid piece of evidence in Open Educational Practices. It is very well written and it is pedagogically sound.\n\nThe arguments are well presented, the literature review is clear and informative, as well as adequate, the method is well presented and described, it presents an innovative approach for teaching and learning, thus this paper can have an impact at informing curriculum design, as it provides clear and practical guidelines for the adoption of open data and open educational resources.\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": "97262", "date": "17 Nov 2021", "name": "Robert Wyttenbach", "expertise": [ "Reviewer Expertise Invertebrate neurophysiology", "lab exercise development", "software for teaching" ], "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 make a persuasive argument for including electrophysiology (specifically EMG, EEG, and ECG) in a biomedical curriculum. The medical (rather than research) focus and cost justify omitting intracellular recording and concentrating on human subjects.\nThe history of their project, while specific to their institution, may be helpful to others in similar positions. However, the core of this report is undoubtedly the open-source equipment, software, and lab exercises. Most of these are linked directly in the text.\nIn other educational contexts, invertebrate preparations would be suitable; a few are mentioned in passing. Most of the equipment and software reported here could be used or adapted for such preparations.\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": [] } ]	1	https://f1000research.com/articles/10-187
https://f1000research.com/articles/9-1186/v1	01 Oct 20	{ "type": "Software Tool Article", "title": "Umpire 2.0: Simulating realistic, mixed-type, clinical data for machine learning", "authors": [ "Caitlin E. Coombes", "Zachary B. Abrams", "Samantha Nakayiza", "Guy Brock", "Kevin R. Coombes", "Caitlin E. Coombes", "Zachary B. Abrams", "Samantha Nakayiza", "Guy Brock" ], "abstract": "The Umpire 2.0 R-package offers a streamlined, user-friendly workflow to simulate complex, heterogeneous, mixed-type data with known subgroup identities, dichotomous outcomes, and time-to-event data, while providing ample opportunities for fine-tuning and flexibility. Mixed-type data is characterized by a combination of continuous (e.g., height, blood pressure, creatinine levels), binary (e.g., gender, pain presence), and categorical (e.g., ethnicity, mental status, pain level) data. Here, we describe how we have expanded the core Umpire 1.0 R-package, developed to simulate gene expression data, to generate clinically realistic, mixed-type data for use in evaluating unsupervised and supervised machine learning (ML) methods. As the availability of large-scale clinical data for ML has increased, clinical data has posed unique challenges, including widely variable size, individual biological heterogeneity, data collection and measurement noise, and mixed data types. Developing and validating ML methods for clinical data requires data sets with known ground truth, generated from simulation. Umpire 2.0 addresses challenges to simulating realistic clinical data by providing the user a series of modules to generate survival parameters and subgroups, apply meaningful additive noise, and discretize to single or mixed data types. Umpire 2.0 provides broad functionality across sample sizes, feature spaces, and data types, allowing the user to simulate correlated, heterogeneous, binary, continuous, categorical, or mixed type data from the scale of a small clinical trial to data on thousands of patients drawn from electronic health records. The user may generate elaborate simulations by varying parameters in order to compare algorithms or interrogate operating characteristics of an algorithm in both supervised and unsupervised ML.", "keywords": [ "machine learning", "unsupervised machine learning", "supervised machine learning", "clustering", "clinical informatics", "mixed data", "mixed-type data", "clinical data" ], "content": "Introduction\n\nAs large clinical databases expand and data mining of the electronic medical record (EMR) improves, the scale and potential of data available for clinical knowledge discovery is increasing dramatically. Expanding size and complexity of data demands new analytics approaches and paves the way for applications of machine learning (ML) in novel clinical contexts1,2. However, clinical data are characterized by heterogeneity, including measurement and data collection noise, individual biological variation, variable data set size, and mixed data types, which raises new challenges for ML analyses1. Clinical data sets vary widely in scale, from early-stage clinical trials with fewer than 100 patients to prospective cohorts following 10,000 patients to large-scale mining of electronic health records. They consist of data collected in the clinical setting, including demographic information, laboratory values, results of physical exams, disease and symptom histories, dates of visits or hospital length-of-stay, pharmacologic medications and dosing, and procedures performed, possibly with associated ICD-9 or -10 codes. The most salient, identifying feature of clinical data is that it is of mixed-type, containing continuous, categorical, and binary data. The result of this heterogeneity is an ML milieu characterized by methodological experimentation, without consensus best methods to apply to challenging clinical data3.\n\nDeveloping and evaluating best practice methodologies for ML on clinical data demands a known validation standard for comparison. Previously, we described an approach using “biological validation”: testing an ML methodology in a disease with well-understood relationships between patient features and outcomes. Thus, we allow known biological truths uncovered (or absent) in a solution to validate a method3. However, biological validation fails to capture interaction effects or allow the validation of emergent discoveries. By far, a superior solution would be to validate novel methods on data with known “ground truth.” Artificial clinical data, simulated with known assignments, can serve to rigorously test and validate ML algorithms.\n\nSimulating realistic clinical data poses challenges. The wide range in feature spaces and sample sizes demands simulation solutions that vary by orders of magnitude. Rather than simulating data of a single type, simulated clinical data must be of mixed-type and must reflect the variable mixtures of types found in clinical scenarios, where one type may predominate over others4–6. In addition, in order to conclusively test algorithms for use in clinical contexts, simulations of clinical data must replicate the noisiness of these data that results from variation of human and technological features in measurement and the biological variation between individuals.\n\nA real need exists for noisy, realistic, clinically meaningful simulated data to advance ML in clinical contexts. The user finds few tools currently available, and those pose problematic restrictions. For example, the KAMILA (k-means for mixed large data) R package can be used to generate complex mixed-type clusters with a high degree of user specificity, but can only be used to generate two clusters7. Because many important problems face the analyst beyond distinguishing two groups in data, the need presents itself in the literature for more comprehensive, mixed-type simulation tools.\n\nHere, we present Umpire 2.0, a tool that facilitates generation of complex, noisy, simulated clinical and mixed-type data sets. Umpire 2.0 provides broad functionality across sample sizes, feature spaces, and data types to allow the user to simulate correlated, heterogeneous binary, continuous, categorical, or mixed type data from the scale of a small clinical trial to data on thousands of patients drawn from the EMR. These realistic clinical simulations are vital for testing and developing superior ML techniques for new clinical data challenges.\n\n\nMethods\n\nThe original Umpire R package (1.0) could be used to simulate complex, correlated, continuous gene expression data with known subgroup identities and both dichotomous and survival outcomes, as previously described8. Two core ideas underlie Umpire. First, biological data are correlated in blocks of variable size, simulating the functioning of genes, tissues, or symptoms in biological networks and pathways. Second, motivated by the multi-hit theory of cancer, subgroups (or clusters) of patients are defined by a number of informative, latent variables called “hits”. Each patient receives a combination of multiple “hits,” simulating population heterogeneity. These latent hits are used to link simulated alterations in patient data to outcome data in the form of dichotomous outcomes and time-to-event data.\n\nUmpire 2.0 expands the Umpire simulation engine for clinical and mixed-type data through a flexible pipeline. Users can vary the characteristics and the number of subgroups, features, hits, and correlated blocks. Using Umpire, they can control the level of patient-to-patient heterogeneity in various configurations of mixed-type data. Users can also generate multiple data sets (for example, training and test) of unlimited sizes from the same underlying distributional models.\n\nUmpire 2.0 enables users to incorporate individual and population heterogeneity in multiple ways. First, as above, latent hits are used to simulate features in correlated blocks, using multivariate normal distributions, with variation between individual members of a subgroup. Second, users can simulate clusters of equal or unequal size. Third, users can apply additive noise modeling measurement error and individual biological variation to simulations.\n\nBecause we know that clusters of equal size are unrealistic (outside of pre-defined case-control studies), we enable users to simulate clusters of equal or unequal sizes. In the equal case, we set the population proportions equal and sample data using a multinomial distribution. In the unequal case, we first sample a vector, r ∼ Dirichlet(α1, . . . , αk), setting the expected proportions of k clusters from the Dirichlet distribution. For small numbers of clusters (k ≤ 8), we set all α = 10. For more clusters (k > 8), we set one quarter each of the α parameters to 1, 2, 4, and 8, respectively, accepting only a vector of cluster sizes r in which every cluster contains at least 1% of patients.\n\nThe initial data simulated by Umpire represents the true, unadulterated biological signal. To these data, Umpire can add noise, mimicking biological variation and experimental random error. Marlin and colleagues9 argue that all clinical data “must be treated as fundamentally uncertain” due to human error in measurement and manual recording, variability in sampling frequencies, and variation within automatic monitoring equipment. Clinical experience teaches us that variability in clinical data arises from many sources, including human error, measurement error, and individual biological variation. However, because clinical measurements are integral to the provision of patient care, demanding high accuracy and reliability, we also assume that many clinical variables have low measurement error, such as tightly calibrated laboratory tests. For a given feature f measured on patient i, we model the clinically observed value Y from additive measurement noise E applied to the true biological signal S as\n\n\n\nWe model the additive noise following the normal distribution E ∼ N(0, τ) with mean 0 and standard deviation τ, where τ follows the gamma distribution τ ∼ Γ (c, b) such that bc = 0.05. Thus, we create a distribution in which most features have very low noise while some are subject to very high noisiness.\n\nUmpire 2.0 generates binary and categorical data by discretizing raw, continuous features along meaningful cutoffs. To convert a continuous feature into a binary vector, we select a cutoff and assign values on one side of this demarcation to “zero” and the others to “one.” We begin by calculating a “bimodality index” (BI) for the continuous vector10. To compute the bimodality index, we model the data as a mixture of two normal distributions, and take:\n\n\n\nHere π is the fraction of members in one population and δ =(µ1 −µ2)/σ is the standardized distance between the two means. The recommended cutoff of 1.1 to define bimodality was determined by simulation10. If the continuous data are bimodal, we split them midway between the means. For continuous features without a bimodal distribution, we partition them to binary features by randomly selecting an arbitrary cutoff between 5% to 35%. Although arbitrariness feels uncomfortable in an informatics sphere, we believe that this approach reflects a fundamental arbitrariness in many clinical definitions. For example, an adult female with a hemoglobin of 12.0 is said to be anemic, even though the clinical presentation and symptoms of a woman with a hemoglobin of 11.9 probably do not differ from those of a woman with a hemoglobin of 12.1. The choice of an arbitrary cutoff reflects these clinical decision-making processes: along a spectrum of phenotype, a value is chosen based on experience to define the edge of the syndrome. By choosing an arbitrary cutoff, we replicate this process. To reduce bias that could result if all low values were assigned “0” and all larger values were assigned “1,” we randomly choose whether values above or below the cutoff are assigned 0. We mark binary features in which 10% or fewer values fall into one category as asymmetric and mark the remainder as symmetric binary features.\n\nTo simulate a categorical feature, we rank a continuous feature from low to high and bin its components into categories, which we label numerically (i.e., 1, 2, 3, 4, 5). Distributing an equal number of observations into each bin does not reflect the realities we see in clinical data, and dividing a continuous feature by values (e.g., dividing a feature of 500 observations between 1 and 100 into units of 1-10, 11-20, etc.) could lead to overly disparate distributions of observations into categories, especially at the tails. Here, for c categories, we model a vector of R sizes along the Dirichlet distribution,\n\n\n\nsuch that we create categories of unequal membership without overly sparse tails. To generate an ordinal categorical feature, we bin a continuous feature and number its bins sequentially by value of observations (e.g., 1, 2, 3, 4, 5). To generate a nominal categorical feature, we number these bins in random order (e.g., 4, 2, 5, 1, 3).\n\nThe user may choose to simulate continuous, binary, nominal, or ordinal data, or any mixture thereof.\n\nUmpire 2.0 has been implemented as a package for R 3.6.3 and R 4.0. It is freely available on RForge and CRAN. Any system (Linux, Windows, MacOS) capable of running R 3.6.3 or R 4.0 is sufficient for implementing Umpire.\n\nUmpire 2.0 provides a 4-part workflow to generate simulations and save parameters for downstream reuse (Figure 1). The original Umpire 1.0 functionality and the Umpire 2.0 extension are arranged as a series of interchangeable modules (e.g., Engines, NoiseModels) within a parallel workflow. For a more thorough, guided introduction to the Umpire functions, please see the package vignettes. For clinical simulations, the user begins by generating a ClinicalEngine, consisting of a correlated block structure to generate population heterogeneity, a model of subgroup membership, and a survival model, which is used to generate a raw (continuous, not-noisy) data set. Next, clinically representative noise is applied. The user discretizes these data to mixed-type. Finally, Engine parameters, the ClinicalNoiseModel, and mixed data definitions are stored in a MixedTypeEngine to easily generate downstream simulations from the same parameter set.\n\nThe user begins by generating a ClinicalEngine to define correlated block structure, latent hits, subgroup prevalences, and a survival model. This is used to generate a raw, continuous data set. The user generates a clinically meaningful ClinicalNoiseModel, and applies it to the raw data. Next, the data are discretized to mixed type. Finally, the parameters of the ClinicalEngine, the ClinicalNoiseModel, and the discretized cutpoints are stored in a MixedTypeEngine to generate future simulations with the same parameters.\n\n\nUse cases\n\nIn this section, we present several examples explaining how Umpire can be used to simulate data relevant to important clinical questions.\n\nUnsupervised machine learning algorithms, designed to discover the subtypes inherent in a given data set, form one of the major branches in the field. In the clinical literature, these algorithms are being applied to data with variable feature sizes, including some studies with fewer than 10 features4,11. The number of subtypes (or clusters) identified in the literature also spans a fairly wide range4,5,12,13. At present, however, there is no consensus on which unsupervised ML algorithms are most effective, nor is it clear if different algorithms work better for different numbers of patients, clusters, features, or mixtures of data types.\n\nSince one idea at the core of Umpire is that cohorts of patients tend to be heterogeneous, it is perfectly positioned to perform simulations to evaluate unsupervised ML algorithms in the clinical context. As an illustration, we start by construcing a ClinicalEngine with four subtypes of patients.\n\n\n\nInternally, the ClinicalEngine simulates latent variables that affect both the expression of the clinical covariates and the outcomes in each of the four patient clusters. You can visualize which latent variables affect which clusters by extracting the “hit pattern” nested inside the ClinicalEngine (Figure 2).\n\n\n\n\n\nBlack pixels mark the presence of latent variables within a cluster.\n\nNote that this heatmap shows the true underlying structure relating the clusters to the latent variables, and not any simulated data sets. By design, however, the ClinicalEngine can only simulate “perfect” continuous data reflecting the true signal. In order to simulate realistic mixed-type data, we must first add noise to these data, and then discretize some of the features to create binary or nominal features.\n\nData collected in the clinic includes many different kinds of values. Demographic values include a small number of nominal demographic values (e.g., ethnicity, marital status) with four to five categories. Physical exam values include binary indicators (such as presence or absence of enlarged liver). Most of the values assessed on patients are continuous, such as heart rate, blood pressure, and laboratory values. We assume that most of these values have low error. For example, clinical laboratory values are tightly calibrated. However, some measurements, such as physical exam values, were assessed by chart review from the medical record. Others, such as blood pressure, were measured by hand and typed into the record at the time of the visit. Thus, a few values may be very prone to measurement and human error. Here we apply a ClinicalNoiseModel to our features that reflects our beliefs about the noisiness of our data, with the standard deviation following a gamma distribution defined by shape and scale parameters. Then, we construct a MixedTypeEngine.\n\n\n\n\n\nNote that the cm slot of the clinical engine is retained as a slot in the mixed-type engine, so the heatmap shown above can be recreated with the command\n\n\n\nAt this point, we still haven’t simulated any actual data. For that purpose, we use the rand method.\n\n\n\nWe now take a look inside the simulated data\n\n\n\n\n\n\n\n\n\nThere are four components:\n\n1. “clinical” contains the subtype, a binary outcome, and a time-to-event outcome represented by the last follow up time (LFU) and a logical indicator of whether the event occurred.\n\n2. “raw” contains the continuous data simulated by the clinical engine.\n\n3. “noisy” contains the same data, with noise added.\n\n4. “binned” contains the mixed type data, after discretization of some features.\n\nNote that using keepall = FALSE will not preserve the raw or noisy components. Also, the raw and noisy components are arranged in the “omics” style, where rows are features and columns are patients. By contrast, the binned component is transposed into the usual clinical style, where rows are patients and columns are features.\n\nAs an illustration, we visualize clusters for the noisy, continuous data compared to the discretized, mixed-type data. We use the daisy function from the cluster R package to compute distances between mixed-type data, and we use the Rtsne package for visualization (Figure 3).\n\n\n\n\n\nBased on the literature results referenced above, we constructed simulation parameters to represent common problems in clinical data (Table 1). These included a range of feature sizes (9–243), patient sizes (200– 3200), and number of clusters (2–16). In essence, this simulation was equivalent to evaluating a set of nested for-loops:\n\n\n\nParameters were chosen to reflect data set sizes from a Phase II clinical trial to a large EHR data set or cohort.\n\n1 A mixture of nominal and ordinal data\n\nOne could also vary other parameters, with the most likely candidates being the “cutpoints” parameters that control the fraction of continuous, binary, or categorical features in the data.\n\nThe primary benefit of these simulations for assessing clustering algorithms is that Umpire generates data with known, gold-standard cluster assignments. Using simulation parameters in Table 2, we examined hierarchical clustering (HC) with Euclidean distance, a method commonly found in the literature5,14–16, We compared HC to partitioning around medoids (PAM) and self-organizing maps (SOM)17,18. We also compared Euclidean distance to the mixed-type distance measure, DAISY. We were able to assess accuracy and quality of each clustering solution against a known ground truth using the Adjusted Rand Index (ARI)19. Summarized results are shown in Figure 4.\n\nThree algorithms (hierarchical clustering with Ward’s criterion, Partitioning Around Medoids, and Self-Organizing Maps) were implemented with a single-distance metric (Euclidean distance) and a mixed-type metric and tested on single- and mixedtype simulations generated with Umpire.\n\nDAISY outperformed all other methods on both balanced (top) and unbalanced continuous (bottom) data mixtures. Algorithm performance varied between data mixtures, with improved performance of hierarchical clustering (HC) and self-orgamnizing maps (SOM) with Euclidean distance on unbalanced continuous data.\n\nTime to response or adverse event is a core clinical question in clinical trials of pharmaceutical and device interventions. Here, we use Umpire to simulate time-to-event data for clinical trials to inform study design or methods development.\n\nWe begin by customizing a SurvivalModel, which in this case will simulate a trial with 5 years of patient accrual and 1 year of follow up. The user may customize length and units of follow up, as well as the base hazard rate. (Internally, Umpire uses this hazard rate in an exponential survival function.)\n\n\n\nHere, we illustrate the impact of altering the base hazard on the simulated mortality rate. We simulate three different survival models, using the default values for accrual and follow up (Figure 5).\n\n\n\n\n\nThe survival model is an argument to the constructor for a ClinicalEngine; if omitted, a default survival model is used. Here we explicitly use our survival model to construct a clinical engine with four balanced subtypes.\n\n\n\nBecause the latent variables affect the survival outcomes (by changing the hazard ratio) in the four patient clusters, you can generate different clinical engines from the same underlying parameters and obtain cohorts with different survival patterns (Figure 6). Note that we create different clinical engines to select differnt latent variables and create different populations. Using the rand function multiple times with the same engine would generate different samples from the same population, which would be useful for creating separate training and test data sets.\n\n\n\n\n\nIt is important to realize that the subtypes generated as part of a clinical engine or a mixed-type engine are unlikely to represent the arms of an actual clinical trial. They are, after all, based on patterns of latent variables that, by definition, would be unobserved by the team running the clinical trial. One might want to view the simulations as a single-arm trial, where different unknown subgroups of patients respond to the therapy differently, and the goal is to use the covariate to identify a subset of patients who respond. In that case, the ability of Umpire to generate another data set from the same mixed-type engine could be used to provide independent validation of the method.\n\nA sensible approach might be to simulate a two-arm clinical trial where one arm receives a placebo (or the current standard-of-care), while the second arm receives a new (or additional) therapy. Again, one possible goal is to identify the subset of patients in the experimental arm with better response. We can achieve this in Umpire by adding a control group.\n\n\n\n\n\nThe control arm is now subtype 1, and the experimental arm is given by the collection of all other (heterogeneous) subtypes.\n\nWe note here that one of the default parameters to the CancerModel constructor that is used inside a clinical engine defines the distribution of the beta-parameters in a Cox proportional hazards model. By default, these are chosen from a normal distribution with mean 0 and standard deviation 0.3. As a consequence, each latent variable is just as likely to make the hazard ratio worse rather than better. For purposes of illustration, we are going to cheat and adjust the beta parameters to bias them toward an improved outcome in the experimental group:\n\n\n\nOf course, the better way to accomplish this goal would have been to set that parameter when we constructed the ClinicalEngine orginally, to something like SURV = function(n) rnorm(n, 0.2, 0.3).\n\nHere is an example of a simulated trial.\n\n\n\n\n\nNow we compute the Cox proportional hazards model that we would see from the two-arm trial.\n\n\n\n\n\nThanks in part to the bias we built into the simulation, the experimental group does significantly better than the control group. We can also fit the model that would be obtained if the trial designers were omniscient and could see the subtypes defined by the latent variables.\n\n\n\n\n\nHere we see that one of the four subtypes is equivalent to the control group, while all three other subtypes appear to do better. Finally, we plot the resulting Kaplan-Meirer curves for both models (Figure 7).\n\n\n\nAs in the first use case, you can run a set of nested loops to vary the parameters of interest. As noted previously, one possible application would be to test algorithms for finding clinical variables that define patient subgroups with better (or worse) responses than the control group.\n\nLarge epidemiological cohorts are a foundational data type in public health research. Here, we simulate an extensive patient cohort and assess for a binary outcome.\n\nEpidemiological cohorts may aggregate data from multiple data collection instruments, possibly including chart review, laboratory data, and survey. Here, we generate mixed type data consisting of continuous laboratory data gathered at time of study entry and an extensive survey, which contains both nominal and ordinal (Likert scale) categorical responses. We simulate a ClinicalEngine with a large feature space and 6 latent clusters of unequal size, taking the default noise and survival models. We generate data for 4,000 patients.\n\n\n\n\n\nNow we are going to fit univariate models to determine which features are associated with the binary outcome. For continuous variables, we perform a t-test comparing the values in the two outcome groups. For discrete variables (binary or categorical), we perform a chi-squared test.\n\n\n\n\n\nTo account for multiple testing, we fit a beta-uniform-mixture (BUM) model to estimate the false discovery rate (FDR)20. We show the results by overlaying the fitted model on a histogram of p-values (Figure 8).\n\n\n\nThere is clear evidence of an enrichment of small p-values indicating features that are associated with the clinical outcome in univariate models. We can determine the number of significant features and the nominal p-value cutoff associated with any given FDR.\n\n\n\nWe can also count the number of significant “discoveries” associated with each block of correlated genes, but this requires some spelunking into the depths of the mixed-type engine.\n\n\n\n\n\nWe can compare this table with a heatmap of the hit pattern (Figure 9). Only 20 of the 27 blocks were included as possible hits, and blocks 2, 4, 11, and 15 were unused. The table shows that none of the identified features were included in any of those blocks, suggesting that we made no false discoveries.\n\n\n\nBlack pixels mark the presence of latent variables within a cluster. Columns are clusters (or subypes); rows are latent variables (or hits).\n\n\nDiscussion\n\nThe Umpire R-package provides a series of tools to simulate complex, correlated, heterogenous data for methods development and testing for omics and clinical data. The Umpire 2.0 package version described here provides an easy, user-friendly pipeline to generate clinically realistic, mixed-type data to interrogate analytic problems in clinical data. Alongside data sets with meaningful noise and complex feature interrelationships, Umpire simulates subgroup or cluster identities with known ground truth and single- and multi-group dichotomous and survival outcomes. Thus, Umpire facilitates the creation of simulations to explore a variety of methodological problems.\n\nUmpire offers the user a streamlined workflow with ample opportunities for fine-tuning and flexibility. Although this paper describes applications for clinical data, we have previously described Umpire’s tools for simulating omics data8. Furthermore, the modules of the package (e.g., Engines, NoiseModels, and make-DataTypes) may be used interchangeably. Thus, the user may choose to generate omics-scale data of noncontinuous type. The user may generate elaborate simulations by varying and increasing parameters (including, but not limited to, subgroup size or number, feature space, sample size, noise, survival model) to target an inquiry.\n\nIn our use cases, we demonstrated the flexibility of Umpire for generating simulations to help evaluate a variety of applications of machine learning to clinical data. These include applications of unsupervised ML to discover subtypes (in Use case 1) and applications of supervised machine learning to find predictive or prognostic factors (in Use cases 2 and 3). The ability of Umpire to evaluate analysis methods is not confined to these use cases. Our use cases illustrating supervised ML did not exploit the fact that, using the parameters saved in a mixed-type engine, Umpire can simulate multiple data sets from the same underlying population, thus providing unlimited test and validation sets. In addition to testing algorithms head-to-head, Umpire can also be used to generate complex simulations to interrogate the “operating characteristics” of an algorithm. For instance, one of the still-unsolved problems in clustering is determining the true number of clusters. A researcher who has developed a new method that claims to solve this problem could simulate mixed-type data with a variety of different cluster numbers, prevalences, feature numbers, and patient sizes to determine which factors influence the accuracy of the method.\n\nWe expect Umpire to have wide applicability as a tool for comparing and understanding the behavior of any ML method that has the potential to be applied to clinical data.\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\nUmpire is freely available at the Comprehensive R Archive Network: https://cran.r-project.org/ web/packages/Umpire/index.html.\n\nSource code is available from R-Forge: https://r-forge.r-project.org/R/?group_id=1831.\n\nArchived source code at time of publication: https://cran.r-project.org/src/contrib/Archive/Umpire/ and https://doi.org/10.5281/zenodo.402310621.\n\nLicense: Apache License, version 2.0.", "appendix": "Author contributions\n\n\n\nCEC contributed in conceptualization, formal analysis, methodology, software, validation, visualization, and writing (both original draft preparation and review & editing). ZBA contributed in methodology, software, and writing – reviewing & editing. GB contributed in supervision and writing – reviewing & editing. KRC contributed in conceptualization, formal analysis, methodology, software, supervision, validation, and writing – reviewing and editing. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work.\n\n\nReferences\n\nRaghupathi W, Raghupathi V: Big data analytics in healthcare: promise and potential. Health Inf Sci Syst. 2014; 2: 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCook JA, Collins GS: The rise of big clinical databases. Br J Surg. 2015; 102(2): e93–e101. PubMed Abstract \| Publisher Full Text\n\nCoombes CE, Abrams ZB, Li S, et al.: Unsupervised machine learning and prognostic factors of survival in chronic lymphocytic leukemia. J Am Med Inform Assoc. 2020; 27(7): 1019–1027. PubMed Abstract \| Publisher Full Text\n\nCastaldi PJ, Benet M, Petersen H, et al.: Do COPD subtypes really exist? COPD heterogeneity and clustering in 10 independent cohorts. Thorax. 2017; 72(11): 998–1006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPikoula M, Quint JK, Nissen F, et al.: Identifying clinically important COPD sub-types using data-driven approaches in primary care population based electronic health records. BMC Med Inform Decis Mak. 2019; 19(1): 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPowers BW, Yan J, Zhu J, et al.: Subgroups of High-Cost Medicare Advantage Patients: an Observational Study. J Gen Intern Med. 2019; 34(2): 218–225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFoss AH, Markatou M: kamila: clustering mixed-type data in R and Hadoop. J Stat Softw. 2018; 83(1): 1–44. Publisher Full Text\n\nZhang J, Coombes KR: Sources of variation in false discovery rate estimation include sample size, correlation, and inherent differences between groups. BMC Bioinformatics. 2012; Suppl 13(Suppl 13): S1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarlin BM, Kale DC, Khemani RG, et al.: Unsupervised pattern discovery in electronic health care data using probabilistic clustering models. In: Proceedings of the 2nd ACM SIGHIT international health informatics symposium. 2012; 389–398. Publisher Full Text\n\nWang J, Wen S, Symmans WF, et al.: The bimodality index: a criterion for discovering and ranking bimodal signatures from cancer gene expression profiling data. Cancer Inform. 2009; 7: 199–216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee JH, Rhee CK, Kim K, et al.: Identification of subtypes in subjects with mild-to-moderate airflow limitation and its clinical and socioeconomic implications. Int J Chron Obstruct Pulmon Dis. 2017; 12: 1135–1144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBose E, Radhakrishnan K: Using Unsupervised Machine Learning to Identify Subgroups Among Home Health Patients With Heart Failure Using Telehealth. Comput Inform Nurs. 2018; 36(5): 242–248. PubMed Abstract \| Publisher Full Text\n\nYan J, Linn KA, Powers BW, et al.: Applying Machine Learning Algorithms to Segment High-Cost Patient Populations. J Gen Intern Med. 2019; 34(2): 211–217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurgel PR, Paillasseur JL, Caillaud D, et al.: Clinical COPD phenotypes: a novel approach using principal component and cluster analyses. Eur Respir J. 2010; 36(3): 531–9. PubMed Abstract \| Publisher Full Text\n\nInohara T, Shrader P, Pieper K, et al.: Association of of Atrial Fibrillation Clinical Phenotypes With Treatment Patterns and Outcomes: A Multicenter Registry Study. JAMA Cardiol. 2018; 3(1): 54–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEgan BM, Sutherland SE, Tilkemeier PL, et al.: A cluster-based approach for integrating clinical management of Medicare beneficiaries with multiple chronic conditions. PLoS One. 2019; 14(6): e0217696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaufman PJ, Rousseeuw L: Finding Groups in Data: An Introduction to Cluster Analysis. John Wiley & Sons, Hoboken, NJ. 1990. Publisher Full Text\n\nKohonen T: Self-Organizing Maps. Springer-Verlag, Berlin; Heidelberg; New York, third edition, 2001. Reference Source\n\nHubert L, Arabie P: Comparing partitions. J Classif. 1985; 2(1): 193–218. 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): 1236–42. PubMed Abstract \| Publisher Full Text\n\nCoombes KR, Zhang J, Coombes CE: Umpire 2.0: An R Package to simulate realistic gene expression and clinical data. 2020. http://www.doi.org/10.5281/zenodo.4023106" }	[ { "id": "75047", "date": "02 Dec 2020", "name": "Julia Wrobel", "expertise": [ "Reviewer Expertise Biomedical imaging", "statistical software development", "wearable technology." ], "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 article introduces Umpire 2.0, an R package that builds on its previous version (Umpire 1.0) and provides a way to simulate complicated clinical data that has survival and genomics components. The package has a wide range of useful capabilities, and will be helpful for researchers who need simulated data with ground truth to assess the performance of their supervised and unsupervised machine learning methods. However, some work can be done to clarify the interpretation and underlying mechanism of the package. A few specific comments are given as bullet points below.\nOne of the key improvements for Umpire 2.0 is the ability to simulate \"mixed-type\" data. However, \"mixed-type\" is not clearly defined when it is introduced. It's meaning becomes clear in later sections, but it would be helpful to the reader to define it more explicitly at the outset.\n\nThe interpretation of Figures 2 and 3 are not well explained. In the text or a caption, explanation of what is learned from these figures should be added. For example with Figure 2, it seems that the dendrogram on top shows the four clusters, but what does the dendrogram on the left side represent? What features are being fed in here? Similarly, how are the two plots in Figure 3 related? Are the clusters assigned using the true underlying cluster values?\n\nIt seems that the package was built using an S4 object-oriented paradigm. It would be helpful to include a paragraph or two about how the package was structured and implemented using this approach.\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? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6343", "date": "05 Mar 2021", "name": "Kevin Coombes", "role": "Author Response", "response": "In response to the comments from Reviewer 1 (Julia Wrobel), we: 1. Added a sentence to the abstract to clarify the meaning of \"mixed type data\". 2. Expanded the captions on Figures 2, 3, and 4 in order to make them easier to understand. 3. Added several paragraphs to the \"Implementation\" section to highlight the use of S4 classes in our R package." } ] } ]	1	https://f1000research.com/articles/9-1186
https://f1000research.com/articles/9-1031/v1	24 Aug 20	{ "type": "Research Article", "title": "An assessment of the autism neuroimaging literature for the prospects of re-executability", "authors": [ "Steven M. Hodge", "Christian Haselgrove", "Leah Honor", "David N. Kennedy", "Jean A. Frazier", "Steven M. Hodge", "Christian Haselgrove", "Leah Honor", "Jean A. Frazier" ], "abstract": "Background: The degree of reproducibility of the neuroimaging literature in psychiatric application areas has been called into question and the issues that relate to this reproducibility are extremely complex. Some of these complexities have to do with the underlying biology of the disorders that we study and others arise due to the technology we apply to the analysis of the data we collect. Ultimately, the observations we make get communicated to the rest of the community through publications in the scientific literature. Methods: We sought to perform a ‘re-executability survey’ to evaluate the recent neuroimaging literature with an eye toward seeing if our publication practices are helping or hindering the overall quest for a more reproducible understanding of brain development and aging. The topic areas examined include availability of the data, the precision of the imaging method description and the reporting of the statistical analytic approach, and the availability of the complete results. We applied the survey to 50 publications in the autism neuroimaging literature that were published between September 16, 2017 to October 1, 2018. Results: The results of the survey indicate that for the literature examined, data that is not already part of a public repository is rarely available, software tools are usually named but versions and operating system are not, it is expected that reasonably skilled analysts could approximately perform the analyses described, and the complete results of the studies are rarely available. Conclusions: We have identified that there is ample room for improvement in research publication practices. We hope exposing these issues in the retrospective literature can provide guidance and motivation for improving this aspect of our reporting practices in the future.", "keywords": [ "Reproducible Science", "Neuroimaging", "Autism", "Data Sharing", "Re-executability" ], "content": "Introduction\n\nThere is concern about the status of reproducibility in science in general and neuroimaging neuroscience in particular (Button et al., 2013; Gorgolewski & Poldrack, 2016). A particularly germane concern was expressed by Kapur and colleagues in lamenting: “a profusion of statistically significant, but minimally differentiating, biological findings; ‘approximate replications’ of these findings in a way that neither confirms nor refutes them” (Kapur et al., 2012). The replication of a specific finding (or reproducibility of a specific analysis), as reflected in a publication, has many details and nuances to it (Kennedy et al., 2019). Often, we are searching for the ‘generalizability’ of a finding: does the finding hold true when using ‘similar’ data and a ‘similar’ analysis. The similarity of data (or analysis) is a fuzzy concept. One could have a population with the same number of subjects with the same diagnosis, having the same mean age and same gender distribution as a target population; however, if the diagnosis in question is a ‘spectrum’-diagnosis (for example, autism, schizophrenia, depression, etc.), despite the ‘sameness’ of my sample in the aforementioned categories, the detailed nature of the characteristics of my sample in the features of the diagnosis itself can still be quite variable. At the level of a biological finding, we typically do not predicate the finding on an exact acquisition protocol, or a specific analysis protocol; rather, it is implicit in our finding that it should hold for other valid acquisitions and analyses of the reported types. There is increasing evidence that this implicit assumption of similarity, when it relates to the specific details of acquisition or analysis, does not necessarily hold (Glatard et al., 2015).\n\nSome have argued that the starting point for the structured exploration of the generalizability of a specific finding (and thus a cornerstone to the quest for reproducibility) lies in the original finding itself being re-executable (Ghosh et al., 2017; Kennedy, 2019). Starting from the re-execution of a finding will allow for the systematic exploration of the generalizability of that finding, over changes in data and analysis. To date, when new studies find different findings from prior studies, it is too easy to simply argue that differences in the subject population or analysis workflow differences account for the discrepancy.\n\nThe potential impact of reproducibility issues become most obvious when trying to make sense of the accumulated literature on specific topic areas (Rane et al., 2015). For this reason, we have chosen a particular area, ‘autism’ as a way to focus the literature for this survey, so that the conclusions we reach can have potential specific implications for that topic area. We feel that the autism focus, however, will generate findings that will have similar implications to other psychiatric and developmental application areas.\n\nIn this paper, we: 1) develop a specification for what constitutes an assessment of the re-executability for a given publication in each of the domains of: data, software, execution environment, statistics and results; 2) codify this assessment in survey form; and 3) apply the survey to a subset of the autism neuroimaging literature published recently (~2018). From the results of this survey, we can begin to generalize the state of the re-executability of the recent autism neuroimaging literature, in order to identify trends and opportunities for the enhancement of the re-executability status in support of greater overall generalizability (and hence reproducibility) of the literature. The survey template could also be applied as part of the publication review process, in order to prospectively attempt to enhance these aspects of reproducibility.\n\n\nMethods\n\nFollowing the concept of a ‘re-executable publication’ (Kennedy, 2019), in order to assess the prospects of re-execution of a given paper, we assess 1) the availability of the starting data, 2) the precision of the analysis description (both data processing and statistical assessment), and 3) the availability of the detailed complete results (in order to verify accuracy of re-execution). Regarding the ‘availability of the starting data’, we assess if the publication indicates how someone1 (other than the authors themselves) could appropriately access the data. The ‘precision of the analysis description’ ultimately asks if a reader who is reasonably skilled in the necessary domains, could precisely carry out the prescribed analysis steps. Specifically, are the software versions, operating system and complete parameters somehow made available to the reader? The ‘detailed complete results’ assesses if the publication indicates how to obtain the complete results, in order to both verify that the re-execution generates the same result and to overcome the limitations of only a selected summary being presented, which impedes a more complete meta-analysis of the literature.\n\nIn each of the three assessment areas, the survey distinguished between the theoretical potential for reproduction (such as complete descriptions of data used, software and commands executed, and statistical tests applied) and the practical potential for reproduction (whether the data is in fact accessible, whether the software is still available and will run). While the survey did not require the raters to actually reproduce the various steps, they were asked to use their professional judgement and past experience to determine the potential reproducibility. In these ‘judgement’ questions we allow responses of ‘Yes’, ‘Approximately’, ‘I’m not sure’, and ‘No’ to allow some degree of confidence in these judgements. For ‘results availability’, we coded ‘Yes’ if all of the results were indicated as being available, ‘Partially’ if some of the results were indicated as being available, and ‘No’ if none of the results were indicated as available or no indication of the results availability was provided.\n\nFigure 1 provides an overview of the survey design.\n\nOS, operating system.\n\nThe survey was constructed in Google Forms. The details of the logic and wording of the survey forms was piloted within our own group, and then released for public comment to the BrainHack Slack2 channel in August, 2018. The final complete (serialized) text of the survey is provided in S1 (see Extended data; Hodge et al., 2020c).\n\nOn January 23, 2019, the following PubMed query was executed:\n\n((\"autistic disorder\"[MeSH Terms] OR (\"autistic\"[All Fields] AND \"disorder\"[All Fields]) OR \"autistic disorder\"[All Fields] OR \"autism\"[All Fields]) AND (\"magnetic resonance imaging\"[MeSH Terms] OR (\"magnetic\"[All Fields] AND \"resonance\"[All Fields] AND \"imaging\"[All Fields]) OR \"magnetic resonance imaging\"[All Fields] OR \"mri\"[All Fields])) AND (\"2014/01/25\"[PDat] : \"2019/01/23\"[PDat] AND \"humans\"[MeSH Terms])\n\nThis is the expansion of the general query for ‘autism AND MRI, qualified to select publications between 1/25/2014 - 1/23/2019 and where the MeSH term includes ‘human’. This query generated 811 resultant publications at the time of the query (see S2, Underlying data; Hodge et al., 2020a). We note that re-running the query today will generate additional results due to publications that have been added to PubMed after the search date but with publication dates within the defined range.\n\nStarting from the most recent publication and working backwards, we reviewed the title and abstract to verify publications that were indeed neuroimaging studies (not a case report or review), in English, related to autism and for which we could access the full text of the article. Working backwards from publication date, we selected the first 50 publications that met the above criteria. Of these 50 publications, 38 were available as free full text on PubMed, three were available as a PDF through a general Google Scholar search (publisher/author provided), two were available in PDF format from ResearchGate, and seven did not seem to be available without institutional access. One of three raters applied the survey to each of these articles. Each of the final results were reviewed by one rater and consensus reached with original rater if discrepancies were found.\n\n\nResults\n\nThe final set of publications used in this report is tabulated in Table 1. The publication dates span from September 16, 2017 to October 1, 2018. Publications from 27 different journals are included. The publications selected covered a number of different MRI-based techniques (structural N=20, task-based fMRI N=14, resting-state fMRI N=13, diffusion MRI N=11 and magnetic resonance spectroscopy N=5)3.\n\nA high-level summary of the survey results is represented in Figure 2. The complete set of question-by-question results are provided in S3 (see Underlying data; (Hodge et al., 2020c).\n\nThe 50 publications are summarized on the main factors of data availability, software specification, statistical specification and results availability.\n\n\nDiscussion\n\nThe recent past literature of autism neuroimaging presents a somewhat consistent picture with respect to the prospects of re-executability with regard to the characteristics we examined in this report.\n\nPublication availability: 38 of the 50 (76%) publications appear to have ‘free full text’ available, according to the PubMed search. Of these, 33 are indexed in PubMed Central. Overall, 43 were freely available through either PubMed Central, Google Scholar or publisher or other websites.\n\nData availability: 16 of the 50 (32%) publications make reference to the availability of the data used in the publication. However, the publications that indicate availability are reusing data from the large repositories, whereas the publications that do not indicate data availability are principally locally conducted studies. Thus, this indicates that a large fraction of the data being used in publications are not available to the community. For the data that is available, the following resources are indicated: ABIDE 1 (Di Martino et al., 2014), ABIDE 2 (Di Martino et al., 2017), FCP/INDI (Mennes et al., 2013), COINS (Scott et al., 2011), LORIS (Das et al., 2012), NITRC (Kennedy et al., 2016), Preprocessed Connectomes Project (Puccio et al., 2016), UKBiobank (Miller et al., 2016), Brain Genomics Superstruct Project (Holmes et al., 2015), ADHD-200 (HD-200 Consortium et al., 2012), and Human Connectome Project (Glasser et al., 2016).\n\nImage analysis: Virtually all of the publications surveyed indicate the imaging analysis software used (44 of 50, 88%). Most publications indicate the use of multiple tools. However, specific tool versions are indicated only about half of the time. While this may seem a minor point, software version can make a difference in results (Glatard et al., 2015; Ghosh et al., 2017). In this collection of 50 papers, 35 different publicly released tools (plus a number of in-house packages) are used. Not surprisingly, the following tools are used in over 10 publications each: SPM (Ashburner et al., 1998), FSL (Jenkinson et al., 2012), and FreeSurfer (Makris et al., 2003). The specific operating system used is rarely reported (1 of 50, 2%). Overall, our raters felt that in 80% of the publications a skilled image analyst could (or might be able to) repeat the analysis.\n\nStatistical analysis: In approximately two thirds of the publications (66%), the statistical software is indicated, again with variable indication of version and no reporting of the operating system upon which the software was running. In summary, our raters felt that in 29 of the 50 papers (58%), a skilled statistical analyst could (or might be able to) repeat the analysis.\n\nThere is a distinct difference between the theoretical and practical ability to reproduce both the image analysis and statistical analysis. While the software packages used for the image analyses are specified in nearly all the cases, only some give the rater confidence that a skilled analyst could actually re-execute the analysis. Similarly, for the cases where the statistical tools are specified, only a handful are descriptive enough to give confidence in re-executability.\n\nResults availability: Availability of the detailed results is fairly rare. All or partial results are available in seven of the 50 publications (14%). Lack of results availability causes a number of problems. Primarily, it is harder to confirm replication (or the degree to which replication was or was not achieved) without the complete set of reported observations, not just the summary tables or figures. Resorting to visual interpretations of ‘similarity’ of published figures remains fraught with issues that can hamper true understanding of new results compared to prior results. Lack of detailed results sharing also compromises subsequent meta-analytic studies that would strive to integrate observations across multiple publications. Finally, lack of complete results exacerbates the publication bias (Jennings & Van Horn, 2012) through focus on the (relatively few) statistically significant observations while not reporting the large set of observations that are not significant.\n\nOther observations: None of the reviewed publications indicated pre-registration (Nosek et al., 2019). This is not surprising as pre-registration is a fairly new phenomenon, and its uptake in the literature can be expected to take a while. However, as a ‘baseline’ observation, it is still important to note, so that changes in the prevalence of the pre-registration practice can be monitored.\n\nThe scope of our survey was rather limited; only 50 publications, and in a selected topic area, autism. However, as a retrospective starting point for evaluation, we believe that it fairly represents the qualitative impressions that investigators have about the nature of neuroimaging publications. We covered numerous neuroimaging subdomains: structural, diffusion, functional; and data and analytic practices in these subdomains can be rather variable.\n\nThe raters (DNK, CH, SMH) we used had over 15 years of neuroimaging research experience each; however, the specialties of each varied from more methodological/statistical to image analytic. This ‘background’ can influence the interpretation of how successfully other ‘reasonably skilled’ investigators could re-execute a given analysis. Familiarity with particular methods can both increase perceived confidence with its reuse (“Of course, everyone knows how to execute that common method”) or decrease confidence (“There are so many details that I know could be varied, how do I know what was really done?”).\n\nFinally, the assessment of each publication is performed on the accessible manuscript as published. It is possible that data and results sharing can have occurred after publication, but this fact may not be represented in the materials reviewed. Indeed, it would be a valuable service to facilitate a more prospective management of these critical re-execution factors that can support authors in making additional supporting data and methods available post publication.\n\n\nConclusions\n\nIn conclusion, we feel that the survey results presented here reflect a state of neuroimaging publication practices that leaves ample room for improvement. While reuse of existing data is good, the majority of new data being collected for use in publications is not made publicly available. While the listing of software used is good, important details for reproducibility, such as version, detailed parameters, operating system, etc. are not fully disclosed. Similarly, statistical assessment details are variably reported, making re-execution problematic and approximate. Finally, as very little of the complete results of a publication are disclosed, assessment of the similarity of future replication attempts is severely hampered. Given the overall state of uncertainty about how reproducible (and representative) specific neuroimaging findings are, it seems prudent to begin to tighten up the variables that we as authors do have in order to better support the effective accumulation of knowledge about conditions we study. Promoting best practices in ethical data sharing, complete analytic approach disclosure, and complete results reporting seem to be critical in integrating the complex set of observations we collectively have published about the brain and how it develops and ages. The implications of these observations are that authors should redouble their efforts to be comprehensive in their reporting, even after the publication, to make as accessible as possible the detailed methods and results that they are reporting on. Specifically, authors, reviewers and editors should insist on the complete declaration of: data source and availability status, all software and versions used for data analysis and statistical assessment, the operating system (and version) for data and statistical analysis, and the disposition of the analytic results. Such a ‘checklist’ would be a valuable asset for the community and will be the subject of future efforts. This future checklist should be developed in conjunction with journal specific guidelines, and other checklists (established in conjunction with the COBIDAS report (Nichols et al., 2017), statistical reporting (Dexter & Shafer (2017), Nature Neuroscience Reporting Checklist, etc.). In such a way, publishers, editors and reviewers can impart more influence in the manuscripts that they encounter, in an effort to increase the transparency and completeness of the published record that they are playing their role in creating. Together, we hope that we can move the field forward and generate a literature that is more amenable to supporting the understanding of how our collective observations fit together in supporting the understanding of the brain.\n\n\nData availability\n\nNITRC: CANDI Neuroimaging Access Point: S2_Raw_pubmed_Query_result.csv. http://doi.org/10.25790/bml0cm.68 (Hodge et al., 2020a)\n\nThis project contains the following underlying data:\n\n- S2_Raw_pubmed_Query_result.csv (complete PubMed query result from 1/23/2019)\n\nNITRC: CANDI Neuroimaging Access Point: S3_CompleteSurveyData.xlsx. http://doi.org/10.25790/bml0cm.67 (Hodge et al., 2020b)\n\nThis project contains the following underlying data:\n\n- S3_CompleteSurveyData.xlsx (complete survey results)\n\nNITRC: CANDI Neuroimaging Access Point: S1_Prospects for Reproducibility Check List_V2 - Google Forms.pdf http://doi.org/10.25790/bml0cm.66 (Hodge et al., 2020c)\n\nThis project contains the following extended data:\n\n- S1_Prospects for Reproducibility Check List_V2 - Google Forms.pdf (complete survey form)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Footnotes\n\n1Although maybe not ‘everyone’, depending on the specific details of the data use agreement.\n\n2Currently archived in the BrainHack Mattermost ‘general’ channel: https://mattermost.brainhack.org/brainhack/channels/general\n\n3A number of publications included data from multiple MRI types.\n\n\nReferences\n\nAbbott AE, Linke AC, Nair A, et al.: Repetitive Behaviors in Autism Are Linked to Imbalance of Corticostriatal Connectivity: A Functional Connectivity MRI Study. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei L, Zhong S, Nie S, et al.: Aberrant Development of the Asymmetry between Hemispheric Brain White Matter Networks in Autism Spectrum Disorder. Eur Neuropsychopharmacol. 2018; 28(1): 48–62. PubMed Abstract \| Publisher Full Text\n\nWhite T, Jansen PR, Muetzel RL, et al.: Automated Quality Assessment of Structural Magnetic Resonance Images in Children: Comparison with Visual Inspection and Surface-Based Reconstruction. Hum Brain Mapp. 2018a; 39(3): 1218–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite T, Muetzel RL, Marroun HE, et al.: Paediatric Population Neuroimaging and the Generation R Study: The Second Wave. Eur J Epidemiol. 2018b; 33(1): 99–125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan W, Rangaprakash D, Deshpande G: Aberrant Hemodynamic Responses in Autism: Implications for Resting State fMRI Functional Connectivity Studies. Neuroimage Clin. 2018; 19: 320–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang F, Savadjiev P, Cai W, et al.: Whole Brain White Matter Connectivity Analysis Using Machine Learning: An Application to Autism. Neuroimage. 2018; 172: 826–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao G, Walsh K, Long J, et al.: Reduced Structural Complexity of the Right Cerebellar Cortex in Male Children with Autism Spectrum Disorder. PLoS One. 2018; 13(7): e0196964. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70168", "date": "22 Sep 2020", "name": "Karsten Specht", "expertise": [ "Reviewer Expertise Neuroimaging", "fMRI", "MRS", "reliability" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary The article by Hodge and co-workers summarises an attempt in assessing the possibility to replicate 50 published neuroimaging studies on autism. The results indicate that the majority of the studies provide only partial information that would be required for replication of the study. In particular, are information about the operating system missing, only a few studies share their data or other files, and the description of the different analysis steps are sparsely described.\n\nAssessment: The article is well written with a clearly described method and results. The study provides a suitable method that could easily be applied to other research topics, as well. However, the conclusions that can be drawn from this study are still limited in my view, since it would have been good to include further information in the survey, which I will list below:\n\nIn my opinion, the authors focus too much on the technical aspects of a study. Although the authors introduce that a “spectrum-diagnosis” might generate further problems, they do not follow this up in the survey. I would like to see at least one additional column that codes whether the diagnostic criteria and sample are replicable, i.e. are the patients well characterised (age, gender, education), are the diagnostic instrument mentioned, cut-off criteria, etc.\n\nI suggest including another column (at least) in the supplementary material S3 that also lists the imaging modality, i.e. structural MRI, fMRI, MRS, DTI, since they also partly represent different disciplines and traditions in publishing. Further, some methods have only a very limited number of software tools, like MRS, which are often restricted to only one (type of) OS. So, reporting the software may make it almost obsolete to report the OS. Therefore, doing a survey across different neuroimaging modalities may show some general deficiencies, but the other disciplines may need to improve on different aspects.\n\nSimilarly, concerning fMRI, it also makes a difference whether studies were analysed as whole-brain studies or as a focused region of interest analyses, and, in the latter case, whether the regions were derived from anatomical images or, for example, simply spheres. It would also be informative to know whether studies applied corrected p-values, and which one, and whether the effect sizes were reported.\n\nDid the authors control how many studies came from the same lab? Some labs might have a kind of “tradition” in reporting results, which could bias the survey.\n\nI think, the headlines of the article are a bit off since the “Discussion” mostly reports the results, and the “Conclusion” primarily discusses the results.\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": "6314", "date": "04 Mar 2021", "name": "David Kennedy", "role": "Author Response", "response": "We would like to thank the reviewer for their thoughtful comments and attention to this manuscript. Here we outline our responses to the comments, and indicate where in the manuscript we have made changes. [...] the authors focus too much on the technical aspects of a study. Although the authors introduce that a “spectrum-diagnosis” might generate further problems, they do not follow this up in the survey. I would like to see at least one additional column that codes whether the diagnostic criteria and sample are replicable, i.e. are the patients well characterised (age, gender, education), are the diagnostic instrument mentioned, cut-off criteria, etc. Response: The survey is indeed focused on the technical ability to reproduce the analytic approach of a study. We try to do a better job of setting up the scope of the survey in the introduction. Specifically, we clarify that there are at least three domains in a publication where sufficient information for re-execution needs to be considered: the subject selection (can another researcher generate a comparable group); the data acquisition (can another researcher collect the same data); and the analysis (can another researcher perform the same analysis). All of these areas are important but we are only addressing the ‘analysis’ aspect in this manuscript. Development and application of a similar re-ascertainment survey for the subject selection is an excellent idea, and we hope to pursue such an endeavour in a future survey that would look at subject characteristics such as age, gender, education, diagnostic instruments, etc. We have augmented the second paragraph of the Introduction to address this (and a similar concern raised by Reviewer #2): “In this paper we concentrate on assessing the technical prospects of re-executability of a publication. As introduced above, there are many other factors that will contribute to the actual generalization of the findings including subject population details, data acquisition details, the nature of the processing and statistics (even if they can be re-executed), the underlying biological effect size, if present, etc. (see Figure 1). Take for example, the subject population. Too often researchers communicate a finding based on a convenience sample without any statement indicating that the results might not generalize to a sample that more accurately reflects human diversity (e.g. DeJesus, Callanan, Solis & Gelman, 20191; Hruschka, Medin, Rogoff & Henrich, 20182; Rad, Martingano & Ginges, 20183; Henrich, Heine, & Norenzayan, 20104). Comprehensive and standardized description of all these additional factors are critical as well, but are beyond the scope of this evaluation. Our groups and others are looking into reporting standards for these areas as well.” I suggest including another column (at least) in the supplementary material S3 that also lists the imaging modality, i.e. structural MRI, fMRI, MRS, DTI, since they also partly represent different disciplines and traditions in publishing. Response: We have added a new column to Table 1 that indicates modality. While the details of the data and analysis procedures will vary by these modalities, the need to fully express the complete analysis should be independent of the specific modality. some methods have only a very limited number of software tools, like MRS, which are often restricted to only one (type of) OS. So, reporting the software may make it almost obsolete to report the OS. Response: While this is certainly true in some situations, we suggest that a good best practice for reporting should be universal (and OS versions change and thus should be disclosed). We have added in the Discussion, third paragraph: “Even if there are currently only limited software options in some analysis domains, which may implicitly implicate the operating system used, such limitations are not guaranteed to persist through time and should not be assumed for the reader.“ doing a survey across different neuroimaging modalities may show some general deficiencies, but the other disciplines may need to improve on different aspects. Response: Again, while this is true, the general best practices and principles we’re trying to elucidate here should be universal. What specific disciplines need to do to support these necessary practices may indeed vary by discipline. We try to elaborate on this in the Limitations section, first paragraph: “We acknowledge that the details of precise description and dissemination of data and methods may indeed vary by discipline. However, we argue that the ‘best practice’ principles that we are suggesting here are universal and domain-specific solutions are currently available.” concerning fMRI, it also makes a difference whether studies were analysed as whole-brain studies or as a focused region of interest analyses, and, in the latter case, whether the regions were derived from anatomical images or, for example, simply spheres. Response: This specific factor is accounted for in the assessors interpretation of how confident they are about re-executing the procedure. If a focused region of interest study is reported, the assessor will determine how confident they are that they could arrive at the ROIs used. It would also be informative to know whether studies applied corrected p-values, and which one, and whether the effect sizes were reported. Response: This is an important distinction that we did not clarify in the original manuscript. Our assessment is aimed at evaluating the quality of the reporting (can I do what was reported), rather than the content (is what was reported the right or best thing to do?). The latter assessment is really the purview of the original reviewers of the article itself, whereas the former (in other words, an attempt to generalize a reported finding) is a function that the community of readers would be engaged in and hence our assessment of the feasibility of this from the article. We’ve added a statement at end of the Survey Design section: “Note that our assessments are not if the analysis or data accessibility is ‘optimum’, or even ‘correct’, but rather if the assessor could redo the approach as described.” Did the authors control how many studies came from the same lab? Some labs might have a kind of “tradition” in reporting results, which could bias the survey. Response: This is a fair point. We have reviewed the author lists of the articles included in the survey and indeed discovered that a number of these articles come from the same groups. This is now explicitly documented in the first paragraph of the Limitations section. “Also, even though fifty publications are included in the survey, a number of these publications share co-authors or originate from the same research groups. Specifically, 15 of these authors are listed on two or more publications, and 14 of the publications have authors that are also authors on other publications in this set.” the headlines of the article are a bit off since the “Discussion” mostly reports the results, and the “Conclusion” primarily discusses the results. Response: The Results, Discussion and Conclusions sections have been updated to better reflect the appropriate content." } ] }, { "id": "70151", "date": "22 Sep 2020", "name": "Travis Riddle", "expertise": [ "Reviewer Expertise Neuroscience", "Psychology", "Computer Science", "Neuroimaging" ], "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 highlights an important concern regarding the quality of the science seen in published literature. We applaud the authors for undertaking this work, and agree with their general conclusions that there are many opportunities for researchers to improve their reporting. However, we feel that it is worth mentioning a few details in the paper that caused us some concern or confusion.\nFirst, the paper leads with a summary of some of the issues surrounding reproducibility of science. We urge the authors to make note of another concern that is widely overlooked. Too often researchers communicate a finding based on a convenience sample without any statement indicating that the results might not generalize to a sample that more accurately reflects human diversity (e.g. DeJesus, Callanan, Solis & Gelman, 20191; Hruschka, Medin, Rogoff & Henrich, 20182; Rad, Martingano & Ginges, 20183; Henrich, Heine, & Norenzayan, 20104). Of course, this paper is about other reasons for reproducibility, but it seems appropriate to mention this, especially in light of the increased attention given to exclusionary social systems in other domains.\nWe also had some concern with the concepts of the 'precision of analysis' (methods paragraph 1). This issue in particular seems difficult to assess reliably, and so there might be a higher degree of measurement error for this concept in comparison to the other concepts. We appreciate that the authors allude to this difficulty later in the paper, when they state that more expertise could also lead to higher levels of measurement error, but here we feel that a more explicit note of caution that these variables in particular should be viewed with additional skepticism.\nThe description of how the assessment was applied to each paper was difficult to follow ('survey application' pg 4: \"one of three raters applied the survey to each of these articles. Each of the final results...\"). Does this mean that each paper was evaluated by 1 reviewer? It seems like it would be useful to have more than one person complete the review. This would allow the reader to have a sense of the degree of inter-rater reliability. If the reliability was low, that would lead us to be a little more credulous with respect to many of the subsequent findings. If there was more than one reviewer per paper, the authors should report some standard inter-rater agreement metrics. If not, an independent assessment by other raters (along with ratings) would be a wonderful addition to the work, if a bit effort-intensive. Adding a column to figure 2 listing which rater assessed which publication would be helpful. This column could be coded for anonymity (Rater 1, Rater 2) if the authors so choose.\nWe additionally found the category of 'results availability' to be a little vague. Especially so since it seems as though papers never reached this cutoff. What does it take for a paper to have complete results availability? Some models might have thousands of parameters or more, and some papers might include dozens or hundreds of fitted models. Does this mean that all parameter values would be reported, confidence intervals, model fit statistics, and so forth would be reported?\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": "6315", "date": "04 Mar 2021", "name": "David Kennedy", "role": "Author Response", "response": "We would like to thank the reviewer for their thoughtful comments and attention to this manuscript. Here we outline our responses to the comments, and indicate where in the manuscript we have made changes. We urge the authors to make note of another concern that is widely overlooked. Too often researchers communicate a finding based on a convenience sample without any statement indicating that the results might not generalize to a sample that more accurately reflects human diversity (e.g. DeJesus, Callanan, Solis & Gelman, 20191; Hruschka, Medin, Rogoff & Henrich, 20182; Rad, Martingano & Ginges, 20183; Henrich, Heine, & Norenzayan, 20104). Of course, this paper is about other reasons for reproducibility, but it seems appropriate to mention this, especially in light of the increased attention given to exclusionary social systems in other domains. Response: As we discuss in response to Reviewer 1 above, the details of the subject pool ascertainment and its’ generalizability is beyond the scope of this manuscript, but as this is an important point, we have included it in our updated introduction. “In this paper we concentrate on assessing the technical prospects of re-executability of a publication. As introduced above, there are many other factors that will contribute to the actual generalization of the findings including subject population details, data acquisition details, the nature of the processing and statistics (even if they can be re-executed), the underlying biological effect size, if present, etc. (see Figure 1). Take for example, the subject population. Too often researchers communicate a finding based on a convenience sample without any statement indicating that the results might not generalize to a sample that more accurately reflects human diversity (e.g. DeJesus, Callanan, Solis & Gelman, 20191; Hruschka, Medin, Rogoff & Henrich, 20182; Rad, Martingano & Ginges, 20183; Henrich, Heine, & Norenzayan, 20104). Comprehensive and standardized description of all these additional factors are critical as well, but are beyond the scope of this evaluation. Our groups and others are looking into reporting standards for these areas as well.” We also had some concern with the concepts of the 'precision of analysis' (methods paragraph 1). This issue in particular seems difficult to assess reliably, and so there might be a higher degree of measurement error for this concept in comparison to the other concepts. We appreciate that the authors allude to this difficulty later in the paper, when they state that more expertise could also lead to higher levels of measurement error, but here we feel that a more explicit note of caution that these variables in particular should be viewed with additional skepticism. Response: In order to help the reader appreciate the cautionary note regarding these assessments, we have updated the notion of ‘precision’ to “perceived completeness” to help remind that the precision assessment is in the mind of the assessor. This is reflected in Methods paragraph one and elaborated upon a little more in Limitations paragraph two: “In the absence of inclusion of explicitly re-executable data and methods in a publication (as in, for example, Ghosh, et al.) the interpretation of the precision and completeness of the description with regard to re-executability will be somewhat imprecise and reader-dependent.” The description of how the assessment was applied to each paper was difficult to follow ('survey application' pg 4: \"one of three raters applied the survey to each of these articles. Each of the final results...\"). Does this mean that each paper was evaluated by 1 reviewer? It seems like it would be useful to have more than one person complete the review. This would allow the reader to have a sense of the degree of inter-rater reliability. Response: We have attempted to clarify the text regarding the validation cases (pilot assessment) and the dual raters for each publication. “The survey was applied to each paper by one of three raters (DNK, SMH, CH). Each of the final results were reviewed by a second rater (DNK or SMH) and consensus reached with the original rater if discrepancies were found.” [related] an independent assessment by other raters (along with ratings) would be a wonderful addition to the work, if a bit effort-intensive. Adding a column to figure 2 listing which rater assessed which publication would be helpful. This column could be coded for anonymity (Rater 1, Rater 2) if the authors so choose. Response: Table 1 now has a column indicating which raters (Rev1, Rev2 or Rev3) reviewed each publication as the ‘primary’ or ‘checking’ reviewer. We additionally found the category of 'results availability' to be a little vague. Especially so since it seems as though papers never reached this cutoff. What does it take for a paper to have complete results availability? Response: We agree that the ‘complete results availability’ was a lofty and somewhat variable goal statement. We have tried to clarify the meaning and ways that this can be satisfied in the updated text of paragraph five in the Discussion: “The complete results availability criterion was rarely met. Lack of results availability causes a number of problems. Primarily, it is harder to confirm replication (or the degree to which replication was or was not achieved) without the complete set of reported observations, not just the summary tables or figures. Resorting to visual interpretations of ‘similarity’ of published figures remains fraught with issues that can hamper true understanding of new results compared to prior results. Lack of detailed results sharing also compromises subsequent meta-analytic studies that would strive to integrate observations across multiple publications. Finally, lack of complete results exacerbates the publication bias (Jennings and Van Horn 2012) through focus on the (relatively few) statistically significant observations while not reporting the large set of observations that are not significant. Examples of complete results availability include when the individual statistical maps for a fMRI annalysis are available in a resource such as NeuroVault, the individual segmentation results of a processing workflow are available at NITRC or Zenodo, etc.”" } ] } ]	1	https://f1000research.com/articles/9-1031
https://f1000research.com/articles/10-178/v1	04 Mar 21	{ "type": "Brief Report", "title": "Body mass index (BMI) and quality of life among long-term survivors of childhood acute lymphoblastic leukemia", "authors": [ "Srilakshmi P. Vankina", "Rachel I. Vogel", "Patricia I. Jewett", "Alexander A. Boucher", "Sanyukta K. Janardan", "Alicia Kunin-Batson", "Karim Thomas Sadak", "Anne Blaes", "Srilakshmi P. Vankina", "Rachel I. Vogel", "Patricia I. Jewett", "Alexander A. Boucher", "Sanyukta K. Janardan", "Alicia Kunin-Batson", "Karim Thomas Sadak" ], "abstract": "Background: Adult survivors of childhood cancers such as acute lymphoblastic leukemia (ALL) are at risk of overweight and related chronic morbidities. As childhood cancer survival has been improving, long-term quality of life (QOL) among cancer survivors becomes more important. We examined the association of body mass index with physical and psychosocial QOL among childhood ALL survivors who returned for long-term follow-up after end of therapy. Methods: Using a cross-sectional survey (2006-2012), we assessed the association between body mass index and quality of life in 58 long-term survivors of childhood ALL (ages 9 to 43 at the time of survey/measurement) using age-appropriate QOL instruments (Health-Related Quality of Life Short Form – SF-36 or Child Health Questionnaire-PF-50). Results: Half of the participants were overweight or obese at the time of survey. Mean QOL scores were similar to population norms. Compared to underweight/healthy weight status, being overweight/obese was not significantly associated with poorer physical QOL, but with poorer psychosocial QOL (47.1±13.2 vs. 54.0±6.0, P=0.01, effect size Cohen’s d=0.67), which remained statistically significant after adjusting for age and sex. Conclusions: Weight management should be the target of timely interventions among survivors of childhood ALL.", "keywords": [ "obesity", "BMI", "leukemia", "cancer survivorship", "quality of life" ], "content": "Introduction\n\nSurvival rates for acute lymphoblastic leukemia (ALL) have improved in the last few decades with five-year survival of standard risk pediatric ALL now exceeding 90%1. Adult survivors of childhood cancer are at increased risk of obesity (11–56%), metabolic syndrome, poor cardiac outcomes, and type-2 diabetes2–10. With improved survival, greater attention has been dedicated to treatment-related late effects and quality of life (QOL).\n\nExcess weight is known to contribute to decreased QOL in individuals with chronic diseases, including adult cancer survivors11,12. Fewer studies have described the relationship between body mass index (BMI) and QOL in long-term pediatric cancer survivors, and in the studies that did, findings were mixed13,14. We examined the relationship between BMI and QOL among childhood ALL survivors who returned for long-term follow-up (LTFU) after completion of therapy. We hypothesized that being overweight/obese compared to a healthy weight at the time of survey completion would be associated with poorer QOL using validated measures.\n\n\nMethods\n\nA cross sectional study was performed of all childhood ALL survivors receiving routine survivor-focused care in the University of Minnesota Childhood Cancer Survivor Program (CCSP) clinic between 2006–201215. Hematopoietic stem cell transplant survivors were excluded because of typically presumed higher rates of complications. At each CCSP visit, participants completed a QOL assessment. As part of their routine care, height and weight measurements were obtained. Height, weight, treatment and medical history were abstracted from the medical record. The University of Minnesota IRB approved this study (protocol number 0002M36181). Participants aged 18 years or older provided written consent; parents/guardians provided written consent for participants younger than 18 years; but participants over age 8 provided assent.\n\nThe primary main outcome of interest was general QOL, measured by the Child Health Questionnaire – PF50 (used for children <18 years, N=38; child’s QOL rated by parents), and the Health-Related Quality of life Short Form – SF-36 for participants > 18 years at survey16–19. Both scales assess multiple QOL domains and can be aggregated into composite physical and psychosocial T-scores that are transformed to a 0–100 scale. Higher scores reflect a better QOL, with a mean of 50 and a standard deviation of 10 in the general population. BMI was measured as percentile for age and sex for those <21 years, and categorized in the following manner for participants aged ≥21 years: Underweight (<5% or <18.5 kg/m2), Healthy (5–85% or 18.5–24.9 kg/m2), Overweight (85–95% or 25–29.9 kg/m2) and Obese (>95% or >30 kg/m2), and further dichotomized into Underweight/Healthy vs. Overweight/Obese.\n\nWe compared patients’ demographics, clinical, and QOL data by weight status using Chi-squared and Fisher’s Exact tests for categorical variables and t-tests and Wilcoxon Rank Sum tests for continuous variables as appropriate. A multivariate linear regression model was used to examine the relationship between BMI and physical and psychosocial QOL T-scores. We had to limit the number of covariates due to small sample size, adjusting for gender and age at time of survey only. P<0.05 was considered statistically significant. Data were analyzed using SAS version 9.3 (Cary, NC).\n\n\nResults\n\nA total of 90 individuals completed the survey; 83 provided QOL data and 58 were eligible for this analysis. Most were non-Hispanic white (97%) and 47% were male (Table 1). The median age was 3.9 years at ALL diagnosis (range: 1.3–19.5) and 21 years at the time of survey (range: 9–43), with a median time of 11 years since diagnosis. Participants with overweight/obesity were further from their diagnosis of ALL than healthy weight/underweight participants (median 15 (range: 5–35) vs. 9 (range: 5–32) years since diagnosis, p=0.15). All survivors were treated with standard of care ALL chemotherapy protocols, and 41% received cranial radiation. Half (51%) of the participants were overweight/obese at time of survey completion vs. 15% at time of diagnosis, with a median BMI at time of survey of 26.2 (adults; range: 19.2–47.4), and a median BMI percentile for children of 78.3 (range: 17.6–99.7). The association of overweight/obesity at time of survey with overweight/obesity at diagnosis (p = 0.05) and with receipt of cranial radiation (p = 0.06) were close to statistically significant.\n\nMean physical (51.5±9.0) and psychosocial (50.4±10.8) QOL scores were consistent with population normative data, suggesting that survivors are functioning well overall (Table 2). Compared to underweight/healthy weight status, obesity/overweight status was associated with significantly poorer psychosocial QOL (47.1±13.2 vs. 54.0±6.0, P=0.01, effect size Cohen’s d=0.67) and this remained statistically significant after adjusting for age and sex (P=0.03). Overweight/obesity at time of survey was associated with poorer physical QOL (compared to underweight/healthy weight status; 49.9±8.6 vs. 53.2±9.3, P=0.17, effect size Cohen’s d=0.37), however this difference was not statistically significant.\n\n* adjusted for age at time of survey and gender.\n\n\nDiscussion\n\nWhile obesity has been recognized as a late effect of pediatric ALL therapy, the extent to which this affects long-term survivors QOL remains unknown. In our population, half of our study participants were overweight/obese at time of survey, compared with 15% at time of diagnosis. QOL scores were similar to general population means across all weight groups, as was the prevalence of overweight/obesity in this population20. Overweight/obesity were associated with poorer physical QOL (unadjusted), and, when adjusted for age and sex, with poorer psychosocial QOL. Estimated effect sizes were small for psychosocial QOL and medium for psychological QOL21.\n\nThe large proportion of long-term ALL survivors who are overweight/obese, and the long-term increase in overweight/obesity after finishing treatment for ALL highlight a need for weight management, nutrition, and exercise interventions in survivors of childhood ALL22. Our findings suggest that such interventions may have co-benefits for survivors’ long-term QOL. Furthermore, participants with overweight/obesity were on average longer out from diagnosis, which suggests that time might be a factor in weight gain after ALL, but we could not test this given the cross-sectional nature of our study. Previous studies have examined the effectiveness of patient weight management interventions in primarily adult cancer populations; such interventions and their long-term effectiveness have been less studied for survivors of childhood cancers23–25. Further research is necessary to evaluate the long-term feasibility, sustainability, and timing of such interventions among childhood cancer survivors, as these may have significant implications for survivors’ long-term QOL.\n\nThis study is consistent with other studies that found associations of obesity with poorer QOL but expands the data to include overweight survivors as well. However, this study has some limitations. This is a cross-sectional analysis and we could not determine the directionality of the associations we measured. Our sample size was small due to the single-site nature of the study, and participants were almost exclusively white, which may not be generalizable to the broader pediatric ALL survivor population. Underweight and healthy weight status were aggregated into one group due to small sample size, but given the understudied nature of underweight status and QOL in survivors of pediatric cancer, future studies may benefit for examining this category separately from healthy weight status.\n\nThese data demonstrate that pediatric ALL treatment carries with it a substantial burden for weight management issues well after therapy is completed, with possible implications for QOL. Weight management interventions are needed to increase awareness and to reduce weight gain in the years following treatment for childhood ALL. In addition to such interventions, future research should address the interplay between treatment for pediatric ALL and other factors, for example diet, physical activity, and sleep, and their combined contribution to unhealthy weight gain during and after ALL.\n\n\nData availability\n\nData for these analyses came from the University of Minnesota Fairview pediatric cancer survivor database. The data for this study were collected at the University of Minnesota; but we cannot share these data due to HIPAA reasons: for privacy reasons the consent form of the project specified that we will not share the data. Access to these data can only be given in the form of deidentified data, or through having an individual who is on the protocol run analyses on the data. For questions, Char Napurski is overseeing the database (bake0257@umn.edu).", "appendix": "References\n\nHunger SP, Lu X, Devidas M, et al.: Improved survival for children and adolescents with acute lymphoblastic leukemia between 1990 and 2005: a report from the children's oncology group. J Clin Oncol. 2012; 30(14): 1663–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrignardello E, Felicetti F, Castiglione A, et al.: Endocrine health conditions in adult survivors of childhood cancer: the need for specialized adult-focused follow-up clinics. Eur J Endocrinol. 2013; 168(3): 465–472. PubMed Abstract \| Publisher Full Text\n\nBrewster DH, Clark D, Hopkins L, et al.: Subsequent mortality experience in five-year survivors of childhood, adolescent and young adult cancer in Scotland: a population based, retrospective cohort study. Eur J Cancer. 2013; 49(15): 3274–3283. PubMed Abstract \| Publisher Full Text\n\nKero AE, Järvelä LS, Arola M, et al.: Late mortality among 5-year survivors of early onset cancer: a population-based register study. Int J Cancer. 2015; 136(7): 1655–1664. PubMed Abstract \| Publisher Full Text\n\nLipshultz SE, Cochran TR, Franco VI, et al.: Treatment-related cardiotoxicity in survivors of childhood cancer. Nat Rev Clin Oncol. 2013; 10(12): 697–710. PubMed Abstract \| Publisher Full Text\n\nLega IC, Pole JD, Austin PC, et al.: Diabetes Risk in Childhood Cancer Survivors: A Population-Based Study. Can J Diabetes. 2018; 42(5): 533–539. PubMed Abstract \| Publisher Full Text\n\nOgden CL, Carroll MD, Kit BK, et al.: Prevalence of childhood and adult obesity in the United States, 2011-2012. JAMA. 2014; 311(8): 806–814. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsner S, Ammann R, Ozsahin H, et al.: Obesity in long-term survivors of childhood acute lymphoblastic leukemia. Pediatr Blood Cancer. 2008; 51(1): 118–122. PubMed Abstract \| Publisher Full Text\n\nMeacham LR, Gurney JG, Mertens AC, et al.: Body mass index in long-term adult survivors of childhood cancer: a report of the Childhood Cancer Survivor Study. Cancer. 2005; 103(8): 1730–1739. PubMed Abstract \| Publisher Full Text\n\nZhang FF, Rodday AM, Kelly MJ, et al.: Predictors of being overweight or obese in survivors of pediatric acute lymphoblastic leukemia (ALL). Pediatric blood cancer. 2014; 61(7): 1263–1269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButtitta M, Iliescu C, Rousseau A, et al.: Quality of life in overweight and obese children and adolescents: a literature review. Qual Life Res. 2014; 23(4): 1117–1139. PubMed Abstract \| Publisher Full Text\n\nKolodziejczyk JK, Gutzmer K, Wright SM, et al.: Influence of specific individual and environmental variables on the relationship between body mass index and health-related quality of life in overweight and obese adolescents. Qual Life Res. 2015; 24(1): 251–261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanellopoulos A, Hamre HM, Dahl AA, et al.: Factors associated with poor quality of life in survivors of childhood acute lymphoblastic leukemia and lymphoma. Pediatr Blood Cancer. 2013; 60(5): 849–855. PubMed Abstract \| Publisher Full Text\n\nNayiager T, Anderson L, Cranston A, et al.: Health-related quality of life in long-term survivors of acute lymphoblastic leukemia in childhood and adolescence. Qual Life Res. 2017; 26(5): 1371–1377. PubMed Abstract \| Publisher Full Text\n\nSadak KT, Bahr TL, Moen C, et al.: The clinical and research infrastructure of a childhood cancer survivor program. J Cancer Educ. 2015; 30(3): 471–476. PubMed Abstract \| Publisher Full Text\n\nDrotar D, Schwartz L, Palermo TM, et al.: Factor structure of the child health questionnaire-parent form in pediatric populations. J Pediatr Psychol. 2006; 31(2): 127–138. PubMed Abstract \| Publisher Full Text\n\nMosconi P, Apolone G, Barni S, et al.: Quality of life in breast and colon cancer long-term survivors: an assessment with the EORTC QLQ-C30 and SF-36 questionnaires. Tumori. 2002; 88(2): 110–116. PubMed Abstract \| Publisher Full Text\n\nReulen RC, Zeegers MP, Jenkinson C, et al.: The use of the SF-36 questionnaire in adult survivors of childhood cancer: evaluation of data quality, score reliability, and scaling assumptions. Health Qual Life Outcomes. 2006; 4: 77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManocchia M: SF-36 Health Survey: Annotated Bibliography;(1988-1996). Health Assessment Lab. 1998.\n\nFlegal KM, Carroll MD, Ogden CL, et al.: Prevalence and trends in obesity among US adults, 1999-2008. JAMA. 2010; 303(3): 235–241. PubMed Abstract \| Publisher Full Text\n\nCohen J: Statistical power analysis for the behavioral sciences. Academic press; 2013. Reference Source\n\nZhang FF, Parsons SK: Obesity in childhood cancer survivors: call for early weight management. Adv Nutr. 2015; 6(5): 611–619. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMishra SI, Scherer RW, Snyder C, et al.: Exercise interventions on health-related quality of life for people with cancer during active treatment. Cochrane Database Syst Rev. 2012; 2012(8): CD008465. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerondi MB, Gualano B, Artioli GG, et al.: Effects of a combined aerobic and strength training program in youth patients with acute lymphoblastic leukemia. J Sports Sci Med. 2012; 11(3): 387–92. PubMed Abstract \| Free Full Text\n\nWong J, Fetters L: Effects of exercise intervention for children with acute lymphoblastic leukemia: a systematic review. Rehabilitation Oncology. 2014; 32(3): 40–51. Publisher Full Text" }	[ { "id": "85986", "date": "01 Jul 2021", "name": "Annalisa Paviglianiti", "expertise": [ "Reviewer Expertise Hematopoietic stem cell transplantation" ], "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 association of BMI and psycological QOL among children survivors was analyzed.\nCompared to underweight/healthy weight status, being overweight/obese was not significantly associated with poorer physical QOL, but with poorer psychosocial QOL (47.1±13.2 vs. 54.0±6.0, P=0.01, effect size Cohen’s d=0.67), which remained statistically significant after adjusting for age and sex.\nA criticism is that the number of patients is limited. A larger cohort would better help with the statistical power.\n\nHow could it be that half of the patients are obese or overweight in such a little population?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "97420", "date": "02 Nov 2021", "name": "Angela Maria Spinola-Castro", "expertise": [ "Reviewer Expertise Endocrine disorders in chronic diseases", "cancer treatment late effects and growth and development" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is about the relationship between body mass index and quality of life among long term survivors of childhood acute lymphoblastic leukemia. The authors have hypothesized that being obese would be associated with poor QOL using validated measures. They selected a population treated for leukemia during childhood. In the introduction they stated that previous studies that evaluated BMI and QOL were too mixed, but they did not explain how it is possible to identify the efffects on QOL of being a cancer survivour from the effects of being obese.\nIn study design we need more information about the group evaluated, age, sex, time after treatment, and protocol, describing when radiation was done, and how many grades were applied. Some of these data were presented in the results. However the authors did not consider important to evaluate height. One can have a high BMI due to weight excess or because of a short stature. If 40 % of the patients were treated with radiation therapy, it is important to know if they have at first place, growth hormone deficiency and if any of those patients had to be treated. It is also helpful for a better understanding of the results to make clear how to interpretate QOL tests results.\nIn the results information about those patients that were not eligible to participate are needed. It's also necessary to explain how many patients irradiated were in the obese group. 15% of patients were obese at time of diagnosis, but we were not informed if QOL was more compromised in this group comparing to those obese in adult life. In fact an appropriate comparison between groups obese and non obese, including characteristics at diagnoses, time after treatment, and life style are also required. Obesity can be a late effect in LLA survivors, but visceral fat increment is usually more frequent than subcutaneous fat. Children usually have adiposity rebound at the end of treatament, that disappear after almost a year and depending on their growth rate. The discussion does not touch all those aspects, It also does not mention the tools to evaluate QOL and their limitations and does not consider the reasons to conclude that obesity is the main cause affecting life quality.\nThe references give a better idea to late effects experienced by patients treated for leukemia and also about obesity. I believe it could be useful to the authors expand some aspects of their work.\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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/10-178
https://f1000research.com/articles/10-176/v1	03 Mar 21	{ "type": "Systematic Review", "title": "Analysis of clinical efficacy of Si Miao decoction combine with acupuncture and cupping treatment for gout: a systematic review and meta-analysis", "authors": [ "Yun Jin Kim", "Muhammad Shahzad Aslam", "Yun Jin Kim" ], "abstract": "This systematic review has been aimed to evaluate the clinical efficacy of the Si Miao decoction combined with acupuncture and cupping treatment for Gout. Three English and Chinese databases were searched for articles related to the effect of the Si Miao decoction combine with acupuncture and cupping therapy on CRP, UA, and ESR in Gout. The time period was limited from 01 January 2010 till 31 August 2019. Meta- analysis was performed using both the random and fixed effects model, and I2 was used to evaluate the heterogeneity. Identification was made through database searching of 238 publications. Three articles were eligible. Following the Si Miao Decoction combined with acupuncture and cupping therapy there was a significant reduction in the clinical parameters (SMD: −0.91, 95% CI: −1.081, −0.741, p = 0.000) (OR: −1.652, 95% CI: −1.960, −1.344, p = 0.000). High heterogeneity tests were indicated (Q=146.548, P = 0.00, I2 = 94.54%). This systematic review and meta-analysis indicated that the Si Miao decoction combined with acupuncture and cupping therapy significantly reduced the circulating levels of UA, ESR, and CRP. There is a need to improve the methodologies of clinical trials on modified Si Miao decoction combined with Acupuncture and Cupping treatment.", "keywords": [ "Si Miao", "Decoction", "Acupuncture", "Cupping" ], "content": "Abbreviations\n\nStd= Standard; CRP= C-reactive protein; ESR= Erythrocyte sedimentation rate; SDM= standard difference in mean; Hg= hedge’s g; OR= odds ratio; SPDM= Standard paired difference; CORR= Correlation; CONSORT= Consolidated Standards of Reporting Trials; OSF= Center for Open Science; M-Si-M= Modified Si Miao; WM= Western medicine; WHO= World Health organization; M-Si-M+Ac+Cu= Ac=Si Miao Decoction combine with acupuncture and cupping therapy; Ac=Acupuncture; Cu= Cupping.\n\n\nIntroduction\n\nArthritis is defined as the swelling of a joint that is accompanied by a limitation of motion, and increase in heat, pain, or tenderness1. Gout is a systemic disease which is also known as podagra and is caused by the presence of crystals of monosodium urate inside the tissues. These crystals trigger the inflammatory response that results in painful swelling mainly over the big toe2. Gouty arthritis can cause significant disability amongst patients that results in the dysfunction of normal self-care and recreational, social, and work activities3.\n\nGout is considered as one of the most prevalent inflammatory diseases. The general prevalence of gout is 1–4% of the general population4. It is affects people in different parts of the world including the UK (2.5% of the total population)5, Korea (1.94% per 1000 persons)6, and New-Zealand (2.69% Aotearoa New Zealand Health Tracker population)7.\n\nSi Miao is the herbal combination of Cortex Phellodendri Chinensis, Rhizoma Atractylodis Lanceae, Radix Achyranthis Bidentatae, and Semen Coicis Lachryma-Jobi. It is also well known as the Four-Marvels Pill8. It is also available in a modified form with some additional herbs in actual formulation and has been used in the treatment of gout and arthritis. Acupuncture has been used to treat health conditions, including pain, for over 3,000 years, yet it has only been in the last half a century that biochemistry and neural imaging advances have allowed for the scientific understanding of its physiological mechanisms9. A number of studies have found the effectiveness of acupuncture in the treatment of gouty arthritis10,11. Lee, et al. have performed a systematic review on acupuncture for gouty arthritis and found it to be an effective complementary medicine12. Cupping therapy, commonly known as Al-hijamah in Islamic medicine, is also well-known amongst traditional practitioners in treating arthritis12–14.\n\nModified Si Miao San extract inhibits an inflammatory response and modulates insulin sensitivity in hepatocytes through an IKKβ/IRS-1/Akt-dependent pathway15. Acupuncture helps in reducing the pain and inflammation through an increase in IL-10 concentrations in the muscle16. Wet cupping helps to remove oxidants and reduce the oxidative stress through oxidative balance17. The imbalance of oxidative stress will result in an increase in the inflammatory response18.\n\n\nMethods\n\nA systematic literature search strategy was prepared according to the given guidelines by Cochrane infectious diseases19. Si Miao, Si Miao San, Si Miao Pian, Si Miao Wan, Gout and arthritis were the main keywords used during the search from different databases, such as PubMed, PubMed Central, and CNKI with the publication range being from 01 January 2010 till 31 August 2019. The literature works identified had been searched for in the English and Chinese languages. The list of keywords used in the English and Chinese versions have been mentioned in Table 1. The initial search syntax for PubMed and PubMed Central consisted of the following list.\n\n1. (Si[All Fields] AND Miao[All Fields]) AND (\"gout\"[MeSH Terms] OR \"gout\"[All Fields]) AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT]); Si Miao[Text Word] AND Gout[Text Word] AND (\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate])\n\n2. (Si[All Fields] AND Miao[All Fields]) AND (\"arthritis\"[MeSH Terms] OR \"arthritis\"[All Fields]) AND (\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate]); Si Miao[Text Word] AND arthritis[Text Word] AND (\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate])\n\n3. (Si[All Fields] AND Miao[All Fields] AND San[All Fields]) AND (\"gout\"[MeSH Terms] OR \"gout\"[All Fields]) AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT]); Si Miao San[Text Word] AND Gout[Text Word] AND (\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate])\n\n4. (Si[All Fields] AND Miao[All Fields] AND San[All Fields]) AND (\"arthritis\"[MeSH Terms] OR \"arthritis\"[All Fields]) AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT]); Si Miao San[Text Word] AND arthritis[Text Word] AND (\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate])\n\n5. (Si[All Fields] AND Miao[All Fields] AND Pian[All Fields]) AND (\"gout\"[MeSH Terms] OR \"gout\"[All Fields]) AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT]); (Si Miao Pian[Text Word]) AND gout[Text Word] AND ((\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate]))\n\n6. (Si[All Fields] AND Miao[All Fields] AND Pian[All Fields]) AND (\"arthritis\"[MeSH Terms] OR \"arthritis\"[All Fields]) AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT]); (Si Miao Pian[Text Word]) AND arthritis[Text Word] AND ((\"2010/01/01\"[PubDate] : \"2019/08/31\"[PubDate]))\n\n7. (Si[All Fields] AND Miao[All Fields] AND Wan[All Fields]) AND gout[All Fields] AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT])\n\n8. (Si[All Fields] AND Miao[All Fields] AND Wan[All Fields]) AND arthritis[All Fields] AND (\"2010/01/01\"[PDAT] : \"2019/08/31\"[PDAT])\n\nA total number of 238 (n=238) articles were identified from different databases (PubMed, PubMed Central, and CNKI). The records were screened in two stages on the basis of number of duplications followed by some initial criteria where irrelevant articles, review articles, and animal and chemical studies had been excluded. After the initial screening, 132 (n=132) articles were identified. Microsoft Excel 201620 and Mendeley version 1.19.221 were used for data deduplication and citation management.\n\nThe included studies were based upon the use of the modified Si Miao (M-Si-M) combined with acupuncture and cupping for the treatment of gout and arthritis. The selected studies included both the Chinese and English languages but most of the studies available were in the Chinese language. The articles that focussed on the Si Miao formulation, modified Si Miao (M-Si-M) or combination of modified Si Miao (M-Si-M) with western medicine were excluded. After the application of the eligibility criteria, the number of articles had been reduced to six (n=6) articles and 126 articles (n=126) had been excluded. One article (n=1) had been removed further for qualitative synthesis and data extraction because of the absence of a randomised clinical trial (RCT). Clinical effectiveness, uric acid (UA), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were accessed as the main outcome whereas the visual analogue Scale (VAS) and symptom scale had been excluded. Two articles (n=2) had been excluded because of unavailable outcome indicators which restrict the use to only three studies (n=3) for the meta-analysis. The flow chart of the study’s selection process according to the PRISMA flow diagram has been mentioned in Figure 1.\n\nPRISMA flow chart of the selection process of Si Miao decoction combine with acupuncture and cupping therapy.\n\nThe Cochrane Handbook for Systematic Reviews of Interventions22 and JBI Systematic Reviews were used as reporting channels to assess the quality of the manuscript23. Two researchers independently evaluated the quality of the included studies, and a third party was consulted for resolution of any disagreement. The study design, participants, intervention, and outcome (SPIO) criteria have been mentioned in Table 2. The authors evaluated the methodological assessment under: aim/hypothesis clearly defined, adequate sample representation, patient care quality assurance, ethical approval protocol number, outcome clearly described, validity and reliability of outcome measure, attempt to blind researcher, follow-up, appropriate statistical analysis, and missing data reported (Figure 2). The bias assessment of the studies included in the qualitative synthesis included random sequence, allocation concealment, selective reporting, blinding of participants and personnel, blinding of outcome assessment, and incomplete outcome data according to the Cochrane guidelines (Table 3)22. The study characteristics of the selected studies were assessed according to the study design, treatment, control, statistical tool used, software to assess the outcome, and final outcome of the findings (Table 4). Uric acid, erythrocyte sedimentation rate, and C-reactive protein were the clinical parameters used after the treatment and further evaluation for the meta-analysis (Table 5). Microsoft Excel 201620 (Microsoft Corporation, Redmond, WA) was used to extract the data, removing study duplications, and Mendeley desktop 1.19.221 was used for importing the references.\n\n\n\nData expressed Mean±SD\n\nComprehensive Meta-Analysis (CMA) V2 software (Biostat, NJ) was used for data synthesis. Heterogeneity was quantitatively assessed using the I2 index. An I2 greater than 50% indicated significant heterogeneity amongst the studies. The results were expressed as mixed methods using both random and fixed effects. Effect sizes were expressed as odds ratio (OR), standard mean difference (SMD), Hedge’s g (HG), Standard paired difference (SPD), Correlation (C), and 95% confidence interval (CI). A Z score was calculated to determine the overall effect. Funnel plot analysis was used to evaluate the presence of publication bias. The extent of variation amongst the effects observed in different studies (between-study variance) is referred to as tau-squared, τ2, or Tau229.\n\nMeta-analysis was conducted using the trial version of Comprehensive Meta-analysis (CMA) version 2.0 software30. A forest plot displaying the standard difference in mean, Hedge’s g, odds ratio, Standard paired difference, and Correlation 95% CIs for the impact of the observed therapy versus the control on different parameters were observed. True heterogeneity amongst the trials was assessed using the tau, T231, and the extent of the inconsistency was measured by the I2 index32.\n\n\nResults\n\nAll the included studies employed clinical effect as the outcome assessment. The clinical parameters included CRP, ESR, and uric acid concentration after treatment with the modified Si Miao decoction combined with acupuncture and cupping therapy. The cumulative effect was observed by the meta-analysis of three randomised clinical treatment arms that suggested a significant reduction in the clinical parameters using the modified Si Miao decoction combined with acupuncture and cupping therapy (SMD: −0.91,95% CI: −1.081, −0.741, p = 0.000) (OR: −1.652, 95% CI: −1.960, −1.344, p = 0.000). The forest plot, funnel plot of Cumulative std difference in means, and Log odds ratio of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control have been expressed in the Extended data (Figures 3 and Figure 4), respectively. A high heterogeneity test was indicated (Q=146.548, P = 0.00, I2 = 94.54%) (Extended data: Figure 5). The estimated between-study variances of the cumulative Log odds ratio of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control were tau= 1.974, Tau-squared = 3.897 with a standard error= 2.129, and variance= 4.533 (Extended data: Figure 5). The estimated between-study variances of the cumulative standard difference in means of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control were tau= 1.088, Tau-squared = 1.184 with a standard error= 0.647, and variance= 0.419 (Extended data: Figure 6).\n\nThree studies measured blood uric acid as the outcome; a comparison between the observed versus the Control post-treatment was calculated. Data extracted from the three individual studies showed that heterogeneity existed (Q=34.154, P = 0.000, I2 = 94.144%; Extended data: Figure 7). The fixed and random effects model was utilised for the statistical analysis. The estimated between-study variances of the std difference in the mean of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control were tau= 1.042, Tau-squared = 1.085 with a standard error= 1.163, and variance= 1.353 (Extended data: Figure 7). The data suggested that the Si Miao decoction combined with acupuncture and cupping therapy could further decrease uric acid as compared to the control (SDM -0.960; 95% CI -1.240, -0.660; P < 0.000) (Hg -0.940; 95% CI -1.227, -0.652; P < 0.000) (OR 0.179; 95% CI 0.105, 0.302; P < 0.000) (SPDM -0.950; 95% CI -1.240, -0.660; P < 0.000) (CORR -0.513; 95% CI -0.597, -0.417; P < 0.000) (Extended data: Figure 8). The funnel plot displaying the (A) standard difference in mean, (B) Hedge’s g, (C) odds ratio, (D) Standard paired difference, and (E) Correlation 95% CIs for the impact of the observed therapy versus the Control on the uric acid concentrations have been represented in Extended data (Figure 9).\n\nThree studies measured blood Erythrocyte Sedimentation Rate (ESR) as the outcome; a comparison between the observed versus the Control post-treatment was calculated. The data extracted from the three individual studies showed that heterogeneity existed (Q=12.092, P = 0.002, I2 = 83.460%; Extended data: Figure 7). Fixed and random effects models were utilised for the statistical analysis. The estimated between-study variances of the std difference in the mean of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control were tau= 0.556, Tau-squared = 0.310 with a standard error= 0.372, and variance= 0.139 (Extended data: Figure 7). The data suggested that the Si Miao decoction combined with acupuncture and cupping therapy could further decrease the ESR as compared to the Control (SDM -0.808; 95% CI -1.087, 0.530; P < 0.000) (Hg -0800; 95% CI -1.076, -0.524; P < 0.000) (OR 0.231; 95% CI 0.139, 0.383; P < 0.000) (SPDM -0.808; 95% CI -1.087, 0.530; P < 0.000) (CORR -0.400; 95% CI -0.500, -0.289; P < 0.000) (Extended data: Figure 10). The funnel plot displaying the (A) standard difference in mean, (B) Hedge’s g, (C) odds ratio, (D) Standard paired difference, and (E) Correlation 95% CIs for the impact of the observed therapy versus the Control on the uric acid concentrations have been represented in the Extended data (Figure 11).\n\nThree studies measured blood C-Reactive Protein (CRP) as the outcome; a comparison between the observed versus the Control post-treatment was calculated. The data extracted from the three individual studies showed that heterogeneity existed (Q=99.391, P = 0.000, I2 = 97.988%; Extended data: Figure 7). Fixed and random effects models were utilised for the statistical analysis. The estimated between-study variances of the std difference in the mean of the Si Miao decoction combined with acupuncture and cupping therapy versus the Control were tau= 2.056, Tau-squared = 4.228 with a standard error= 4.483, and variance= 20.099 (Extended data: Figure 7). The data suggested that the Si Miao decoction combined with acupuncture and cupping therapy could further decrease the CRP as compared to the Control (SDM -3.892; 95% CI -4.637, -3.146; P < 0.000) (Hg -0.940; 95% CI -1.227, -0.652; P < 0.000) (OR 0.179; 95% CI 0.105, 0.302; P < 0.000) (SPDM -0.950; 95% CI -1.240, -0.660; P < 0.000) (CORR -0.513; 95% CI -0.597, -0.417; P < 0.000) (Extended data: Figure 12). The funnel plot displaying the (A) standard difference in mean, (B) Hedge’s g, (C) odds ratio, (D) Standard paired difference, and (E) Correlation 95% CIs for the impact of the observed therapy versus the Control on theuric acid concentrations have been represented in Extended data (Figure 13).\n\n\nDiscussion\n\nFive studies were included for methodological assessment and inconsistencies were found in the methodology. Yu, et al. (2013)28 was the only study in compliance with the key indicators for the methodological assessment followed by Deng, et al. (2017)27. The Cochrane bias assessment also validated the results produced from the methodological assessment with most of the indicators being unclear and reporting of high risk of bias in random sequencing generation and blinding of the outcome assessment in Deng, et al. (2017)27. Three (n=3) studies were considered for meta-analysis because the other two (n=2) studies were unable to comply with the eligibility criteria. This research has focused on comparing the randomised clinical trial of studies consisting of the combination of the Modified Si Miao (M-Si-M) with acupuncture (Ac) and cupping (Cu) treatment (M-Si-M+Ac+Cu) against that of western medicine. The clinical parameters of uric acid (UA)33, Erythrocyte sedimentation rate (ESR), and C-Reactive protein (CRP) are important indicators in the treatment of arthritis and gout34. The combination of the Modified Si Miao (M-Si-M) with acupuncture (Ac) and cupping (Cu) treatment was found to be better as compared with the western medicine (WM) but high heterogeneity was observed amongst all of the clinical parameters. High heterogeneity was observed because of methodological issues, such as problems with randomisation, early termination of trials, use of absolute rather than relative measures of risk, and publication bias35. An improvement in the symptoms was observed when compared between the two treatments without any adverse effects.\n\nThis review is the first systematic review comparing the modified Si Miao decoction combined with acupuncture and cupping therapy (M-Si-M+Ac+Cu) against western medicine. There were a few limitations of this study, such as small sample size, lack of available research, and that most of the articles were published in China. Language was another barrier to report in this research as most the articles had been published in the Chinese language. There was also a high uncertainty in the methodology that led to publication bias. This evidence had been supported and verified in another systematic review on cupping therapy versus acupuncture for pain‑related conditions36. Further research on the same formulation is recommended with an improved clinical protocol using Consolidated Standards of Reporting Trials (CONSORT)37. It is also recommended to register the protocol with ClinicalTrials.gov38, Trial Registration by WHO39 or ISRCTN registry40. To increase the transparency and openness in the research, Science Europe40 launched plan S40 on 4 September 2018 that acknowledged the implementation of open archives and repositories to all journals. Therefore, it is recommended to increase the openness, integrity, and reproducibility of scientific research using the Centre for Open Science (OSF)41.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: Analysis of Clinical efficacy of Si Miao decoction combine with Acupuncture and Cupping treatment for Gout: A systematic review and meta-analysis, https://doi.org/10.17605/OSF.IO/PUCX642.\n\nThis project contains the following extended data:\n\n- Figure 3: (A) Forest plot of Si Miao decoction combine with acupuncture and cupping therapy versus Control: cumulative std difference in means (B) Funnel plot of Si Miao decoction combine with acupuncture and cupping therapy versus Control: cumulative std difference in means\n\n- Figure 4: (A) Forest plot of Si Miao Decoction combine with acupuncture and cupping therapy versus Control: Log odds ratio (B) Funnel plot of Si Miao Decoction combine with acupuncture and cupping therapy versus Control: Log odds ratio\n\n- Figure 5: Heterogeneity and tau squared cumulative Log odds ratio of Si Miao Decoction combine with acupuncture and cupping therapy versus Control\n\n- Figure 6: Heterogeneity and tau squared cumulative std difference in means of Si Miao Decoction combine with acupuncture and cupping therapy versus Control\n\n- Figure 7: Heterogeneity and tau squared std difference in means of Si Miao Decoction combine with acupuncture and cupping therapy versus Control on outcomes parameters (A) ESR (B) CRP (C) Uric Acid\n\n- Figure 8: Forest plot displaying (A) standard difference in mean for the impact of observed therapy versus control on uric acid concentrations. (B) Hedge’s g for the impact of observed therapy versus control on uric acid concentrations. (C) Odds ratio for the impact of observed therapy versus control on uric acid concentrations. (D) Standard paired difference for the impact of observed therapy versus control on uric acid concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on uric acid concentrations.\n\n- Figure 9: Funnel plot displaying (A) Standard difference in mean for the impact of observed therapy versus control on uric acid concentrations. (B) Hedge’s g for the impact of observed therapy versus control on uric acid concentrations. (C) Odds ratio for the impact of observed therapy versus control on uric acid concentrations. (D) Standard paired difference for the impact of observed therapy versus control on uric acid concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on uric acid concentrations.\n\n- Figure 10: Forest plot displaying (A) Standard difference in mean for the impact of observed therapy versus control on ESR concentrations. (B) Hedges’ g for the impact of observed therapy versus control on ESR concentrations. (C) Odds ratio for the impact of observed therapy versus control on ESR concentrations. (D) Standard paired difference for the impact of observed therapy versus control on ESR concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on ESR concentrations.\n\n- Figure 11: Funnel plot displaying (A) Standard difference in mean for the impact of observed therapy versus control on ESR concentrations. (B) Hedge’s g for the impact of observed therapy versus control on ESR concentrations. (C) Odds ratio for the impact of observed therapy versus control on ESR concentrations. (D) Standard paired difference for the impact of observed therapy versus control on ESR concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on ESR concentrations.\n\n- Figure 12: Forest plot displaying (A) Standard difference in mean for the impact of observed therapy versus control on CRP concentrations. (B) Hedge’s g for the impact of observed therapy versus control on CRP concentrations. (C) Odds ratio for the impact of observed therapy versus control on CRP concentrations. (D) Standard paired difference for the impact of observed therapy versus control on CRP concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on CRP concentrations.\n\n- Figure 13: Funnel plot displaying (A) Standard difference in mean for the impact of observed therapy versus control on CRP concentrations. (B) Hedge’s g for the impact of observed therapy versus control on CRP concentrations. (C) Odds ratio for the impact of observed therapy versus control on CRP concentrations. (D) Standard paired difference for the impact of observed therapy versus control on CRP concentrations. (E) Correlation 95 % CIs for the impact of observed therapy versus control on CRP concentrations.\n\nOpen Science Framework: PRISMA checklist for ‘Analysis of clinical efficacy of Si Miao decoction combine with acupuncture and cupping treatment for gout: a systematic review and meta-analysis’, https://doi.org/10.17605/OSF.IO/PUCX642.\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\nSpecial thanks to Aishah Rose Marie for her perseverance in proofreading the manuscript.\n\n\nReferences\n\nDifferential Diagnosis. In: Pediatric Clinical Advisor. Elsevier; 2007; 613–32. Publisher Full Text \| Free Full Text\n\nSo AK, Martinon F: Inflammation in gout: mechanisms and therapeutic targets. Nat Rev Rheumatol. 2017; 13(11): 639–47. PubMed Abstract \| Publisher Full Text\n\nten Klooster PM, Vonkeman HE, van de Laar MAFJ: Disability due to gouty arthritis. Curr Opin Rheumatol. 2012; 24(2): 139–44. PubMed Abstract \| Publisher Full Text\n\nKuo CF, Grainge MJ, Zhang W, et al.: Global epidemiology of gout: prevalence, incidence and risk factors. Nat Rev Rheumatol. 2015; 11(11): 649–62. PubMed Abstract \| Publisher Full Text\n\nAbhishek A, Roddy E, Doherty M: Gout – a guide for the general and acute physicians. Clin Med (Lond). 2017; 17(1): 54–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim JW, Kwak SG, Lee H, et al.: Prevalence and incidence of gout in Korea: data from the national health claims database 2007–2015. Rheumatol Int. 2017; 37(9): 1499–506. PubMed Abstract \| Publisher Full Text\n\nWinnard D, Wright C, Taylor WJ, et al.: National prevalence of gout derived from administrative health data in Aotearoa New Zealand. Rheumatology (Oxford). 2012; 51(5): 901–9. PubMed Abstract \| Publisher Full Text\n\nLi Y, Wei W: Chief physician Zhang Tang-fa’s experience of acupuncture for gouty arthritis. J Acupunct Tuina Sci. 2018; 16(3): 145–9. Publisher Full Text\n\nVickers AJ, Linde K: Acupuncture for Chronic Pain. JAMA. 2014; 311(9): 955. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou R, Zhang HX, Zhang TF, et al.: Treatment to acute gouty arthritis with electro-acupuncture at different frequencies versus medication. Chinese J Clin Rehabil. 2006; 10: 188–9. Reference Source\n\nZhou L, Xu QF, Zhang WS: [Comparative observation of the efficacy on acute gouty arthritis between acupuncture combined with infrared irradiation and western medicine]. Zhongguo Zhen Jiu. 2011; 31(9): 787–9. PubMed Abstract\n\nLee WB, Woo SH, Min BI, et al.: Acupuncture for gouty arthritis: a concise report of a systematic and meta-analysis approach. Rheumatology (Oxford). 2013; 52(7): 1225–32. PubMed Abstract \| Publisher Full Text\n\nKabir Dar A, Hussain Lone A: Therapetic application of Al-Hijamah (Cupping therapy) in osteoarthrtis of the Knee. Int J reserch Dev Pharm life Sci. 2015; 4(3): 1540–4. Reference Source\n\nAkhtar H, Rashid M, Siddiqi M, et al.: Role of Hijama’ (Cupping Therapy) in the Management of Niqras (Gouty Arthritis). J Arthritis. 2017; 06(06). Publisher Full Text\n\nLiu K, Luo T, Zhang Z, et al.: Modified Si-Miao-San extract inhibits inflammatory response and modulates insulin sensitivity in hepatocytes through an IKKβ/IRS-1/Akt-dependent pathway. J Ethnopharmacol. 2011; 136(3): 473–9. PubMed Abstract \| Publisher Full Text\n\nda Silva MD, Bobinski F, Sato KL, et al.: IL-10 Cytokine Released from M2 Macrophages Is Crucial for Analgesic and Anti-inflammatory Effects of Acupuncture in a Model of Inflammatory Muscle Pain. Mol Neurobiol. 2015; 51(1): 19–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTagil SM, Celik HT, Ciftci S, et al.: Wet-cupping removes oxidants and decreases oxidative stress. Complement Ther Med. 2014; 22(6): 1032–6. PubMed Abstract \| Publisher Full Text\n\nChatterjee S: Chapter Two - Oxidative Stress, Inflammation, and Disease. In: Oxidative Stress and Biomaterials. Elsevier; 2016; 35–58. Publisher Full Text\n\nLutje V: Guide to the search strategy. 2013. Reference Source\n\nMicrosoft Office 2016. Microsoft Corporation, Redmond, WA; 2015. Reference Source\n\nMendeley. Mendeley Desktop Reference Software, version 1.19.2. Reference Source\n\nHiggins JP, Green S: Cochrane Handbook for Systematic Reviews of Interventions: Cochrane Book Series. Cochrane Handbook for Systematic Reviews of Interventions: Cochrane Book Series. 2008. Publisher Full Text\n\nThe Joanna Briggs Institute: Checklist for Systematic Reviews and Research Syntheses. The Joanna Briggs Institute. 2016.\n\nJianshe Z, Ruihua C, Ying D, et al.: Navel Needling Combined with Modified Si Miao Powder in Treatment of Gout with Damp-heat Syndrome. Acta Chinese Med. 2019; 34(251).\n\nShijian J, Xiaoying X, Lingmei Y: Clinical Observation of Applying Blood－letting Therapy Combined with Jiawei Simiao Decoction in the Treatment of Acute Aout Arthritis. J Sichuan Tradit Chinese Med. 2016; 34(1): 75.\n\nChen ZD: Jiawei Si Miao San oral administration and cold foot liquid external washing treatment of damp heat accumulation type of acute gouty arthritis observation. J new Chinese Med. 2015; 47(8): 125.\n\nDeng P, Bin Z: Pricking and cupping bloodletting combined with Si Miao San for acute gouty arthritis. CJGMCM. 2017; 32(23): 3445.\n\nXiaozhong Y, Dinggang H, Xiaozhu W: Treatment of 32 Cases of Acute Gouty Arthritis with Modified Si Miao Powder and Auricular Point Pressing. J Gansu Coll Tcm. 2013; 30(3): 69.\n\nDeeks JJ, Higgins JP, Altman DG: Analysing Data and Undertaking Meta-Analyses. Cochrane Handbook for Systematic Reviews of Interventions: Cochrane Book Series. 2008. Publisher Full Text\n\nBorenstein M, Hedges L, Higgins J, et al.: Comprehensive Meta-Analysis Software (CMA). Comprehensive Meta-Analysis Version 2. 2005. p. Engelwood, NJ, Biostat. 2005. Reference Source\n\nJoanna Briggs Institute: Tau-squared for random effects model meta-analysis. 2019; [cited 2019 Sep 19].\n\nJulian PT Higgins and Sally Green: Identifying and measuring heterogeneity. Cochrane Handbook for Systematic Reviews of Interventions, Version 5.1.0; 2011; [cited 2019 Sep 19]. Reference Source\n\nSpiga R, Marini MA, Mancuso E, et al.: Uric Acid Is Associated With Inflammatory Biomarkers and Induces Inflammation Via Activating the NF-κB Signaling Pathway in HepG2 Cells. Arterioscler Thromb Vasc Biol. 2017; 37(6): 1241–9. PubMed Abstract \| Publisher Full Text\n\nHaasnoot AJW, van Tent-Hoeve M, Wulffraat NM, et al.: Erythrocyte Sedimentation Rate as Baseline Predictor for the Development of Uveitis in Children With Juvenile Idiopathic Arthritis. Am J Ophthalmol. 2015; 159(2): 372–377.e1. PubMed Abstract \| Publisher Full Text\n\nFletcher J: What is heterogeneity and is it important? BMJ. 2007; 334(7584): 94–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang YJ, Cao HJ, Li XL, et al.: Cupping therapy versus acupuncture for pain-related conditions: A systematic review of randomized controlled trials and trial sequential analysis. Chin Med. 2017; 12(1): 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Schulz KF, Altman DG: The CONSORT Statement: Revised Recommendations for Improving the Quality of Reports of Parallel-Group Randomized Trials. Ann Intern Med. 2001; 134(8): 657. PubMed Abstract \| Publisher Full Text\n\nClinicalTrials.gov: National Library of Medicine. [cited 2019 Sep 19]. Reference Source\n\nWHO Trial Registration. World health organization. World Health Organization; 2018 [cited 2019 Sep 19]. Reference Source\n\nISRCTN Registry. [cited 2019 Sep 19]. Reference Source\n\nNosek BA: Strategic Plan. Center for Open Science. 2017; [cited 2019 Sep 19].\n\nAslam MS, kim YJ: Analysis of Clinical efficacy of Si Miao decoction combine with Acupuncture and Cupping treatment for Gout: A systematic review and meta-analysis. 2021. http://www.doi.org/10.17605/OSF.IO/PUCX6" }	[ { "id": "82473", "date": "16 Apr 2021", "name": "Jun Yong Choi", "expertise": [ "Reviewer Expertise Traditional Medicine (eastern asian)." ], "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 systematic review&meta-analysis of the effect of modified Si Miao decoction combined with acupuncture and cupping therapy compared to western medicine on gout. There are several points to be clear for improving this manuscript.\nPlease add a table for the information of every herb contained in the modified Si Miao decoction in each study. Also, acupuncture points used and specific names of western medicines in the control group in every analysed study should be contained in this table.\n\nSafety must be considered when assessing the efficacy of every medical intervention. So, add the information of safety of included studies.\n\nFrom the result of this study, modified Si Miao decoction plus acupuncture and cupping therapy showed significant effects of lowering blood uric acid level as well as ESR and CRP over western medicine. Please provide suggestive mechanisms of these TCM interventions on lowering these biomarkers in the discussion section with relevant references.\n\nAlso, there should be a note of caution in the manuscript about the use of these TCM interventions against the use of standard therapy of gout before firm clinical evidence of TCM is established.\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": [] }, { "id": "123022", "date": "22 Feb 2022", "name": "Yen-Ying Kung", "expertise": [ "Reviewer Expertise traditional Chindese medicine and acupuncture" ], "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 used meta-analysis to evaluate the efficacy of Si Miao decoction combined with acupuncture and cupping treatment for gout in this manuscript. It is interesting but it still has some issues to be addressed as follows:\nFrom the result, Si Miao decoction combined with acupuncture and cupping therapy reduced the levels of UA, ESR and CRP in patients with gout. However, it is hard to distinguish whether the efficiency in treating gout is only due to Si Miao decoction itself or the interaction between Si Miao decoction and acupuncture (or cupping). Researchers should find out the possible mechanism of Si Miao decoction in treating gout and the interaction between herbal medicine and acupuncture.\n\nThe methodology is well-defined for Systematic Review and Meta-analysis; however, the including studies as the materials in this article have many methodological flaws, such as randomization, double-blind and allocation concealment. The results and conclusion could not be judged solidly, so it could not be recommended to TCM practitioners worldwide.\n\nMore solid and well-defined scientific studies should be included for analysis.\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": [] } ]	1	https://f1000research.com/articles/10-176
https://f1000research.com/articles/10-175/v1	03 Mar 21	{ "type": "Research Article", "title": "Retrospective analysis of the use of osteoporosis medication at the presentation of non-vertebral fragility fractures in a predominantly Hispanic population.", "authors": [ "Annelyn Torres-Reveron", "Michael Serra-Torres", "Michael Serra-Torres" ], "abstract": "Background: Despite the high incidence of osteoporosis, many patients at risk of fragility fractures may not initiate treatment due to concerns about side effects, cost or under-diagnosis, such as the case of vertebral fractures. We aimed to identify whether the patient population with non-vertebral fragility fractures where already receiving prophylactic treatment for osteoporosis at presentation within a regional hospital in the southernmost region of the United States. This region is characterized by a high number of patients from Hispanic/Latino heritage (80%) and reduced access to healthcare services. Methods: We conducted a three-year, retrospective cohort study of patients presenting with low impact fractures of the humerus or the shoulder griddle, lower end of radius or ulna and forearm, hip fractures (femoral neck, intertrochanteric/ subtrochanteric), and ankle fractures. Male and female subjects of 50 years or older were included. Demographic data and information on medications reported at fracture presentation were extracted from electronic medical records. Results: We found that 42% of the patients were taking at least one medication to prevent osteoporosis. The predominant combination was vitamin D plus calcium and bisphosphonates. If patients taking only vitamin D plus calcium are excluded, 16.7% of the sample took osteoporosis medications at the fragility fracture presentation. The likelihood of taking osteoporosis medication was increased by age and type of health insurance (Medicare/private insurance), and concomitant diagnosis of impaired gait and mobility. The percentage of the patients taking prophylactic medications for osteoporosis at the time of a fragility fracture was comparable to reported national standards and associated with increased age and health insurance coverage. Conclusion: In a predominantly Hispanic/Latino patient population living in a medically underserved region, there is substantial recognition and prevention strategies for osteoporosis.", "keywords": [ "osteoporosis", "prevention", "bisphosphonates", "calcium", "vitamin D", "fragility", "fractures" ], "content": "Introduction\n\nOsteoporosis is a systemic bone disease characterized by a decrease in bone strength reflected by a combination of bone mineral density and bone quality, which includes architecture, turnover, damage accumulation, matrix mineralization, and collagen composition1. It affects one in four women and one in eight men over the age of 50 years. It is estimated that 44 million people have either osteoporosis or low bone mass in the United States, representing 55% of the population 50 years and older2. The annual cost of osteoporosis fractures in 2018 is estimated at $57 billion dollars and is projected to reach $95 billion by the year 20403–5. Osteoporosis can be primarily influenced by lifestyle changes and the use of prophylactic bone-forming/preserving agents. Examining the frequency and use of prophylactic medications to prevent osteoporotic/fragility fractures can help design interventions to reduce negative impacts on the elderly.\n\nOsteoporosis leads to fragility fractures most commonly observed in the vertebrae, distal radius, proximal humerus, proximal femur, and ankle that result from minimal trauma, such as a fall from a standing height6. While vertebral fragility fractures are the most common manifestation of osteoporosis, only 25% of these fractures are diagnosed, since they are often asymptomatic or mildly asymptomatic7. Fragility fractures can lead to loss of functionality, pain, limitation of activities of daily living (ADLs), increased morbidity, disability, and significant adverse effect on the quality of life8. These consequences present a high burden on healthcare systems due to increased hospitalizations, surgeries, utilization requirements (e.g., rehabilitation/physical therapy, home care, nursing), and medical costs9. A study comparing the hospitalization burden due to fragility fractures in women 55 years or over found that annual cost for fragility fracture admissions was more significant than the cost of myocardial infarction, stroke, or breast cancer10. It is estimated that the global number of fragility fractures was nine million annually in 2000, including 1.6 million hip fractures, 1.7 million wrist fractures, and 1.4 million spine fractures2. It is predicted that this number will continue to increase due to the changes in life expectancy and to change demographics globally. Moreover, by the year 2050, it is estimated that 70% of all patients with hip fractures will be located in Asia, Latin America, and the Middle East11.\n\nIn 2020, the United States population of patients over the age of 50 years old is between 13–20%. This number is projected to increase to 28–40% by 2050. Hispanics, according to the 2000s United States-Census, represent the largest minority group at 12.5% of the total population, and this ethnic group is anticipated to increase to 25% by the year 2050 (https://www.census.gov/data/tables/2014/demo/popproj/2014-summary-tables.html). Concurrently, the incidence of hip fractures in Latin Americans is projected to increase to 12.5% by 2050, representing an incidence increase of 5.5% from that in 19903. Despite these projections, there is a lack of research and data assessing the risk factors for osteoporosis and osteopenia in Hispanics12–14. The risk factors for osteoporosis can be classified as modifiable, such as smoking, low body weight, hormone deficiency, alcohol intake, physical activity, dietary intake, falls, medications, chronic conditions, social clinical and medical insurance status, and non-modifiable risk factors: race, age, sex, dementia, past medical and family history of fractures15. Understanding the interaction of modifiable and non-modifiable factors in Hispanics is crucial to design and implement appropriate prevention programs for osteoporosis.\n\nOsteoporosis continues to be under-recognized and under-treated despite its massive economic cost and impact on morbidity and mortality16. The viability of health systems payer (insurance) and economic conditions has been related to quality in health care provided in Latin American regions3. A recent study showed that in four countries from Latin America, the baseline number of postmenopausal women using any type of osteoporosis medication was 70.9%, contrasting with that for the East Asia (63.3%) and Middle East (19.9%) sub-cohorts before any intervention occurred17–19.\n\nMost medications for treatment of osteoporosis work by either decreasing bone resorption, (bisphosphonates, selective estrogen receptor modulators, Denosumab- RANK ligand inhibitors) or increasing bone formation (recombinant parathyroid hormone)20. Using a patient cohort from the southernmost border of the United States composed of approximately 80% Hispanics, we aim to recognize whether the population presenting with non-vertebral fragility fractures is receiving prophylactic treatment for such condition. Based on prior reports from Latin America, we hypothesized that use of prophylactic medications for osteoporosis is significantly lower in our cohort than previously reported national studies in the United States. Our target is to improve the knowledge of osteoporosis treatment use in the Hispanic population at presentation with a fragility fracture and increase early interventions in communities at risk.\n\n\nMethods\n\nThe study conformed to the Declaration of Helsinki and the U.S. Federal Policy for the Protection of Humans Subjects and was approved by the Institutional Review Board; protocol number 1542366-6. This retrospective study received approval on February 3, 2020, by the DHR Health Institute for Research and Development IRB under the expedited mechanism of review. A full waiver of consent to participate was submitted and approved by the IRB, specifying the retrospective nature of the study and the age of the participants to be included. All data was collected and analyzed de-identified.\n\nThis is a three-year, retrospective cohort study of patients from a single hospital, Level 2 Trauma Center in South Texas. The retrospective period of chart review was set from January 1, 2017, to December 31, 2019.\n\nMale and female participants 50 years and older presenting to any of the clinics or emergency rooms at our health system were included in the study. The electronic medical record at the hospital links with that of primary care providers and other clinics within the health system (including urgency room, orthopedic, endocrinology, and radiology depts., etc.), allowing for prescriptions of multiple providers to be captured in the patient’s medical record. The following types of fractures were included: upper end of the humerus or the shoulder griddle, lower end of radius or ulna and forearm, displaced or non-displaced femoral neck, intertrochanteric/ subtrochanteric fractures, the lower end of tibia, medial malleolus, lateral malleolus, or lower leg. Patients were excluded from the study if the onset of the injury or presentation was outside the study period, if the fracture was already treated at another institution (e.g., referred to a rehabilitation hospital), or if the fracture was nonunion. Additional exclusion criteria were based on the mechanism of injury: fractures produced as a result of projectiles, motor vehicle accidents, or falls from a height higher than one meter were excluded from the cohort. Fractures due to diagnosed/ documented cancer were excluded as well. Fragility fractures of the vertebrae (as defined by the International Classification of Disease-10 as M80.08) were initially considered in this study. However, the incidence of such fractures, as documented in the medical records, was lower than any of the other types of fragility fractures included herein. Therefore, fractures of the vertebrae were underdiagnosed, similar to what has been previously reported for this type of injuries21,22. To reduce the introduction of information bias, these fractures were purposefully excluded from data extraction or analysis23.\n\nThe demographic variables included were age, sex, ethnicity, body mass index, date of arrival, presentation site, and health insurance or self-pay. From the medical record, we obtained the onset of the injury, the primary diagnosis and secondary diagnosis, the mechanism of injury, treatment received and date, intensive care unit (ICU) usage, comorbidities, home medications use at the time of presentation, zip code of primary residency, discharge disposition, and history of prior dual-energy X-ray absorptiometry (DXA) scan. The type and number of drugs for osteoporosis treatment and prevention were classified based on their mechanism of action: biphosphonates, RANK ligand inhibitors, estrogen, selective estrogen receptor modulators, sclerostin inhibitor, parathyroid hormone analog, calcium, and vitamin D. As a surrogate for patient’s mortality at six months and 12 months, we used the last known reported activity at the hospital, regardless of services requested. In some cases, detailed notes from clinicians indicated the patient’s status.\n\nPatients meeting inclusion criteria were identified via an electronic report from the hospital trauma registry, and the business intelligence department at the hospital system. The trauma registry is populated from information directly extracted from the patient’s medical record and is used for purposes of quality improvement and trauma level certification by the American College of Surgeons. The first author has unlimited access to generate reports from the trauma registry using the software DI Report Writer (DI Data Management System) and the second author has unlimited access to the electronic medical record used in the hospital. Home medications, diagnosis of osteoporosis and occurrence of a DXA scan were not part of the trauma registry, and were extracted via a report from the hospital business intelligence department. The files were filtered and processed using the Pandas package for Python (https://pandas.pydata.org/). Each medication was classified using string processing of its generic and the brand names as published by the National Osteoporosis Foundation (https://www.nof.org/patients/treatment/medicationadherence/), plus calcium and vitamin D. The resulting classifications were thenanalyzed for frequencies. The process was verified by the first author to ensure that no data was missing and all possible medication classes (grouped by their mechanism of action) were accounted. All presented information was part of the participant’s standard of care as documented in their medical record, and no data were collected directly from the patient. The orthopedic surgeon (second author) provided oversight of the data collection process and designed the group comparisons.\n\nThe fractures were identified using the International Classification of Diseases 10th revision (ICD10) as follows: fractures of the upper end of the humerus (S42.2) or the shoulder griddle (S42.9); fractures of the lower end of radius (S52.5), lower end of ulna (S52.60), fractures of the forearm (S52.9), displaced or non-displaced femoral neck, intertrochanteric/ subtrochanteric fractures (S72.0, S72.1, and S72.2), fracture of the lower end of tibia, medial malleolus, lateral malleolus or lower leg (S82.3, S82.5, S.82.6, and S82.8). All modifiers to the above fractures based on the ICD10 were also included. Once all patients presenting with these fractures were identified, data was further filtered to retain only those patients who were older than 50 years of age at the time of injury. Subsequently, participants were excluded if the mechanism of injury was other than a fall from a height greater than one meter. Both exclusion rules were done using the parameters reported in the trauma registry. The exclusion criteria was validated by randomly verifying 20 patients from each ICD10 classification (S42, S52, S72, S82) using the clinical notes documented in the patient’s medical record.\n\nDescriptive statistics were used for the entire study population. Frequencies and column percentages were used to summarize categorical variables. The normal distribution of continuous variables was measured using the Shapiro-Wilk goodness-of-fit test. Non-normally distributed variables were analyzed using the Wilcoxon test, and normally distributed variables were analyzed using the Student t-test for independent samples. Chi-square or Fisher exact tests were used for categorical variables. Multinomial regression analyses were used to explore the changes in medication across injury types, taking into consideration the age and sex of the patients. The statistical analyses were two-sided and conducted using JMP 15.0 (SAS Institute, Inc, Carry, NC, USA). The statistical significance was set at p < 0.05.\n\nAn earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.12.24.424289).\n\n\nResults\n\nA total of 864 cases of non-vertebral fractures in patients older than 50 years of age were identified: 121 shoulder cases, 230 wrist cases, 297 hip cases, and 216 ankle cases. Most of the excluded clinical cases were due to the mechanisms of high energy injuries: pedestrian-MVA injuries, motor vehicle accidents, and falls from a height greater than one meter. A handful of cases were excluded due to inaccurate coding of fracture. The final cohort consisted of 719 patients.\n\nTable 1 describes the number of cases included in each cohort grouped by the site of injury. Patients were predominantly female (>60%), except for shoulder injuries, where 16% of the cases were females. 81% of the patients self-reported as Hispanic/Latino ethnicity, which is representative of the demographic distribution for the region. The average age of the cohort was 74.03 ± 11.90 years. Patients with ankle injuries were the youngest in the cohort, with an average age of 66.72 ± 11.08. The length of stay in the hospital was highest for hip injuries (3.19 ± 5.05 days) and lowest for wrist injuries (0.32 ± 1.87 days). Only 62 patients (8.6%) had a prior diagnosis of osteoporosis. The highest percentage of prior diagnosis was in patients with hip fractures and the lowest was in patients with ankle fractures (13.1% and 5.3%, respectively). Of the patients with prior diagnosis, 51 (82%) were taking osteoporosis medications, while 11 (17.7%) were not taking any osteoporosis medication. 98% of the patients diagnosed with osteoporosis were female, while only one patient was male. 10% of the cohort (72 patients) had a dual-energy X-ray absorptiometry (DXA) scan, from which 66 patients were female, and only six were male. The highest percentage of patients who received a DXA presented with shoulder fractures (12%). The following three classes of medications were not reported being used by any of the participants: sclerostin inhibitor (Romosozumab), parathyroid hormone analog (Teriparatide), and parathyroid hormone-related protein analog (Abaloparatide); hence these were not included in any of the results tables.\n\nN, number; Dx, diagnosis; SD, standard deviation; DXA, dual energy X-ray absorptiometry, LOS, length of stay.\n\nPatients were stratified based on the number of medications for osteoporosis they were taking (zero to four). 42% of the patients, regardless of the fracture site, were taking at least one medication, while 58% were not taking any medication for osteoporosis at the time of fracture (Table 2). Within those taking medications, the vast majority took one or two. The most common medications were a combination of Vitamin D plus calcium and bisphosphonates (Table 3). Other than Vitamin D plus calcium, only 16.7% were on medications for osteoporosis. The percentage of patients taking medications also varied by fracture site, with the highest percentage of patients in the hip fracture group (58.9%) and the lowest in the ankle fractures group (27.8%). Patients who had Medicare as the principal payer constituted 70% of the cohort, which is expected due to the age group. The type of health insurance influenced whether the patients took osteoporosis medication (X2= 66.78, d.f.= 4, p< 0.001; Figure 1). The odds of taking osteoporosis medication for patients on Medicare compared to self-pay was 6.84 (95% CI: 2.62 to 17.85). The odds were even higher for patients in Medicare than other payment types grouped together (government, charity, indigent): 11.76 (95% CI: 3.56 to 38.86). However, the odds of taking osteoporosis medications for those patients in Medicare compared to private insurance was similar at 1.49 (95% CI: 0.55 to 4.08; Figure 1).\n\n“% of Total” refers to the total number of patients included in the study (n= 719); % Meds refers to the total number of patients that were taking medications at fracture presentation (n= 308).\n\nPatients who took medications for osteoporosis (“YES” column) had predominantly Medicare or Medicaid. However, at ages younger than 66, many patients were not taking any medications for osteoporosis (“NO” column) irrespective of the high incidence of private insurance. Data from 24.\n\nBesides a documented diagnosis for osteoporosis, 10 other comorbidities were collected from the patient charts: hypertension, obesity, impaired gait or mobility, diabetes, hyperlipidemia, osteoarthritis, other cardiovascular conditions besides hypertension, thyroid-related disease, renal disease, and any cancer. The three most frequent comorbidities in the cohort were: hypertension (59.1%), obesity (47.7%), and impaired gait or mobility (45.2%). Neither hypertension nor obesity influenced whether the patient was taking any type of osteoporosis medication. However, patients with impaired gait or mobility and patients with thyroid-related disease had an increased odds ratio of receiving osteoporosis medications at 2.56 (95% CI, 1.80 to 3.65) and 5.33 (95% CI, 3.04 to 9.36), respectively. The remaining comorbidities did not influence osteoporosis medication patterns.\n\nTo further understand osteoporosis medication use patterns across sex, data were stratified based on three age groups: 50–65, 66–83, and 83 years and older (Figure 2). The age groups were constructed using the 25th quartile (65 years) and 75th quartile (83 years) for age. Regardless of fracture site and age, females had a significantly higher odds of using osteoporosis medications than males: 1.77 (95% CI, 1.21 to 2.58). Patients presenting with hip fractures at any age had the highest odds of taking osteoporosis medication compared to patients with fractures in any other body area: 2.66 (95% CI, 1.94 to 3.68). The odds for patients presenting with shoulder fractures to be on osteoporosis medications was very similar to other fractures at 1.13 (95% CI, 0.73 to 1.73). The older the patient, the higher the probability of patients taking osteoporosis medications. The odds for osteoporosis medication use in patients in the 84 years and older group compared to patients in the 50–65 years group were 4.92 (95% CI, 3.12 to 7.82). Similarly, patients in the 66–83 years group had odds of taking medication of 3.75 (95% CI, 2.29 to 5.19) than the younger group.\n\nZero (0) value in the figure represents patients that were not taking any medications for osteoporosis. It is evident that males were using fewer medications (if any) than females and this was obvious for hip fractures. Females with shoulder fractures had the largest increase across age groups in the percentage of patients taking medications. Please refer to the Results sections for odds ratios. Data from 24.\n\n\nDiscussion\n\nContrary to our hypothesis, the use of medications to prevent osteoporosis in patients that present non-vertebral fragility fractures were comparable to previously reported national averages25. Within the set of patients taking osteoporosis medications, the use of calcium, vitamin D, and bisphosphonates constituted more than 80% of the consumption. When calcium and vitamin D were not considered, the percent of patients on osteoporosis medications dropped by 25% (from 42 to 16.7%) but remained above the national averages of 10% for years 2010–201125. Females were more likely to take osteoporosis medications, and this probability increased with age. In our population, the number of male patients presenting with fractures and treated with osteoporosis medications was very low. This finding is similar to previous reports, which is partially explained due to underdiagnosed and under-treatment of males for osteoporosis26,27. To our knowledge, this is the first time that a study identifies the use of medications for osteoporosis within the southernmost region of the United States, with a high prevalence of Hispanic/Latino heritage.\n\nWhen we examine socioeconomic status and self-reported race/ethnicity, reports have shown that individuals at an extreme socioeconomic disadvantage are very vulnerable to relatively low bone mineral density28. The Rio Grande Valley (southernmost Texas, USA) population is well documented to have low socioeconomic status with an individual median income of only $22,302 dollars (median for the state of Texas, USA: $30,596). The Rio Grande Valley is also classified as a medically underserved population with a prevalence of 30% for uninsured/underinsured patients29. Despite these well documented socioeconomic conditions, the use of osteoporotic medication was not lower than the national average. However, it is essential to consider that vitamin D and calcium, which can be obtained at a low cost and without prescription, were the predominant medications used in our population. Within our cohort, patients taking medications for osteoporosis were older and had a higher prevalence of comorbidities than patients not taking medications for osteoporosis. The need for closer multidisciplinary medical treatment for patients with increased age and multiple comorbidities produce more medical interventions and closer monitoring, allowing for identification of the patient is at risk of osteoporosis and medical treatment interventions.\n\nA study looking at bone turnover in Mexican Americans who also have type 2 diabetes found lower bone turnover in men with diabetes and poor glycemic control30; hence, screening for osteoporosis in Mexican Americans to prevent fractures should be highly considered31. Native Americans, White and Hispanic women remain among the highest for fracture risk than other ethnic groups (Black, Asian)32. Given that the Rio Grande Valley population is medically underserved and has a high prevalence of type 2 diabetes, we believe that there could be an increased risk of osteoporosis-related fractures in our community. However, this information has never been collected nor reported.\n\nWe observed a discrepancy for prior osteoporosis diagnosis within our data with the number of patients taking the medications. This discrepancy might be produced by a lack of proper documentation within the medical records or simply by the retrospective nature of the data. It is possible that patients might have been diagnosed at a primary care facility not associated with our hospital system; thus, documentation of DXA scans or diagnosis is not within our system’s electronic medical record. However, it should be noted that in the mid-nineties, the use of bone mineral density scans for osteoporosis was not recommended33, but this view has been challenged, and DXA remains the “gold standard” for osteoporosis diagnosis34,35. An additional possibility to consider is that the patients initiate the reported use of calcium and vitamin D on their own, as part of a multi-vitamin regime, or only as a physician’s recommendation due to their advanced age. The latter is supported by a previous study indicating that for Hispanics, information regarding medication use and adherence is more readily received from the doctor than from any other source of information: “the doctor is still king”36. Future prospective studies should address the process of diagnosis and reporting, both by primary care and specialty doctors.\n\nRetrospective cohort studies provide a quick estimate when no previous data on the topic exist, especially for specific populations, but it also carries a series of limitations. Information regarding the length that the patients have been taking the medication was not collected, nor the effectiveness of such medications. While the number of patients taking medications in the current study is not suggestive of underreporting, it is always possible that patients are taking calcium plus vitamin D as part of their regular multi-vitamins but do not consider vitamins as “medicine” or “treatment.” Hence the possibility of underreporting is impossible to rule out. An attempt to verify previous fracture history was initiated, but we ruled out acquiring this information given that our region has three large hospitals. The possibility of patients seeking care for a prior fracture at a different facility is high. Thus, future studies should consider a multi-institutional design, allowing the recording of this type of information. We acknowledge an intrinsic bias in the study design by selecting patients that already present with a fracture. However, the presence of a fragility fracture is one of the critical factors for the diagnosis of osteoporosis7. Based on the new practice guidelines for endocrinologists, the current patient population would fall on the very high-risk group based on the presence of a fracture within the previous 12 months35. Despite the limitations, the current study sets the stage for designing prospective interventions in high-risk groups for osteoporotic fractures.\n\n\nConclusions\n\nOsteoporosis continues to be an underdiagnosed and undertreated disease. In our cohort, despite the high use of prophylactic medication among the most elderly patients, the usage of medications in the younger population continues to be minimal. Within the male population, the usage and diagnosis of osteoporosis continue to be almost nonexistent. Despite slightly higher use of prophylactic medication than national standards, the percentage of patients taking medication still falls under desired levels, especially considering that only 16% of the patients took medication once vitamin D plus calcium were removed from the comparison. It is essential to recognize there is still significant work to promote consciousness, improve diagnoses, and encourage early use of prophylactic medications.\n\n\nData availability\n\nZenodo: Retrospective analysis of the use of osteoporosis medication at the presentation of non-vertebral fragility fractures in a predominantly Hispanic population. https://doi.org/10.5281/zenodo.452630624.\n\nThe project contains the following underlying data:\n\n- Full data oseto-de-identfied.csv (This is a de-identified data set including 719 patients older than 50 years of age. The patients have a fragility fracture of the hip, shoulder, wrist and ankle. Comorbidities and medications taken at the time of presentation are documented.)\n\nZenodo: STROBE checklist for “Retrospective analysis of the use of osteoporosis medication at the presentation of non-vertebral fragility fractures in a predominantly Hispanic population”. http://doi.org/10.5281/zenodo.455785437.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0).", "appendix": "Acknowledgments\n\nWe acknowledge the contributions of DHR Health employees from the Quality Department in some aspects of data extraction, and an intern from the Education Department for providing help with data mining. We appreciate the valuable comments on the manuscript from Dr. Lisa Trevino and Mr. Peter Roberge from DHR Health Institute for Research and Development.\n\n\nReferences\n\nNIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy: Osteoporosis prevention, diagnosis, and therapy. JAMA. 2001; 285(6): 785–795. PubMed Abstract \| Publisher Full Text\n\nJohnell O, Kanis JA: An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int. 2006; 17(12): 1726–1733. PubMed Abstract \| Publisher Full Text\n\nAlbergaria BH, Chalem M, Clark P, et al.: Consensus statement: osteoporosis prevention and treatment in Latin America-current structure and future directions. Arch Osteoporos. 2018; 13(1): 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarvey N, Dennison E, Cooper C: Osteoporosis: impact on health and economics. Nat Rev Rheumatol. 2010; 6(2): 99–105. PubMed Abstract \| Publisher Full Text\n\nHanson D, Bazell C, Pelizzari P, et al.: Medicare costs for osteoporosis-related fractures. PharmacoEcon Outcomes News. 2019; 839(1): 26. Publisher Full Text\n\nKanis JA, Oden A, Johnell O, et al.: The burden of osteoporotic fractures: a method for setting intervention thresholds. Osteoporos Int. 2001; 12(5): 417–427. PubMed Abstract \| Publisher Full Text\n\nvan Oostwaard M: Osteoporosis and the Nature of Fragility Fracture: An Overview. In: Hertz K, Santy-Tomlinson J, eds. Cham (CH); 2018; 1–13. PubMed Abstract \| Publisher Full Text\n\nIbrahim NI, Ahmad MS, Zulfarina MS, et al.: Activities of Daily Living and Determinant Factors among Older Adult Subjects with Lower Body Fracture after Discharge from Hospital: A Prospective Study. Int J Environ Res Public Health. 2018; 15(5): 1002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecker DJ, Yun H, Kilgore ML, et al.: Health services utilization after fractures: evidence from Medicare. J Gerontol A Biol Sci Med Sci. 2010; 65(9): 1012–1020. PubMed Abstract \| Publisher Full Text\n\nSinger A, Exuzides A, Spangler L, et al.: Burden of illness for osteoporotic fractures compared with other serious diseases among postmenopausal women in the United States. Mayo Clin Proc. 2015; 90(1): 53–62. PubMed Abstract \| Publisher Full Text\n\nDhanwal DK, Dennison EM, Harvey NC, et al.: Epidemiology of hip fracture: Worldwide geographic variation. Indian J Orthop. 2011; 45(1): 15–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CH, Elsalmawy AH, Ish-Shalom S, et al.: Study description and baseline characteristics of the population enrolled in a multinational, observational study of teriparatide in postmenopausal women with osteoporosis: the Asia and Latin America Fracture Observational Study (ALAFOS). Curr Med Res Opin. 2019; 35(6): 1041–1049. PubMed Abstract \| Publisher Full Text\n\nRiggs BL, Parfitt AM: Drugs used to treat osteoporosis: the critical need for a uniform nomenclature based on their action on bone remodeling. J Bone Miner Res. 2005; 20(2): 177–184. PubMed Abstract \| Publisher Full Text\n\nSeeman E, Delmas PD: Bone quality--the material and structural basis of bone strength and fragility. N Engl J Med. 2006; 354(21): 2250–2261. PubMed Abstract \| Publisher Full Text\n\nLems WF, Raterman HG: Critical issues and current challenges in osteoporosis and fracture prevention. An overview of unmet needs. Ther Adv Musculoskelet Dis. 2017; 9(12): 299–316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurtis JR, Arora T, Matthews RS, et al.: Is withholding osteoporosis medication after fracture sometimes rational? A comparison of the risk for second fracture versus death. J Am Med Dir Assoc. 2010; 11(8): 584–591. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen CH, Lim SJ, Oh JK, et al.: Teriparatide in East Asian Postmenopausal Women with Osteoporosis in a Real-World Setting: A Baseline Analysis of the Asia and Latin America Fracture Observational Study (ALAFOS). Clin Interv Aging. 2020; 15: 111–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElsalmawy AA, Al-Ali NS, Yaghi Y, et al.: Middle East experience from the Asia And Latin America Fracture Observational Study (ALAFOS): Baseline characteristics of postmenopausal women with osteoporosis using teriparatide. J Int Med Res. 2020; 48(8): 300060520940855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCunha-Borges JL, Mier GM, Casas N, et al.: Baseline characteristics of postmenopausal women with osteoporosis treated with teriparatide in a real-world setting in Latin America: a subregional analysis from the Asia and Latin America Fracture Observational Study (ALAFOS). Adv Rheumatol. 2019; 59(1): 46. PubMed Abstract \| Publisher Full Text\n\nTu KN, Lie JD, Wan CKV, et al.: Osteoporosis: A Review of Treatment Options. P T. 2018; 43(2): 92–104. PubMed Abstract \| Free Full Text\n\nBottai V, Giannotti S, Raffaetà G, et al.: Underdiagnosis of osteoporotic vertebral fractures in patients with fragility fractures: retrospective analysis of over 300 patients. Clin Cases Miner Bone Metab. 2016; 13(2): 119–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarongiu G, Congia S, Verona M, et al.: The impact of magnetic resonance imaging in the diagnostic and classification process of osteoporotic vertebral fractures. Injury. 2018; 49 Suppl 3: S26–S31. PubMed Abstract \| Publisher Full Text\n\nHammer GP, du Prel JB, Blettner M: Avoiding bias in observational studies: part 8 in a series of articles on evaluation of scientific publications. Dtsch Arztebl Int. 2009; 106(41): 664–668. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorres-Reveron, Serra-Torres: Retrospective analysis of the use of osteoporosis medication at the presentation of non-vertebral fragility fractures in a predominantly Hispanic population. F1000Res. 2021. http://www.doi.org/10.5281/zenodo.4526306\n\nSarpong EM: Statistical Brief #442. Changes in Osteoporosis Medication Use and Expenditures among Women (Age ≥ 50), United States, 2000 to 2011. Statistica. Rockville, Maryland: Agency for Healthcare Researcha and Quality; 2014. PubMed Abstract\n\nChau YT, Nashi N, Law LSC, et al.: Undertreatment of osteoporosis following hip fracture: a retrospective, observational study in Singapore. Arch Osteoporos. 2020; 15(1): 141. PubMed Abstract \| Publisher Full Text\n\nPorcelli T, Maffezzoni F, Pezzaioli LC, et al.: Management Of Endocrine Disease: Male osteoporosis: diagnosis and management - should the treatment and the target be the same as for female osteoporosis? Eur J Endocrinol. 2020; 183(3): R75–R93. PubMed Abstract \| Publisher Full Text\n\nDu Y, Zhao LJ, Xu Q, et al.: Socioeconomic status and bone mineral density in adults by race/ethnicity and gender: the Louisiana osteoporosis study. Osteoporos Int. 2017; 28(5): 1699–1709. PubMed Abstract \| Publisher Full Text\n\nTorres S: Health Care Access in the Rio Grande Valley: The Specialty Care Challenge. Edinburg, Texas; 2018. Reference Source\n\nRianon NJ, Smith SM, Lee M, et al.: Glycemic Control and Bone Turnover in Older Mexican Americans with Type 2 Diabetes. J Osteoporos. 2018; 2018: 7153021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBroussard DL, Magnus JH: Risk assessment and screening for low bone mineral density in a multi-ethnic population of women and men: does one approach fit all? Osteoporos Int. 2004; 15(5): 349–360. PubMed Abstract \| Publisher Full Text\n\nCauley JA, Wu L, Wampler NS, et al.: Clinical risk factors for fractures in multi-ethnic women: the Women’s Health Initiative. J Bone Miner Res. 2007; 22(11): 1816–1826. PubMed Abstract \| Publisher Full Text\n\nMarshall D, Johnell O, Wedel H: Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. BMJ. 1996; 312(7041): 1254–1259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeeta, Harinarayan CV, Marwah R, et al.: Clinical practice guidelines on postmenopausal osteoporosis: An executive summary and recommendations. J Midlife Health. 2013; 4(2): 107–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCamacho PM, Petak SM, Binkley N, et al.: American Association of Clinical Endocrinologists/American College of Endocrinology Clinical Practice Guidelines For The Diagnosis And Treatment of Postmenopausal Osteoporosis- 2020 Update Executive Summary. Endocr Pract. 2020; 26(5): 564–570. PubMed Abstract \| Publisher Full Text\n\nSchroeder DG, Alaoui M, Keefe-Oates B: Hispanic Medication Adherence Study. HolaDoctor. 2016. Reference Source\n\nTorres-Reveron A: STROBE & RECORD checklist for the article entitled: Retrospective analysis of the use of osteoporosis medication at the presentation of non-vertebral fragility fractures in a predominantly Hispanic population. (Version Version1). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4557854" }	[ { "id": "84854", "date": "01 Jun 2021", "name": "Nicole C. Wright", "expertise": [ "Reviewer Expertise Musculoskeletal research", "osteoporosis epidemiology", "racial disparities in osteoporosis management", "outcomes research" ], "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 evaluated the use of osteoporosis medications in a predominantly Hispanic population. This is important to the field as data is lacking on the use of osteoporosis medications in Hispanics and other people of color. The study was derived from hospital claims, which provides high level of specificity for fractures; however, there were a series of methodologic concerns that are needed to be corrected.\n\n1) The paper subheadings need to be reorganized, particularly when it comes to exposure and outcome variables. I would suggest the following:\n\nEthics statement. Study design and setting - can probably include description of participants here. Fracture identification and respective codes; including position in the record where codes were derived. Medication identification - again which meds and where identified in the record. Covariates of interest. Statistical Analysis.\n\n2) Fracture identification - there are published algorithms for identification that use ICD code, surgical procedure codes, as well as imaging codes (Wright et al., 20191). Consider utilizing these algorithms, particularly for clinical vertebral fractures. This could potentially increase the number of fractures in the population.\n\n3) A flowchart of exclusions is needed to show the reader visually the sample size determination\n4) How good of a surrogate is the \"last known reported activity\"? If there is data around this, please provide validity estimates. If not, I would reconsider phrasing this as mortality unless you have vital status information.\n\n5) Sentence structure: I would avoid starting sentences with numbers.\n\n6) Tables 1 and 2 could have group comparisons using chi-square test and ANOVA.\n7) I would reconsider the shading in figures for those who are printing article in black and white.\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "89853", "date": "28 Jul 2021", "name": "Kok-Yong Chin", "expertise": [ "Reviewer Expertise osteoporosis", "animal models of osteoporosis & osteoarthritis", "tocotrienol", "vitamin E" ], "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 reported the use of osteoporosis medications in Hispanic populations with low sociodemography admitted for fragility fracture in a single centre in USA. The data are valuable in providing insights into osteoporosis preventive practices among this population. The manuscript is well written and all limitations were acknowledged by the authors. I only have minor comments on the manuscript.\nTitle: osteoporosis medications.\nAbstract: The predominant combination was vitamin D plus calcium and bisphosphonates - please provide the percentage.\n\nIntroduction:\n\nThe prevalence of osteoporosis as indicated in ref 2 is quite outdated. Please replace with updated data.\n\nThe annual cost of osteoporosis fractures - are the authors referring to global or US data?\n\nThe entire paragraph 2 can be summarised and integrated into paragraph 1.\n\nResults:\n\n\"121 shoulder cases, 230 wrist cases, 297 hip cases, and 216 ankle cases\" - please also provide the percentage.\n\n\"81% of the patients\" - please do not start the sentence with numerical.\n\nGeneric drug names such as romosozumab, teriparatide and abaloparatide should not be capitalised.\n\nPlease provide the percentage for the following statement \"Within those taking medications, the vast majority took one or two. The most common medications were a combination of Vitamin D plus calcium and bisphosphonates\".\n\nSubheading: \"Outcomes-based on sex and age group\" should be modified as outcomes may be misinterpreted as \"fracture outcomes\". It is actually analysis based on sex and age group.\n\nDiscussion: \"... in patients that present non-vertebral fragility fractures\" - replace 'that' with 'who'.\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/10-175
https://f1000research.com/articles/9-1264/v1	22 Oct 20	{ "type": "Software Tool Article", "title": "TicTimer Web: software for measuring tic suppression remotely", "authors": [ "Jonathan K. Black", "Jonathan M. Koller", "Kevin J. Black", "Jonathan M. Koller" ], "abstract": "Woods and Himle developed a standardized tic suppression paradigm (TSP) for the experimental setting, to quantify the effects of intentional tic suppression in Tourette syndrome. We previously provided a computer program to facilitate recording tic occurrence and to automate reward delivery during the several experimental conditions of the TSP. The present article describes a web-based program that performs the same functions. Implementing this program on the web allows research sessions to be performed remotely, in tandem with a video calling program. Relevant data for each session, such as the timing of tics and dispensed rewards, are stored in plain text files for later analysis. Expected applications include research on Tourette syndrome and related disorders.", "keywords": [ "tc disorders", "Tourette syndrome", "reward", "reinforcement (psychology)", "software" ], "content": "Introduction\n\nIn a previous article1 we mentioned the tic suppression paradigm (TSP) developed by Woods and Himle for use in the experimental setting to demonstrate and quantify the effects of intentional tic suppression on tic frequency in Tourette syndrome (TS) and other tic disorders2–6. In this paradigm, each participant is observed during several experimental conditions: usually baseline and differential reinforcement of zero-rate ticcing (DRO), and sometimes also verbal instruction to suppress tics and/or noncontingent reinforcement (NCR).\n\nIn the course of conducting a longitudinal study of children with Provisional Tic Disorder7, we found that tic suppression is seen within the first few months after a child’s first tic8. We also found that the TSP required substantial investigator effort, so we wrote a simple program to facilitate record keeping and reward delivery during research sessions1. It required an expert observer to press a button to record each tic observed, and in the DRO and NCR conditions it delivered reward tokens at the appropriate times by connecting to a relay module and a token dispenser box. This software improved convenience for the investigator and accuracy of record-keeping.\n\nBecause of enforced social distancing in 2020, the need arose for sessions to be performed remotely. A video calling program allowed us to observe the subjects, but we still needed a way to deliver rewards during the DRO and NCR conditions of the TSP. Previously, we had created a web-based program called TicTrainer for behavioral therapy9, and we decided to expand the functionality of that program so it could be used for TSP research. We present the software here10 to facilitate its use by others.\n\n\nMethods\n\nTicTimer Web10 uses the node.js server that was made for TicTrainer. It adopts that program’s structure, with user accounts for research subjects and an admin account for the rater. Details on account registration, data storage, and logging on were described previously9.\n\nA new field was added to user accounts so that a research ID (different from the ID used to log on) can be set for research subjects.\n\nSessions for TicTimer Web use separate but simultaneous connections to a server from a “user” and from a rater. TSP DRO sessions deliver rewards after every 10-s tic-free interval. To deliver rewards at the appropriate times, the user page checks in with the server periodically to see if it has been 10 seconds since the last tic was signaled by the rater. If it has not, the server responds with the time remaining until the next reward is due, and the user page uses that number, adjusted for the lag time of the round trip, to schedule when to check back with the server. Using this method, reward timing is synchronized so that awards are delivered usually within 50 ms of the target time. The previous, in-person version of TicTimer used a token dispenser box to automatically deliver rewards at the appropriate times. In an attempt to approximate the user experience of the physical token dispenser, TicTimer Web delivers rewards by displaying coin images on the subject’s screen along with a chime sound.\n\nAt the end of each session, a summary is generated and appended at the bottom of the session log file, which is then archived with the date and time of the session in the filename.\n\nSetup. First, node.js is installed on the server. We used an Amazon EC2 instance, but the program can operate on any computer with node.js (e.g. a laptop).\n\nThe researcher and subject need only a modern web browser to interface with TicTimer Web once the server is running. The browser must support JavaScript and HTML5. We have tested TicTimer Web with current versions of Chrome and Edge.\n\nTo perform sessions with TicTimer Web, the researcher creates a user account if needed, then if desired uses the admin interface to assign the user a research ID number to identify the subject for later data analysis.\n\nUse. The researcher and subject sign in on their respective TicTimer session pages and the rater begins the session by selecting one of the four experimental conditions.\n\nDuring a session, the rater watches the subject. We have used a separate video calling program for this observation, but a video camera or one-way mirror could be used for in-person visits. The rater records any observed tics by immediately pressing the “Tic Detected” button, the spacebar, or the letter “T”. If the session type includes rewards (DRO and NCR), they are dispensed appropriately. The session ends after the predetermined duration, or when the rater presses “End Session,” or when either rater or subject closes their browser window early.\n\nFor the NCR condition, the rater first chooses a log file previously created with the current subject, and rewards are delivered to the user at the same times (relative to the session start) that they were delivered in the specified session.\n\nArchived session log files can be downloaded from the admin interface, or they can be copied over directly from the server itself.\n\n\nUse cases\n\nThe video file (Extended data, Supplementary File 1)11 demonstrates the operation of TicTimer Web10 from a researcher’s perspective. The sessions performed here were test sessions with no human subjects being observed. Extended data, Supplementary Files 2–411 are the session log files created in that video.\n\n\nConclusions\n\nThe TicTimer Web10 program allows for remote implementation of the TSP, while maintaining the benefits of earlier versions of the software1: ease and accuracy of record keeping and automated reward delivery. TicTimer Web, while designed for our purposes in tic disorder research, may find other uses. The most obvious of these may be for research on traditional habit disorders; for instance, hair pulling and skin picking appear in the “Obsessive-compulsive and related disorders” section of DSM-512. The most obvious application to the clinical setting may be in documenting suppression ability before and after treatment. Another potential future modification would be to add machine detection of tics, e.g. by online video analysis, surface EMG or accelerometry; such an improvement would be quite welcome but is difficult to reduce to practice.\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: TicTimer Web: software for measuring tic suppression remotely: Supplementary Files. http://doi.org/10.5281/zenodo.402313411.\n\nThis project contains the following extended data:\n\nttw_demo.mp4. (Supplementary File 1: Video Demonstration of Operation. A video demonstrating how to operate TicTimer Web from the researcher’s perspective.)\n\nau5_20200622-153043_baseline.ttsd. (Supplementary File 2: Sample Log, baseline. Log file for the baseline session performed during the video demonstration.)\n\nau5_20200622-153125_DRZ.ttsd. (Supplementary File 3: Sample Log, DRO. Log file for the DRO session performed during the video demonstration.)\n\nau5_20200622-153214_NCR.ttsd. (Supplementary File 4: Sample Log, NCR. Log file for the NCR session performed during the video demonstration.)\n\nLicense: MIT License.\n\n\nSoftware availability\n\nThe source code for TicTimer Web is available at: https://github.com/jonkb/TicTrainer-node.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.399047410.\n\nLicense: MIT License.", "appendix": "References\n\nBlack JK, Koller JM, Black KJ: TicTimer software for measuring tic suppression [version 2; peer review: 2 approved]. F1000Res. 2017; 6: 1560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoods DW, Himle MB: Creating tic suppression: comparing the effects of verbal instruction to differential reinforcement. J Appl Behav Anal. 2004; 37(3): 417–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHimle MB, Woods DW: An experimental evaluation of tic suppression and the tic rebound effect. Behav Res Ther. 2005; 43(11): 1443–51. PubMed Abstract \| Publisher Full Text\n\nHimle MB, Woods DW, Bunaciu L: Evaluating the role of contingency in differentially reinforced tic suppression. J Appl Behav Anal. 2008; 41(2): 285–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLyon GJ, Samar SM, Conelea C, et al.: Testing tic suppression: comparing the effects of dexmethylphenidate to no medication in children and adolescents with attention-deficit/hyperactivity disorder and Tourette’s disorder. J Child Adolesc Psychopharmacol. 2010; 20(4): 283–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConelea CA, Wellen B, Woods DW, et al.: Patterns and Predictors of Tic Suppressibility in Youth With Tic Disorders. Front Psychiatry. 2018; 9: 188. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack KJ, Kim S, Schlaggar BL, et al.: The New Tics study: A novel approach to pathophysiology and cause of tic disorders. J Psychiatr Brain Sci. 2020; 5: e200012. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreene DJ, Koller JM, Robichaux-Viehoever A, et al.: Reward enhances tic suppression in children within months of tic disorder onset. Dev Cogn Neurosci. 2015; 11: 65–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack JK, Black KJ: Software for web-based tic suppression training [version 2; peer review: 3 approved]. F1000Res. 2018; 6: 2150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack JK, Black KJ: TicTrainer and TicTimer Web v4.0.2 (Version 4.0.2).Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3990474\n\nBlack JK, Koller JM, Black KJ: TicTimer Web: software for measuring tic suppression remotely: Supplementary Files. F1000research. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4023134\n\nAmerican Psychiatric Association: Obsessive-Compulsive and Related Disorders. In: Diagnostic and Statistical Manual of Mental Disorders. Fifth Edition. Arlington, VA: American Psychiatric Association; 2013. Publisher Full Text" }	[ { "id": "73543", "date": "20 Jan 2021", "name": "Jennifer Schild", "expertise": [ "Reviewer Expertise I have 15 years of research regarding Tourette's disorder and have utilized the tic detector paradigm in my own 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 a timely and valuable tool for assessing tic suppression remotely in clinical and research settings. This web-based program is based on Woods and Himle’s standardized tic suppression paradigm (TSP) and performs the same function as a computer program previously developed by the authors to record tics and partially automate delivery of rewards. This paper is particularly useful during this time in which remote data collection for research is relevant and necessary, and it provides a helpful overview of a program that can be easily understood by other tic researchers to apply in their own research. The paper could benefit from elaboration in multiple areas to help readers better understand the broader relevance of TSP as well as the research utility of TicTimer Web. Elaboration in the introduction section, as well as a more detailed description of potential applications of this software and how this software helps to overcome previous limitations of the TSP are needed. We recommend that this article be revised and resubmitted. Below are some suggestions for the authors to consider.\nPlease provide a clearer, more detailed explanation of the broad importance of TSP and related concepts in the introduction section. This will be helpful for readers who are less familiar with tic suppression to understand what insights this paradigm have yielded and how this software program could be beneficial to future investigation. For example, the TSP is described as requiring “substantial investigator effort,” but it is not clear what exactly is entailed. Those who used the old tic-detector boxes know how laborious running a participant was, but those less familiar will not appreciate the leap forward TicTimer Web provides.\n\nA short description of the TicTrainer web-based program is also needed in the introduction section. This will be helpful for orienting the reader to the new program that is described in this article, especially considering how similar this older program is to TicTimer Web.\n\nIn the methods section, there is interchangeable usage of the term “reward” and “award.” The word “awards” in paragraph 3 of the implementation subsection of the methods should be replaced with “rewards.”\n\nIn the methods section, elaboration is needed on what each experimental condition is (e.g., differential reinforcement of zero-rate ticcing (DRO), noncontingent reinforcement (NCR). Alternatively, this can be elaborated upon in the introduction section as it is also mentioned there.\n\nA separate section before the conclusion discussing the strengths and future applications of this software would be useful. This is somewhat addressed in the conclusion section, but further elaboration in a devoted section would do a better job of highlighting the potential of this software program.\n\nNext steps: It would be helpful to provide another sentence or two regarding the potential benefits of using artificial intelligence (AI) and machine learning (ML) to do this job as well as barriers to taking this approach.\n\nOverall, this paper provides a concise and useful overview of a web-based program for remote implementation of the TSP. With some clarification in the areas highlighted above, this paper offers a useful contribution to tic research and is especially applicable given the current relevance of remote methods for data collection. We strongly encourage the authors to revise and resubmit this manuscript for consideration.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6378", "date": "23 Feb 2021", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Ms. Schild and Dr. Specht for their thoughtful review, and will edit the report as they suggest." }, { "c_id": "6386", "date": "03 Mar 2021", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "Author responses are below, marked by the > sign and bold face, intercalated between sections of the original review. APPROVED WITH RESERVATIONS This paper describes a timely and valuable tool for assessing tic suppression remotely in clinical and research settings. This web-based program is based on Woods and Himle’s standardized tic suppression paradigm (TSP) and performs the same function as a computer program previously developed by the authors to record tics and partially automate delivery of rewards. This paper is particularly useful during this time in which remote data collection for research is relevant and necessary, and it provides a helpful overview of a program that can be easily understood by other tic researchers to apply in their own research. > Thank you for these positive comments. The paper could benefit from elaboration in multiple areas to help readers better understand the broader relevance of TSP as well as the research utility of TicTimer Web. Elaboration in the introduction section, as well as a more detailed description of potential applications of this software and how this software helps to overcome previous limitations of the TSP are needed. We recommend that this article be revised and resubmitted. Below are some suggestions for the authors to consider. Please provide a clearer, more detailed explanation of the broad importance of TSP and related concepts in the introduction section. This will be helpful for readers who are less familiar with tic suppression to understand what insights this paradigm have yielded and how this software program could be beneficial to future investigation. For example, the TSP is described as requiring “substantial investigator effort,” but it is not clear what exactly is entailed. Those who used the old tic-detector boxes know how laborious running a participant was, but those less familiar will not appreciate the leap forward TicTimer Web provides. > Done (first 2 paragraphs). A short description of the TicTrainer web-based program is also needed in the introduction section. This will be helpful for orienting the reader to the new program that is described in this article, especially considering how similar this older program is to TicTimer Web. > Done (last paragraph of Intro). In the methods section, there is interchangeable usage of the term “reward” and “award.” The word “awards” in paragraph 3 of the implementation subsection of the methods should be replaced with “rewards.” > Thank you. Corrected. In the methods section, elaboration is needed on what each experimental condition is (e.g., differential reinforcement of zero-rate ticcing (DRO), noncontingent reinforcement (NCR). Alternatively, this can be elaborated upon in the introduction section as it is also mentioned there. > Done (in first paragraph of Intro). A separate section before the conclusion discussing the strengths and future applications of this software would be useful. This is somewhat addressed in the conclusion section, but further elaboration in a devoted section would do a better job of highlighting the potential of this software program. > I elaborate on these in the first paragraph of the revised Conclusions section. Next steps: It would be helpful to provide another sentence or two regarding the potential benefits of using artificial intelligence (AI) and machine learning (ML) to do this job as well as barriers to taking this approach. > Done (last paragraph of Conclusions). Overall, this paper provides a concise and useful overview of a web-based program for remote implementation of the TSP. With some clarification in the areas highlighted above, this paper offers a useful contribution to tic research and is especially applicable given the current relevance of remote methods for data collection. We strongly encourage the authors to revise and resubmit this manuscript for consideration. > Thank you." } ] }, { "id": "77056", "date": "17 Feb 2021", "name": "Valerie Brandt", "expertise": [ "Reviewer Expertise Tic 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\nThe report describes the development of TicTimer Web, a nifty software that can be used to record tics, tic timings, and distribute reinforcement tokens during online experiments are therapeutic sessions. The authors describe that they adapted the TicTrainer programme to an online version that allows patients to login to a user account and therapists / tic raters to login as administrators. User and rater are then connected to the server simultaneously. The tic rater can record tics via button press and the software stores the timing of that tic. After every tic-free 10 second interval, the software delivers a reward. The software also delivers a summary at the end of the session. In order for the rater to watch tics, a separate programme, or a webcam can be used. This allows an accurate reward delivery, even remotely.\nThe source code of TicTimer is available on GitHub, the link is provided in the manuscript. Furthermore, an explanatory video shows how TicTimer can be used. The video is simple and clear and provides step-by-step explanations. Examples of demo files are also provided on Zenodo.\nThe manuscript is clearly written, the software is demonstrated in a video, examples and the source code are provided.\nI have no suggestions for improvement.\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": "6377", "date": "23 Feb 2021", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Prof. Brandt for her kind comments." } ] } ]	1	https://f1000research.com/articles/9-1264
https://f1000research.com/articles/9-1337/v1	16 Nov 20	{ "type": "Research Article", "title": "Bottled water brands are contaminated with multidrug resistant bacteria which are associated with companies handling procedures in Nairobi, Kenya", "authors": [ "Safia Adam Mohamed", "Andrew Nyerere", "Willie Kipkemboi Sang", "Musa Ngayo", "Safia Adam Mohamed", "Andrew Nyerere", "Willie Kipkemboi Sang" ], "abstract": "Background: The demand for water has necessitated the proliferation of bottled water companies in Kenya. This study evaluated if retailed bottled water in Nairobi Kenya complies with both local and international reference criteria. Methods: A total of 42 different water brands (25 approved by Kenya Revenue Authority (KRA) and 17 banned brands) were analyzed for both physicochemical and bacteriological quality. Spread plate method was used to obtain the total plate count of bacteria, while the membrane filter method was used to obtain total coliform count (TCC) and fecal coliform count (FCC). Structured interviews were used to gather company-related information. Results: Overall, 16% of KRA-approved and 35.3% of banned bottled water were contaminated with heterotrophic bacteria. Of the approved water brands, 4% were positive for total coliforms, compared with 17% of the banned brands. Similarly, 4% and 17% approved and banned water brands were positive for fecal coliforms, respectively. Escherichia coli (19.1%), Pseudomonas spp. (9.5%) and Klebsiella spp. (4.8%) were the most common bacterial types isolated from all water brands, most of which exhibited multidrug resistance. In multivariable analysis, water companies that cleaned pipework and bottles using chlorine-based disinfectants (OR 0.08, 95% CI 0.01 to 0.8), those that had food safety programs (OR 0.1, 95% CI 0.019 to 0.9), had standard operating procedures (SOP) for water sourcing (OR 0.1, 95% CI 0.012 to 0.9) and SOP for contamination protection (OR 0.1, 95% CI 0.02 to 0.9) remained independently associated with bottled water brands exceeding WHO TCC limits. Conclusions: A number of bottled water brands were contaminated with one or more types of indicator bacteria, some of which were multidrug resistant. Water bottling companies’ processes contribute to contamination; rigorous regulation and monitoring will improve on water quality and safety.", "keywords": [ "Bottled water brands", "Bacteriological quality", "multi-drug resistant bacteria", "role of water companies handling procedures" ], "content": "Introduction\n\nWater is essential for the human body and mental functions1–3 as well as for chronic disease prevention4. Water is essential for thermoregulation, protection and cushioning of body vital organs, as well as for breathing and transporting nutrients and oxygen throughout the body2. It is not surprising therefore that water constitute 50–60% of the human body2. Inevitably therefore, adequate total water intake of between 2 to 2.5 liters per day is recommended2.\n\nAchieving and maintaining good health requires the availability and consumption of clean, potable (drinkable) water. This requires that water must be devoid of pathogens, dissolved toxins, and disagreeable turbidity, odor, color and taste5. The current concerns about palatability and microbial and chemical contaminants in tap water6, have led to the proliferation in the consumption of bottled water reaching historical high accounting for billion gallons in consumption6. Bottled water offers a handy source of water for consumption both within and outside household settings. In developing countries such as Kenya, bottled water is habitually sold and consumed in hotel industries, markets places, streets, schools, and during mass gatherings such as wedding and spotting activities, workplaces, health care facilities, and emergency situations7. Unfortunately, bottled water is not always as sterile as perceived. Several reports are available showing contamination bottled water with heterotrophic bacteria and coliforms counts exceeding the national and international standards8,9. Studies have isolated various bacterial contamination from bottled water such as Vibrio cholera and Salmonella spp.10,11, Pseudomonas spp., Acinetobacter spp., Citrobacter spp. and C. violaceum9. As a result, several waterborne illnesses such as diarrhea are accountable for significant morbidity and mortality among the young and the aged as well as immunocompromised populations12,13.\n\nThe bottled drinking water in Kenya should meet the following minimum requirements: be free from pathogens and chemicals; clear (i.e. low turbidity); none saline and should not have offensive taste or smell14. The Kenyan Bureau of standard (KS EAS 153: 2014) reference criteria for packaged water requires the absence of total coliforms, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus faecalis, Shigella and Salmonella in 100 ml of water14. Microbiological parameters of drinking water can have an immediate and significant impact on human health and must therefore be analyzed frequently. Among the factors reported to influence the microbiological quality of bottled water include material of bottles, color of bottles and the length of storage15. This study investigated the bacteriological quality of bottled water and the association with the processes and handling practices of water bottling companies sold in Nairobi Kenya.\n\n\nMethods\n\nThis study was approved by Kenyatta National Hospital and University of Nairobi Ethical Review Committee (KNH-UoN ERC-P971/12/2016). Before recruitment to this study, all patients provided written informed consent for study participation.\n\nThis descriptive cross-sectional study was carried out at the Center for Microbiology Research (CMR), Kenya Medical Research Institute (KEMRI) (International Organization for Standardization [ISO] 9001:2008– certified) between February 2019 and January 2020.\n\nSample size. There are about 40 registered water bottling companies in Nairobi (http://www.businesslist.co.ke/category/bottled-water/city:nairobi). Further, the Kenya revenue authority (KRA) has listed about 369 banned water bottling companies at https://www.slideshare.net/starwebmaster/list-of-368-water-brands-banned-by-kebs. To select the bottled water samples in this study, we used the 26% failure rate of the bottled water brands in Nigeria16 to meet the United States Environmental Protection Agency (USEPA) and World Health Organization (WHO) requirement for drinking water standard of 100 total coliforms/ml water. Applying the formula for estimating the population proportion with specified relative precision described by Lemeshow et al.17, setting the α at 0.05, a total of 288 bottled water samples were collected to achieve 0.90 power. This number of bottled water samples was divided equally among the 40 brands sold in Nairobi. Therefore, a total of seven bottles per brand were sampled.\n\nData collection. At the time of the study, due to availability, 25 water brands approved by KRA were purchased from major retail outlets in Nairobi. The other 17 brands non-approved by KRA were purchased by the roadside or from small retail shops in the streets of Nairobi. All the brands (25 KRA-approved and 17 banned bottled water brands) were sought without preferential treatment of any brands or retail outlets. Seven bottles of each bottled water brand from the same batch were purchased at different retail outlets and shipped in cool box to the laboratory for microbiological analysis within 6 hours of purchasing.\n\nStructured interviews. To investigate the role of manufacturing handling and packaging process on the microbiological quality of water, randomly, this study visited the premises of all the available 25 registered and 17 banned water bottle and packaging companies located in Nairobi. Those consenting (see Extended data for the consent form18) underwent a structured 20–30-minute face-to-face discussion within the premises at secluded and secured offices to gather information including the following information: type of abstraction, pipe work materials, bottling process, staff training, policies and procedures and microbiological quality of bottled water in Nairobi adopted from the WHO/UNICEF Joint Monitoring Programme) (see Extended data for a blank copy of the survey18).\n\nThe water temperature and pH were measured immediately after purchase using the HACH Sensionþ MM150 Portable Multi-Parameter Meter (Hach Company, Loveland, CO), according to the manufacturer’s instructions.\n\nEach of the seven water samples per brand were analyzed separately. Bacterial contamination in these water samples were achieved using total plate count by the spread plate method and total coliform count and fecal coliform count by membrane filter method as described by WHO,19. Briefly, 100 mL of water samples were filtered through a 0.22-µm-pore-size membrane filter (Millipore Corp., Bedford, MA), and filters placed on membrane Fecal Coliform (m-FC) agar plates were incubated at 37 and 44°C for 18 to 24 h to determine total coliform (TC) and fecal coliform (FC) counts, respectively.\n\nThe bacteria isolates were subsequently cultured onto bile esculin agar, eosine methylene blue agar, m-endo agar les, and plate count agar. These were then identified using colony morphology, Gram’s staining, biochemical tests and further characterized using the VITEK 2 system, version 0.8.01 (bioMerieux, Inc., Hazelwood, MO).\n\nEach of the bacterial isolates were tested for susceptibility to antimicrobials by a controlled disk diffusion technique of Kirby-Bauer incubated at 35°C for 18 hours. The isolates were tested for susceptibility to the following 11 antibiotics (OXOID, England): amoxicillin (10 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (30 μg), chloramphenicol (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), doxycycline (30 μg), erythromycin (30 μg), ofloxacin (30 μg), ceftriaxone (30 μg) and kanamycin (30 μg). These tests were done according to guidelines set by the Clinical Laboratory Standards Institute20. E. coli ATCC 25922 (with known minimum inhibitory concentrations) was used as a reference strain in the disk diffusion susceptibility tests.\n\nFrequency (%), mean and standard deviation, were used to describe the qualitative and laboratory parameters. Chi-square or Fisher’s exact test were used to test for significance where applicable. The association between the presence of TCC >100 CFU/ml contaminating bottled water and companies water handling and processing characteristics were calculated using Poisson regression. Manual backward elimination method was used to reach the most parsimonious model in multivariate analysis. This included factors that were associated with contamination with TCC >100 CFU/ml at the significance level of P≤0.05. All statistical analyses were performed using STATA v13 (StataCorp LP, College Station, TX, USA).\n\n\nResults\n\nThe buying price (mean ± SD) of KRA-approved brands was slightly higher than the banned bottled water: 37.8 ± 16.95 Kenyan shillings (Kshs) versus 29.6 ± 13.32 Kshs (p = 0.0208). The temperatures (mean ± SD) for KRA approved and banned bottled water were not significantly different 16.09 ± 0.85°C versus 16.1 ± 1.21°C, respectively (p = 0.869). On the contrary, the pH (mean ± SD) of KRA approved and banned bottled water were statistically different, at 6.8 ± 0.23 versus 7.1± 0.36, respectively (p = 0.0002). The (mean ± SD per ml) total plate count, total coliform count and fecal coliform count KRA approved bottled water were found to be lower than those from KRA banned bottled water, with values of 18.5 ± 32.89 versus 56.9 ± 122.06 (p = 0.0373), 6.9 ± 14.42 versus 33.5 ± 64.37 (p = 0.0058), and 1.02 ± 3.01 versus 14.5 ± 29.51 (p = 0.0019), respectively (Table 1). Characteristics of each sample are available as Underlying data18.\n\nKshs, Kenyan Shillings\n\nBased on the WHO recommended criteria for drinking water, there were no KRA-approved bottled water brands exceeding the recommended pH limit of 6.5 to 7.5 while 35.3% banned bottled water exceeded the limit. With regards to total plate counts, there were 4 (16%) KRA-approved and 6 (35.3%) KRA-banned bottled water exceeding WHO criteria for drinking water. Similarly, on the basis of TCC and FCC there were 1 (4%) KRA approved and 3 (17.6%) KRA-banned bottled water brands exceeding WHO criteria for drinking water (Table 2).\n\nE. coli was the most common bacteria type found contaminating four (16%) different KRA-approved bottled water brands and four (23.5%) of the banned brands. Other bacteria isolated from the KRA-approved bottled water brands included Pseudomonas spp. (n=4, 16%), Enterobacter spp. (n=1, 4%), Klebsiella spp. (n=1, 4%) and Proteus spp. (n=1 (4 %). With regards to KRA banned bottled water samples apart from the E. coli, other isolated bacteria were Enterobacter spp. (n=1, 5.9%), Klebsiella spp. (n=1, 5.9%) and Aeromonas spp. (n=1, 5.9%).\n\nSusceptibility testing showed that all the bacterial isolates were resistant to at least one type of antibiotics. All the isolates were susceptible to ceftriaxone and ofloxacin. Most of the bacterial isolates 19 out of 28 (67.9%) were resistant to amoxicillin. The bacteria isolates were also resistance to erythromycin (14/28; 50%), trimethoprim-sulfamethoxazole (8/28; 28.6%), doxycycline (7/28; 25%), tetracycline (5/28; 17.9%), gentamycin (4/28; 14.3%), chloramphenicol (4/28; 14.3%), kanamycin (3/28; 10.7%) and ciprofloxacin (3/28; 10.7%).\n\nMost bacteria from KRA-banned bottled waters were resistant to gentamycin and erythromycin. The bacteria from the KRA approved brands were mostly resistant to trimethoprim-sulfamethoxazole, Amoxicillin, tetracycline and doxycycline. Most of multidrug resistance (resistant to more than three drugs) E. coli and Klebsiella spp. were from KRA-banned bottled water, while multidrug resistance Pseudomonas spp. from KRA-approved brands (Table 3).\n\nEach row represents one different bacterial isolate. GEN, gentamicin; SXT, trimethoprim-sulfamethoxazole; C, ceftriaxone; AML, amoxicillin; K, kanamycin; TE, tetracycline; OFX, ofloxacin; CIP, ciprofloxacin; E, erythromycin; DXT, doxycycline; CHLO, chloramphenicol\n\nIn multivariable analysis, bottled water brands that used chlorine-based disinfectants for cleaning pipework/tankers and bottling equipment were less likely to exceed WHO TCC limits compared to those that did not use any detergent for cleaning (OR 0.08, 95% CI 0.007 to 0.8). Companies that had food safety programs (OR 0.1, 95% CI 0.019 to 0.9), procedures for water sourcing (OR 0.1, 95% CI 0.012 to 0.9) and procedures for contamination protection (OR 0.1, 95% CI 0.02 to 0.9) (Table 4). Self-reported company details are available as Underlying data18.\n\nOR, odds ratio; CI, confidence interval; NS – not significant/done; uOR, Unadjusted OR; aOR, adjusted OR.\n\n\nDiscussion\n\nEvaluation of bacteriological quality of bottled drinking water is important and urgent in Kenya given the current upsurge of different brands of bottled water, most of which are not regulated. This study was unique and among the first in Kenya to evaluate the role of the practices used by water bottling companies in relation to the bacterial quality of water in line with the WHO acceptability criteria. This was compared between those bottled waters approved and banned brands by Kenya Revenue Authority (KRA). The bacteriological quality of bottled water from approved brands was found to be better than those of banned brands. The total coliforms and fecal coliform present in 100 ml of water were detected cumulatively in 4/42 (9.5%) of all brands, and in 4% of KRA-approved and 17.6% of banned bottled water brands. The proportion of bottle water brands unacceptable in line with WHO limits were lower than the 50% reported in Bangladesh21, 37.5% in India22, 26% reported in Nigeria16 and 25% in Nepal9. On the contrary, the proportion of unacceptable bottled water in our study was higher than the 4.6% reported in Tanzania23 the 9% in Sri Lanka24 and 0% reported in Saudi Arabia25. Although KRA approval is based on tax payment rather than on scientific basis, the high number of KRA-banned bottled water brands points to the possibilities of ineffectiveness of the disinfection processes used in these brands. In a process likely to be mainly for financial benefit by the bottled water manufacturers, studies have cited the improper practice of filling the bottle directly from tap water and sealing it without any prior treatment as among the reasons responsible for higher brands of bottled water beyond the acceptable limits of bacteriological quality9. Longer storage periods, especially of already-contaminated bottled water, have been shown to worsen the bacteriological quality. As in many developing countries, the laxity by the government body responsible for monitoring the quality of bottled water has been shown to account for higher levels of unacceptable bottled water brands9.\n\nWith regards to total plate count or heterotrophic bacteria, in this study, a total of 10 (n=42; 23.8%) of the bottled water brands (40% KRA-approved and 60% banned brands) were contaminated. In other settings, higher percentages of between 20% to 100% of heterotrophic bacteria contamination of bottled drinking water9,26,27. Studies have associated long storage duration with high levels of bacterial concentration mainly due to larger surface area for growth, higher temperature, and the nutrients arising in the container28. This quantity of heterotrophic bacteria is shown to correlate with water pH. There were 35.5% of banned bottled water brands with a pH below the pH 6.5 minimum level recommended by WHO, which could be accounted for the higher numbers of heterotrophic bacteria per milliliter detected in these brands. Similar results were also reported by Pant et al.9.\n\nThe presence of total coliforms and fecal coliforms in 4% KRA-approved and 17.6% KRA-banned bottled water brands exceeding WHO criteria is similar to that observed by other studies23,29. This is worrying and a pointer to either poor water processing, introducing flakes of human skin or indigenously acquired by filling the bottles directly from the natural sources or taps23.\n\nE. coli (in 19.1%) was the most common bacteria found contaminating the bottled water brands. Others included Pseudomonas spp. (9.5%), Enterobacter spp. (4.8%), Klebsiella spp. (4.8%) and Proteus spp. (2.3%) and Aeromonas spp. (2.3%). In Nepal, Pant et al.9 isolated more of Pseudomonas spp. (87.5 %) and Acinetobacter spp. (87.5 %). In Iran Momtaz et al.,10 isolated E. coli, while in Brazil, Vasconcellos et al.30 isolated Salmonella spp., and V. cholerae from bottled water.\n\nAlthough all bacteria in our investigation were susceptible to ceftriaxone and ofloxacin, resistance to erythromycin, trimethoprim-sulfamethoxazole, doxycycline, tetracycline, gentamycin, chloramphenicol, kanamycin and ciprofloxacin were noted. Multidrug resistance (resistant to more than three drugs) in E. coli, Klebsiella spp. and Pseudomonas spp. from all bottle brands were also detected. The presence of different species of bacteria, including multidrug resistant strains, in supposedly bacteria-free bottled water is of important public health problem. Pathogenicity notwithstanding, their presence in bottled waters heavily consumed by those including the elderly, children and the immunocompromised, the hazards of contamination, and health risks to consumers should not be taken for granted10,30.\n\nThis study incorporated a unique feature by investigating the manufacturing practices potentially associated with contamination of bottled water. In multivariable analysis, companies that used chlorine-based disinfectants for cleaning pipework/tankers and bottling equipment were less likely to have water brands exceeding WHO TCC limits compared to that that did not use any detergent for cleaning. Zamberlan et al.31 showed the importance of disinfection processes used by the water bottling companies as playing a key determinant of bacterial concentration in bottled water. In Nepal, Pant et al.9 showed that failure to disinfect water represents an important avenue for bacterial entry and colonization of water processing systems. Our study further showed that companies that had food safety programs, procedures for water sourcing and procedures for contamination protection were less likely to have unacceptable bottled waters. As expected, if companies are set up in line with guidelines set by regulatory authorities, then the end product will be devoid or have a reduced microbial contamination. In this study, although more of companies producing approved brands had recommended water collection and transportation systems, water treatment procedures (filtration, UV and chlorination and reverse osmosis), packaged water using polycarbonate containers, used machine during bottling process, had batch tracing system, routinely tested their products according to the WHO guidelines and having recommended handling standard procedures than banned ones, these were not factors associated with the contamination of bottled water. To the best of our knowledge, this study is among the first to investigate the possible role of water company procedures and operations associated with to water contamination in Kenya.\n\nOur study had some limitations. First, due to limited resources available, the study could not process large enough numbers of the samples to include all brands sold in the country. Second, owing to the limited laboratory methods used, we were not able to identify all the potential pathogens that contaminated the water, including other pathogenic bacteria, viruses, fungi, and parasites. Third, the cross-sectional nature of our study only allowed us to describe associations between water company processes and procedures and bacterial quality and not a causal conclusion. Such outcomes can be confirmed in a longitudinal study. These limitations notwithstanding, one of the key outcomes of this investigation is the capacity to show that the perceived safe bottled water brands, including the top-selling and most expensive brands in Kenya, could be contaminated with bacteria beyond the WHO recommended limits. Additionally, some of these bacteria associated with significant disease outbreaks were multidrug-resistant. The study also showed that water bottling companies’ operations and processes are key avenue for bacterial water contamination. The Kenya Bureau of Standards is the Kenyan regulatory and monitoring authority for all water and packaged foods. Our results may suggest, however, that concerted efforts must be made to improve the ability of national governments to properly regulate and monitor these products which has been shown to improve product quality and safety32,33.\n\n\nData availability\n\nFigshare: Bottled water brands are contaminated with multidrug resistant bacteria which are associated with companies handling procedures in Nairobi Kenya. https://doi.org/10.6084/m9.figshare.13046534.v218\n\nThis project contains the following underlying data:\n\nSafia Company Response F1000R.xlsx. (Company responses to each question of the survey.)\n\nSafia Water property F1000 Data 1.xlsx. (Properties of each bottled water sample analyzed in this study.)\n\nFigshare: Bottled water brands are contaminated with multidrug resistant bacteria which are associated with companies handling procedures in Nairobi Kenya. https://doi.org/10.6084/m9.figshare.13046534.v218\n\nThis project contains the following extended data:\n\nSafia F1000_Consent.docx. (Informed consent form.)\n\nSafia F1000 Interview guide.docx. (Survey used in the present study.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgement\n\nWe would like to thank the study participants (heads of water bottling companies) who shared with us their operations and experience. We wish to acknowledge the Director KEMRI, all the staff of the CMR, Nairobi, Kenya.\n\n\nReferences\n\nPopkin BM, D’Anci KE, Rosenberg IH: Water, hydration, and health. Nutr Rev. 2010; 68(8): 439–458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEFSA Panel on Dietetic Products Nutrition and Allergies (NDA): Scientific Opinion on the substantiation of health claims related to water and maintenance of normal physical and cognitive functions (ID 1102, 1209, 1294, 1331), maintenance of normal thermoregulation (ID 1208) and “basic requirement of all living things. EFSA J. 2011; 9(4): 2075. Publisher Full Text\n\nPross N, Demazières A, Girard N, et al.: Influence of progressive fluid restriction on mood and physiological markers of dehydration in women. Br J Nutr. 2013; 109(2): 313–321. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuckelbauer R, Sarganas G, Grüneis A, et al.: Association between water consumption and body weight outcomes: A systematic review. Am J Clin Nutr. 2013; 98(2): 282–299. PubMed Abstract \| Publisher Full Text\n\nGazan R, Sondey J, Maillot M, et al.: Drinking Water Intake Is Associated with Higher Diet Quality among French Adults. Nutrients. 2016; 8(11): 689. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKohnen W, Teske-Keiser S, Meyer HG, et al.: Microbiological quality of carbonated drinking water produced with in-home carbonation system. Int J Hyg Environ Health. 2005; 208(5): 415–423. PubMed Abstract \| Publisher Full Text\n\nWilliams AR, Bain RE, Fisher MB, et al.: A Systematic Review and Meta-Analysis of Fecal Contamination and Inadequate Treatment of Packaged Water. PLoS One. 2015; 10(10): e0140899. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSemerjian LA: Quality assessment of various bottled waters marketed in Lebanon. Environ Monit Asses. 2011; 172(1–4): 274–85. PubMed Abstract \| Publisher Full Text\n\nPant ND, Poudyal N, Bhattacharya SK: Bacteriological quality of bottled drinking water versus municipal tap water in Dharan municipality, Nepal. J Health Popul Nutr. 2016; 35(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMomtaz H, Dehkordi FS, Rahimi E, et al.: Detection of Escherichia coli, Salmonella species, and Vibrio cholerae in tap water and bottled drinking water in Isfahan, Iran. BMC Public Health. 2013; 13: 556. Published 2013 Jun 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeuret C, Kohler D, Baumgartner A, et al.: Norwalk-LikeVirus Sequences in Minelral Waters: One-Year Monitoring of Three Brands. Appl Environ Microbiol. 2002; 68(4): 1925–1931. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO, UNICEF: Progress on drinking water and sanitation: 2015 update and MDG assessment. 2015. Reference Source\n\nWHO: Mortality and burden of disease from water and sanitation. 2018; Accessed January, 2020. Reference Source\n\nKenya Bureau of Standards: Kenya standard Potable water — Specification. 2015; accessed on 15th November, 2019. Reference Source\n\nAkond MA, Alam S, Shil A, et al.: Bacteriological quality of bottled water available commercially in Bangladesh. Journal of Environmental Sciences (Dhaka). 2006; 4: 47–52. Reference Source\n\nIgbeneghu OA, Lamikanra A: The bacteriological quality of different brands of bottled water available to consumers in Ile-Ife, south-western Nigeria. BMC Res Notes. 2014; 7: 859. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLemeshow S, Hosmer DW Jr, Klar J: Sample size requirements for studies estimating odds ratios or relative risks. Stat Med. 1988; 7(7): 759–764. PubMed Abstract \| Publisher Full Text\n\nMohamed SA, Nyerere AS, Willie K, et al.: Bottled water brands are contaminated with multidrug resistant bacteria which are associated with companies handling procedures in Nairobi Kenya. figshare. Dataset 2020. http://www.doi.org/10.6084/m9.figshare.13046534.v2\n\nWorld Health Organization: Guidelines for drinking-water quality. 4th ed. Geneva: WHO; 2011; accessed January 2020. Reference Source\n\nClinical and Laboratory Standard Institute (CLSI): Performance Standards for Antimicrobial Disk Susceptibility Tests. 13th ed. Wayne, Pennsylvania 19087 USA, 2018. Reference Source\n\nIslam S, Begum HA, Nili NY: Bacteriological safety assessment of municipal tap water and quality of bottle water in Dhaka city: health hazard analysis. Bangladesh J Med Microbiol. 2010; 4(1): 9–13. Publisher Full Text\n\nJoseph N, Bhat S, Mahapatra S, et al.: Bacteriological Assessment of Bottled Drinking Water Available at Major Transit Places in Mangalore City of South India. J Environ Public Health. 2018; 2018: 7472097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKassenga GR: The health-related microbiological quality of bottled drinking water sold in Dares Salaam, Tanzania. J Water Health. 2007; 5(1): 179–85. PubMed Abstract \| Publisher Full Text\n\nSasikaran S, Sritharan K, Balakumar S, et al.: Physical, chemical and microbial analysis of bottled drinking water. Ceylon Med J. 2012; 57(3): 111–116. PubMed Abstract \| Publisher Full Text\n\nShahaby AF, Alharthi AA, El Tarras AL: Bacteriological Evaluation of Tap Water and Bottled Mineral Water in Taif, Western Saudi Arabia. Int J Curr Microbiol App Sci. 2015; 4(12): 600–615. Reference Source\n\nKhaniki GRJ, Zarei A, Kamkar A, et al.: Bacteriological evaluation of bottled water from domestic brands in Tehran market, Iran. World Appl Sci J. 2010; 8(3): 274–8. Reference Source\n\nMajumder AK, Islam KN, Nite RN, et al.: Evaluation of microbiological quality of commercially available bottled water in the city of Dhaka, Bangladesh. Stamford J Microbiol. 2011; 1(1): 24–30. Publisher Full Text\n\nWarburton DW: The microbiological safety of bottled waters. In: Farber JM, Ewen ED T, editors. Safe handling of foods. New York: Marcel Dekker; 2000.\n\nYasin N, Shah N, Khan J, et al.: Bacteriological status of drinking water in the peri-urban areas of Rawalpindi and Islamabad-Pakistan. Afr J Microbiol Res. 2012; 6(1): 169–75. Reference Source\n\nVasconcellos L, Medeiros V, Rosas C, et al.: Occurrence of total coliforms, Escherichia coli and Cronobacter species in commercially available 20 l bottled drinking water sold in Rio de Janeiro State, Brazil. Applied Microbiology. 2019; 69(6): 431–437. Publisher Full Text\n\nZamberlan da Silva ME, Santana RG, Guilhermetti M, et al.: Comparison of the bacteriological quality of tap water and bottled mineral water. Int J Hyg Environ Health. 2008; 211(5–6): 504–9. PubMed Abstract \| Publisher Full Text\n\nDada AC: Packaged water: optimizing local processes for sustainable water delivery in developing nations. Global Health. 2011; 7: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStoler J, Weeks JR, Fink G: Sachet drinking water in Ghana’s Accra-Tema metropolitan area: Past, present, and future. J Water Sanit Hyg Dev. 2012; 2(4): 223–40. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75673", "date": "20 Jan 2021", "name": "Ahmad Zarei", "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 discusses Bottled water brands contaminated with multidrug resistant bacteria in Nairobi, Kenya. Water is essential for the human body and mental functions. This requires that water must be devoid of pathogens, dissolved toxins, and disagreeable turbidity, odor, color and taste. In my opinion, it is a good article to be accepted for indexing in F1000Research only after doing the following comments:\nComments:\nThe English language of the paper should be improved.\n\nFor the paper title I propose use “Contamination of bottled water brands with multidrug resistant bacteria in Nairobi, Kenya” instead.\n\nI think it is better to remove “role of water companies handling procedures” from “Keywords”.\n\nReference 17 is before 1998. Please replace with a newer one (if possible).\n\nDid the authors consider the production date of the bottled water brands? If yes, how many bottles with different production dates were analyzed?\n\nIn Introduction. The authors can use the following paper:\n\nChemical and microbial quality of bottled drinking water in Gonabad city, Iran: Effect of time and storage conditions on microbial quality of bottled waters\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": "6381", "date": "03 Mar 2021", "name": "Musa Ngayo", "role": "Author Response", "response": "Response to reviewer’s comments Dr. Ahmad Zarei Comments: Comment: The English language of the paper should be improved. Respond: The paper has been re-read and the language moderated. We would that the reviewer points out exactly which section or how they would prefer the paper English language changed. Comment: For the paper title I propose to use “Contamination of bottled water brands with multidrug-resistant bacteria in Nairobi, Kenya” instead. Respond: The title suggestion has been adopted Comment: I think it is better to remove “role of water companies handling procedures” from “Keywords”. Respond: The key outcome of the paper actually addresses the role of water companies handling procedures on contamination of drinking water. We believe is key to this paper Comment: Reference 17 is before 1998. Please replace it with a newer one (if possible). Response: This reference by Lemeshow S, Hosmer DW Jr, Klar J: Sample size requirements for studies estimating odds ratios or relative risks. Stat Med. 1988;7(7):759–764. 3406603 10.1002/sim.4780070705 is still easily available online and describes sample size calculations for many scenarios including the formula adopted in this study. We are unable to access new reference Comment: Did the authors consider the production date of the bottled water brands? If yes, how many bottles with different production dates were analyzed? Response: All bottled water brands were of the same date and batch. Comment: In Introduction. The authors can use the following paper: Response: We have read the suggested paper but of the opinion that the introduction is adequate unless the reviewer has specific issues to be addressed" } ] }, { "id": "74869", "date": "12 Feb 2021", "name": "Lucy A. Semerjian", "expertise": [ "Reviewer Expertise Water quality", "water and wastewater treatment and reuse", "emerging contaminants", "PPCPs", "environmental pollution control" ], "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 research study investigates the microbial quality of selected bottled waters marketed in Kenya as well displays survey results from water bottling companies. The title needs revising as it is as a conclusion in its current language. The study is a basic investigation of microbial contamination in bottled water, drug resistance patterns of encountered microbes, and data collection on practices and processes of water bottling companies. In the Methods section, the term \"patients\" is invalid and needs revising as the study is not related to patients. The Sections \"Results\" and \"Discussion\" can be combined and restructured to avoid repetition of ideas and findings. Statistical analysis and conclusions could be more detailed and justified to give the research more scientific value.\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?\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": "6382", "date": "03 Mar 2021", "name": "Musa Ngayo", "role": "Author Response", "response": "Reviewer Prof. Lucy A. Semerjian The research study investigates the microbial quality of selected bottled waters marketed in Kenya as well displays survey results from water bottling companies. Comment: The title needs revising as it is as a conclusion in its current language. Response: title changed to read. Contamination of bottled water brands with multidrug-resistant bacteria in Nairobi, Kenya. Comment: The study is a basic investigation of microbial contamination in bottled water, drug resistance patterns of encountered microbes, and data collection on practices and processes of water bottling companies. Response: This is true Comment: In the Methods section, the term \"patients\" is invalid and needs revising as the study is not related to patients. Respond: The term patients dropped and replaced with study participants Comment: The Sections” Results\" and \"Discussion\" can be combined and restructured to avoid repetition of ideas and findings. Respond: The section maintained as separate but modified to minimize repeating results in the discussion section Comment: Statistical analysis and conclusions could be more detailed and justified to give the research more scientific value. Response: These sections have been clarified and improved where necessary" } ] } ]	1	https://f1000research.com/articles/9-1337
https://f1000research.com/articles/10-174/v1	03 Mar 21	{ "type": "Research Article", "title": "Medical negligence in healthcare organizations and its impact on patient safety and public health: a bibliometric study", "authors": [ "Saad Dahlawi", "Ritesh G Menezes", "Mohammad Ajmal Khan", "Abu Waris", "- Saifullah", "Mirza Muhammad Naseer", "Ritesh G Menezes", "Mohammad Ajmal Khan", "Abu Waris", "- Saifullah", "Mirza Muhammad Naseer" ], "abstract": "Background: Medical negligence is an increasing public health concern among healthcare providers worldwide as it affects patient safety. It poses a significant risk of patient injury, disease, disability, or death. The WHO has recognized deficiencies in patient safety as a global healthcare issue to be addressed. This study aimed to analyze various components of medical negligence research literature. Methods: Bibliographic data visualizations tools like Biblioshiny (RStudio) and VOSviewer were used besides MS Excel to examine the types of documents, annual scientific production, top contributing authors and their impact, authorship patterns and collaboration, top contributing countries and organizations, most significant sources of publication, most cited documents, and most frequently used keywords. Bibliometric methods were used to analyze the bibliographic records of research output on medical negligence downloaded from the Web of Science Core Collection. Results: The annual productivity of medical negligence research was increasing gradually. The most productive period for medical negligence research was 2011-2020. Bird contributed the highest number of publications to medical negligence literature while Brennan emerged as the highly cited author. Single-authored publications on medical negligence were not highly cited. The United States was the highest contributing country and the University of South Florida was the highest contributing organization while Harvard University was a highly cited organization. Nine out of the top ten contributing organizations were academic institutions and most of them belonged to the United States. The most important sources of publication on this topic were The Lancet and British Medical Journal. Localio et al. was the most important research article on medical negligence research. Conclusion: Due to increasing attention on this topic, there was a sharp increase in the research output on medical negligence. This is of significance as the WHO set in motion a patient safety program almost two decades ago.", "keywords": [ "Medical negligence", "medical malpractice", "medical errors", "patient safety", "bibliometric", "scientometric" ], "content": "Introduction\n\nMedical negligence (also known as medical malpractice, medical errors, tort system) is an increasing public health concern among healthcare providers worldwide. The most comprehensive definition is “an act of omission or commission in planning or execution that contributes or could contribute to an unintended result” (Grober & Bohnen, 2005; Thavarajah, Saranya & Priya, 2019). Medical negligence occurs when a healthcare professional selects the wrong method or procedure or improperly executes an appropriate method to treat or diagnose the patient (Chukwuneke, 2015). There is no clear definition of medical negligence due to lack of nomenclature, overlapping of definitions, and lack of a standardized method to measure it (Rodziewicz, Houseman & Hipskind, 2020).\n\nAll clinical practitioners and healthcare providers (e.g. physicians, nurses, medical technicians, paramedics, and other healthcare professionals) are responsible for any mistakes that could lead to medical negligence. There are several areas where medical negligence can arise, such as technical errors during surgical procedures, misdiagnosis of the disease, or prescribing the wrong medicine or incorrect dose (Tariq, Vashisht, Sinha & Scherbak, 2020). These practices pose a significant risk of patient injury, disease, disability, or death. Subsequently, it may give rise to criminal and financial liabilities on hospitals and healthcare institutions (He et al., 2015; Ramanathan, 2014). Medical negligence lawsuits are focused on the medical professional's damage, injury, or failure to the patient. In general, medical negligence relief is given by means of penalties, i.e. monetary compensation (Cheluvappa & Selvendran, 2020; Tumelty, 2020).\n\nIt is not easy to estimate the annual cost of liabilities and compensations on hospitals and public healthcare organizations. However, many studies show that this could be in billions of dollars per year. A study by the National Health Service in the United Kingdom estimated that the annual cost is around $1.20 billion (Mathew, Asimacopoulos & Valentine, 2011). Medical negligence has been recognized for a long time by many researchers from different backgrounds. Several previous studies focused on the economic burden of medical negligence either on clinical practitioners as individuals or healthcare organizations as a management system. However, due to the complicity of this issue, it is not easy to estimate the exact cost of liabilities and compensations on doctors, hospitals, and healthcare organizations (Mello, Chandra, Gawande, & Studdert, 2010).\n\nThe patient or the claimant has the right to file a lawsuit against clinicians by proving the following: the clinician owes a duty of care, there was a breach of that duty, and that breach caused the injury or damage (Beran, Devereaux & Buchanan, 2020; Connelly & Serpell, 2020; Phillips, Thorne, Casey & Russo, 2021). Many previous studies focus on the estimation of annual cost and the financial liabilities on both the public and the private healthcare systems. The cost is not only the direct monetary expense that the doctors must pay but it also includes indirect costs such as physician’s time, stress, and loss of reputation (Albano et al., 2019). Furthermore, Wilson et al. (1995) reported that about 16% of 14000 hospitalization cases in Australia resulted in adverse disability due to medical negligence with legal implications. Moreover, doctors and medical professionals face lawsuits due to the cases filed against them due to negligen.\n\nA study in Wuhan city in China performed 519 autopsies between 2004 and 2013 to evaluate medical negligence. The study showed that 36.6% of the death cases were due to medical malpractice (He et al., 2015). Every year, thousands of cases are filed in the courts against healthcare professionals due to tort cases (Sohn, 2013). Despite the high occurrence of these cases, medical negligence is claimed to be under-reported in most healthcare settings (Wu, 2000). It is, therefore, difficult to provide accurate statistics about medical negligence cases due to difficulties in analyzing and evaluating such type of data (Jena, Seabury, Lakdawalla & Chandra, 2011; Rodziewicz, Houseman & Hipskind, 2020). There are many reasons for the limited availability of data related to medical negligence since not all hospitals have a clear policy for reporting every single medical error during routine medical procedures. Moreover, patients suffering from medical negligence may recover from damage and therefore may not be considered a medical negligence case thereafter.\n\nAs a result of criminal and financial liabilities arising due to medical negligence and the increasing demand to improve patient safety and quality care, there is an increased international focus on improving patient outcomes, safety, and quality of care that has led stakeholders, policymakers, and healthcare organizations to adopt standardized processes for evaluating healthcare organizations. Hospitals and healthcare organizations are now adopting standardized processes and an international accreditation system (Alkhenizan & Shawb, 2011). The accreditation and certification system provide recommended guidelines and international standards to improve healthcare and patient safety in hospitals. The result is certification by an independent external auditor. Despite the national and international strategies for pushing hospitals and healthcare centers to be certified by recognized accreditation bodies, patient safety remains below the acceptable levels. Many studies proved that the effectiveness of such accreditation and certification is limited. Brubakk et al. (2015) claim that accreditation has little effect on patient outcomes, organizational culture, and reliability. Many other researchers argue that there was no convincing evidence on improving output quality and patient safety due to accreditation and certification (Grepperud, 2015; Bogh, et al., 2017).\n\nNevertheless, it is challenging to provide consistent solutions to eliminate or minimize recurrent events and work toward improving patient safety (Oyebode 2013). Furthermore, it is essential that the governing bodies for the healthcare system should enforce hospitals to establish a litigation system by providing guidelines and steps to resolve the matter either by out of court settlement or a full court trial. This system should include effective policy and procedure to ensure high standards of effectiveness, transparency, and justice for all the involved parties (Alkhenizan & Shafiq, 2018).\n\nThis research paper aimed to summarize the previous research done in this area and to determine the existing practice to control such issues. The trends of previously published research on this topic have been highlighted by emphasizing highly cited authors, international collaboration, keywords used, and analysis of future trends. Although several review articles on medical negligence have been published that summarize previous work (Epstein, 2020; Connelly and Serpell, 2020; Birch and Todd, 2020), no bibliometric study has been published to date to analyze the research conducted in this field. In this study, a thorough evaluation of previously published literature on medical negligence and tort cases was conducted. Research output published in Web of Science were retrieved and analyzed to classify and determine the next steps and find out research gaps.\n\nThe bibliometric study is a quantitative analysis and statistical assessment to analyze the published articles using different parameters such as the leading authors and co-authors, keywords co-citations, document co-citations, institutes performance, international collaboration, etc. There is a notable growth trend in publication output along with more participation and collaboration of countries and institutes. The purpose of this type of analysis is to focus on the emerging trends and the knowledge structure on a topic. Using bibliometric tools, it is possible to generate easy to follow visual representations of complex correlations. This article provides a clear overview and general trends of research conducted on medical negligence over the last 67 years. It will highlight the highly cited publications and classify the existing literature into groups and clusters based on the latest developments and future trends.\n\nThe present study aimed to fulfill the following objectives relating to medical negligence research:\n\nTo determine the types of documents\n\nTo know the annual scientific production\n\nTo find the top contributing authors and their impact\n\nTo examine authorship and collaboration patterns\n\nTo recognize top contributing countries and organizations\n\nTo identify the most relevant sources of publication\n\nTo discover the most cited documents\n\nTo detect the most frequently used keywords\n\n\nMethods\n\nThe bibliographic records of research output on medical negligence research were downloaded from the Web of Science Core Collection (WOSCC) using the e-resources portal of Imam Abdulrahman Bin Faisal University (IAU). Web of Science (WOS) has been recognized as the most accurate and consistent indexing and abstracting database used by researchers worldwide and it has comprehensive coverage (Birkle, et al., 2020; Khan et al., 2020; Tahira, Alias & Bakri, 2013). Data were downloaded on October 25th, 2020 using WOSCC category topic search (TS) with the following query:\n\nTS= (\"medical negligence\") Refined by: [excluding] DOCUMENT TYPES: (NEWS ITEM OR NOTE OR MEETING ABSTRACT OR CORRECTION) Timespan: All years. Indexes: SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC.\n\nThe present study was limited to publications on “medical negligence” indexed in the WOS database only; no other databases were used for bibliographic data. Therefore, the results of this study should be considered keeping in view the limitations of the study.\n\nAs compared to other search queries, we found the highest number of records for download with the search query above without applying any filter for time limit, country, or language. The total number of records downloaded and analyzed was 464. All publications relating to medical negligence were selected without any filter. Data were screened for duplication through Endnote Desktop X8 with matching options title, author, and year, which found zero duplicate records.\n\nBibliometric methods were applied for the data analysis. Variables for which data analysis and visualization were performed included the following:\n\nAnnual scientific production: Number of scientific publications produced in a year.\n\nTop contributing authors and their impact: Authors who contributed the most in the field of study and impact of their research in the field in terms of citations received.\n\nAuthorship and collaboration patterns: Pattern of working of authors and how they collaborate with others to conduct the studies.\n\nTop contributing countries and organizations: Countries and organizations who contributed the most in the field of study.\n\nMost relevant sources of publication: Journals or sources where maximum number of documents were published relating to the field of study.\n\nMost cited documents: Documents which received highest number of citations.\n\nTypes of documents: Forms of output of the documents like article, conference proceedings paper, review paper etc.\n\nFrequently used keywords: Keywords which were used more frequently by the authors.\n\nBibliographic data analysis and visualization tools Biblioshiny (RStudio, Version 1.2.5033) and VOSviewer (Version 1.6.13) were used in addition to MS Excel. Biblioshiny was used to determine the annual scientific production, top contributing authors and their impact, top contributing countries and organizations, most relevant sources of publication, most cited documents, and types of documents. MS Excel was used to determine the authorship and collaboration patterns while VOSviewer was used to visualize the frequently used keywords in medical negligence research.\n\n\nResults and discussion\n\nTable 1 shows that the total number of documents was 464, out of which 304 documents (65.52%) were research articles, 66 documents (14.23%) reviews, and 49 documents (10.56%) were editorials. Research articles obtained the highest global citations with 3,374 citations (87.66%); reviews received 343 (8.92%) citations, and editorials received 108 (2.81%) citations. Overall, most of the documents were published as articles, which received more citations as compared to the other types of publications on the research topic.\n\nOur results cover medical negligence research for 67 years. Table 2 displays the annual scientific productivity and citations per document on medical negligence research, and shows that annual productivity of medical negligence research has increased gradually. Research output was very low in the beginning with only nine research papers published from 1954 to 1980 with an accumulated percentage of 1.94%. Documents published from 1954 to 1980 did not receive any citations. However, research productivity significantly increased in the last two decades (2001-2020). From 2001 to 2010, 118 research documents were published, with an accumulated percentage of 25.43%. These publications received 9.09 citations per document. In the last decade (2011-2020), 216 research documents were published with an accumulated percentage of 46.55%, which received 3.70 citations per document. This shows that the most productive period for medical negligence research was 2011-2020, with about half of the total research output. Citation analysis showed that 70 documents were published on medical negligence research in 1991-2000, which received the highest number of citations (1,915) at a rate of 27.36 citations per document. Data revealed that no document was published in the years 1955 to 1957, 1959 to 1961, 1963 to 1975, and 1977 to 1978.\n\nAnalysis of authors’ productivity revealed contributions from 974 authors to research on medical negligence. Table 3 presents the top ten authors who contributed to research on medical negligence along with their productivity. The analysis revealed that Bird S. (from MDA National, Sydney, Australia) contributed the highest number of documents (nine) to medical negligence research but received only 18 citations, with a citation impact of two. Samuels A. (from University of Southampton, United Kingdom) contributed five publications and received only three citations, while Brahams D. (from Lincoln's Inn, London, United Kingdom) contributed four publications and received only one citation. It demonstrates that most productive authors in the field of medical negligence are not highly cited. Brennan T. A. (from CVS, Woonsocket, United States) contributed only three publications but received the highest number of citations (754) with a citation impact of 251 per paper. Brennan was followed by Studdert D. M. and Fenn P. who contributed four documents each and received 275 and 86 citations, respectively.\n\nFigure 1 describes the authorship pattern of medical negligence research. For authorship pattern, frequency of collaborating authors for each publication was analyzed, which ranged from a single author to 13 authors. There were 228 single-author publications, which obtained 724 citations while 86 publications were written by two authors and obtained 899 citations. It was revealed that single-authored publications on medical negligence were not highly cited. There were 55 publications with three authors and 34 publications with four authors, which obtained 771 and 150 citations, respectively. The frequencies of publications contributed by seven or more authors remained in single digits. Overall, the authorship pattern showed that most publications on medical negligence research (51%) were contributed by more than one author, which showed that authors contributing to medical negligence research were inclined towards collaborative research.\n\nCountry-wise analysis showed that 51 countries contributed 464 medical negligence research. The top ten contributing countries have been presented in Table 4. The United States was the highest contributing country with 96 occurrences. The publications affiliated with the United States obtained 1,735 citations, having a citation impact of 18.07 per paper. The United Kingdom was in second place with 83 occurrences and 625 citations, with a citation impact of 7.53 per paper. Australia was in the third position with 210 occurrences and 5.83 citation impact. Italy contributed only 17 documents but attained a good number of citations (119) and citation impact (7.00). Contributions from Japan and the Netherlands were in single digits, but the Netherlands obtained 77 citations with a citation impact of 12.83, which was the second-highest citation impact after the United States.\n\nResults of some previous studies were similar to this study, which exhibited that the United States was the most productive country in different areas of research, such as like Carpal Tunnel Syndrome, Coronavirus, Middle East Respiratory Syndrome (MERS), m-health, Sudden Infant Death Syndrome, and regulatory T-cell (Bonilla-Aldana et al., 2020; Danesh & Ghavidel, 2020; Menezes et al., 2020; Ram, 2019; Ram, 2020; Sweileh et al., 2017; Wang et al., 2016; Zongyi, et al., 2016; Zyoud, 2016).\n\nAnalysis for organizations that participated in research on medical negligence revealed that 546 organizations contributed to the literature on the subject. In Table 5, the top ten contributing organizations have been presented along with their publications, citations, and citation impact. University of South Florida (United States) was the highest contributing organization with 16 publications, 58 citations, and 3.63 citation impact. Harvard University (United States) and the University of Melbourne (Australia) ranked second, each contributing nine publications. Harvard University obtained the highest number of citations (762) with 84.67 citation impact. This indicates that the most contributing organization was not widely cited in the area of medical negligence research. University of Padua (Italy) and Queensland University of Technology (Australia) ranked fourth, each contributing seven publications, but the University of Padua received more citations (29 with 4.14 citation impact) as compared to the Queensland University of Technology, which received only six citations with 0.86 citation impact. Other organizations included in the top ten contributed six publications each.\n\nOur analysis showed that nine out of the top ten contributing organizations were academic institutions and most of them (six) were based in the United States, which also confirmed the results of the country-wise analysis. Analogous results were exhibited during earlier studies, which found that universities contributed much of the research related to MERS, coronavirus, and regulatory T-cells (Danesh & Ghavidel, 2020; Ram, 2020; Wang et al., 2016; Zongyi, Dongying & Baifeng, 2016).\n\nPublication source analysis disclosed that 464 documents on medical negligence research were published in 274 sources. Most of the documents were published in journals. In Table 6, the top 10 sources of publication have been presented. The top six sources published medical negligence literature in double-digits while less than ten documents were published by the remaining sources. The most important sources of publication for medical negligence research were The Lancet and British Medical Journal, which published 23 documents each and received 439 and 198 citations, respectively. These were followed by Medicine Science and The Law with 16 publications, which received nine citations. Cardiology in The Young, Australian Family Practitioner, and Trial contributed 10 publications each with 112 citations, 21 citations, and one citation, respectively.\n\nThe results of this study found that the most important sources of publication for medical negligence research were The Lancet and British Medical Journal. Some earlier studies discovered that the most favored journals for research on Carpal Tunnel Syndrome, Coronavirus, and MERS were the Journal of Hand Surgery-American Volume, and Journal of Virology, respectively (Ram, 2019; Ram, 2020; Wang et al., 2016; Zyoud, 2016).\n\nIn order to determine the status of research publications and the efficiency of researchers in any area of research, citations are a tool for comparative analysis of publications. The number of citations received specifies the standing of any publication in its field (Ram, 2020). Table 7 presents the name of the first author, year of publication, and source of publication for the ten most cited research publications on medical negligence research. The study revealed that the research paper titled “Relation between Malpractice Claims and Adverse Events Due to Negligence — Results of the Harvard Medical Practice Study III” by Localio et al. published in The New England Journal of Medicine (doi:10.1056/NEJM199107253250405) was the most important research article on medical negligence research, which obtained 521 citations at a rate of 17.37 citations per year. The article titled “Why do people sue doctors? A study of patients and relatives taking legal action” by Vincent et al., published in The Lancet (doi:10.1016/s0140-6736(94)93062-7) was the second most important research paper, which received 396 citations at a rate of 14.67 citations per year. The article titled “Assessments of Noneconomic Damage Awards in Medical Negligence: A Comparison of Jurors with Legal Professionals” by Vidmar, N. and Rice, Jeffrey J., published in Iowa Law Rev, volume 78, number 4, 1993, pp. 883-912, gained 48 citations at a rate of 1.71 citations per year.\n\nFigure 2 displays the author keyword co-occurrence analysis of 798 keywords on medical negligence research using the full counting method. A threshold occurrence value of two was set for the analysis. In total, 92 keywords met the threshold value, but some items were not connected to each other. The largest set of connected items consisted of 85 items having ten clusters identified by different colors in Figure 2. The analysis revealed that 85 items, having 10 clusters, generated 279 links while the total link strength was 817. Most commonly keywords used by authors were medical negligence, malpractice, negligence, litigation, and patient safety.\n\n\nConclusion\n\nBased on the findings of this study, it can be understood that medical negligence research is published in various types of sources and a variety of output formats. We can conclude that research on medical negligence is getting the attention of the researchers, which has resulted in a sharp increase in the research output during the last two decades. This is of significance as the WHO set in motion a patient safety program almost two decades ago in the year 2004 recognizing deficiencies in patient safety as a global healthcare issue to be addressed (WHO, 2004). Research on medical negligence is mostly concentrated in developed countries and contributing authors are inclined towards collaborative research. The study concludes that the accumulation of citations does not depend on the productivity of an author. It is recommended to replicate this study after ten years to observe future research trends in the field.\n\n\nData availability\n\nOpen Science Framework: Medical negligence in healthcare organizations and impacts on the patient safety: A bibliometric study, https://doi.org/10.17605/OSF.IO/DR3NZ (Dahlawi et al., 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\nAlkhenizan A, Shawb C: Impact of accreditation on the quality of healthcare services: A systematic review of the literature. Ann Saudi Med 2011; 31(4): 407–416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlkhenizan A, Shafiq M: The process of litigation for medical errors in Saudi Arabia and the United Kingdom. Saudi Med J 2018; 39(11): 1075–1081. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "82341", "date": "29 Mar 2021", "name": "Ghulam Murtaza RAFIQUE", "expertise": [ "Reviewer Expertise Information behavior", "Knowledge sharing", "Bibliometric analysis", "Information literacy" ], "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 \"Medical negligence in healthcare organizations and its impact on patient safety and public health: a bibliometric study\" addresses a topic in public health that is very timely. Medical negligence and its impact on patient safety and public health are areas that would be of interest to other professionals in the field of medical health. The article identifies why the topic was chosen, mentions how the area of medical negligence needed further research and study, and outlines the research objectives studied and the research design followed. The tables included in the article were clear to follow and were referenced properly in the article. I recommend its indexing in the journal.\nOverall, I enjoyed reading the article and think the findings of the study can shed light on the of literature on the topic and its contribution in the field.\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": "6520", "date": "31 Mar 2021", "name": "Saad Dahlawi", "role": "Author Response", "response": "Dear reviewer Thanks for reading our manuscript and giving us your valuable feedback. We appreciate your quick and positive comments. Thank you very much. Saad Dahlawi The corresponding author" } ] }, { "id": "85963", "date": "26 May 2021", "name": "Rijen Shrestha", "expertise": [ "Reviewer Expertise research methodology", "statistical analysis", "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\nThis is an excellent bibliometric research on the output of research on medical negligence. The manuscript is well written, comprehensive and has relevant citations. This article does very well to inform the readers of the relevance of different types of publications, platforms, authorship etc.\nI found 'one' (1) typographical mistake - last word in fourth paragraph of Introduction (negligen - negligence).\n\nAdditionally, 'one' (1) minor edit that would provide clarity:\nReplace \"clear definition\" with \"universally accepted definition\" [last sentence of first paragraph in Introduction]\nFinally, in the section on Results and Discussion, perhaps elaborate a little on the findings and discuss the relevance, interpretation of the findings as well.\nFor example, Table 1 shows that articles are cited more frequently. Perhaps discuss in brief the relevance and inference of this finding. With 87.66% of citations, does it mean that it is being read 10X?\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/10-174
https://f1000research.com/articles/10-173/v1	03 Mar 21	{ "type": "Research Article", "title": "Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness", "authors": [ "Hoda Atef Abdelsattar Ibrahim", "Rasha Abdel-Raouf", "Ahmed S. Zeid", "Eman H. Elsebaie", "Shaimaa Abdalaleem", "Aya A. Amin", "Hanna Aboulghar", "Rasha Abdel-Raouf", "Ahmed S. Zeid", "Eman H. Elsebaie", "Shaimaa Abdalaleem", "Aya A. Amin" ], "abstract": "Background: Nutritional screening, intervention and assessment in patients with undernutrition are key components of any nutritional care. The goal of any nutritional assessment is to determine the specific nutritional risk(s). Presently, there are no guidelines on any ideal screening tool to be used on admission for identification of children that are at risk of developing malnutrition during their hospital stay. The objective of the study was to develop a valid and simple nutritional screening tool which can be used on hospital admission to identify pediatric patients at risk of malnutrition.\nMethods: This study was cross sectional analytical that enrolled children (n:161) admitted with acute illness to the general wards at Cairo University Children Hospitals (CUCH). The answers to the developed questionnaire were compared to the Subjective Global Assessment (SGA), those with high accuracy (≥80%) were used for validity with anthropometric measures. Results: In the ‘less than two years of age’ group, the simple and valid nutritional screening tools were the following questions: (Is there a problem during breast-feeding?), (Is there scanty breast milk?), (Is there appetite loss?). The simple and valid nutritional screening tools during the ‘early childhood’ group were the following questions: (Is there appetite loss?), (Is there any skipping of meals?), (Are they watching TV, videotapes and/or playing computer games for more than two hours/day?). The simple and valid nutritional screening tools during the ‘late childhood’ group were the following questions: (Is there appetite loss?), (Are they watching TV, videotapes and/or playing computer games for more than two hours/day?).\nConclusion: The simple and valid nutritional screening tools differ according to age groups. The one which is valid in all ages is the question about the appetite loss.", "keywords": [ "Nutritional screening tools", "appetite loss", "acute illness", "SGA", "children" ], "content": "Introduction\n\nNutrition is an essential factor in the development, growth, and functioning of any child. Good nutritional status provides energy and nutrients which are essential to maintain our life and promote social, physical, emotional, and cognitive development1.\n\nChildhood malnutrition is known to be a global health concern as it is complicated with poor development and growth, as well as reduced educational outcomes of children and can have negative implications on their adulthood2,3.\n\nThe incidence of the undernutrition among the inpatients is seeming to be more than that noticed among the community; malnourished children have a comorbid association, and are vulnerable to developing further medical complications. In addition, evidence indicates that the nutritional state of the admitted ill children deteriorates during the hospital stay. Besides, the absence of any known nutritional screening tool in these circumstances could result in an underestimation of that condition4,5. The nutritional screening tool tries to identify the patients that are at nutritional risk, including not only the children who are undernourished, but also children who may develop any form of undernutrition during their hospital stay, and, the children whose prognosis can be improved as a result of the nutrition intervention6.\n\nThe Subjective Global Nutritional Assessment (SGNA) is a systematic nutritional assessment method which had been validated. It contains items of a physical examination and nutrition-focused medical history, and it is the closest nutrition tool that is available for assessing the nutritional state in pediatrics. Although it is a systematic nutritional evaluation and screening tool, it does not give a chance for a simple and rapid pediatric nutritional screening tool owing to the time that is required for its completion7. The characteristics of a nutritional screening tool are (1) sensitivity, specificity, and validity; (2) easy to use, simple, and without any need for training; (3) cheap, noninvasive and quick8.\n\n\nMethods\n\nA cross sectional analytical study. Study population and setting: The current study was conducted at Cairo University Children Hospitals (CUCHs) for all children admitted with acute illness in the general wards over a period of 18 months.\n\nA convenient sample of all children (n =161) who were admitted to CUCHs were recruited for this study.\n\nInclusion criteria: 1-Children with acute illness, in the first two days of admission (to avoid the effect of hospitalization and possible weight loss, to prevent bias). 2- Both male and female genders. 3- Approval of the parent or legal guardian.\n\nExclusion criteria: 1- Children who have gross physical disabilities, mental retardation, and chronic illnesses. e.g., sickle cell disease and cystic fibrosis. 2- Patients admitted for more than two days.\n\nA semi-structured questionnaire form was used while interviewing the patients. The answers were compared with Pediatric Subjective Global Assessment (PSGA) validated tool7. Answers with accuracy more than 80% were validated with the anthropometric measures. It covered the following items in the questionnaire : Appetite, Weight loss, Food allergies, Morbidity evidenced by recurrent hospitalizations, Enforcement to eat or not, Family eating together or not, Vitamins and minerals supplementations and Maternal deprivation, with focus on certain points in each stage as:\n\nInfancy: breast-feeding, feeding on colostrum, any problems during breast-feeding, developmental history, adverse reactions to vaccines, and feeding disorders. N.B The criteria for successful breast feeding was checked to exclude those if there were actual breastfeeding problems e.g., scanty breast milk.\n\nEarly childhood (2–8 years): feeding disorders, eating out, skipping meals, child abuse, school achievement, functional impairment, mealtime atmosphere and watching TV or video-tapes and/or playing computer games for more than two hours per day.\n\nLate childhood and early adolescence (8–12 years): skipping meals, child abuse, school achievement, functional impairment, mealtime atmosphere and watching TV, or video-tapes and/or playing computer games for more than two hours per day.\n\nSubjective global assessment evaluates the nutritional state which is based on the components of the history (any change in the weight, any change in the dietary intake, any gastrointestinal symptoms which had continued for more than two weeks, any change in the functional ability) in addition to the examination (subcutaneous fat loss, any wasting of muscles, edema in sacral/ankle area, and presence of ascites). Results were categorized subjectively into a global evaluation, where children had been rated into ῾well-nourished category᾽ (SGA A); ῾moderately malnourished category᾽ (SGA B); or ῾severely malnourished category᾽ (SGA C). The SGA is known to be the “gold standard” in comparison with the nutritional screening questions, for assessment of any malnutrition. The nutritional screening questions which had the highest accuracy, positive predictive and negative predictive values (≥80%) at predicting nutrition state (according to the comparison with SGA) were considered as the final nutrition screening tool. The Predictive Validity of our nutritional screening tool was established through comparison of the nutritional screening tool with anthropometric measures. When the tool is confirmed to be valid, it can be considered as a screening tool of the nutritional status.\n\nUnderweight: when the weight for age is less than the mean by two standard deviations (SD) of the World Health Organization (WHO) Child Standards for growth or less than 5th centile for age.\n\nStunting: when the height for age is less than the mean by two standard deviations of the WHO Child Standards for growth or less than the 5th centile for age.\n\nWasting: when the weight for the height age is less than the mean by two standard deviations of the WHO Child Standards for growth or Body Mass Index (BMI) is less than the 5th centile for age.\n\nOverweight: when the weight for the height is more than the mean by two standard deviations of the WHO Child Standards for growth or Body Mass Index( BMI )is more than 85th centile for age\n\nObesity: when the weight for the height is more than the mean by three standard deviations of the WHO Child Standards for growth or BMI is more than 95th centile for age.\n\n1. The study was done on the admitted children in the first two days to avoid the effect of the hospital admission due to the possible weight loss owing to the possible appetite loss.\n\n2. We excluded children with chronic illness as they may have external factors for malnutrition due to chronic disease, so we enrolled only patients with acute illnesses such as bronchiolitis, or glomerulonephritis as these diseases are acute and haven’t yet affected the nutritional state.\n\nAfter the step of data collection, the questionnaires with their answers were revised for the completeness and the logical consistency. The pre-coded data was subsequently entered on the computer using the Program of the Microsoft Office Excel for Windows. Data were then double checked and transferred to The Statistical Package of the Social Science, Version 21 (SPSS-V21). The provided graphs were consequently constructed using SPSS Program. All the statistical analysis was done using the two tailed tests and the alpha error of 0.05. P value less than or equal to 0.05 was identified to be statistically significant. Simple descriptive statistics (arithmetic mean and standard deviation) are used for the summary of the quantitative data and frequencies are used for qualitative data. Bivariate relationship was identified and displayed in cross tabulations and a subsequent comparison of proportions was performed through the chi-square and Fisher’s exact tests.\n\nThe study was revised and approved by the scientific research committee and ethics of Cairo University, Faculty of Medicine (ethical clearance number, Ι-071017) and the study was done in accordance with Cairo University's laws for human research. Written informed consent for participation and publication of the patient's details was obtained from parent/guardians/relative of the patients. The study was done after the explanation of its importance and the objectives of the study to the participants. Only subjects who clearly agreed were enrolled and those who refused after the explanation were excluded.\n\n\nResults\n\nThe present study was conducted in CUCHs on children admitted with acute illnesses in the general wards. The answers to the developed questionnaire were compared to SGA, those with high accuracy (≥80%) were used for validity with anthropometric measures. Table 1 shows the characteristics of the study participants.\n\n* SD: Standard Deviation. The percentage is raw percentage. *The percentage is column percentage\n\nThere were (n = 66) patients in this group, 40 males,and 26 females. Comparison of the answers of the developed questionnaire to those of the SGA was done to detect the questions which have the highest sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and level of accuracy. Table 2 summarizes the comparison results. Table 3 illustrates the validity. Table 4 illustrates the tool.\n\nThere were (n = 66) patients in this group, 37 male, and 29 female. The questionnaire was answered. Then the questions were compared with SGA to detect the questions which have the highest specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and level of accuracy. Table 5 summarizes the comparison results. Table 6 illustrates the validity. Table 7 illustrates the tool.\n\nThere were (n = 29) patients in this group, 13 male and 16 female. The questionnaire was answered. Then the questions were compared with SGA to detect the questions which have the highest specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and level of accuracy. Table 8 summarizes the comparison results. Table 9 illustrates the validity. Table 10 illustrates the tool.\n\nThe nutrition screening tools for all age groups are illustrated in Table 11.\n\n\nDiscussion\n\nThe nutritional state of any child is a determinant of the body composition and the functional state. Deficient states badly affect the patient's outcomes, mortality, morbidity, hospital stay, and re-admission rates. Thus, screening for risk factors which are known to be present with the deficiencies should be part of the evaluation of any child on admission.9,10.\n\nThe hospitalized children have high risks to develop severe malnutrition. The nutritional risk screening is an essential tool to maintain the nutritional status in any hospitalized patient owing to different reasons. For example, the energy need is increased and the subsequent decreased appetite is problematic.11\n\nIt is important to know the inpatients children who are with nutrition risks so that the appropriate timely nutritional intervention and the planned treatment can be performed and the nutritional deterioration prevented to improve the health outcomes12.\n\nHartman et al. recommended that “a valid and a simple nutritional screening tool appeared highly needed to improve the early cost effective identification of pediatrics who will get benefits later from the nutrition intervention”13.\n\nThere are many nutritional screening tools which are currently being used in hospitals and the community, but most of them are difficult and sophisticated8. In this work, we aimed to develop an ideal nutritional pediatric screening tool which can be easily and rapidly implemented, and with a high level of sensitivity ,specificity and good accuracy in the identification of the nutritional risks together with the nutrition-related outcomes.\n\nUnfortunately, there is no previous study that has used the Pediatric Subjective Global Assessment as a gold standard method to compare the screening tools on hospital admission of pediatric patients to detect the tools which have the highest sensitivity, specificity, and accuracy. There is a lot of debate among doctors and professionals on the way to validate the nutritional screening tools, especially if they have higher accuracy, which can predict the present nutritional status. Some hospitals have validated their nutritional screening tools using a full nutrition evaluation. Nevertheless, it is questionable if this is the standard tool, especially as not all countries have sufficient dieticians and their role might be different depending on the country14. In this study, SGA was used as the gold standard to identify the nutritional screening tools with the highest accuracy in order to be validated with anthropometric measures. The Pediatric Subjective Global Assessment consists of both objective and subjective items in a physical examination and detailed questionnaire; then each child is categorized into (1) well-nourished category, (2) moderately malnourished category, or (3) severely malnourished category15.\n\nOur study resulted in a simple, valid and effective nutritional pediatric screening tool for the early identification of at-risk admitted children who require thorough nutritional assessment and subsequent individualized nutritional intervention. In this study, we tried to detect the ability of the tool to pick up those children who have malnutrition.\n\nIn all age groups, the screening tool question about lost appetite was one of the most sensitive, specific, and accurate questions in prediction of the nutritional risk.\n\nOur present study is in line with a study done on a simple nutritional screening tool for hospitalized pediatric children after checking the accuracy of a new, rapid, and simple pediatric nutritional screening method. The question (Has child been feeding less during the last weeks?) was one of the most sensitive and specific questions in detection of the patients with nutrition risks. On the other hand, it disagrees with the rest of questions screening tool as they were (Has the child lost weight unintentionally?), and (Has the child had poor or unsatisfied weight gain during the last months?). This difference may be due to the accuracy in weight detection objectively by the mothers16.\n\nIn this study, we found that one of the nutritional screening tools was about the appetite loss. There was a study that used STAMP (Screening Tool Assessment of Malnutrition in Pediatrics) for the determination of the malnutrition and malnutrition risks in the pediatric primary health care setting. One of the items of this screening tool was (what is the nutritional intake of the child) which may be related to his appetite. However, it incorporated weight and height (anthropometric measures) in this screening tool unlike the case in our study. That is because we aimed to find a quick screening tool. Thus, we didn’t consider the anthropometric measures in the tool17.\n\nThis study agrees with a previous study that found that one of the nutritional screening tools among the adult hospitalized patients was (Have you had poor eating because of the presence of a decreased appetite?), but disagrees with that the other tools were (Have you lost weight unintentionally?), (If yes, how much weight have you actually lost?). This difference may be due to the age group difference18.\n\nThis study is very near to a previous study that was worked on the evaluation of the screening tool for further assessment of malnutrition in children. Significant predictors of the nutritional risks were decreased dietary intakes which was due to a recent poor appetite. Other predictors found were reported19.\n\nThis study agrees with a study that was done to detect the nutritional screening tool. One of the main principles of nutritional screening was about reduced intake due to a poor appetite. It disagrees with it in that the other principle was about the severity of the disease whether acute or chronic. This disagreement may be due to that in our present study we intentionally removed the effect of the chronic element on the disease which may affect the nutritional status. That is why we enrolled admitted patients with acute illnesses only20.\n\nIn the ῾less than two years age᾽ group, it was found that the presence of any problems during breast- feeding or scanty breast milk were predictors of nutritional risks. They affect the nutritional status as seen in the underweight and the stunted groups. There was a significant association (P-value <0.001) between the presence of any problems or scanty breast milk and the prediction of any nutritional risk.\n\nThis study agrees with a study in which a relationship was found between the breast- feeding practices of mothers in Nairobi, and the nutritional state of their children aged between 0 to 24 months. There was a significant and noticeable association between the continuation of breast-feeding for a child less than 24 months and those underweight and stunting. Children who had stopped breast-feeding were more than three or four times likely to be underweight in comparison with those who continue breastfeeding21.\n\nThis study is closely located to a previous study that was done in a rural area, under administrative control of the tertiary care hospital. All lactating mothers who have babies up to one year were participants in the study. They found a negative relationship between exclusive breast feeding and the prevalence of underweight and stunted children. Their conclusion was based on assumption that there is no breastfeeding problems in nourished children receiving exclusive breast feeding22.\n\nThe current study found that, in the early childhood group, one of the predictors of nutritional risks was the question about skipping meals\n\nThis study agrees with a previous one which found that more subjects of the non- breakfast skippers were found to have BMI in the normal range than breakfast skippers. Unlike our study, they found that more of the breakfast skippers were overweight than the non-breakfast skippers. This may be a result of more consumption of junk food, i.e., high in saturated fat, by the breakfast skippers23.\n\nThis study is similar to a previous one which found that there is an association between meal skipping and malnutrition. Among the skippers, 12.1% of children were in the underweight category and 10.3% of the children were overweight. The malnutrition is represented in the underweight and the overweight groups24.\n\nIn the present study it was found that in the early and late childhood groups, there is a significant association between watching TV, video-tapes, and/or playing computer games for more than two hours per day and the presence of a nutritional risk with (P -value <0.001).\n\nThis agrees with a study which found that the older age of the child, the presence of multiple televisions at home, having her or his own television ,and number of hours spent inside watching television at the weekend were essentially associated with increased risk of childhood obesity25.\n\nThe current study also agrees with a previous study that was done on an Egyptian cross-sectional survey on children aged between 6 and 17 years in Manshit El Gamal region of Fayoum Governorate. Increasing age and not eating breakfast were associated risks for stunting, whereas the incidence of obesity was higher in the children who eat while watching TV26.\n\nIn the present study, no significant association was found between history of weight loss and the nutritional status of the child.\n\nThis disagrees with a previous study in that they aimed to detect a score to be used on hospital admission to pick up patients who are at risk of acute malnutrition during the hospital stay27. The nutritional risk was evaluated within 48 hours of admission. The nutritional predictor score consisted of history of weight loss, decreased food intake, disease severity divided into Grades 1, 2 and 3 and pain. This disagreement may be owed to the fact that the present study didn’t use a scoring system in the questionnaire.\n\nSome limitations to our study should be mentioned which, may in turn lead to the limitation of its generalizability to the total population. For example, the study sample was limited to Cairo University Children Hospitals; a broader geographic sample may lead to different results. Furthermore, this study has used a cross sectional design, thus a rather prospective one can help in making other predictions through follow up. One limitation of the Subjective Global Assessment to be mentioned is the limitations of attempts to only categorize undernutrition. Obese patients are effectively categorized as normal.\n\n\nConclusion\n\nThis study has established the validity of a simple nutrition screening tool that can be implemented on infants and children at the time of admission to hospital. In the ῾less than two years of age᾽ group: the simple and valid nutritional screening tools were: (Is there a problem during breast- feeding?), (Is there scanty breast milk ?), (Is there appetite loss?).In ῾the early childhood group᾽: the simple and valid nutritional screening tools were: (Is there appetite loss?), (Is there any skipping of meals?), (Are they watching TV, video-tapes and/or playing computer games for more than two hours/day). The ῾late childhood᾽ group: the simple and valid nutritional screening tools were: (Is there appetite loss?), (Are they watching TV, video-tapes and/or playing computer games for more than two hours/day).\n\n\nData availability\n\nOpen Science Framework: Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness, https://doi.org/10.17605/OSF.IO/6YTMN28\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 Pediatric Department and the Pediatric Clinical Nutrition Department, Faculty of Medicine, Cairo University.\n\n\nReferences\n\nPerkins JM, Kim R, Krishna A, et al.: Understanding the association between stunting and child development in low- and middle-income countries: Next steps for research and intervention. Soc Sci Med. 2017; 193: 101–109. PubMed Abstract \| Publisher Full Text\n\nPhyu LE, Kriengsinyos W, Rojroongwasinkul N, et al.: Validation of a Pediatric Nutrition Screening Tool in Hospital Outpatients of Myanmar. Asian J Clin Nutr. 2020; 2: 9–14. Reference Source\n\nBecker PJ, Bellini SG, Vega MW, et al.: Validity and Reliability of Pediatric Nutrition Screening Tools for Hospital, Outpatient, and Community Settings: A 2018 Evidence Analysis Center Systematic Review. J Acad Nutr Diet. 2020; 120(2): 288–318.e2. PubMed Abstract \| Publisher Full Text\n\nKramer CV, Allen S: Malnutrition in developing countries. Paediatr Child Health. 2015; 25(9): 422–427. Publisher Full Text\n\nBalhara KS, Silvestri DM, Tyler Winders W, et al.: Global Emergency Medicine Literature Review Group (GEMLR). Impact of nutrition interventions on pediatric mortality and nutrition outcomes in humanitarian emergencies: A systematic review. Trop Med Int Health. 2017; 22(12): 1464–1492. PubMed Abstract \| Publisher Full Text\n\nPérez-Solís D, Larrea-Tamayo E, Menéndez-Arias C, et al.: Assessment of Two Nutritional Screening Tools in Hospitalized Children. Nutrients. 2020; 12(5): 1221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSecker DJ, Jeejeebhoy KN: Subjective global nutritional assessment for children. Am J Clin Nutr. 2007; 85(4): 1083–1089. PubMed Abstract \| Publisher Full Text\n\nHershkovich S, Stark AH, Levi CS, et al.: A tailored automated nutrition screening tool for rapid identification of risk in acute-care hospital settings. Eur J Clin Nutr. 2017; 71(12): 284–286. PubMed Abstract \| Publisher Full Text\n\nCorreia MIT, Waitzberg DL: The impact of malnutrition on morbidity, mortality, length of hospital stay and costs evaluated through a multivariate model analysis. Clin Nutr. 2003; 22(3): 235–239. PubMed Abstract \| Publisher Full Text\n\nCorreia MI, Perman MI, Waitzberg DL: Hospital malnutrition in Latin America, a systematic review. Clin Nutr. 2016; 36(4): 958–967. PubMed Abstract \| Publisher Full Text\n\nNjike V, Smith T, Shuval O, et al.: Snack Food, Satiety, and Weight. Adv Nutr. 2016; 7(5): 866–878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCao J, Peng L, Li R, et al.: Nutritional risk screening and its clinical significance in hospitalized children. Clin Nutr. 2014; 33(3): 432–436. PubMed Abstract \| Publisher Full Text\n\nHartman C, Shamir R, Hecht C, et al.: Malnutrition screening tools for hospitalized children. Curr Opin Clin Nutr Metab Care. 2012; 15(3): 303–9. PubMed Abstract \| Publisher Full Text\n\nJoosten KFM, Hulst JM: Nutritional screening tools for hospitalized children: methodological considerations. Clin Nutr. 2014; 33(1): 1–5. PubMed Abstract \| Publisher Full Text\n\nMoeeni V, Day AS: Nutritional risk screening tools in hospitalized children. Int J Child Health Nutr. 2012; 1: 39–43. Publisher Full Text\n\nWhite M, Lawson K, Ramsey R, et al.: Simple Nutrition Screening Tool for Pediatric Inpatients. JPEN J Parenter Enteral Nutr. 2016; 40(3): 392–98. PubMed Abstract \| Publisher Full Text\n\nWahed MAA, Nassar M, Ahmed AK, et al.: Nutritional Screening for 2-5 Years Old Children in Urban and Rural Outpatient Settings. Med J Cairo Univ. 2020; 88(4): 1767–1775.\n\nFerguson M, Capra S, Bauer J, et al.: Development of a valid and reliable malnutrition screening tool for adult and acute hospital patients. Nutrition. 1999; 15(6): 458–464. PubMed Abstract \| Publisher Full Text\n\nMcCarthy H, Dixon M, Crabtree I, et al.: The development and evaluation of the Screening Tool for the Assessment of Malnutrition in Paediatrics (STAMP) for use by healthcare staff. J Hum Nutr Diet. 2012; 25(4): 311–18. PubMed Abstract \| Publisher Full Text\n\nWonoputri N, Djais J, Rosalina I: Validity of Nutritional Screening Tools for Hospitalized Children. J Nutr Metab. 2014; 2014: 143649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuchina EN, Waithaka PM: Relationship between breastfeeding practices and nutritional status of children aged 0-24 months in Nairobi, Kenya. African J Food Agric Nutr Dev. 2010; 10(4): 1–21. Reference Source\n\nKumar A, Singh VK: A Study of Exclusive Breastfeeding and its impact on Nutritional Status of Child in EAG States. J Stat Appl Pro. 2015; 4(3): 435–445. Reference Source\n\nGarg M, Rajesh V, Kumar P: Effect of the breakfast skipping on nutritional status and school performance of 10-16 years old children of udupi district. Health Popul Perspect Issues. 2014; 37(3–4): 98–117. Reference Source\n\nBae H, Kim M, Hong S: Meal skipping children in low-income families and community practice implications. Nutr Res Pract. 2008; 2(2): 100–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Ghamdi S: The association between watching television and obesity in children of school-age in Saudi Arabia. J Family Community Med. 2013; 20(2): 83–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdel Wahed W, Hassan S, Eldessouki R: Malnutrition and Its Associated Factors among Rural School Children in Fayoum Governorate, Egypt. J Environ Public Health. 2017; 2017: 4783791. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSermet-Gaudelus I, Poisson-Salomon A, Colomb V, et al.: Simple pediatric nutritional risk score to identify children at risk of Malnutrition. Am J Clin Nutr. 2000; 72(1): 64–70. PubMed Abstract \| Publisher Full Text\n\nAtef H: Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness. 2021. http://www.doi.org/10.17605/OSF.IO/6YTMN" }	[ { "id": "80762", "date": "08 Mar 2021", "name": "Andrew Day", "expertise": [ "Reviewer Expertise paediatric gastroenterology and nutrition" ], "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 MS describes the development of a short nutritional risk screening tool, with comparison to a larger tool\nSpecific Comments:\nThe population is relatively small - larger numbers required to clearly demonstrate the utility of this tool. Especially with the three subgroups\n\nThere was no depiction of the current nutritional state, of the underlying medical issues, the length of stay or the change in weight during the stay. NRS are focused on identifying those at risk of nutritional compromise during admission - this doesn't include that focus\n\nThe METHODS should describe how the study was done. The number of subjects is a result and should be moved accordingly\n\nThere are a large number of tables - are these all required? some could be supplementary instead and some deleted\n\nThe DISCUSSION should be shortened and more focused. When using et al, the reference number must follow directly afterwards. Some sentences are awkward and could be improved with revision\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6480", "date": "23 Mar 2021", "name": "Hoda Atef", "role": "Author Response", "response": "Dear : Andrew S Day Thank you for your valuable comments 1.Regarding the point number 1: the sample size was dependent on the time of the study. In addition, we excluded patients with chronic illnesses to exclude bias as the chronic nature could cause malnutrition. These could be the causes of a smaller sample size as the inclusion criteria were very precise Of course, further studies are needed to validate the tool and our study may be the point of the start for these further researches. 2. Regarding the point number 2: There was a depiction for the current nutritional status . You can get it in the supplementary files. For example, there were underweight, wasted and short status groups. Regarding the length of hospital stay, it wasn’t our aim as the study aim is to find a tool on hospital admission. Further studies are required to apply it on the hospital length stay. 3. Regarding the point number 3, please explain more, what do you mean? 4. Regarding the point number 4, our aim was to explain all data but the tables could be further summarized 5. Regarding the point number 5, we tried to comment on the results as much as possible and compare them with the relevant articles. However, the discussion could be further shortened. Thank you so much Sincerely Hoda Atef" } ] }, { "id": "80755", "date": "22 Mar 2021", "name": "Osama El-Asheer", "expertise": [ "Reviewer Expertise Professor Of Pediatrics - Assiut University & Head of clinical nutrition unite - Assiut University children hospital" ], "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 document from my Egyptian colleagues with great interest. This paper is well written and addresses an important topic which is the development of a simple and valid nutrition screening tool in pediatric hospitalized patients with acute illness. The article is clear and of relevance also because there is a need for this important tool to improve the prognosis and outcomes of pediatric diseases. Furthermore, the tool respects the age groups and also respects the time as it is rapid (unlike the subjective global assessment and the anthropometric measures) and applicable.\nAlthough the reliability of this tool was not achieved, there are several important conclusions from this article. For example, appetite loss is the most consistent tool in all age groups. However, a scoring system for this appetite loss would have been done to improve the quality of this tool. Very important: watching television and computer games for more than 2 hours per day could represent a major nutritional risk for older children. Besides, respecting the meal time and the avoidance of skipping meals can greatly keep children away from malnutrition. Problems in breastfeeding can negatively affect the growth of infant and have earlier implications on the nutritional parameters. Correctly the authors exclude the chronic diseases to exclude bias as the chronic course could cause organic failure to thrive.\nTo improve paper readability, I suggest the following:\nAbstract: Detailed questionnaire should be removed from Results\n\nPlease clarify the method of getting the sample size.\n\nPlease clarify why you did not compare with other screening tools like STAMP or STRONG Kids scoring in addition to SGNA\n\nPlease clarify the base of age classification 2-8 and 8-12\n\nResults: tables are so much and should be summarized into 4-5 tables\n\nThe discussion should be shorter and more focused to the point. It should be more aimed more than this. You can remove the first six paragraphs in the discussion as you mentioned similar ones in the introduction.\n\nI suggest also to update the references.\n\nAs a further question: Did the authors observe the nutritional lab assessment?\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": "6485", "date": "23 Mar 2021", "name": "Hoda Atef", "role": "Author Response", "response": "Dear Professor , DR : Osama El-Asheer Thank you for your valuable comments Regarding the results : we added the detailed questionnaire to collectively gather the ways for the aimed tool Regarding the sample size : it depended on the time of study and all patients who fulfilled the inclusion criteria were enrolled in the study Regarding the comparison with other tools like STAMP or STRONG Kids scoring, we didn’t use them as this wasn’t our aim of the study . Our aim was to find a simple and a valid tool . The process of validity required high sensitivity and specificity which needed a comparison with any valid tool such as SGA Regarding the base of age classification . WHO (World Health Organization ) defines the early childhood group from 0-8 years . I further categorized the first two years as a separate group as they have different feeding (e.g. breast feeding ) and its problems are present during this period which is a distinctive period in the childhood Regarding the tables , our aim was to explain in details .However, they could be further shortened Regarding the discussion ,we intended to introduce our work before comparing it with the similar articles in the discussion . However, it could be further shortened. Regarding the references , we tried to update them till 2020 Regarding the lab comments , they weren’t observed as this wasn’t the aim . The aim of the study was to develop a rapid and simple tool. The labs would take time." } ] }, { "id": "83395", "date": "22 Apr 2021", "name": "Ali M. El-shafie", "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 your kind invitation to be a reviewer for this study.\nThe study investigated a nutritional screening tool for pediatrics. The researchers tried to reveal the sensitivity, specificity and validity of this tool. Although the results of the study are intriguing, few flaws remain in the paper.\nTitle: It would be better that more specific term would have been chosen for the title of study. I suggest you describe the tool as it is rapid. In addition, I suggest you describe those pediatrics as having acute malnutrition as this way of description is more specified. Thus, the title would be (Development of a rapid and valid nutritional screening tool for pediatric hospitalized patients with acute malnutrition).\nAbstract: The first sentence would have been removed as the paper didn’t incorporate any methods for nutritional intervention.\n\nIntroduction: The subjective global assessment is needed to be further explained. In addition, the basis of choosing the questionnaire for each group and previous studies that used similar questionnaires would have been better introduced.\nMethod: The logic for calculating the sample size is missed in this study. The calculation of sample size or a power analysis is better to be added. The acute illnesses would have been prescribed and correlations between them and types of malnutrition would have been addressed. Regarding the SGA, its method is better to be mentioned clearly.\n\nResults: I noticed that there are no graphs to explain the groups of malnutrition revealed in the study. I think it was better to add these graphs. Concerning the applied questionnaire, what is meant by \"weight loss measured\", it would have been better explained. Furthermore, the question of \"food allergy\" is not clear as symptoms of food allergy that you asked should be associated with the questionnaire\n\nDiscussion: The first four paragraphs could be removed as they are repeated. The aim of the study should have been mentioned at the end of the Introduction part instead of at the beginning of the Discussion one.\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": "83394", "date": "23 Apr 2021", "name": "Areej Alkhaldy", "expertise": [ "Reviewer Expertise 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\nIt has been a great honour to review the article entitled “Development of a simple and valid nutrition screening tool for pediatric hospitalized patients with acute illness.” The paper aims to develop a valid and simple nutritional screening tool which can be used on hospital admission to identify pediatric patients at risk of malnutrition. The paper is interesting; however, there are some comments that need to be addressed to improve it.\nAbstract:\n\nThe age, sex, and groups need to be indicated in the abstract.\n\nThe results need to be improved and expanded along with numbers.\n\nThe conclusion needs to be rewritten. What is the main message regarding the use of these screening tools?\n\nIntroduction:\n\nThe introduction was written in a consistent manner, yet you need to add more information including recent research in the area of SGA and pediatrics. There is a need to focus more on the nutrition screening tool for pediatric patients than general ones for adults.\n\nAt the end of the last paragraph, the objectives need to be expressed in a clear manner and include the rational of conducting the study.\n\nMethods:\n\nYou need to include more information in the sample size section. How was the sample size calculated in terms of population size, source of the data, and references of the equation or website, or program you used to calculate the sample size?\n\nExplain the validation process in more detail.\n\nBriefly explain the questionnaire (for example: sections and numbers of questions)\n\nDefine abbreviations at first mention; the abbreviation should always be used in the rest of the manuscript instead of the complete term. All group abbreviations need to be indicated and explained in the methods.\n\nResults:\n\nTable 2, 5, and 8 could be combined and compared\n\nFootnotes need to be added to all tables (example PPV and NPV)\n\nDiscussion:\nYou need to discuss your findings in the context of the other studies in the same area.\n\nThe manuscript requires minor language editing throughout.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-173
https://f1000research.com/articles/10-172/v1	03 Mar 21	{ "type": "Research Article", "title": "Determination of field capacity in the Chibunga and Guano rivers micro-basins", "authors": [ "Benito Mendoza", "Manuel Fiallos", "Sandra Iturralde", "Patricio Santillán", "Nelly Guananga", "Jaime Bejar", "Daniel A. Lowy", "Imre Vágó", "Zsolt Sándor", "Benito Mendoza", "Manuel Fiallos", "Sandra Iturralde", "Patricio Santillán", "Nelly Guananga", "Jaime Bejar", "Daniel A. Lowy", "Imre Vágó" ], "abstract": "Background: The micro-basins of the Chibunga and Guano rivers are located within the sub-basin of the Chambo River, which starts at the thaw of the Chimborazo, crosses the cities of Guano and Riobamba, and ends in the Chambo River. These rivers are considered fluvial hydrological forces and geological limits of the aquifer, located in this sub-basin. For this reason, our investigation addressed the field capacity in the micro-basins of Chibunga and Guano rivers, to determine the maximum retention potential, i.e., the saturation of water in the soil. Methods: We investigated the change of precipitation to runoff through the correlations between the characteristics of the soil and its vegetation. We applied the Curve Number (CN) method introduced by the United States Soil Conservation Service (USSCS); this represents an empirical model, which relates the vegetation cover to the geological and topographic conditions of the soil. Along with the geographic information system, the model allows to represent the variation of runoffs for each micro-basin, according to the different land use categories, over the time frame from 2010 to 2014. Results: We found that the maximum retention potential is directly affected by CN values, representing the runoff potential. Highest values of 100 belong to the wetlands, urban area, snow, and water, as rain is converted directly into runoff, being impervious areas. The Guano river micro-basin possesses clay soil with CN of 78, the soil texture for eucalyptus forest is clay loam, and its CN value, 46, is the lowest of the data set. Knowledge of field capacity allows to properly evaluate the storage capacity of soil and water conservation. Conclusions: Results of this work will be useful in the quantification of the water balance, to determine the water supply and demand.", "keywords": [ "Field capacity", "maximum retention potential", "precipitation-runoff relationship", "curve number method" ], "content": "Introduction\n\nThe most important natural resources are soil (Bautista et al., 2018; Mátyás et al., 2018; Singla et al., 2018; Sukiasyan & Kirakosyan, 2020), air (DRC, 2020), and water (Bersosa et al., 2019). Therefore, protection of natural resources and their rational use are essential to provide sustainable environmental and agricultural management practices (Mátyás et al., 2020; Sándor et al., 2020; Shomana et al., 2020). The hydrological cycle is the process of water circulation in the earth, therefore, it is important to study surface and underground water, to understand the cycle in a global way and its interrelation with the environment (Pacheco Moya et al., 2018).\n\nAlso, the management of a basin must be oriented to the rational use of its resources, i.e., to order and spatially distribute their use (Sánchez-Cohen et al., 2015). Management is important, as a basin has environmental, social, economic, and cultural implications in the distribution of water resources (Sánchez-Cohen et al., 2015). As of today, in Ecuador, most of the hydrographic basins do not have proper conservation management, causing alterations in the hydrological cycle and generating for a given area little retention of humidity, erosion, and flooding over periods of rain and water scarcity in dry periods (Lizárraga-Mendiola et al., 2019).\n\nSurface runoff is part of the hydrological cycle; it occurs as a result of excess water, which that does not filter off, and collects in the secondary channels, and finally ends up in the main channel, until it reaches the mouth of the river (Francés & Montalvo, 2017). When this process is not adequately controlled, it becomes a critical environmental problem, as it favors water erosion of the soil, rivers overflow, increasing the sediment load, and, by dragging, it causes water contamination (Alonso-Brito, 2016). Various study techniques have been developed, both empirically and in the field (Maidment et al., 1994), which allow determination of the volume of runoff in a given area, for providing adequate water management (Amy, 1974). These techniques enable appropriate design structures for the use and control of water resources. Surface and underground runoffs depend on the hydraulic characteristics of the subsoil, such as hydraulic conductance and porosity, which are distributed according to the geological and topographic soil conditions (Lee et al., 2015).\n\nAs a result, runoff calculations can be derived from precipitation events, using correlations between the characteristics of the soil and its vegetation, and also taking into account the interaction of different components of the natural system (Pacheco Moya et al., 2018). Nevertheless, the hydro-meteorological information in the hydrographic basins is limited in terms of a rainfall and river database of several years, as there is no good hydro-meteorological network in Ecuador (Ochoa-Tocachi et al., 2018), and this data is needed for hydrological modeling of water balance, when calculating water supply and demand (Zubieta et al., 2018).\n\nTherefore, one can apply empirical models that provide approximate data on the hydrological reality of an area, e.g., for runoff the Curve Number (CN) method is used (Uwizeyimana et al., 2019).\n\nThe goal of this work is to determine the field capacity, considering the physical characteristics of the environment and the use of the soil and vegetation cover. We aim to obtain quantitative values for CN, and to determine the maximum potential retention and the precipitation-runoff relationship. These calculations are done for the Chibunga and Guano rivers micro-basins, located in the province of Chimborazo, Ecuador. This work should be helpful in quantifying the water balance in a basin, and to determine water supply and demand in a more efficient manner.\n\n\nMethods\n\nOur study was conducted from January 7 to March 29, 2019, in the area of interest, namely, the micro-basins of the Chibunga and Guano rivers, which are part of the sub-basin of the Chambo River, in the province of Chimborazo. Economic activity, which predominates in the region is agriculture, providing living means for most families residing there. Chibunga river originates from the slopes of the Chimborazo Volcano and descends through the Arenal moorland, until it reaches agricultural areas in San Juan town, and the communities including Chimborazo, Shobol Llinllin, with the name arising from Rio Chimborazo. Next, it joins the Cajabamba river at 3,238 m above sea level, and becomes Chibunga river (Mayorga & Carbonel, 2018). This micro-basin extends over 38 km, extends from northwest to southeast, and is the main tributary of the Chambo River at the Northern extremity of the Amazon region of Ecuador (at 680 km from the Amazonas River) (CNRH, 2007). The Guano River micro-basin is born on the slopes of the Chimborazo Volcano and receives contributions to its channel from streams, such as Cascajal, Chuquipogio, Abras, Puluchaca, Patulú, Igualata, Asaco, and others (Cevallos Gaibor, 2015). Its approximate area is 37,061 ha, and most of its water is used in the Guano Canton (Mendoza, 2016) until it eventually flows into the Chambo River.\n\nSampling points were identified by the over position of geology and elevation layer, where the most representative categories concerning land use were identified: Pa (moorland), Hu (wetlands), Bs (forest), Na (snow and water), Z (urban area), C (crops), Se (eroded soil), and Ps (grass).\n\nSubsequently, the following criteria were considered to determine the sampling points:\n\nAreas with steep slopes and poor accessibility were excluded\n\nAreas with a high degree of water for field tests were included\n\nCategories of urban area, snow, and water were excluded because of their impervious surface.\n\nThe CN parameter measured in the field (in-situ) and laboratory. First, the infiltration capacity was determined by infiltrometer (model 09.04, SDEC France, France). Second, soil characterization was done for permeability, texture, porosity, and organic matter (Tailor & Shrimali, 2016).\n\n\nIn-situ analysis\n\nAt each sampling point, infiltration tests were performed with by means of a double ring infiltrometer (Model 09.04, SDEC France, France). Briefly, two concentric rings were set up on a portion of soil and water was poured into the outer ring. Variation of the water level in the inner ring was measured to determine infiltration of the soil (Carreras Nampulá et al., 2015). Samples should be as little disturbed as possible, for not altering the results.\n\nFigure 1 shows a total of 23 points corresponding to the Chibunga river micro-basin and 21 points for the Guano river micro-basin, which were used for sampling. Five soil samples were taken from each sampling point, with the excavation being every 0.40 m to a maximum depth of 2.0 m, within a circular area 0.50 m in diameter. A total of 2 kg of soil were taken by a tubular soil sampler (15”L x ¾”Dia, Accuproducts International, USA) for each point, were placed in sampling bags (125 mm × 225 mm, Geology Superstore, United Kingdom), and transported to the Environmental Services Laboratory to determine the permeability, texture, porosity, and organic matter.\n\nThe red pins correspond to the Chibunga and Guano river micro-basins. Figure 1 was adapted from satellite images to identify the categories of land use and vegetation cover. These images were downloaded from the Copernicus website, using the Sentinel 2A satellite (Immitzer et al., 2016) (data is open source once a user registers for access).The images are from 2013, 2014, and 2018.\n\nSoil texture was determined according to the USDA triangle of textural classes (Bautista Carrascosa & Oliver Talens, 2017), applying the organoleptic method. Briefly, numerical values were assigned within the range of 1–12 to define granulometry: lower numbers (1–5), represent fine granulometry, such as clay and loam clay type; mid numbers (6–9) correspond to medium granulometry, such as loams and silts soils; while high numbers (10–12), are for coarse granulometry, such as sands.\n\nPorosity was obtained by a literature method (Liu et al., 2017), which relies on the real and apparent density of the soil. Calculations were based on Equation (1), which can be applied to each soil class, identified in the study area.\n\n\n\nWhere:\n\nP = Total porosity of the soil sample, [%]\n\nDa = Apparent density of the soil, [g/cm3]\n\nDr = Real particle density, [g/cm3]\n\nPermeability or hydraulic conductivity (Papierowska et al., 2018) was calculated using a permeameter (with internal diameter D = 6.4 cm, height L = 15.3 cm) (Cotecno Falling Head Permeameter, Santiago, Chile), in which h values are measured, obtained for various times elapsed from the beginning of the test. Then, the soil permeability coefficient, k, is calculated using Darcy's Law, according to Equation (2) and Equation (3), (Wang et al., 2020):\n\n\n\n\n\nWhere: Q = flow, expressed in m3/s; L = length of sample (m); k = Darcy's permeability coefficient, variable as a function of the sample material, expressed in m/s; A = cross-sectional area of the sample (m2); H3 = height, above the water level in reference tube placed at the entrance of the filter layer; H4 =height, above the reference level, that reaches the water in a tube placed at the out of the filter layer.\n\nOrganic matter was determined via calcination of the samples to later calculate the weight loss, which corresponds to the organic compounds present in the samples (Papierowska et al., 2018). The following procedure was used: crucibles with samples were measured in triplicate. Samples were subjected to 105 °C for 2 h; then, the temperature was raised to 550 °C for 2h. At the end, samples were cooled for 2 h inside the muffle. Subsequently, samples are returned to the oven at 105 °C for 30 min, to stabilize their temperature and eliminate moisture remained in the samples, then cooled in the desiccator, and finally weighed for determining the weight loss of each sample. Soil organic matter (SOM) was determined gravimetrically and calculated according to (Equation 4).\n\n\n\nCN was determined according to the SCS method, a technique developed for infiltration proofs depending on the runoff generating properties. The procedure considered: the hydrological group of soil (HGS), the previous humidity condition, the use of the land and treatment within the hydrographic basin. A value is calculated, which estimates the soil condition with regard to its usefulness (Satheeshkumar et al., 2017). CN is a curve number, obtained from cross-tabulation in spatial digital format, processed by soil and geomorphology maps, generating the hydrological groups (GH), as described in the SCS Handbook of Hydrology (NEH-4), Section-4 (USDA 1972). CN can be expressed by Equation (5).\n\n\n\nwhere:\n\n\n\nTaking as the input values of the CN parameter, the field capacity or maximum retention potential (S) was determined as the relatively constant amount of water contained by a saturated soil, after 48 h of drainage (Wróbel & Boczoń, 2020). Hence, there is a relationship between the soil and the coverage conditions within the micro-watersheds, via CN values. Numbers are shown on a scale from 0 to 100; the greater the number, the greater the direct runoff volume that can be expected from a storm (Lian et al., 2020).\n\nPrecipitation values were obtained from the interpolation by the kriging method, by using data from 6 meteorological stations (Table 1), over the time encompassing years 2010–2015. Precipitation values can be calculated using Equation (7) (Naranjo, 2014)\n\n\n\nZ (si) = the value measured at location\n\nλi = an unknown weight for the value measured at location\n\nS0 = the location of the prediction\n\nN = the number of measured values\n\nSimilarly, for runoff, the maximum retention potential was analyzed as a function of yearly precipitation. By this, one obtained the total flow, based on the vegetation cover (Sikorska et al., 2018).\n\nFor the multitemporal study the following methodology was used, all methods described below being applied for each sampling site:\n\nSupervised Classification Criteria: Study areas should match the number of categories that are intended to be analyzed (TELEDET, 2017). The areas must be correctly identified to cover all classes and must be homogeneous. Also, in supervised classification uniband statistical analysis verifies that values resemble normal distribution. To meet these criteria, we used the following classic algorithms: classifier by minimum distance, by parallelepipeds, and by maximum probability (Acosta, 2017).\n\n\nClassification methods\n\nWith this classifier collected data is used to determine the mean of the classes selected at the study sites. This approach assigns each unidentified pixel to the class with the closest mean. For the closest mean values one can use the Euclidean distance between the pixel and the center of each class. Although simple from the computational standpoint, this algorithm has certain limitations, including the insensitivity to different degrees of variance in the spectral responses (TELEDET, 2017).\n\nML or maximum probability assumes that the reflectivity values in each class follow a multivariate normal probability distribution. The vector of means and the variance-covariance matrix are then used to estimate the probability by which one given pixel belongs to all classes. Finally, pixels were assigned to the class to which it belonged with the highest probability (Tso & Mather, 2009). A Bayesian approach was used and considered was the a priori probability, whether a pixel belonged to a certain class. The greater the surface area, the greater the probability that a pixel belongs to it (Toro et al., 2013).\n\nIt consists of a double-entry table, which compares the real values with results obtained in the classification. The diagonal of the matrix reveals the number of real pixels and the classification that coincides by category, while the remaining pixels indicate the ones that may be confused with other categories. One represents vertically the percentage of real pixels that were confused in the truth-terrain, while horizontally one shows the percentage of all pixels (Borràs et al., 2017).\n\nThe accuracy measures considered by us are simple to use, being based on either the main diagonal, or the rows and columns of the contingency table. In some cases, a totally random distribution of pixels in the classes can yield apparently correct results in the contingency table (TELEDET, 2017).\n\n\nResults and discussion\n\nThrough the multi-temporal study and the application of spectral signatures, eight categories of vegetation cover were identified for each micro-basin: Pa (moorland), Hu (wetlands), Bs (forest), Na (snow and water), Z (urban area), C (crops), Se (eroded soil), and Ps (grass). The 26 sampling points were in the Chibunga river micro-basin and 23 points in the Guano river micro-basin.\n\nPhysical characteristics of the soil from each sampling point were determined, including porosity, permeability, and infiltration capacity (Table 2). These unique values are required to determine vegetation cover and soil texture, necessary to calculate CN.\n\nPa (moorland), Hu (wetlands), Bs (forest), Na (snow and water), Z (urban area), C (crops), Se (eroded soil), and Ps (grass). Null values mean 0 or minute areas.\n\nAbbreviations: Poro., porosity; Perm., permeability; Cap. Infil., infiltration capacity.\n\nTable 3 lists the values for CN and HSG (hydrological group) depending on the state of conservation and conditions present in the sampling area.\n\nAbbreviations: Poro., porosity; Perm., permeability; Cap. Infil., infiltration capacity.\n\nIn Table 3 one finds different combinations of vegetation cover and the HSGs of the soil in the micro-basin of the Chibunga River, where each combination corresponds to a CN that represents the runoff potential, in which the highest values belong to the wetlands, urban area, snow, and water, assigning them the value of 100. In these areas rain is converted directly to runoff, as they are impervious areas. By contrast, the Guano River micro-basin revealed that the moorland class had clay soil with CN of 78, the soil texture for eucalyptus forest was silty clay, and its CN was the lowest of the data set (46).\n\nIn Figure 2, the “S” values (Lian et al., 2020) are shown for the Chibunga River micro-basin for years 2013 and 2014. One can observe a low CN value (orange color) in the range of 1–52 mm of water retained in the soil. Highest values of the water retention potential in the study area are highlighted in yellow; they correspond to moorlands and coniferous forests. In the micro-basin of the Guano River, we uncovered water retention potential values of 0 mm (red color). Such areas are characterized by the presence of urbanization, bodies of water, and snow. On the opposite end, highest values of water retention correspond to areas with eucalyptus forests (blue color), which are located in the middle part of the micro-basin. One should emphasize that the vegetation cover in this area has not changed over the three years when the multitemporal study was conducted.\n\nIn Figure 3, the variation of precipitation is shown over the 3 years of our multitemporal study. Our findings agree with a prior study of Chidichimo and co-workers, according to which highest precipitation occurs over the months of March, April, and May, while minimum precipitation values are recorded in January, November, and December (Chidichimo et al., 2018).\n\nThe values of precipitation-runoff in the micro-basins were obtained via the curve number method (CN). Values of maximum retention potential were found to the vegetation cover for years 2013, 2014, and 2018.\n\nIn Figure 4, the precipitation-runoff relationship is much higher for an urban area in 2018 with a similar value of 44.6 mm, and a CN of 100, due to its retention capacity which is 0 . On the other hand, we obtained for vegetable crops and eucalyptus forests a similar value of 13 mm of runoff in 2014 and the retention capacity was of 60 and 68 mm of water in the soil, respectively. Therefore, it was determined that the grass category in 2013 had the lowest runoff at 4.4 mm and a CN of 79, demonstrating the saturation of porous medium in silty clay soil.\n\nFigure 5 reveals that in 2013, vegetable crops have a runoff of 12.7 mm and an average annual rainfall of 32.5 mm, while in 2014 and 2018, the urban area, wetlands, and eroded soil was in the range of 27–44 mm of runoff, showing retention of water of 0 mm in the soil. In addition, the greatest values of precipitation were found in 2018 for moorland category with 59.2 mm and runoff of 21.7 mm in the precipitation-runoff conversion, while the retention value is 72 mm of water, indicating the saturation of the porous medium in the soil.\n\n\nConclusion\n\nThe maximum retention potential is directly affected by the CN values that are recorded according to the conditions and use of the vegetation cover on the land. The categories snow, and water, wetlands, and urban area showed null retention of water in the soil because their transformation is directly from precipitation to runoff. This trend was opposite for the other categories, with the highest values in the maximum retention potential, while their values of runoff decreased.\n\nTo better understand the water storage capacity, we had to adequately interpret the values of field capacity. This was done by relating the water storage capacity to permeability, porosity, and the amount of organic matter. Obtained values allowed the formulation of adequate recovery or conservation measures to achieve better retention of water in the soil.\n\n\nData availability\n\nFigshare: GPS Coordinates of Sampling Points in Guano and Chibunga basins, https://doi.org/10.6084/m9.figshare.13874696.v1 (Mendoza et al., 2021).\n\nFigshare: Raw data Chibunga, https://doi.org/10.6084/m9.figshare.13875050.v1 (Melendez, 2021).\n\nFigshare: Supporting data: Guano (Recuperado).xlsx, https://doi.org/10.6084/m9.figshare.13299686.v1 (Dama Research Center Limited, 2020).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAcosta Gutiérrez, M: Alternativa de ejercicios físicos en agua para la Rehabilitación en pacientes hemipléjicos por infarto Cerebral en el municipio gibara. MS thesis. Facultad de Cultura Física, 2017. 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D. thesis, University of Calabria, Rende, Italy. 2016.\n\nMendoza BDama Research Center limited; Ltd., Genesis Sustainable Future: GPS Coordinates of Sampling Points in Guano and Chibunga basins. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13874696.v1\n\nNaranjo C: Anuario meteorológico. Quito, Ecuador: Instituto Nacional de Meteorología e Hidrología (INAMHI). 2014; 130. Reference Source\n\nOchoa-Tocachi BF, Buytaert W, Antiporta J, et al.: High-resolution hydrometeorological data from a network of headwater catchments in the tropical Andes. Sci Data. 2018; 5: 180080. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPacheco Moya RM, Quiala Ortiz E, Martínez Hernández I: Determinación del parámetro número de curva en la cuenca Las Coloradas en un entorno SIG. Ingeniería Hidráulica y Ambiental. 2018; 39(2): 60–71. Reference Source\n\nPapierowska E, Matysiak W, Szatyłowicz J, et al.: Compatibility of methods used for soil water repellency determination for organic and organo-mineral soils. Geoderma. 2018; 314: 221–231. Publisher Full Text\n\nSánchez-Cohen I, Velásquez-Valle MA, Esquivel-Arriaga G, et al.: Minimum hydrologic characterization for research in experimental watersheds with limited availability of information in arid lands. Revista Chapingo Serie Zonas Áridas. 2015; 14(2): 185–208. Publisher Full Text\n\nSatheeshkumar S, Venkateswaran S, Kannan R: Rainfall–runoff estimation using SCS–CN and GIS approach in the Pappiredipatti watershed of the Vaniyar sub basin, South India. Model Earth Syst Environ. 2017; 3(1): 24. Publisher Full Text\n\nSándor Zs, Tállai M, Kincses I, et al.: Effect of various soil cultivation methods on some microbial soil properties. DRC Sustainable Future. 2020; 1(1): 14–20. Publisher Full Text\n\nShomana T, Botha DE, Agachi PS: The water retention properties of biochar derived from broiler poultry litter as applied to the Botswana soil”. DRC Sustainable Future. 2020; 1(1): 67–72. Publisher Full Text\n\nSikorska AE, Viviroli D, Seibert J: Effective precipitation duration for runoff peaks based on catchment modelling. J Hydrology. 2018; 556: 510–522. Publisher Full Text\n\nSingla A, Bautista G, Mátyás B, et al.: Altitudinal variations in H and Al ions interchange along with Fe content in Amazonian rainforest soil. La Granja: Revista de Ciencias de la Vida. 2018; 28(2): 42–50. Publisher Full Text\n\nSukiasyan A, Kirakosyan A: Ecological evaluation of heavy metal pollution of different soil-climatic regions of Armenia by biogeochemical coefficients. DRC Sustainable Future. 2020; 1(2): 94–102. Publisher Full Text\n\nTailor D, Shrimali NJ: Surface runoff estimation by SCS curve number method using GIS for Rupen-Khan watershed, Mehsana district, Gujarat. J Indian Water Resour Soc. 2016; 36(4): 1–5. Reference Source\n\nTELEDET: «TELEDET - Percepción Remota,» Imágenes satelitales - clasificación supervisada, [En línea]. 2017. Reference Source\n\nToro EF: Riemann solvers and numerical methods for fluid dynamics: a practical introduction. Springer Science & Business Media, 2013. Publisher Full Text\n\nTso B, Mather P: Classification Methods for Remotely Sensed Data, CRC Presst. ISBN 1-4200-9072-0, 2009. Publisher Full Text\n\nUwizeyimana D, Mureithi SM, Mvuyekure SM, et al.: Modelling surface runoff using the soil conservation service-curve number method in a drought prone agro-ecological zone in Rwanda. International Soil and Water Conservation Research. 2019; 7(1): 9–17. Publisher Full Text\n\nWang L, Tang L, Wang Z, et al.: Probabilistic characterization of the soil-water retention curve and hydraulic conductivity and its application to slope reliability analysis. Comput Geotech. 2020; 121: 103460. Publisher Full Text\n\nWróbel M, Boczoń A: Determining the potential retention of a forest catchment based on the CN parameter. Model Earth Syst Environ. 2020; 1–4. Publisher Full Text\n\nZubieta R, Laqui W, Lavado W: Modelación hidrológica de la cuenca del río Ilave a partir de datos de precipitación observada y de satélite, periodo 2011-2015, Puno, Perú-Hydrological modeling using observed and satellite precipitation datasets for the Ilave River basin, 2011-2015 period, Puno, Peru. Tecnología y ciencias del agua. 2018; 9(5): 85–105. Publisher Full Text" }	[ { "id": "80799", "date": "23 Mar 2021", "name": "Holger Benavides-Muñoz", "expertise": [ "Reviewer Expertise Hydraulic and environmental 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\nGeneral comments: The article is within the scope of the F1000 Research Open for Science.\nThe abstract clearly explains the goal and the methodology as well as the flow of the paper. The method is well defined and well-illustrated as it is.\nThis reviewer believes that the authors are using pertinent equations with relevant variables which are clear to understand; besides, the authors detailed the laboratory procedures used in analyzing the associated analytical data.\nSpecific comments.- On page 6, “The 26 sampling points were in the Chibunga river micro-basin and 23 points in the Guano river micro-basin.” should be replaced by “The 23 sampling points were in the Chibunga river micro-basin and 21 points in the Guano river micro-basin.” Please, the authors should verify these numbers.\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": "92491", "date": "26 Nov 2021", "name": "Dwita Sutjiningsih", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title should be added with the name of the country.\nIn general, the work is clearly and accurately presented and cites the current literatures as well, except the presentation of tables and figures. The table should be accompanied by the unit dimension of the variables involved, for example in Table 1, precipitation is in mm. Figure 1 up to 3 are clear, but Figure 4-5 are a bit difficult to interpret, maybe it will be clearer if for each type of land cover, a relationship is made between precipitation and runoff.\nThe study design is appropriate, and the work is also technically sound. Sufficient details of methods and analysis are provided to allow replication by others.\nAlthough it is presented in Spanish, the source data underlying the results are available and accessible to ensure full reproducibility. The conclusions drawn however are very general, so without conducting any research, one can make such a statement. Conclusions should be made more specific. Also should be synchronized with the conclusion section in the abstract.\nNote: Comment from Reviewer 1 that is not yet responded: Which one is used? A total of 23 points corresponding to the Chibunga river micro-basin and 21 points for the Guano river micro-basin (in “Analysis in the Laboratory”) OR The 26 sampling points were in the Chibunga river micro-basin and 23 points in the Guano river micro-basin (in “Results and Discussion”).\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": [] } ]	1	https://f1000research.com/articles/10-172
https://f1000research.com/articles/10-171/v1	02 Mar 21	{ "type": "Opinion Article", "title": "Relations between the EU, Turkey, and Japan: dissonances in the strategic triangle", "authors": [ "Atsuko Higashino" ], "abstract": "This paper considers how the three sets of bilateral relations, between the European Union (EU) and Japan, the EU and Turkey, and Turkey and Japan, have developed (or been underdeveloped) and how the three have failed to form a strategic triangle that could potentially be beneficial for dealing with regional and international problems more efficiently. One of the main arguments is that, although all three sides of this triangle have developed significant economic relationships, their political relationships are less consolidated. Such a phenomenon is largely illustrated by the following three elements of this triangle: a deteriorated relationship between the EU and Turkey, an underutilised relationship between Japan and the EU, and an extant relationship between Japan and Turkey. This paper analyses the elements that have impeded or continue to hinder constructive political dialogue. It concludes that the potential for improvement in the three sets of bilateral relations is slight, in the short term, with Japan, in particular, finding it increasingly difficult to strike a good balance between developing the relationship with the EU while maintaining historical ties with Turkey.", "keywords": [ "European Union (EU)", "Turkey", "Japan", "Economic Partnership Agreement (EPA)", "Strategic Partnership Agreement (SPA)", "Customs Union", "Accession", "Connectivity" ], "content": "Introduction\n\nThe early version of this paper was presented at the 6th biannual European Union in International Affairs conference in 2018 (Higashino, 2018). On 8 December 2017, the EU and Japan finalised the EU–Japan Economic Partnership Agreement (EPA) after nearly five years of negotiations. The agreement was signed on 17 July 2018 and came into force on 1 February 2019, ahead of the original target date of 31 October 2019. Currently, the largest free trade agreement (FTA) in the world, which covers over 600 million people, the EU–Japan EPA, is set to have considerable global economic impact. The EU also concluded parallel negotiations on a Strategic Partnership Agreement (SPA) with Japan on 25 April 2018, which was signed on 17 July together with the aforementioned EPA. The SPA aims to reinforce the overall partnership between the two countries from a strategic perspective and to facilitate common solutions to shared challenges such as climate change, energy supply, and security threats.\n\nThe strengthening of the relationship between the EU and Japan has already affected their respective relationships with other countries. For instance, in light of the EU–Japan EPA negotiations and the EU–Turkey Customs Union that has been in place since 1995, the EPA between Japan and Turkey was negotiated in 2013. Stronger ties between the EU, Turkey, and Japan are, in principle, to be welcomed. Since all play leading roles in the G20, freer trade between them and closer consultation on various international affairs would enhance their respective positions as global actors, potentially enabling them to form an influential strategic triangle, in terms of politics, economy, and security. Such a triangle would be an important element for stability in the face of the uncertainties, such as those posed by the Trump administration in the US, as well as Brexit in the UK. As it stands, however, things are not developing in that direction. The relationship between the EU and Turkey has deteriorated, largely because of the significant democratic setbacks in Turkey over the past few years (e.g., Danforth, 2017; Rubin, 2017), such as oppression of freedom of speech, the press, and assembly. Mutual condemnation between the Erdoğan government and the EU is becoming ever harsher, and Turkey’s EU accession prospects are on the verge of collapse. Another side of the triangle does not seem to be promising either—the EU–Japan relationship requires consolidation before both sides can discuss some tricky and delicate issues facing them, including the democratic crisis in Turkey and how best to manage it together. Despite relations between Japan and Turkey rarely having had any problems, endeavours to substantiate their political and diplomatic relations remain largely undeveloped.\n\nThis paper first provides the background for the commencement of the Turkey–Japan EPA negotiations, explaining that it is a logical extension of the EU–Turkey Customs Union and the EU–Japan EPA negotiations. It then considers how the three sets of bilateral relations have developed (or been underdeveloped) and how the three have failed to form a strategic triangle. One of the main arguments is that all three sides of this triangle have developed strong economic relationships, but their political relationships are less consolidated. Such a phenomenon is largely illustrated by the following three elements of this triangle: first, the relationship between the EU and Turkey has deteriorated to an unprecedented level (referred to as deteriorated below); not only is the accession process at a standstill—at the time of writing the EU was considering sanctions over Turkey’s increasingly aggressive foreign policy in the East Mediterranean region—but the renewal of the Customs Union is also unlikely to materialise in the near future. Second, despite their long-standing endeavour to develop political dialogue, and despite repeated declarations that both sides share fundamental values and norms, the relationship between the EU and Japan has not reached a point of discussion and alignment over the trickiest problems facing them (referred to as underutilised below). Third, while historical ties and friendship have often been underscored, and recent endeavours to upgrade their economic relations have been significant, the relationship between Turkey and Japan is still dependent on the events and discourses of the past, falling far short of creating ties that enable the two countries to consult over more than bilateral issues (referred to as extant below).\n\nThere has been a rich quantity and quality of literature on the EU–Turkey relationship, a significant part of which focuses on the process of Turkey’s accession process to the EU and difficulties pertaining to it (e.g., Aydın-Düzgit & Tocci, 2015; Buzan & Diez, 1999; Pererini, 2017). With regard to the literature on the Japan–EU relationship, there has been a considerable shift in the tone assessing the relationship between the two sides, notably before the signing of the EPA/SPA and after. Although many studies used to problematise the rather inactive political relationship between the EU and Japan (e.g., Higashino, 2016; Tsuruoka, 2008), the emphasis of studies which followed the materialisation of the EPA/SPA has been on how to better substantiate and consolidate the relationship between the two parties, with more of an optimistic tone than in previous years (e.g., Heijmans, 2019; Söderberg, 2020; Tsuruoka, 2019; Tsuruoka, 2020). In terms of the Turkey–Japan relationship, although there have been some excellent overviews of the relations between the two countries (Pehlivanturk, 2011-12; Yilmaz, 2012), the quantity of the research is less than the studies on EU–Turkey and EU–Japan relations, and it has rarely been built upon. However, as this paper emphasises, the three relationships have mutually affected one another and the strengthening and worsening of the relationship of one side of the triangle (e.g., EU–Turkey) inevitably affects the other two (e.g., EU–Japan and Japan–EU). Such intertwining has not been explicitly discussed in previous research. One of the main purposes of this paper is therefore to explore and discuss the interconnectedness of the three sets of relationships. It should be noted that the strengthening of one side of this EU–Japan–Turkey relationship does not necessarily lead to positive implications on the other sides; as this paper shows, for example, strengthening the EU–Japan relationship via its EPA could potentially have negative impacts on Turkey’s trade with other countries, if the endeavours to modernise the Customs Union between the EU and Turkey are not realised.\n\nIn addition to the analyses mentioned above, this paper considers a potential implication of the deteriorating relationship of one side of the triangle—the EU and Turkey—shedding light on Turkey’s recent rapprochement with non-Western organisations and frameworks, inter alia, the Shanghai Cooperation Organisation (SCO). Erdoğan’s repeated references to Turkey’s intention to seek formal membership in the SCO has raised concerns both from the EU and, to a lesser extent, from Japan, as it has largely been seen as an indication that Turkey is moving farther away from the Western community and closer to a Russia-China-led regional/international order.\n\n\nBackground: bilateral trade agreements\n\nThe current Turkey–Japan EPA negotiation is a logical extension of two important trade agreements: The Turkey–EU Customs Union came into force in 1996 and the EU–Japan EPA came into force in 2019. Turkey’s relations with the EU are based on the Ankara Agreement establishing an Association between the European Economic Community (EEC) and Turkey, thus creating the Customs Union between the two. EU Customs Union Decision 1/95 requires Turkey to align its commercial policy with the EU’s Common Commercial Policy, which includes, inter alia, the EU’s preferential trade agreements (Official Journal of the European Communities, 1996). In this context, Turkey has been expected to negotiate agreements on a mutually advantageous basis with other countries with which the EU already has an FTA (Joint Study Group, 2013: 4). This means that Turkey and the EU should have ‘parallel FTAs in force’ (Joint Study Group, 2013: 4). To this end, Turkey has initiated a negotiation process and concluded FTAs with third countries at almost the same time as the EU (Joint Study Group 2013: 5).\n\nTherefore, the commencement of EPA/SPA negotiations between the EU and Japan in March 2013 necessitated FTA negotiations between Turkey and Japan. At the G20 Summit in November 2011, Turkish President Recep Tayyip Erdoğan told the then Prime Minister Yoshihiko Noda of Japan that he expected an EPA/SPA agreement to be negotiated between Japan and Turkey. After almost a month, in December 2011, Turkish Deputy Prime Minister, Ali Babacan, expressed Turkey’s intention to commence negotiations for a Japan–Turkey EPA/SPA in parallel with the EU–Japan EPA negotiations In July 2012, both sides agreed to launch a Joint Study Group for a Turkey–Japan EPA. Following the publication of the Report of the Joint Study Group in July 2013, formal negotiations began in December 2014. As of January 2021, 17 rounds of negotiations had been conducted (no negotiation tool place in 2020). However, as will be discussed below, such developments on each side of the EU–Turkey–Japan triangle in trade agreements have not led to the construction of more open, solid, and constructive political relationships between the three. The following sections of this paper will introduce the respective problems that characterise each relationship.\n\n\nEU–Turkey relationship: renewed Customs Union for a lesser evil\n\nSince it has already been widely understood that Turkey’s accession to the EU is not likely to happen for years to come (Blockmans & Yilmaz, 2017; Wagstyl & Chazan, 2017), one of the few options that could reinforce the relationship between the EU and Turkey is the renewal of the existing EU–Turkey Customs Union. As it stands, it is a rather limited agreement that covers all industrial goods and processed agricultural products. Still, it has contributed to the expansion of trade between the EU and Turkey; in 2019, Turkey was the EU’s 5th-largest trading partner, export market, and provider of imports, while the EU is Turkey’s most important trading partner (European Commission, 2020a). However, it is a trade agreement concluded 20 years ago and without a doubt needs an overhaul: the 1995 Customs Union was intended to be ‘an interim process, and not an end in itself’ (Vesterbye & Akman, 2017: 5).\n\nIn recent times, Turkey has been seriously affected by the conclusions of the several FTA/EPA by the EU with third countries. Turkey is expected to grant tariff-free access to goods from a third country with which the EU has negotiated an FTA, without receiving any reciprocal access. Further, Turkey does not have any influence over when the EU negotiates an FTA/EPA with third countries. Unless Turkey manages to conclude a similar deal with a third country, it could be forced to suffer unilaterally from a disadvantageous trade position. For instance, when the Transatlantic Trade and Investment Partnership (TTIP) was negotiated between the US and the EU before the inauguration of the Trump administration, Turkey was concerned that the TTIP could create discrimination against Turkey since the country could well have been forced to adjust its external tariffs to the US according to the rules of the Customs Union, while Turkey was not expected to have an equal share in the benefits stemming from the agreement (Yalcin et al., 2016). While the TTIP negotiations have been halted since early 2017, such concerns could well be raised whenever the EU negotiates to conclude new trade deals with third countries. Such a situation has already occurred in the case of the EU–Mexico EPA, in which Mexico unilaterally enjoys tariff-free access to Turkey’s market while Turkey cannot have equivalent access to the Mexican market. Turkey has therefore demanded that the EU begin negotiations for the upgrading of the Customs Union.\n\nThe EU, for its part, finds the current Customs Union unsatisfactory for solving bilateral trade problems with Turkey (European Commission, 2016). The European Commission condemns Turkey for the fact that it ‘has introduced various trade and market access barriers which also affect European companies. A number of these measures are in breach of the Customs Union rules’ (European Commission, 2017: 77–78). The Commission considers that such problems can largely be solved if both sides renew the Customs Union to remodel it as one that is more appropriate for current problems and realities. Johannes Hahn, the then Commissioner for European Neighbourhood Policy and Enlargement Negotiations, clearly states that a modernisation of the Customs Union would ‘bring big benefits for the EU’ (Vytiska, 2018).\n\nThe EU’s response to the renewal of the Customs Union has developed in three stages: (i) the basic agreement between the European Commission and Turkey to modernise the Customs Union (May 2015) and the recommendation by the European Commission to start the negotiations (December 2016), (ii) the EU’s announcement not to continue to work towards the modernisation of the Customs Union until the situation within Turkey improves (June 2018), and (iii) the European Council’s decision to launch a positive political agenda with Turkey, including the modernisation of the Customs Union. The first stage was when Turkey and the European Commission agreed in principle in May 2015 to modernise and expand the existing Customs Union between the two parties (European Commission, 2020b). This objective was reiterated in the ‘EU-Turkey Statement’ in March 2016, when both parties agreed to deal with the massive flood of migrants and asylum seekers mainly from Syria and other Middle Eastern states who risked their lives crossing the Mediterranean Sea, hoping to reach EU member states such as Germany and Sweden (European Council, 2016). On 21 December 2016, the European Commission adopted the recommendation for a Council decision authorising the opening of negotiations with Turkey on an agreement for the extension of the scope of the bilateral preferential trade relationship and the modernisation of the Customs Union (European Commission, 2020b). The main element of the recommendation was to further liberalise agricultural products and cover services, public procurement, and sustainable development. The proposal also included better dispute settlement mechanisms between the two. In addition to providing for a common external tariff for the products covered, the Customs Union foresaw Turkey aligning with the acquis communautaire in several essential internal market areas, notably concerning industrial standards. It was originally the intention of the Maltese Presidency to provide the Commission with the mandate within their term by the end of June 2017 (Vesterbye & Akman, 2017: 8). However, several member states, including Germany, were strongly opposed to starting any new trade deals with Turkey. So far, there is no sign that the EU member states will give a mandate to the European Commission to begin the negotiation.\n\nThe second stage was the decision and announcement by the General Affairs Council of the EU on 26 June 2018. This claimed that ‘Turkey has been moving further away from the European Union’ and ‘Turkey’s accession negotiations have therefore effectively come to a standstill’; thus, the Council ruled out not only the opening of the new chapters of the accession negotiations but also further works towards the modernisation of the Customs Union (Council of the European Union, 2018, para. 35). This policy was endorsed by the European Council on 28–29 June. Since that announcement, the approach of Brussels was that there were two conditions for the start of talks on the modernisation of the Customs Union and progress in the membership process: i) initiatives towards democratisation and improving rule of law and ii) greater alignment with the EU’s foreign policy towards third countries (Adar et al., 2020). However, this policy by the EU did not leverage a review of the domestic and international stance by the Erdoğan administration; as a result, the bilateral relations between the two reached a stalemate.\n\nA slightly more positive development was seen in the third stage, when on 1 October 2020 the European Council agreed to launch ‘a positive political EU-Turkey agenda with a specific emphasis on the modernisation of the Customs Union’ (European Council, 2020, para. 19). However, the latest Commission’s report on Turkey, which was published just a few days after the declaration of the European Council, contained negative assessments of Turkey’s political situation and foreign policy and did not include a means to restart the Customs Union negotiations (European Commission, 2020b).\n\nIt should be noted, however, that both European and Turkish experts argue that it is destructive for the EU to block the overhaul of the Customs Union process since it would be ‘tantamount to eliminating the most promising avenue of engagement with Turkey aimed at fostering greater rule-based governance’ (Ülgen, 2017; for similar assessment, also see Adar et al., 2020). Some even argue that upgrading the Customs Union would contribute to Turkey’s integration into the world economy (Vesterbye & Akman, 2017: 9) and that a wider and deeper agreement could spill over into areas like the rule of law, freedoms, and the judiciary (Vesterbye & Akman, 2017: 12). Having an updated and comprehensive trade deal is congruent with the global trade strategy that has been pursued by the EU in recent years. As it stands, however, many member states of the EU consider that starting negotiations to upgrade the Customs Union is tantamount to overlooking or tolerating the current authoritarian Turkish regime (Adar et al., 2020). The upgrading of the Customs Union has therefore become yet another issue of discord between Turkey and the EU.\n\nThis means that the conclusion of the EU–Japan EPA could, in theory, have an inevitable negative impact on Turkey, no matter how welcoming it may appear to be. In the scenario in which the renewal of the EU–Turkey Customs Union does not happen in the near future, and Japan–Turkey EPA negotiations need more time to be concluded, the Japan–EU EPA could be yet another example of an arrangement between the EU and a third country harming the Turkish economy. Even though such damages can partially be covered by the bilateral efforts of Japan and Turkey, such endeavours would be less effective in the absence of the upgraded Customs Union between the EU and Turkey. It should also be noted that Japan does not seem to be aware of the negative impact that a Japan–EU EPA could have on Turkey.\n\n\nEU–Japan strategic partnership: underutilised\n\nAlthough the EU designated Japan as one of its Strategic Partners in 2003 when the European Security Strategy was published—and although Japan and the EU have declared that they are partners that share principal values and norms—in-depth consultations regarding specific problems involving the EU and Japan have not been sufficiently conducted so far (for more positive views on EU-Japan relationship, see Tsuruoka, 2008; Tsuruoka, 2019; Tsuruoka, 2020). While I have pointed out elsewhere that Japan and the EU have largely been unsuccessful in discussing and dealing with the issues concerning security in East Asia (Higashino, 2016), the same phenomenon can be observed for the issues of the non-EU European countries1. Among the thorniest issues are the deterioration of democracy in Turkey, particularly after the attempted coup in July 2016, and how to (or rather whether or not to) continue Turkey’s EU accession process against this background. However, the communiqués and declarations after various high-level meetings and summits give no hint that the EU and Japan discussed the situation in Turkey in detail. This implies that, from the EU perspective, the democratic crisis in Turkey and deteriorating EU−Turkey relationship are ‘European’ problems, which are not to be shared or discussed with Japan. Japan, for its part, does not have any political will to bring these issues to the discussion table with the EU. Considering that the EU and Japan have concluded the SPA, whose core objective is to establish consensus in various fields of cooperation, based on their shared fundamental values such as democracy, rule of law, and human rights, it is desirable to engage in dialogue concerning delicate issues such as the political situation in Turkey. After the provisional application of the SPA was agreed for 1 February 2019, the Joint Committee under the SPA was held twice, on 25 March 2019 and 31 January 2020. In neither of these meetings, however, were issues concerning Turkey raised (Ministry of Foreign Affairs of Japan, 2019; Ministry of Foreign Affairs of Japan, 2020).\n\nOne of the relatively new arrangements that has the potential to foster EU–Japan cooperation but that has not yet been fully utilised is the Partnership on Sustainable Connectivity and Quality Infrastructure (henceforth ‘Connectivity Partnership’) that was signed between the EU and Japan in September 2019 (European External Action Service, 2019). This partnership aimed to facilitate cooperation between the EU and Japan, particularly on connectivity and infrastructure, including digital, transport, energy and people-to-people exchanges in regions such as ‘the Western Balkans, Eastern Europe, Central Asia, Indo-Pacific, as well as in Africa’ (European External Action Service, 2019, para. 2). As Shinzo Abe, the then Prime Minister of Japan, reiterated in his keynote speech at the Europa Connectivity Forum, this partnership forms an important part of the framework of the Japan–EU SPA, which would enable both sides to work on international affairs with shared values and principles (Abe, 2019). The document that inaugurates this partnership claims that both sides intend to promote ‘free, open, rules-based, fair, non-discriminatory and predictable regional and international trade and investment, transparent procurement practices, the ensuring of debt sustainability and the high standards of economic, fiscal, financial, social and environmental sustainability’. Furthermore, it was largely regarded as an approach to counter the Belt and Road Initiative (BRI) by China, which raised concerns about the so-called debt spirals in the countries in regions such as Central Asia and the Western Balkans (Emmott, 2019; Heijmans, 2019; Söderberg, 2020). Therefore, the Connectivity Partnership between the EU and Japan should not be viewed simply as an economic project, but as a reaction to the sense that the growing influence of China is a threat.\n\nThere could have been at least two vectors of potential for this Connectivity Partnership to fortify the Japan–EU relationship in their approach to Turkey: first, by positioning Turkey as a beneficiary of this Partnership, along with the regions listed, such as the Western Balkans and Eastern Europe, and second, by bringing Turkey’s expertise and experience into the projects undertaken within the framework of the partnership, in such regions as Central Asia, where Turkey has a much longer history of commitment. However, as it stands, it is highly unlikely that these potentials will be explored by either the EU or Japan because many of the EU Member States would not accept the positioning of Turkey as a beneficiary of newly developed EU projects, not least because of Turkey’s democratic backslide and its assertiveness in its foreign policy (see above). As for Japan, the current priority of the Connectivity Partnership is to mobilise this new form of cooperation between the EU and Japan as early as possible and counter Chinese influence in the targeted regions. It is therefore unthinkable for Japan to raise any delicate issues, including the idea of liaison with Turkey, as it could hinder this nascent partnership.\n\n\nTurkey−Japan political relationship: extant\n\nOn the surface, the relationship between Japan and Turkey has been ‘virtually free of serious friction or controversies’ (Pehlivanturk, 2011-12). Particularly in Japan, there has been a long-rooted discourse in which Turkey is sympathetic to Japan. Such discourse is based upon the history of mutual assistance between the two countries. This ranges from the tragedy of the Ertugrul frigate that sank off the coast of Kushimoto in 1890 to the 1985 operation by the Turkish government and Turkish Airlines to rescue around 200 Japanese nationals who were left behind in Tehran during airstrikes in the Iran−Iraq war. Even the experience of mutual assistance at the time of major earthquakes, such as those in Izmit in 1999 and Van in 2011 in Turkey, and the Great East Japan Earthquake in March 2011, have served as the basis of a deep friendship between Turkey and Japan (Higashino, 2014). Furthermore, the Abe administration placed special emphasis on strengthening the economic ties with Turkey, beginning with the previously negotiated EPA between Turkey and Japan.\n\nAs it stands today, there are two distinct problems concerning the Turkey−Japan relationship. First, the current Turkey−Japan relationship is still heavily dependent on the discourses of the ‘past’. In other words, rather contradictorily, by overemphasising their historical ties, the two countries have not made a sufficiently serious endeavour to renew and strengthen their relationship in a more up-to-date form2. What is lacking is an effort to establish a solid and constructive policy dialogue, concerning global issues, between the two. Thus, the previous talks between the Turkish and Japanese governments have focused almost exclusively on the EPA negotiations and other kinds of economic cooperation, rarely touching upon the more delicate political and diplomatic issues, including the recent deterioration of the EU−Turkey relationship. Even though the worsening relationship between the EU and Turkey is not beneficial to Japan, Japan has not developed an effective way to mitigate such tensions between its most significant partners.\n\nSecond, Japan does not necessarily share the same level of concern as other Western societies, namely within the EU as well as others, about the authoritarian trend of Turkish politics and the growing isolation of Turkey in world politics. Even though Erdoğan is now widely described as a ‘dictator’, or at least, one of the ‘illiberal leaders’ along with Vladimir V. Putin of Russia or Viktor Orban of Hungary (Guriev & Treisman, 2015), the frequency with which such concerns are expressed and reported in the Japanese media is distinctly low. Behind this lack of serious concern about current Turkish politics are several intertwining elements. Discourses that describe Turkey as a precious long-term friend of Japan have tended to restrict negative reporting of Turkey. There is a widespread tendency to see Turkey as a ‘victim’ of ‘discriminatory’ Western attitudes (e.g., Naito, 2020). Turkey’s stalemated EU accession process is largely seen as, rather simplistically, a false promise and expression of anti-Islamic sentiment by European countries. According to such a view, the hardening of Erdoğan’s attitude towards the EU has been regarded with a certain level of sympathy. Inevitably, such understandings circumvent balanced analyses of the ongoing political oppression by the Erdoğan administration.\n\nFurthermore, the timing of the notable destabilisation of Turkish politics coincided with important moments for the Turkey−Japan EPA (i.e., the publication of the Report of the Joint Study Group for a Turkey−Japan EPA in July 2013 or the fifth round of EPA negotiation in early 2016). The relevant events included: mass protests at the Gezi Park and the overreaction of the Erdoğan administration in early summer 2013; the attempted coup in July 2017; and the subsequent large-scale suspension of officials and academics who were alleged to have ‘Gülenist links’. The causal relationship between EPA-related developments and Japan’s reserved position towards the obvious worsening of the political situation in Turkey requires further detailed analysis. Still, we can assume that Japan was less motivated to problematise Turkish politics while the EPA negotiations were proceeding soundly.\n\n\nTurkey’s relationship with other regional organisations: destabilising effects\n\nIt has been widely discussed within Europe in recent years that, at least partly because of the deteriorating relationship between Turkey and the EU, Turkey is drifting farther from the Western community and is rapidly approaching non-Western international frameworks. One indication of this is Turkey becoming a founding member of the Asian Infrastructure and Investment Bank (AIIB) in January 2016. Turkey’s recent approach to join the SCO has also attracted interest and raised concerns (Chaziza, 2016; Wang, 2016; also see Antonenko, 2007). This reflects Turkey’s rapidly deepening interests towards China (Çolakoğlu, 2010; Çolakoğlu, 2013; Çolakoğlu, 2014; Çolakoğlu, 2015; Çolakoğlu, 2018) and approaching China-led international institutions can be interpreted as Turkey’s diplomatic endeavour to strengthen its relationship with China both bilaterally and multilaterally. Turkey had already become a ‘dialogue partner’ of the SCO, as the first NATO member and EU candidate to acquire such a position, and in recent years Erdoğan mentioned his intention to upgrade Turkey’s relationship with the SCO several times; this stirred speculation that Turkey will discard the long sought after path of joining the EU. For instance, Erdoğan repeatedly mentioned that Turkey should not be ‘fixated’ on the idea of joining the EU and could seek full membership in the SCO (Hürriyet Daily News, 2016). Judging from his statement, ‘[i]f we get into the SCO, we will say good-bye to the European Union’ (cited in Pantucci & Petersen, 2013), Erdoğan seems to believe that membership in the SCO is an alternative to membership in the EU (Keck, 2013) and that approaching the former does mean deviating from the latter (Hürriyet Daily News, 2013). Such statements may well be regarded as a retaliation against the EU, which would not make any tangible advancement in terms of the accession process to the EU, or as a bargaining chip in talks with the West (Gaspers, 2017). There is also the view that Turkey sees the SCO as a useful economic platform, ‘on which infrastructure projects are being implemented with the participation of China’ (Stepanov, 2016), but not as an international organisation that Turkey can finally settle into instead of the EU. At the same time, however, the consistency that can be observed in the series of Erdoğan’s statements concerning potential SCO membership should not be neglected.\n\nAs it stands, Beijing has verbally welcomed Erdoğan’s positive remarks about the SCO (Ministry of Foreign Affairs of the Peoples Republic of China, 2016), but it seems to maintain a reserved stance towards the potential for Turkish accession to the organisation (Gaspers & Huotari, 2017). Russia has also been reserved, with Putin refraining from mentioning anything positive or negative about the potential Turkish accession to the SCO.\n\nThese developments suggest that it is unlikely that Turkey’s integration into the SCO will go beyond the current status of dialogue partner. Also, as many experts point out, the Turkey–China relationship is not without its problems. According to Selçuk Çolakoğlu, diplomatic relations between the two countries have soured because (i) China lost the competition over the Sinop nuclear power plant tender vis-à-vis the Japanese–French consortium in 2013, (ii) the Chinese government never declared the Kurdistan Workers’ Party (PKK) to be a terrorist organisation, (iii) considerable differences exist between the Turkish and Chinese governments in terms of their treatment of the Uyghur diaspora, and (iv) Beijing’s imposition of new visa restrictions on Turkish citizens as of February 2016 made it almost impossible for Turkish citizens to visit China (Çolakoğlu, 2018). However, after the attempted coup in July 2016, the Turkish government has tried to intensify its relationship with China. Diplomatic as well as economic channels at various levels have been expanded, especially strengthening the relationship between the Communist Party of China (CPC) and the Justice and Development Party (AKP).\n\nHowever, it should be noted that, even though the EU regards the realisation of Turkish accession to the EU as unlikely, Turkey’s quest for closer ties to the SCO is not a welcome or desirable development. It is largely regarded as destabilising for transatlantic security, and intelligence sharing among Turkey and the EU/NATO would become increasingly difficult. Erdoğan’s rapprochement with China coincides with the timing of the EU’s concerns towards China and its increasing international influence (Godement & Vasselier, 2017). Japan also considers that Turkey’s closer ties to China and the SCO would be detrimental to the sound bilateral relationship between Japan and Turkey. Japan has experienced significant economic competition with China, particularly concerning infrastructure in South-East and South Asia, and Turkey’s approach to China would only fuel more rampant economic competition between Japan and China, caused by the very country with which Japan has enjoyed an exceptionally sound relationship for many years.\n\n\nConclusion\n\nThis paper has highlighted several problems pertaining to the EU–Turkey–Japan triangle. The intertwining of the three sides of this triangle and problems pertaining to these dyadic relationships have not been explicitly discussed in previous research. This paper sought to explore how the strengthening and worsening of the relationship of one side inevitably affects the other two. It also emphasised that the strengthening of one side of this triangle does not automatically lead to the strengthening of the other two sides; for instance, as this paper showed, the strengthening of the EU–Japan relationship through the recent EPA could potentially have negative impacts on Turkey’s trade with third countries. Also, as discussed in this paper, having a new cooperation framework like the Japan–EU Connectivity Partnership may not, at least in the short term, necessarily lead to a taboo-free relationship and widen the scope of the issues to be discussed between the two. In contrast, having this nascent framework may well have a hidden impact at least on the behaviour of Japan, as it tends to avoid bringing in delicate issues such as Turkey’s political situation being discussed between the EU and Japan.\n\nFor the time being, it is not realistic to expect a rapid and drastic amelioration of the EU–Turkey relationship, when the possibility of halting accession negotiations has been hinted at by both sides. The relationship between Japan and the EU has largely been underutilised; further steps are needed to conduct substantial and constructive dialogues about how to anchor Turkey firmly in the norms of the rule of law and democracy. Relations between Turkey and Japan need considerable upgrading, and much effort is also necessary to go beyond the comfortable discourse of the extant ‘old friendship’ and to have a more substantial political and diplomatic relationship. This paper also analysed how Turkey’s recent endeavour to build closer ties with the SCO is regarded with suspicion by Japan and the EU.\n\nAchieving all these aims is an ambitious task. If Japan intends to maintain a sound and constructive relationship with both the EU and Turkey, it will immediately face a considerable dilemma; as long as Japan considers itself one of the global actors that shares fundamental principles with the EU, it should not ignore the destabilisation of Turkish politics. However, considering the long-rooted discourses on historical ties with Turkey, it is highly unlikely that straightforward criticism of the Erdoğan administration would be a feasible option for Japan. Striking the best balance between the two will be a tightrope walk for Japanese diplomacy in the years to come.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nI am grateful to Jan Völkel who kindly helped me to work on the title of this article.\n\n\nFootnotes\n\n1 So far, Japan has been eager to raise issues such as threats provoked by North Korea and the conduct of China in the South and East China Seas. The EU has raised issues such as the Ukraine crisis and migration crisis.\n\n2 Compare, for instance, the ‘Turkey-Japan relationship’ and ‘Turkey-China relationship’ webpages at the Ministry of Foreign Affairs of Turkey. The latter contains much more concrete detail concerning recent political and economic cooperation. 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Reference Source" }	[ { "id": "80816", "date": "08 Mar 2021", "name": "Selçuk Çolakoğlu", "expertise": [ "Reviewer Expertise International relations", "security studies", "foreign policy", "and global governance." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is original in subject which analyzes the EU, Japan, and Turkey relations in the triangle concept. Although there are plenty of academic publications analyzing relations between the EU and Japan, the EU and Turkey, Japan and Turkey separately, triangular relationship analyses are exceedingly rare in the related literature.\nIf the author clarifies the outcomes of the new development between Turkey and Japan, the paper will be more updated. As creating a “trust gab” issue between Ankara and Tokyo, it can be elaborated the Carlos Ghosn escape from Japan to Lebanon via Istanbul by a private Turkish jet company in December 2019. The Turkish criminal court just found guilty two pilots and one manager of the Turkish jet company in February 2021. However, the current outcome of Turkish legal investigation is far from being satisfactory for the Japanese government considering that the Carlos Ghosn escape was only possible with some high-level help from the Turkish government. The author should clarify whether the Ghosn case has the potential to poison the bilateral relations between Ankara and Tokyo.\nThere are some corrections to some dates and others in the manuscript. The corrections are in bold formats:\nPage 3, paragraph 2, “… EU–Turkey Customs Union that has been in place since 1996 …”. Page 3 paragraph 4, …(e.g., EU–Japan and Japan–Turkey). Page 4, paragraph 3, At the G20 Cannes Summit in November 2011, … Page 7, paragraph 3, the attempted coup in July 2016; and...\n\nAfter doing these minor revisions/corrections, the paper can be indexed.\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": "6455", "date": "16 Mar 2021", "name": "Atsuko Higashino", "role": "Author Response", "response": "Professor Çolakoğlu, thank you for your insightful comments. I agree with your kind suggestions for correction concerning dates and other issues and will make the necessary revisions. Your comments on the potential change in Japan’s perception of Turkey because of the Ghosn case are interesting and certainly worth mentioning in my paper. My current conclusion, however, is that the series of events concerning Ghosn will not affect Japan-Turkey relations significantly. Having read your comments, I consulted articles from: Japanese newspapers, Turkish newspapers (in English, online), and International newspapers that covered the Ghosn case, particularly the recent judgement by the criminal court of Turkey. In the context of your pointing out that the judgement could be considered unsatisfactory by the Japanese government, I consider that the way the issue was reported in (1) is particularly relevant for my paper. What struck me as noteworthy in all cases was the scarcity of media coverage concerning this incident. All the articles that I consulted reported the contents of the judgement by the Turkish court very briefly. Furthermore, none of the articles in category (1) implied any potential discord between Japan and Turkey due to this judgement[1]. I predict that this case will not have a significant negative impact on the bilateral political relationship between Japan and Turkey for the following reasons; Japanese public perception about the case is that Ghosn himself is responsible. Some implicate the Lebanese authorities in his escape from Japan[2], but I found no articles that place the blame on the Turkish authorities. After the flight to Lebanon, the Japanese public gradually lost interest in the case. This could explain the scarcity of media coverage concerning the judgement by the Turkish criminal court. One exceptional example is the strong reaction of the Japanese government towards the Opinion adopted on 20 November 2020 by the Working Group on Arbitrary Detention of the United Nations, which concluded that the measures that had been applied to the defendant Carlos Ghosn by the Government of Japan had constituted arbitrary detention. The Japanese government deemed the Opinion ‘totally unacceptable’ and ‘not legally binding[3]’. Needless to say, this Opinion had nothing to do with the Turkish government. As you mention, some — in Japan or Turkey — believe that Ghosn’s flight could have been possible only ‘with some high-level help from the Turkish government’; however, I could not find any media reports that blamed the Turkish government for this. I therefore consider that, rather than implying a potential crisis for their bilateral relationship, this silence by the Japanese media concerning potential involvement by Turkish authorities might even indicate the ‘resilience’ of the relationship. In the context of my article, it might be possible to assert that even the Japan-Turkey relationship is not easily affected by incidents of this kind. I will consider whether I should refer to the Ghosn case in my article. I reiterate my sincere gratitude for your kind comments, which have stimulated my thoughts on this issue. [1] See, for example, https://www.tokyo-np.co.jp/article/88034; https://news.yahoo.co.jp/articles/25b8d813f7198677e1e50e52337a59f843b45081 [2] https://www.bbc.com/news/world-50964040 [3] https://www.mofa.go.jp/press/release/press3e_000135.html" } ] }, { "id": "80819", "date": "17 Mar 2021", "name": "Paul M. Bacon", "expertise": [ "Reviewer Expertise The European Union", "EU-Japan relations", "the EU as an Indo-Pacific actor", "Brexit", "human security." ], "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 was an enjoyable and interesting paper. It was well researched and written, and I learned a lot about three significant bilateral relationships. In my opinion, it is clear that the article deserves to be published, and to be published with minor revisions, that is to say 'Approved', using the F1000 terminology.\nThe author offers sophisticated, clear and detailed analysis of three relationships: EU-Japan, EU-Turkey, and Turkey-Japan. The significance of each of the relationships is established, as are the problems and potentials. A helpful distinction is drawn between economic factors such as trade and connectivity, and political factors such as human rights and democracy, with the latter being correctly regarded as more problematic. The sources that the author uses are well-respected experts on each of the three relationships, and are also up to date, covering recent developments comprehensively. The author also uses governmental texts very well, correctly identifying the most relevant official political and economic statements and other communications, to explain the evolving relationships of each of the parties.\nOne dimension of the paper that I would like see further explored in subsequent work, which is beyond the scope of this valuable paper, is the idea of a ‘strategic triangle’. This concept features in the title, the abstract and the conclusion of the paper under discussion, and is used effectively in this paper, but descriptively rather than analytically. This is fine. However, the concept has a rich history in both game theory and strategic analysis (see for example Tatu, 19701; Brown, 19762; Pillsbury, 19773; Gottlieb, 19774; Garrett, 19795; Dittmer, 19816; and Paul, 20197), and can be explored in more detail.\nDittmer (1981) for example, argues that: The preconditions for a triangular relationship are that each player recognize the strategic salience of the three principals, and the relationship between any two will be affected by each player's relationship to the third. Within the triangle, there are three distinct pattern dynamics: the ménage à trois, consisting of mutually positive relationships among all three; the stable marriage, consisting of a bilateral relationship excluding the third, and the romantic triangle, consisting of one pivot player playing off two suitors. There are two dimensions to this definition; the first is the mutual recognition of strategic salience, the second is the type of dynamic among the three principals. This first begs the question to what extent the three principals in the paper recognize each other as strategically salient. The IR literature on strategic triangles has often focused on the most powerful states in the system: China/Russia/US in the context of the Cold War, and China/India/US in the emerging international context. So, in order to have ‘triangleness’ as an attribute, the three principals have to meet two criteria: to be objectively important in their own right, and to recognize each other as such.\nDoes Turkey meet either criterion, both in terms of absolute or relational power? At one level, the cognitive premise of the paper appears to be that this triangular relationship is significant: why isn’t the obvious potential for a ménage à trois being realized? But the author doesn’t really explain why we should think of Turkey and Japan as being objectively important, other than both being in the G20, and could also say more about the relative significance of the economic relationships.\nIn fact, the EU-Japan and EU-Turkey trade relationships are highly important, and of roughly equal significance, while the Turkey-Japan relationship is quite insignificant by comparison. According to the European Commission (2019, https://trade.ec.europa.eu/doclib/docs/2006/september/tradoc_122530.pdf), EU-Turkey trade volume was 138 billion Euros, and EU-Japan trade volume 124 billion Euros, compared to a volume of under 4 billion Euros for Japan-Turkey trade (Daily Sabah, 2019, https://www.dailysabah.com/business/2019/07/02/turkey-japan-economic-cooperation-more-important-in-times-of-trade-protectionism).\nIn the main body of the paper the author explains in a detailed and sophisticated way what the obstacles are to deeper political cooperation and alignment between the three principals. She also explains expertly how timing has been a factor; how Turkey’s worst human rights abuses have taken place at sensitive times with regard to the EU-Japan negotiations, meaning that Japan elected to tread carefully and to prioritize the EU relationship. So at one level the paper invites us to share an initial premise that this triangular relationship is important, but then proceeds to explain forensically and expertly why it hasn’t fulfilled its potential, and that in fact the EU-Japan side of the triangle has been more important. We could therefore, logically, argue that there is no strategic triangle, because Turkey isn’t important enough. Or, we could say that Turkey is important, but that the relationship can be characterized as a ‘stable marriage’ between the EU and Japan, somewhat at the expense of Turkey, as outlined in the Dittmer quote above. If she wishes to pursue this second option in future work, the author could perhaps say more to establish why Turkey is potentially important, emphasizing the significance of the EU-Turkey trade relationship, and the strategic significance of Turkey as a civilizational swing state with a natural leadership role in Central Asia, an area of great significance to Japan and China (Huntington, 19938). Although the author does talk about Turkey’s possible membership of the SCO, it is not really explained why that is important, or indeed why Turkey can be a highly significant player in the region even if it does not fully join the SCO.\nI do think that a case can be made that Turkey is highly important strategically and civilizationally, but the author doesn’t really set out this positive case. This argument needs to be made more systematically at the start of the discussion in subsequent work. This would then establish the credentials of Turkey as a legitimate part of an EU-Japan-Turkey strategic triangle, and this then allows us to identify a puzzle: that Turkey is potentially important strategically, but Japan has largely acquiesced in the EU’s practice of marginalizing Turkey, meaning that Turkey is the junior partner in the triangle.\nIn short, I would suggest that the triangular premise and its attributes need to be explored in more detail in subsequent work. The triangular dimension lies at the heart of what makes this paper an important contribution. And the triangle concept, used analytically rather than descriptively, can shed still more light on this particular case, and in turn show the approach could be profitably used in other cases, for example in the analysis of EU-US-China, or EU-China-Japan. EU Studies scholarship on the Asian region has in the past arguably been a little one-dimensional, focusing bilaterally on the EU’s strategic partnerships, when it can be far richer and more profitable to take a triangular approach. For example, it is difficult to gain a meaningful understanding of the EU-Japan relationship, without understanding its relation to the EU-China relationship (see Berkofsky et al., 20199) for a useful exception to this general rule, which looks at EU-Japan relations in the shadow of China). I look forward to reading future work on these issues from the author, which build on the foundations in this valuable paper.\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": "80820", "date": "22 Mar 2021", "name": "Kohei Imai", "expertise": [ "Reviewer Expertise International Relations", "Turkish foreign policy", "Middle East Studies" ], "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 examined Turkey-EU, EU-Japan, Japan-Turkey relations well. And the item of triangular relations is an undeveloped field in International Relations. Therefore, this article has a good viewpoint.\nMeanwhile, this article should clarify the following questions for a better explanation:\nPlease explain the reason of case selection (why did you choose Japan-Turkey-EU triangle relations as a case?).\n\nThree bilateral relations are not meaningfully connected to each other in this article. It is necessary that this article attempts to adopt frameworks of trilateral relations like “strategic triangle” or “fateful triangle”.\n\nThis manuscript focused on Turkey-SCO relations. However, historically, Turkish decision makers have taken care of great powers and major institutions. So, this article should also examine Turkey’s diplomacy toward great powers and other regional organizations like NATO. In other words, it is necessary that this paper should discuss Turkey’s balancing behaviors.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-171
https://f1000research.com/articles/9-1246/v1	15 Oct 20	{ "type": "Software Tool Article", "title": "TreeSummarizedExperiment: a S4 class for data with hierarchical structure", "authors": [ "Ruizhu Huang", "Charlotte Soneson", "Felix G.M. Ernst", "Kevin C. Rue-Albrecht", "Guangchuang Yu", "Stephanie C. Hicks", "Mark D. Robinson", "Ruizhu Huang", "Charlotte Soneson", "Felix G.M. Ernst", "Kevin C. Rue-Albrecht", "Guangchuang Yu", "Stephanie C. Hicks" ], "abstract": "Data organized into hierarchical structures (e.g., phylogenies or cell types) arises in several biological fields. It is therefore of interest to have data containers that store the hierarchical structure together with the biological profile data, and provide functions to easily access or manipulate data at different resolutions. Here, we present TreeSummarizedExperiment, a R/S4 class that extends the commonly used SingleCellExperiment class by incorporating tree representations of rows and/or columns (represented by objects of the phylo class). It follows the convention of the SummarizedExperiment class, while providing links between the assays and the nodes of a tree to allow data manipulation at arbitrary levels of the tree. The package is designed to be extensible, allowing new functions on the tree (phylo) to be contributed. As the work is based on the SingleCellExperiment class and the phylo class, both of which are popular classes used in many R packages, it is expected to be able to interact seamlessly with many other tools.", "keywords": [ "SummarizedExperiment", "tree", "microbiome", "hierarchical structure" ], "content": "Introduction\n\nBiological data arranged into a hierarchy occurs in several fields. A notable example is in microbial survey studies, where the microbiome is profiled with amplicon sequencing or whole genome shotgun sequencing, and microbial taxa are organized as a tree according to their similarities in the genomic sequence or the evolutionary history. Also, a tree might be used in single cell cytometry or RNA-seq data, with nodes representing cell subpopulations at different granularities1. Currently, phyloseq2 and SingleCellExperiment3 are popular classes used in the analysis of microbial data and single cell data, respectively. The former supports the information pertaining to the hierarchical structure that is available as the phylo class (e.g., phylogenetic tree), and the latter is derived from the SummarizedExperiment class (defined in the SummarizedExperiment package4), which is widely used as a standardized container across many Bioconductor packages. Since the data structures in these fields share similarities, we were motivated to develop an S4 class5, TreeSummarizedExperiment, that not only leverages the facilities from the SummarizedExperiment class, but also bridges the functionality from the phylo class, which is available from the ape6 package and has been imported in more than 200 R packages.\n\nWe define TreeSummarizedExperiment by extending the SingleCellExperiment class, so that it is a member of the SummarizedExperiment family, and thus benefits from the comprehensive Bioconductor ecosystem (e.g., iSEE7, SEtools8, and ggbio9). At the same time, all slots of the phyloseq class have their corresponding slots in the TreeSummarizedExperiment class, which enables convenient conversion between these classes. Furthermore, we allow the link between profile data and nodes of the tree, including leaves and internal nodes, which is useful for algorithms in the downstream analysis that need to access internal nodes of the tree (e.g., treeclimbR1).\n\n\nMethods\n\nR version: R version 4.0.2 (2020-06-22)\n\nBioconductor version: 3.11\n\nPackage: 1.4.8\n\nThe structure of TreeSummarizedExperiment. The structure of the TreeSummarizedExperiment class is shown in Figure 1.\n\nThe rectangular data matrices are stored in assays. Each matrix usually has rows representing entities (e.g., genes or microbial taxa) and columns representing cells or samples. Information about rows and columns is stored in rowData and colData, respectively. The hierarchy structure on rows or columns is stored in rowTree or colTree respectively, and the link information between rows/columns and nodes of the row/column tree is in row/Links colLinks.\n\nCompared to the SingleCellExperiment objects, TreeSummarizedExperiment has four additional slots:\n\nrowTree: the hierarchical structure on the rows of the assays.\n\nrowLinks: the link information between rows of the assays and the rowTree.\n\ncolTree: the hierarchical structure on the columns of the assays.\n\ncolLinks: the link information between columns of the assays and the colTree.\n\nThe rowTree and/or colTree can be left empty (NULL) if no trees are available; in this case, the rowLinks and/or colLinks are also set to NULL. All other TreeSummarizedExperiment slots are inherited from SingleCellExperiment.\n\nThe rowTree and colTree slots require the tree to be an object of the phylo class. If a tree is available in an alternative format, it can often be converted to a phylo object using dedicated R packages (e.g., treeio10).\n\nFunctions in the TreeSummarizedExperiment package fall in two main categories: operations on the TreeSummarizedExperiment object or operations on the tree (phylo) objects. The former includes constructors and accessors, and the latter serves as “components” to be assembled as accessors or functions that manipulate the TreeSummarizedExperiment object. Given that more than 200 R packages make use of the phylo class, there are many resources (e.g., ape) for users to manipulate the small “pieces” in addition to those provided in TreeSummarizedExperiment.\n\nWe generate a toy dataset that has observations of 6 entities collected from 4 samples as an example to show how to construct a TreeSummarizedExperiment object.\n\n\n\n\n\nThe information of entities and samples are given in the row_data and col_data, respectively.\n\n\n\n\n\n\n\nThe hierarchical structure of the 6 entities and 4 samples are denoted as row_tree and col_tree, respectively. The two trees are phylo objects randomly created with rtree from the package ape. Note that the row tree has 5 rather than 6 leaves; this is used later to show that multiple rows in the assays are allowed to map to a single node in the tree.\n\n\n\nWe visualize the tree using the package ggtree 2.2.411. Here, the internal nodes of the row_tree have no labels as shown in Figure 2.\n\n\n\nThe node labels and the node numbers are in orange and blue text, respectively.\n\nThe col_tree has labels for internal nodes as shown in Figure 3.\n\n\n\nThe node labels and the node numbers are in orange and blue text, respectively.\n\nThe TreeSummarizedExperiment class is used to store the toy data generated in the previous section: assay_data, row_data, col_data, col_tree and row_tree. To correctly store data, the link information between the rows (or columns) of ssay_data and the nodes of the row_tree (or col_tree) can be provided via a character vector rowNodeLab (or colNodeLab), with length equal to the number of rows (or columns) of the assays; otherwise the row (or column) names are used. Tree data takes precedence to determine entities included during the creation of the TreeSummarizedExperiment object; columns and rows with labels that are not present among the node labels of the tree are removed with warnings. The link data between the assays tables and the tree data is automatically generated during the construction.\n\nThe row and column trees can be included simultaneously during the construction of a TreeSummarized-Experiment object. Here, the column names of assay_data can be found in the node labels of the column tree, which enables the link to be created between the column dimension of assay_data and the column tree col_tree. If the row names of assay_data are not in the node labels of row_tree, we would need to provide their corresponding node labels (row_lab) to rowNodeLab in the construction of the object. It is possible to map multiple rows or columns to a node, for example, the same leaf label is used for the ﬁrst two rows in row_lab.\n\n\n\n\n\n\n\n\n\n\n\n\n\nWhen printed on screen, TreeSummarizedExperiment objects display information as the parent SingleCell-Experiment class followed by four additional lines for rowLinks, rowTree, colLinks and colTree.\n\nSlots inherited from the SummarizedExperiment class can be accessed in the traditional way (e.g., assays(), rowData(), colData() and metadata()). These functions are both getters and setters. To clarify, getters and setters are functions for users to retrieve and to overwrite data from the corresponding slots, respectively.\n\nFor new slots, we provide rowTree (and colTree) accessors to retrieve the row (column) trees, and rowLinks (and colLinks) to retrieve the link information between assays and nodes of the row (column) tree. Currently, these functions are getters but not setters. If the tree is not available, the corresponding link data is NULL.\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nThe link data objects are of the LinkDataFrame class, which extends the DataFrame class with the restriction that it has at least four columns:\n\nnodeLab: the labels of nodes on the tree\n\nnodeLab_alias: the alias labels of nodes on the tree\n\nnodeNum: the numbers of nodes on the tree\n\nisLeaf: whether the node is a leaf node\n\nMore details about the DataFrame class could be found in the S4Vectors R/Bioconductor package.\n\nA TreeSummarizedExperiment object can be subset in two different ways: [ to subset by rows or columns, and subsetByNode to retrieve row and/or columns that correspond to nodes of a tree. To preserve the original clustering information, the rowTree and colTree are kept identical after subsetting, while rowLinks and rowData are updated accordingly.\n\n\n\n\n\n\n\n\n\nTo subset by nodes, we specify the node by its node label or node number. Here, entity1 and entity2 are both mapped to the same node t3, so both of them are retained\n\n\n\nSubsetting simultaneously in both dimensions is also allowed.\n\n\n\nThe current tree can be replaced by a new one using changeTree. If the hierarchical information is available as a data.frame with each column representing a taxonomic level (e.g., row_data), we provide toTree to convert it into a phylo object that is further visualized in Figure 4.\n\n\n\n\n\nIf the nodes of the two trees have a different set of labels, a vector mapping the nodes of the new tree must be provided in rowNodeLab.\n\n\n\n\n\n\n\nSince it may be of interest to report or analyze observed data at multiple resolutions based on the provided tree(s), the TreeSummarizedExperiment package offers functionality to flexibly aggregate data to arbitrary levels of a tree.\n\nThe column dimension. Here, we demonstrate the aggregation functionality along the column dimension. The desired aggregation level is given in the colLevel argument, which can be speciﬁed using node labels (orange texts in Figure 3) or node numbers (blue texts in Figure 3). Furthermore, the summarization method used to aggregate multiple values can be speciﬁed via the argument FUN.\n\n\n\nThe rowData does not change, but the colData is updated to reflect the metadata information that remains valid for the individual nodes after aggregation. For example, the column group has the A value for GroupA because the descendant nodes of GroupA all have the value A; whereas the column gg has the NA value for GroupA because the descendant nodes of GroupA have different values, (1 and 2).\n\n\n\n\n\n\n\n\n\nThe colLinks is also updated to link the new rows of assays tables to the corresponding nodes of the column tree (Figure 3).\n\n\n\nThe row dimension. Similarly, we can aggregate rows to phyla by providing the names of the internal nodes that represent the phylum level (see taxa_one below).\n\n\n\n\n\n\n\nUsers are nonetheless free to choose nodes from different taxonomic ranks for each ﬁnal aggregated row. Note that it is not necessary to use all original rows during the aggregation process. Similarly, it is entirely possible for a row to contribute to multiple aggregated rows.\n\n\n\n\n\nBoth dimensions. The aggregation on both dimensions could be performed in one step using the same function speciﬁed via FUN, in which case the aggValue function aggregates rows ﬁrst, and columns second. If different functions are required for different dimensions, or if columns need to be aggregated before rows, users should perform the aggregation in two steps.\n\n\n\nAs expected, we obtain a table with 3 rows representing the aggregated row nodes 7, 8 and 9 (rowLevel = 7:9) and 2 columns representing the aggregated column nodes 6 and 7 (colLevel = c(6, 7)).\n\n\n\nNext, we highlight some functions to manipulate and/or to extract information from the phylo object. Further operations can be found in other packages, such as ape6, tidytree12. These functions are useful for users who wish to develop more functions for the TreeSummarizedExperiment class.\n\nTo show these functions, we use the tree shown in Figure 5.\n\n\n\nConversion of the node label and the node number The translation between the node labels and node numbers can be achieved by the function convertNode.\n\n\n\n\n\n\n\n\n\nFind the descendants To get descendants that are at the leaf level, we could set the argument only.leaf = TRUE for the function findDescendant.\n\n\n\n\n\nWhen only.leaf = FALSE, all descendants are returned.\n\n\n\nMore functions. We list some functions that might also be useful in Table 1. More functions are available in the package, and we encourage users to develop and contribute their own functions to the package.\n\nHere, we show examples of how to write custom functions for TreeSummarizedExperiment objects. To extract data corresponding to specific leaves, we created a function subsetByLeaf by combining functions working on the phylo class (e.g., convertNode, keep.tip, trackNode) with the accessor function subsetByNode. Here, convertNode and trackNode are available in TreeSummarizedExperiment, and keep.tip is from the ape package. Since the numeric identifier of a node is changed after pruning a tree with keep.tip, trackNode is provided to track the node and further update links between the rectangular assay matrices and the new tree.\n\n\n\nThe row tree is updated; after subsetting, it has only two leaves, t2 and t3.\n\n\n\n\n\n\n\nThe TreeSummarizedExperiment package can be installed by following the standard installation procedures of Bioconductor packages.\n\n\n\nMinimum system requirements is R version 3.6 (or later) on a Mac, Windows or Linux system. We highly recommend to use the latest versions of R (currently, 4.0.2) and Bioconductor (at time of writing, 3.11) to gain access to the latest version of this package.\n\n\nUse cases\n\nTo demonstrate the functionality of TreeSummarizedExperiment, we use it to store and manipulate a microbial dataset. We further show exploratory graphics using the available functions designed for SummarizedExperiment objects in other packages (e.g., scater), or customized functions from popular visualization packages (e.g., ggplot213).\n\n\n\n\n\nThe Human Microbiome Project (HMP) 16S rRNA sequencing data, v35, is downloaded using the R package HMP16SData14, which contains survey data of samples collected at five major body sites in the variable regions 3–5.v35 is available as a SummarizedExperiment object via the ExperimentHub.\n\n\n\nWe store the phylogenetic tree as the rowTree. Links between nodes of the tree and rows of assays are automatically generated in the construction of the TreeSummarizedExperiment object, and are stored as rowLinks. Rows of the assays matrices that do not have a match to nodes of the tree are removed with warnings.\n\n\n\nHere, we show TreeSummarizedExperiment working seamlessly with SEtools (v. 1.2.0) to prepare data for the exploratory graphics. Since all operational taxonomic units (OTUs) in the sample belong to Bacteria in the SUPERKINGDOM level, we can calculate the sequencing depths by aggregating counts to the SUPERKINGDOM level. The resultant TreeSummarizedExperiment object agg_total is further converted into a data frame df_total with selected columns (HMP_BODY_SITE and RUN_CENTER) from the column data.\n\n\n\nTo make harmonized figures with ggplot2 (v. 3.3.2)13, we customized a theme to be applied to several plots in this section.\n\n\n\nFrom Figure 6, we note that more samples were collected from the oral site than other body sites.\n\nSamples collected at different body sites (HMP_BODY_SITE) are in different colors.\n\n\n\nFigure 7 shows that the sequencing depth of each sample across different coordination centers are quite similar. Within the coordination center, samples collected from Skin are more variable in the sequencing depth than those from other body sites.\n\n\n\nSamples collected at different body sites are in different colors.\n\nWe visualize samples in reduced dimensions to see whether those from the same body site are similar to each other. Three dimensionality reduction techniques are available in the package scater (v. 1.16.2), including principal component analysis (PCA)15, t-distributed Stochastic Neighbor Embedding (t-SNE)16, and uniform manifold approximation and projection (UMAP)17. Since TreeSummarizedExperiment extends the SingleCellExperiment class, functions from scater18 can be used directly. Here, we first apply PCA and t-SNE on data at the operational taxonomic unitl (OTU) evel, and select the one better clustering the samples to apply on data aggregated at coarser taxonomic levels to see whether the resolution affects the separation of samples.\n\nThe PCA is performed on the log-transformed counts that are stored in the assays matrix with the name logcounts. In practice, data normalization is usually applied prior to the downstream analysis, to address bias or noise introduced during the sampling or sequencing process (e.g., uneven sampling depth). Here, the library size is highly variable (Figure 7) and non-zero OTUs vary across body sites. It is difficult to say what is the optimal normalization strategy, and the use of an inappropriate normalization method might introduce new biases. The discussion of normalization is outside the scope of this work. To keep it simple, we will visualize data without further normalization.\n\nIn Figure 8, we see that the Oral samples are distinct from those of other body sites. Samples from Skin, Urogenital Tract, Airways and Gastrointestinal Tract are not separated very well in the first two principal components.\n\nThe first two principal components (PCs) are plotted. Each point represents a sample. Samples are coloured according to the body sites.\n\n\n\nThe separation is well improved with the use of t-SNE in Figure 9. Samples from Oral, Gastrointestinal Tract, and Urogenital Tract form distinct clusters. Skin samples and airways samples still overlap.\n\nThe first two t-SNE components are plotted. Each point represents a sample. Samples are coloured according to the body site.\n\n\n\nNotably, there are two well-separated clusters labelled as oral samples. The smaller cluster includes samples from the Supragingival Plaque and Subgingival Plaque sites, while the larger cluster includes samples from other oral sub-sites (Figure 10).\n\n\n\nThe two t-SNE components computed are plotted. Each point is a sample. Samples from the ‘supragingival or subgingival Plaque‘ are in orange, and those from other oral sub-sites are in blue.\n\nTo organize data at different taxonomic levels, we first replace the phylogenetic tree with the taxonomic tree that is generated from the taxonomic table. Due to the existence of polyphyletic groups, a tree structure cannot be generated. For example, the Alteromonadaceae family is from different orders: Alteromonadales and Oceanospirillales.\n\n\n\n\n\nTo resolve the loops, we add a suffix to the polyphyletic genus with resolveLoop. For example, Ruminococcus belonging to the Lachnospiraceae and the Ruminococcaceae families become Ruminococcus_1 and Ruminococcus_2, respectively. A phylo tree is created afterwards using toTree.\n\n\n\nThe separation of samples from different body sites appears to be worse when the data on broader resolution is used (Figure 11).\n\nThe two t-SNE components computed are plotted. Each point is a sample. Samples are colored according to the body sites.\n\n\n\nSpecifically, we loop over each taxonomic rank and generate a t-SNE representation using data aggregated at that taxonomic rank level.\n\n\n\n\n\n\nSummary\n\nTreeSummarizedExperiment is an S4 class in the family of SummarizedExperiment classes, which enables it to work seamlessly with many other packages in Bioconductor. It integrates the SummarizedExperiment and the phylo class, facilitating data access or manipulation at different resolutions of the hierarchical structure. By providing additional functions for the phylo class, we support users to customize functions for the TreeSummarizedExperiment class in their workﬂows.\n\n\nData availability\n\nHuman Microbiome Project data (v35) was used for the presented use cases. The data can be downloaded using the R package HMP16SData14.\n\n\nSoftware availability\n\nThe TreeSummarizedExperiment package is available at:\n\nhttps://doi.org/doi:10.18129/B9.bioc.TreeSummarizedExperiment\n\nSource code of the development version of the package is available at:\n\nhttps://github.com/fionarhuang/TreeSummarizedExperiment\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.404609619\n\nLicense: MIT", "appendix": "Acknowledgments\n\nWe thank Héctor Corrada Bravo, Levi Waldron, Hervé Pagès, Martin Morgan, Federico Marini, Jayaram Kancherla, Domenick Braccia, Vince Carey, Kasper D Hansen, Davide Risso, Daniel van Twisk, Marcel Ramos and other members of the Bioconductor community for their helpful suggestions.\n\n\nReferences\n\nHuang R, Soneson C, Germain PL, et al.: treeclimbR pinpoints the data-dependent resolution of hierarchical hypotheses. bioRxiv. 2020. Publisher Full Text\n\nMcMurdie PJ, Holmes S: phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013; 8(4): e61217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLun A, Risso D, Korthauer K, et al.: SingleCellExperiment: S4 Classes for Single Cell Data. 2020. Publisher Full Text\n\nMorgan M, Obenchain V, Hester J, et al.: SummarizedExperiment: SummarizedExperiment container. 2020. Publisher Full Text\n\nWickham H: Advanced r. CRC press, second edition. 2019. Reference Source\n\nParadis E, Schliep K: ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019; 35(3): 526–528. PubMed Abstract \| Publisher Full Text\n\nRue-Albrecht K, Marini F, Soneson C, et al.: iSEE: Interactive SummarizedExperiment Explorer [version 1; peer review: 3 approved]. F1000Res. 2018; 7: 741. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGermain PL: SEtools: tools for working with SummarizedExperiment. 2020. Publisher Full Text\n\nYin T, Cook D, Lawrence M: ggbio: an R package for extending the grammar of graphics for genomic data. Genome Biol. 2012; 13(8): R77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang LG, Lam TTY, Xu S, et al.: Treeio: An R Package for Phylogenetic Tree Input and Output with Richly Annotated and Associated Data. Mol Biol Evol. 2019; 37(2): 599–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu G, Smith DK, Zhu H, et al.: ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol. 2017; 8(1): 28–36. Publisher Full Text\n\nYu G: tidytree: A Tidy Tool for Phylogenetic Tree Data Manipulation. 2020. Reference Source\n\nWickham H, Chang W, Henry L, et al.: ggplot2: Create Elegant Data Visualisations Using the Grammar of Graphics. 2020. Reference Source\n\nSchiffer L, Azhar R, Shepherd L, et al.: HMP16SData: Efficient Access to the Human Microbiome Project Through Bioconductor. Am J Epidemiol. 2019; 188(6): 1023–1026. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWold S, Esbensen K, Geladi P: Principal component analysis. Chemometr Intell Lab. 1987; 2(1-3): 37–52. Publisher Full Text\n\nVan Der Maaten L, Hinton G: Visualizing Data using t-SNE. J Mach Learn Res. 2008; 9: 2579–2605. Reference Source\n\nMcInnes L, Healy J, Melville J: UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. arXiv. 2018. Reference Source\n\nMcCarthy DJ, Campbell KR, Lun AT, et al.: Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics. 2017; 33(8): 1179–1186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHUANG R, Soneson C, Ernst F, et al.: ﬁonarhuang/TreeSummarizedExperiment: v1.4.8 TreeSummarizedExperiment (Version v1.4.8). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4046096" }	[ { "id": "73188", "date": "19 Oct 2020", "name": "Leo Lahti", "expertise": [ "Reviewer Expertise Bioinformatics", "open research software", "R/Bioconductor", "microbiome research", "statistical 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\nThis software article introduces TreeSummarizedExperiment, a S4 class for hierarchically structured data in R. This provides a very generic data structure that serves for instance the single cell and microbiome bioinformatics communities, and has already gathered remarkable attention with a growing user base. The package is mature and has been available via Bioconductor for some time already.\nThe rationale for developing the new software tool has been clearly explained, and sufficient examples are provided. It extends the popular SingleCellExperiment class structure by bringing in tree info on data rows and cols (based on the phylo class). The new extended class has potentially many new applications, for instance in microbiome research; concrete examples are provided. The new class combines and extends other common class structures, which is very beneficial for the overall compatibility. Many tools for manipulation and use already exist based for instance on related work on the SummarizedExperiment family of classes, phylo tree structure, and the phyloseq class.\nThe overall description of the software is technically sound and follows standard conventions in the R/Bioconducor community. Sufficient details have been provided to allow replication of the software development and its use by others; the documentation and examples are sufficient for getting started with and interpreting outputs of the new class for anyone who has the technical skills that are needed to utilize this work.\nMajor\nEfficiency of the new method could be discussed further; does this scale up to population level cohorts that have thousands of samples are increasing hierarchical resolutions?\n\nHow easy it would be to incorporate further supporting information on the rows and columns, for instance on DNA/RNA sequence information?\n\nThe class is very generic; is the idea that this package can be used as such in (hierarchical) single-cell experiments, microbiome research, and potentially other fields that have little overlap currently? Or is this package meant to be a fundamental structure that can be further extended in more specific application domains? Some more discussion on these aspects could help to contextualize the new class.\nMinor\n\" ssay_data\" -> \"assay_data\"\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": "6409", "date": "03 Mar 2021", "name": "Ruizhu Huang", "role": "Author Response", "response": "Thank you for reviewing our work. Efficiency of the new method could be discussed further; does this scale up to population level cohorts that have thousands of samples are increasing hierarchical resolutions? TreeSummarizedExperiment (TSE) inherits the slots of the SummarizedExperiment (SE) & SingleCellExperiment (SCE) classes, and adds new slots like rowTree, colTree, rowLinks, colLinks, referenceSeq. For operations involving the inherited slots, TSE works similarly as SE and SCE. For the new slots, the data manipulation depends on the functions that users have applied on the tree object (of class phylo). These functions might be from TSE or outside TSE. For functions from TSE, either those working on the phylo tree (e.g., findDescendant, convertNode, matTree, addLabel) or those working on TSE (e.g., rowTree, colTree, rowLinks, colLinks, changeTree), takes only seconds even for a tree with up to 100,000 nodes. How easy it would be to incorporate further supporting information on the rows and columns, for instance on DNA/RNA sequence information? TSE now has a slot referenceSeq to store the sequence information of features ( rows). The class is very generic; is the idea that this package can be used as such in (hierarchical) single-cell experiments, microbiome research, and potentially other fields that have little overlap currently? Or is this package meant to be a fundamental structure that can be further extended in more specific application domains? Some more discussion on these aspects could help to contextualize the new class. Currently, there is not much overlap in the community across fields, e.g, single-cell experiments, microbiome research. But, we do see that they share similarities in data structures, and can potentially share synergies in data visualization or analysis. We provide TSE as a standalone R package like SummarizedExperiment and SingleCellExperiment, and propose it as a convenient starting point to create R packages for downstream analysis or visualization of data with tree structures. We are open to update our work or receive pull requests if new features (or slots) required in a specific field are feasible to be integrated to TreeSummarizedExperiment. For example, a new optional slot referenceSeq(), which was requested mainly for microbiome data to store RNA/DNA sequencing information, has been developed by Félix G.M. Ernst, and the PR has been accepted in TreeSummarizedExperiment. \" ssay_data\" -> \"assay_data\" The typo is fixed." } ] }, { "id": "73184", "date": "12 Nov 2020", "name": "Shila Ghazanfar", "expertise": [ "Reviewer Expertise statistics", "high throughput genomics data analysis", "single cell genomics analysis", "spatial gene expression 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\nHuang and colleagues have written a software article presenting TreeSummarizedExperiment [currently version 1.6.0], a Bioconductor package aimed at providing an S4 class for omics data with hierarchical tree structure. The TreeSummarizedExperiment class builds on the popular SingleCellExperiment object class, with additional slots in which hierarchical structure, in the form of phylo objects, can be added for features (rows) and observations (columns). In addition to the object class, the package contains several functions for manipulating these objects, ranging from getting/setting/resetting the tree slots, aggregating across rows and/or columns, and various analytical tasks operating on the phylo objects.\nThe article is well written with clear motivation and description of the package, and addresses an important problem of performing analysis of high dimensional hierarchically structured data using object-oriented programming. I have a few further comments and questions that may improve the breadth of use of TreeSummarizedExperiment by the research community.\n\nIs there a way to simply include an argument for aggValue() that would swap the order to columns first and rows second, rather than requiring the user to perform two distinct operations?\n\nThe new slots, rowTree, colTree, rowLinks and colLinks are 'getter' accessors but not currently 'setter' functions. I can imagine a popular use-case among users with an already constructed object of class SummarizedExperiment or SingleCellExperiment would be to simply use as(, \"TreeSummarizedExperiment\") and then attempt to add the additional slots, for example as the output of hclust(). I would suggest prioritising converting these functions to both 'getter' and 'setter', or perhaps adding a constructor usage for TreeSummarizedExperiment for objects that are already SummarizedExperiment or SingleCellExperiment, if possible.\n\nI'm interested in how TreeSummarizedExperiment would work in the case where the hierarchical structure is not a typical single tree, but comprising of multiple distinct tree structures. An example of such is single cell (or single clone) lineage data where there exists a tree structure within each experimental condition, but not between cells from different conditions. Would the colTree slot correspond to a list of trees in this case?\n\nHow would one go about combining different TreeSummarizedExperiment objects? Do the typical cbind() and rbind() operations have meaning here? In which cases are they not to be used?\n\nI would be interested in getting to a clustered heatmap as an example of visualisation for the TreeSummarizedExperiment, either implemented using ggplot2/ggtree, or other packages like ComplexHeatmap?\n\nHow would the tree structure information storage scale in terms of the number of rows and columns, or in the hierarchical structures?\nMinor/cosmetic\ntypo in affiliation 5.\n\nlegend cut off in Figures 6, 7, and 8.\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": "6410", "date": "03 Mar 2021", "name": "Ruizhu Huang", "role": "Author Response", "response": "Thank you for your comments! swap the order to columns first and rows second, rather than requiring the user to perform two distinct operations? aggValue() is now deprecated and replaced by a new function, aggTSE(), that allows users to swap the order of aggregation and define different functions for the row and the column dimension. The new slots, rowTree, colTree, rowLinks and colLinks are 'getter' accessors but not currently 'setter' functions. I can imagine a popular use-case among users with an already constructed object of class SummarizedExperiment or SingleCellExperiment would be to simply use as(, \"TreeSummarizedExperiment\") and then attempt to add the additional slots, for example as the output of hclust(). I would suggest prioritising converting these functions to both 'getter' and 'setter', or perhaps adding a constructor usage for TreeSummarizedExperiment for objects that are already SummarizedExperiment or SingleCellExperiment, if possible. rowTree and colTree are now both setters and getters. When the row/column tree is replaced, the rowLinks/colLinks is updated automatically. To avoid breaking links between assays and trees, we don't recommend users to modify the rowLinks/colLinks data. Therefore, rowLinks/colLinks are still kept as getters. I'm interested in how TreeSummarizedExperiment would work in the case where the hierarchical structure is not a typical single tree, but comprising of multiple distinct tree structures. An example of such is single cell (or single clone) lineage data where there exists a tree structure within each experimental condition, but not between cells from different conditions. Would the colTree slot correspond to a list of trees in this case? Yes, it's possible to have a list of trees in the rowTree/colTree. In the rowLinks/colLinks, we have added a new column (whichTree) to give information about which row/column tree a row/column is mapped to. We have also added a new vignette describing how to combine multiple TSEs. (https://www.bioconductor.org/packages/devel/bioc/vignettes/TreeSummarizedExperiment/inst/doc/The_combination_of_multiple_TSEs.html) How would one go about combining different TreeSummarizedExperiment objects? Do the typical cbind() and rbind() operations have meaning here? In which cases are they not to be used? rbind() and cbind() are now implemented for TreeSummarizedExperiment objects. To rbind() multiple TSEs successfully, it's required that the TSEs agree in the column dimension to have the same colTree() and colLinks(). Similarly, cbind() would require TSEs to have the same rowTree() and rowLinks(). More detailed information is available in the new vignette about combining multiple TSEs. (https://www.bioconductor.org/packages/devel/bioc/vignettes/TreeSummarizedExperiment/inst/doc/The_combination_of_multiple_TSEs.html) I would be interested in getting to a clustered heatmap as an example of visualisation for the TreeSummarizedExperiment, either implemented using ggplot2/ggtree, or other packages like ComplexHeatmap? We have added a new use case of TSE on CyTOF data, and customized a visualization function based on ggtree, ggplot2 and ggnewscale to plot a clustered heatmap. How would the tree structure information storage scale in terms of the number of rows and columns, or in the hierarchical structures. We store the tree structure as a phylo object. The size of a phylo object is quite small even for a tree with 106 leaves (about 90 Mb). To set up the link between rows/columns to a tree, it takes only a few seconds even for 106 rows to a tree with 106 leaves. typo in affiliation 5. legend cut off in Figures 6, 7, and 8. The typo and the legend cut off are fixed." } ] }, { "id": "73185", "date": "23 Nov 2020", "name": "Matthew Ritchie", "expertise": [ "Reviewer Expertise Transcriptomics (bulk and single cell)" ], "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\nHuang et al. describe the TreeSummarizedExperiment package, which provides well-designed S4 infrastructure that couples the phylo and SingleCellExperiment classes to create a container for high-throughput data that can be organised in a tree-like structure.\n\nThe article is structured like a vignette, providing an overview of the class (Figure 1) and stepping the reader through the process of setting up a TreeSummarizedExperiment object and accessing and assigning data to its various slots, firstly for a toy data set and then for data from the Human Microbiome Project.\nThe article is very clearly written, and the authors demonstrate the ability to use TreeSummarizedExperiment objects in conjunction with other established software for dealing with trees (e.g. ggtree and tidyTree) or dimensionality reduction of high-throughout data (e.g. scater). One topic that I was interested to read more about was its potential use in a single cell RNA-seq analysis. Perhaps use cases for such applications can be added as future work. The TreeSummarizedExperiment package has been available from Bioconductor since May 2019 and it has been downloaded > 2.4K times, which indicates it is being taken up by the community.\n\nMinor issues:\nAffiliation 5: missing 'O' in 'Oxford'.\n\n'Functions operating on the phylo object.' section, sentence 2, missing word: 'such as ape [and] tidytree.'\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": "6408", "date": "03 Mar 2021", "name": "Ruizhu Huang", "role": "Author Response", "response": "Affiliation 5: missing 'O' in 'Oxford'. Thank you. The typo is fixed. 'Functions operating on the phylo object.' section, sentence 2, missing word: 'such as ape [and] tidytree.' The missing word is added now." } ] } ]	1	https://f1000research.com/articles/9-1246
https://f1000research.com/articles/9-1236/v1	14 Oct 20	{ "type": "Research Article", "title": "Combined vitrectomy, near-confluent panretinal endolaser, bevacizumab and cyclophotocoagulation for neovascular glaucoma — a retrospective interventional case series", "authors": [ "Piotr Strzalkowski", "Alicja Strzalkowska", "Winfried Göbel", "Nils A. Loewen", "Jost Hillenkamp", "Alicja Strzalkowska", "Winfried Göbel", "Nils A. Loewen", "Jost Hillenkamp" ], "abstract": "Background: Neovascular glaucoma (NVG) is a severe, potentially blinding disease and a therapeutic challenge. The purpose of this study was to evaluate the safety and efficacy of an integrative surgical approach to neovascular glaucoma. Methods: Retrospective analysis of a one-year follow-up of a consecutive interventional case series of NVG. Eyes underwent transscleral cyclophotocoagulation, pars plana vitrectomy, near-confluent panretinal photocoagulation, and intravitreal bevacizumab. Phakic eyes underwent concomitant cataract surgery. Best-corrected visual acuity (BCVA, logMAR), intraocular pressure (IOP, mmHg), number of glaucoma medication, visual analog pain scale (VAPS, 0-10) were recorded at baseline, and 1, 3, 6, and 12 months. Blind eyes were excluded. Results: Seventy-seven eyes of 77 patients (45 male, 32 female, mean age 73.6±12.2 years) were included. NVG underlying conditions included retinal vein occlusion (41.6%), proliferative diabetic retinopathy (35.1%), central retinal artery occlusion (19.5%), and ocular ischemic syndrome (3.9%). Mean IOP decreased postoperatively from 46.3±10.1 mmHg to 14.5±7.9 mmHg (p<0.001), glaucoma medication from 4.7±1.3 to 1.8±1.8 (p<0.001), and VAPS from 6.0±1.8 to 0. BCVA remained unchanged. Postoperative intraocular inflammation had resolved in all eyes at the one-month follow-up. 71.4% (55/77) eyes did not require additional major interventions during follow-up. Conclusions: A single, comprehensive surgery session lowered IOP significantly, reduced GMS, and controlled pain.", "keywords": [ "Neovascular glaucoma", "integrative surgical approach", "iris neovascularization" ], "content": "Introduction\n\nNeovascular glaucoma (NVG) is a serious complication of a variety of ocular and systemic conditions. Neovascularization is the formation of abnormal blood vessels in an abnormal location triggered by an imbalance of anti-angiogenic and proangiogenic factors caused by retinal ischemia1. NVG accounts for 3.9 to 9.2% of all new glaucoma diagnoses2–4. According to the Federal Statistical Office of Germany, NVG's age-specific incidence in Germany in the age group from 45 to 64 years is 8 per 100,000. It increases to 24 per 100,000 in subjects older than 64 years5. The incidence of NVG varies depending on the etiology of retinal ischemia. For central retinal vein occlusion (CRVO) the reported incidence is 16%6, for proliferative diabetic retinopathy (PDR) 21.3%7, for central retinal artery occlusion (CRAO) 14.5%8, and ocular ischemic syndrome (OIS) 12.9%9, respectively. Carotid artery obstructive disease and fistulas are additional, extraocular vascular causes of retinal ischemia10. Early recognition and treatment of NVG are imperative to prevent aggressive evolution with severe vision loss and intractable pain that can require enucleation within a few months11. NVG also carries a poor prognosis for general health: remarkably, the expected lifespan of patients with NVG decreased by 52% compared to an age-correlated normal population, which corresponds to 6.5 years. In diabetics with NVG, the expected lifespan was reduced even more significantly by 72% (5.1 years in this subgroup)12.\n\nIn 1994, Miller et al. showed that retinal laser coagulation of primates could lead to CRVO and subsequent retinal ischemia with iris neovascularization13. In this model, neovascularization was mediated by vascular endothelial growth factor (VEGF) and correlated to its concentration. Several members of the VEGF family have since been identified, including VEGFA, VEGFB, VEGFC, VEGFD, and the placental growth factor (PLGF) with specific receptor-binding patterns. VEGFA primarily binds to VEGFR2, the activation of which stimulates neovascularization, relevant to NVG, and angiogenesis, the formation of normal blood vessels in normal development. In the eye, VEGF-A is produced by the retinal pigment epithelium, retinal ganglion cells, astrocytes, the endothelium, photoreceptors, and Müller cells14–17. Retinal hypoxia upregulates VEGF primarily via the hypoxia-inducible factor (HIF-1α and HIF-2α). The concentration of HIF increases when hydroxylases are inhibited during hypoxia so that HIF is not degraded by the proteasome18. HIF binds to the hypoxia-responsive element (HRE) of the VEGF gene in the nucleus leading to its upregulation19.\n\nAlthough the mechanism by which NVG emerges is hence relatively well understood, there is no consensus on how to best initiate treatment to address the different aspects. Treatment steps include lowering of IOP (topical and systemic glaucoma medications, glaucoma drainage implants, cyclodestruction)20–24, anti-inflammatory treatment (topical or intraocular steroids), reduction of retinal ischemia (panretinal photocoagulation (PRP))25,26, and inhibition of VEGF27–29. Vitrectomy with PRP and silicone oil tamponade may also reduce IOP in eyes with NVG30,31.\n\nHere, we evaluated the safety and efficacy of an integrative combined surgical approach for evolving NVG that combined pars plana vitrectomy, near-confluent full-scatter panretinal photocoagulation, off-label use of intravitreal bevacizumab, and transscleral cyclophotocoagulation in a single surgical session. We hypothesized that such a combined surgical approach for NVG would reduce IOP, medication, pain, reduce the number of necessary outpatient visits, and prevent the necessity of further surgeries.\n\n\nMethods\n\nOur retrospective study was reviewed and approved by the Ethics Committee of the University of Würzburg (reference: 9/17-sc, dated February 3, 2017) and a clearance certificate for retrospective data evaluation was issued (reference: 20180108 02, dated January 30, 2018). For retrospective anonymized data analysis, patient’s consent was not necessary. The Declaration of Helsinki, the Good Clinical Practice (GCP) guidelines and applicable data protection regulations were complied with.\n\nWe included all patients between October 2014 and August 2019 with NVG who met the inclusion criteria in this consecutive interventional case series.\n\nInclusion criteria were: 1) neovascularization of iris (NVI) or neovascularization of the angle (NVA), 2) IOP > 21 mmHg, 3) best-corrected visual acuity (BCVA, logMAR) ≧ light perception, and 4) 18 years or older.\n\nAll patients were treated at the University Eye Hospital in Würzburg, Germany. The combined intervention was part of routine patient care at our clinic and all patients with decompensated neovascular glaucoma and preserved visual function received this intervention. BCVA, intraocular pressure (IOP, mmHg), the number of glaucoma medication, visual analog pain scale (VAPS, 0-10) was recorded at baseline and at follow-up visits at one, three, six, and 12 months as part of routine care. Iris and anterior chamber neovascularization were graded using the Weiss and Gold rubeosis grading system32. Hypotony was defined as IOP ≤ 5 mmHg with hypotonous maculopathy, choroidal folds, or optic neuropathy33. Phthisis bulbi34 was defined as IOP ≤ 5 mmHg in a shrunken eye with worse than hand motion vision with or without pain containing atrophic and disorganized intraocular structures. Success was defined as lOP ≤ 21 mmHg or IOP reduction ≥ 30% from baseline, with or without glaucoma medication and without vision loss35.\n\nAll patients underwent decimal visual acuity testing, which was converted to a logMAR scale. Counting fingers (CF), hand movements (HM), light perception (LP), and no light perception (NLP) were converted into logMAR units 1.9, 2.3, 2.7, and 3.0, respectively36.\n\nWe retrospectively analyzed all patients treated with neovascular glaucoma at our clinic via the electronic hospital information system SAP®. The medical records of the treated patients were analyzed individually. The initial diagnosis of neovascular glaucoma and hospitalization served as the starting point. The cardiovascular risks were taken from the anesthesia premedication form.\n\nSurgical technique. All eyes underwent transscleral cyclophotocoagulation (810 nm diode laser, 360 degrees treatment to pop threshold with 20 spots and leaving out 3 and 9 o'clock), standard 3-port 23 gauge pars plana vitrectomy with a detachment of the posterior vitreous if not already present, near-confluent full-scatter panretinal photocoagulation (PRP) applied under indentation in all four quadrants from the vascular arcades to the ora serrata, intravitreal application of 0,1 mL of bevacizumab (Avastin®️ 25 mg/1 mL, Roche Pharma, Switzerland), and air tamponade. Phakic eyes underwent concomitant cataract surgery. The operation was carried out either with a retrobulbar block or general anesthesia.\n\nRetreatment. At follow-up, all eyes with elevated IOP were treated following an escalation scheme. First, glaucoma medications were increased to what was maximally tolerated. Eyes were then treated with transscleral cyclophotocoagulation (810 nm diode laser, 360 degrees treatment to pop threshold with 20 spots). Eyes that failed to respond to cyclophotocoagulation with a significant IOP reduction and had retained ambulatory visual acuity underwent tube shunt surgery. Repeated vitrectomy, including fill-in PRP, transscleral cyclophotocoagulation, and off-label use of intravitreal bevacizumab was applied to eyes with elevated IOP and dense vitreous hemorrhage. Fill-in PRP was applied when PRP could not be completed during surgery in eyes with extensive intraretinal hemorrhage. Further intravitreal injections of VEGF inhibitors were only applied for clinically significant non-ischemic macular edema and BCVA ≥1.1 logMAR.\n\nPatients lost to follow-up. All patients who failed to attend follow-up visits completed a standardized telephone survey that contained the following questions: What was the main reason for not following up? What is your current vision function? How many glaucoma drops are you using? Do you have any eye pain? Are you overall satisfied with the outcome of the treatment?\n\nData analysis was performed using Statistica 13.1 (Tulsa, Oklahoma, United States). The frequency of observations described categorical variables. Continuous variables were described as mean with standard deviation (SD) or median with range (minimum-maximum). Friedman test and Wilcoxon signed-rank test were used to compare data measured on an ordinal scale and continuous variables with non-normal distribution. Evaluation of data normality was performed using the Shapiro-Wilk test. Welch's t-test for unequal variances was used for IOP between pain versus no-pain group comparison. Categorical variables of the relationship between neovascularization stage and type of intervention were compared using the χ2 test with odds ratio (OR) measurement. Kaplan–Meier curve and log-rank test were used for success analysis. P-values <0.05 were considered significant.\n\nAn earlier version of this article can be found on medRxiv (doi: https://doi.org/10.1101/2020.01.19.20017889)\n\n\nResults\n\nAll 77 patients (45 male, 32 female, mean 73.6±12.2 years, range 29–91 years) completed a one-year follow-up and were included in the retrospective analysis (Figure 1). Conditions that lead to NVG included CRVO 41.6% (32/77), PDR 35.1% (27/77), CRAO 19.5% (15/77), and ocular ischemic syndrome 3.9% (3/77)37 (Table 1).\n\nEyes with no light perception and patients with a history of glaucoma other than neovascular glaucoma were excluded. *Telephone survey with all patients lost to follow-up.\n\nBMI, body mass index; CRAO, central retinal artery occlusion; CRVO, central retinal vein occlusion; PDR, proliferative diabetic retinopathy; OIS, ocular ischemic syndrome.\n\nThe most common cardiovascular risk factor was arterial hypertension 87.0% (67/77), followed by hyperlipidemia 67.5% (52/77), diabetes mellitus 59.7% (46/77), and smoking 11.7% (9/77). While one patient did not have any cardiovascular risk factors, 24.7% (19/77) had at least one risk factor, and 74.0% (57/77) had multiple risk factors.\n\nThe mean body mass index (BMI kg/m²) for the study group was 28.7±5.0 (underweight ≤ 18.5, normal weight = 18.5–24.9, overweight = 25–29.9, obesity ≥ 3038). 1.3% (1/77) were underweight, 24.7% (19/77) had a normal weight, 40.3% were overweight (31/77) and 33,8% (26/77) obese.\n\nMean logMAR BCVA was 1.9±0.7 at baseline,1.7±0.8 at one month, 1.8±0.8 at three months, 1.8±0.8 at six months, and 1.8±0.8 at 12 months (p=0.47, Figure 2). At baseline, 35.1% (27/77) of patients and at 12 months, 45.5% (35/77) of patients had an ambulatory visual acuity (≥ logMAR 1.7), respectively, but was not statistically significant (p=0.0931). One eye worsened from LP to NLP at one week, three eyes at six months, and three eyes at 12 months.\n\nIOP decreased significantly from 46.3±10.1 mmHg at baseline to 21.4±10.9 mmHg at one month, 18.6±10,7 mmHg at three months, 15.1±7.6 mmHg at six months, and 14.5±7.9 mmHg at 12 months (p<0.001; Figure 3). At one-year follow-up, 89.6% (n=69) of patients had an IOP ≤ 21 mmHg. IOP ≤ 5 mmHg was found in 7.8% (n=6) and tolerated without complications. 96.1% (n=74) presented at baseline with IOP≥30 mmHg. The number of glaucoma medication decreased from 4.7±1.3 at baseline to 1.9±1.9 at one month, 1.8±1.7 at three months, 1.8±1.5 at six months, and 1.8±1.8 (p<0.001; Figure 4) at 12 months.\n\nWhile 32.5% (n=29) of patients complained of ocular pain at baseline (VAPS: 6.0±1.8), all patients were without pain at all follow-up visits. Patients with pain had a significantly higher baseline IOP of 49.9±9.0 mmHg than patients without pain 44.1±10.3 mmHg (p<0.01).\n\nEarly postoperative complications (day 1 to four weeks) included intraocular fibrin 67.5% (52/77), hyphema 20.8% (16/77), choroidal detachment 16.9 (13/77), and corneal erosion 14.3% (11/77). Late postoperative complications (> 1 months) included retinal detachment in 3.9% (3/77) and painless phthisis in 3.9% (3/77) (Table 2).\n\nAt the one-year follow-up, surgical intervention was required in 16.9% (22/77) eyes. Of these eyes, 63.6% (14/22) received vitrectomy, 22.7% (5/22) a Baerveldt glaucoma drainage implant, 4.5% (1/22) a trabeculectomy with mitomycin C and 4.5% (1/22) a keratoplasty. One painful and blind eye needed enucleation.\n\nRepeat transscleral cyclophotocoagulation was performed in 11.6% (15/77). 3.9% (5/77) of patients with a mean BCVA 1.1±0.3 logMAR received an additional 4.0±0.8 anti-VEGF injections.\n\nAt baseline, 11.7% (9/77) had an anterior chamber neovascularization stage (rubeosis (R) stage) of 2, 33.8% (26/77) stage 3 and 54.5% (42/77) stage 4. The combined average stage was 3.4±0.7 (range 2–4). Patients with stage 4 needed significantly more major interventions compared with stage 3 (OR 25.0, 95% CI 3.09-201.7, p=0.003) and stage 2 (OR 19.0, 95% CI 1.03-347.3, p=0.047). There was no statistically significant difference between the number of interventions required at stage 2 and stage 3 (OR 0.89, 95% CI 0.03-23.9, p=0.9471). Kaplan-Meier analysis showed a probability of success of 65% at one-year follow-up (Figure 5).\n\n21 patients were lost to follow-up. 10 patients died during the one-year follow-up period. All 21 patients lost to follow-up completed a telephone survey. The main reasons for missed visits were general health issues in 52.4% (11/21), long-distance to our clinic 38.1% (8/21), and family-related reasons in 9.5% (2/21). 57.1% (12/21) of patients had retained at least ambulatory vision. 71.4% (15/21) used 1.7±1.2 glaucoma eye drops. 95.2% (21/22) reported overall satisfaction with the treatment. All patients were pain-free.\n\n\nDiscussion\n\nBy 1871, NVG was known as “glaucoma haemorrhagicum et apoplecticum” and feared as a consequence of ischemia that quickly led to enucleation due to “hefty ciliary neuralgia”39. In 1963, using improved equipment, Weiss et al. found that emerging neovascularization and fibrovascular membranes of the iris and the angle could be observed well before the onset of advanced NVG40, hinting at a window to initiate treatment. Today, the ability to detect neovascularization early is complemented by improved interventions that address both the underlying pathology and elevated IOP. However, these interventions need to be implemented during the early stages of the disease because NVG continues to have a rapid evolution and a poor prognosis for both the eye and the patient12. One study showed that the expected lifespan of patients with NVG decreased by 52% compared to an age-correlated normal population, corresponding to a loss of 6.5 years. In diabetics with NVG, the lifespan was reduced even more by 72%12. In our study, 74% (57/77) of patients had multiple cardiovascular risk factors and were overweight or obese. During follow-up, ten patients died. The high mortality rate is a reminder that any treatment strategy of NVG should aim at keeping the number of necessary follow-up visits and additional interventions at a minimum. We tried to address this need by combining several interventions in a single surgical session.\n\nRetinal ischemia is the primary cause of NVG10,41. Accordingly, PRP is the standard of care to reduce posterior pole oxygen demand and angiogenic drive while vitrectomy is performed to increase the partial pressure of vitreous oxygen42,43. We performed lens extraction, pars plana vitrectomy, and endoscopic PRP because of significant media opacities and because endoscopic laser through the pars plana approach facilitates the delivery of 360° near-confluent peripheral retinal laser treatment out to the ora serrata. In our clinical experience, even relatively small areas of retinal ischemia may cause further NVG progression. Therefore, we took care to apply 360° PRP to near-confluence up to the ora serrata. Such extensive treatment would be challenging or impossible using standard PRP with an indirect ophthalmoscope or at the slit lamp.\n\nIn the healthy eye, the vitreous body and the iris-lens diaphragm form a relative diffusion barrier that maintains a higher oxygen partial pressure in the posterior chamber than the vitreous overlying the posterior pole. Concurrently, it reduces the diffusion of angiogenic mediators. Recreating a relative diffusion barrier after vitrectomy44 is beneficial and reduces NVI occurrence45. A relative barrier can be achieved with silicone oil46 that reduces the incidence of NVG46,47. Following this concept, Bartz-Schmidt et al. treated 32 NVG patients with pars plana vitrectomy, retinal, and ciliary body photocoagulation, and silicone oil tamponade as eyes were left aphakic. This approach controlled IOP (defined as an IOP between 8 and 21 mmHg) in 72% of patients for at least one year30. In our study we achieved an IOP control in 77.9% and all eyes were pseudophakic and without silicone oil at the conclusion of the surgery, thereby suggesting that silicone oil as a diffusion barrier may not be necessary30.\n\nOur success rate of 65%, defined as an IOP <22 mmHg, with or without glaucoma medication and without vision loss after a one-year follow-up, is similar to success rates reported for glaucoma drainage devices, which range from 62% to 66.7%24,48–50. One study reported a 73% success rate in 38 eyes that received a glaucoma drainage device and had relatively few postoperative complications51. However, this report is the exception as others have found24,48–50. Our integrative surgical approach avoided tube-specific complications (e.g., tube exposure, retraction, corneal touch, obstruction), ranging from 13% to 26% in NVG over five years24,48–51. It delivers both retina and glaucoma treatment in a single surgical session and reduces the patient and health care system burden by simplifying postoperative care and follow-up. Our telephone survey with all patients lost to follow-up revealed that the main reasons for missed visits were other (general) health issues in 52.4% and long distance to the clinic in 38.1% but a high subjective satisfaction rate.\n\nPatients with stage 4 anterior chamber neovascularization needed significantly more major interventions than stage 3 or stage 2, but there was a difference between stage 2 and stage 3. This finding suggests that early and comprehensive treatment may be more beneficial than a stepwise treatment strategy.\n\nIt is worth noting that IOP reduction can also be achieved without cyclodestruction applying only pars plana vitrectomy, lensectomy with a preserved anterior capsule, and panretinal endophotocoagulation as reported by Kinoshita et al.31. However, this study included only 13 eyes with a lower mean preoperative IOP of 29 mmHg as in our study. By contrast, other studies that only used anti-VEGF agents as primary treatment28,52 reported failure rates up to 88%52. Anti-VEGF agents may be best used as an adjuvant treatment29. Consistent with our findings, a study that used pars plana vitrectomy, endoscopic peripheral panretinal photocoagulation, and endocyclophotocoagulation (ECP) also achieved an IOP reduction which was more effective than panretinal photocoagulation, intravitreal bevacizumab, pars plana vitrectomy, and trabeculectomy with mitomycin C or Ahmed valve placement. However, the authors reported a higher phthisis rate of 7.4%53.\n\nBecause of the underlying ocular disease, visual function remained low in most eyes in our study.\n\nThere are limitations to our study. Because the integrative surgical approach described here was the primary practice pattern, there was no control group. This limited us to an intragroup comparison of before versus after treatment data. As a retrospective study, it can only inform on future prospective studies' parameters and design and help formulate, but not answer, hypotheses about associations between treatment and outcomes.\n\nIn conclusion, this study shows that NVG can be controlled in most cases by an integrative surgical approach delivered in a single surgical session that combines transscleral cyclophotocoagulation, cataract removal, pars plana vitrectomy, near-confluent full-scatter panretinal photocoagulation, and intravitreal bevacizumab. Patients with advanced iris neovascularization required significantly more additional interventions. The described approach lowered IOP significantly, reduced the number of glaucoma medications, and controlled pain.\n\n\nData availability\n\nOpen Science Framework: Combined vitrectomy, near-confluent panretinal endolaser, bevacizumab and cyclophotocoagulation for neovascular glaucoma — a retrospective interventional case series. https://doi.org/10.17605/OSF.IO/QTCGV37.\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\nKaraman S, Leppänen VM, Alitalo K: Vascular endothelial growth factor signaling in development and disease. Development. 2018; 145(14): dev151019. PubMed Abstract \| Publisher Full Text\n\nLiao N, Li C, Jiang H, et al.: Neovascular glaucoma: a retrospective review from a tertiary center in China. BMC Ophthalmol. 2016; 16(1): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDrobec P: Das hämorrhagische Sekundärglaukom. Klinische Monatsblätter für Augenheilkunde. 1982; 180(02): 138–40.\n\nMocanu C, Barăscu D, Marinescu F, et al.: [Neovascular glaucoma--retrospective study]. Oftalmologia. 2005; 49(4): 58–65. PubMed Abstract\n\nGesundheitsberichterstattung des Bundes (GBE Bund): Diagnosedaten der Krankenhäuser ab 2000 (Eckdaten der vollstationären Patienten und Patientinnen). Reference Source\n\nNatural History and Clinical Management of Central Retinal Vein Occlusion.The Central Vein Occlusion Study Group. Arch Ophthalmol. 1997; 115(4): 486–91. PubMed Abstract \| Publisher Full Text\n\nNielsen NV: The prevalence of glaucoma and ocular hypertension in type 1 and 2 diabetes mellitus. Acta Ophthalmol (Copenh). 1983; 61(4): 662–72. PubMed Abstract \| Publisher Full Text\n\nRudkin AK, Lee AW, Chen CS: Ocular neovascularization following central retinal artery occlusion: prevalence and timing of onset. Eur J Ophthalmol. 2010; 20(6): 1042–6. PubMed Abstract \| Publisher Full Text\n\nBrown GC, Magargal LE, Schachat A, et al.: Neovascular glaucoma. Etiologic considerations. Ophthalmology. 1984; 91(4): 315–20. PubMed Abstract \| Publisher Full Text\n\nHavens SJ, Gulati V: Neovascular Glaucoma. Dev Ophthalmol. 2016; 55: 196–204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSetlur VJ, Parikh JG, Rao NA: Changing causes of enucleation over the past 60 years. Graefes Arch Clin Exp Ophthalmol. 2010; 248(4): 593–7. PubMed Abstract \| Publisher Full Text\n\nBlanc JP, Molteno ACB, Fuller JR, et al.: Life expectancy of patients with neovascular glaucoma drained by Molteno implants. Clin Exp Ophthalmol. 2004; 32(4): 360–3. PubMed Abstract \| Publisher Full Text\n\nMiller JW, Adamis AP, Shima DT, et al.: Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994; 145(3): 574–84. PubMed Abstract \| Free Full Text\n\nPenn JS, Madan A, Caldwell RB, et al.: Vascular endothelial growth factor in eye disease. Prog Retin Eye Res. 2008; 27(4): 331–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFord KM, Saint-Geniez M, Walshe T, et al.: Expression and role of VEGF in the adult retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2011; 52(13): 9478–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVempati P, Popel AS, Mac Gabhann F: Extracellular regulation of VEGF: isoforms, proteolysis, and vascular patterning. Cytokine Growth Factor Rev. 2014; 25(1): 1–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoewen N, Chen J, Dudley VJ, et al.: Genomic response of hypoxic Müller cells involves the very low density lipoprotein receptor as part of an angiogenic network. Exp Eye Res. 2009; 88(5): 928–37. PubMed Abstract \| Publisher Full Text\n\nSchofield CJ, Ratcliffe PJ: Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol. 2004; 5(5): 343–54. PubMed Abstract \| Publisher Full Text\n\nTsuzuki Y, Fukumura D, Oosthuyse B, et al.: Vascular Endothelial Growth Factor (VEGF) Modulation by Targeting Hypoxia-inducible Factor-1α → Hypoxia Response Element → VEGF Cascade Differentially Regulates Vascular Response and Growth Rate in Tumors Cancer Res. 2000. Reference Source\n\nSchlote T, Derse M, Rassmann K, et al.: Efficacy and safety of contact transscleral diode laser cyclophotocoagulation for advanced glaucoma. J Glaucoma. 2001; 10(4): 294–301. PubMed Abstract \| Publisher Full Text\n\nDelgado MF, Dickens CJ, Iwach AG, et al.: Long-term results of noncontact neodymium:yttrium-aluminum-garnet cyclophotocoagulation in neovascular glaucoma. Ophthalmology. 2003; 110(5): 895–9. PubMed Abstract \| Publisher Full Text\n\nOguri A, Takahashi E, Tomita G, et al.: Transscleral cyclophotocoagulation with the diode laser for neovascular glaucoma. Ophthalmic Surg Lasers. 1998; 29(9): 722–7. PubMed Abstract\n\nShchomak Z, Cordeiro Sousa D, Leal I, et al.: Surgical treatment of neovascular glaucoma: a systematic review and meta-analysis. Graefes Arch Clin Exp Ophthalmol. 2019; 257(6): 1079–89. PubMed Abstract \| Publisher Full Text\n\nZhou M, Xu X, Zhang X, et al.: Clinical Outcomes of Ahmed Glaucoma Valve Implantation With or Without Intravitreal Bevacizumab Pretreatment for Neovascular Glaucoma: A Systematic Review and Meta-Analysis. J Glaucoma. 2016; 25(7): 551–7. PubMed Abstract \| Publisher Full Text\n\nChuang LH, Wang NK, Chen YP, et al.: Vitrectomy and panretinal photocoagulation reduces the occurrence of neovascular glaucoma in central retinal vein occlusion with vitreous hemorrhage. Retina. 2013; 33(4): 798–802. PubMed Abstract \| Publisher Full Text\n\nBudzynski E, Smith JH, Bryar P, et al.: Effects of photocoagulation on intraretinal PO2 in cat. Invest Ophthalmol Vis Sci. 2008; 49(1): 380–9. PubMed Abstract \| Publisher Full Text\n\nYazdani S, Hendi K, Pakravan M: Intravitreal bevacizumab (Avastin) injection for neovascular glaucoma. J Glaucoma. 2007; 16(5): 437–9. PubMed Abstract \| Publisher Full Text\n\nLüke J, Nassar K, Lüke M, et al.: Ranibizumab as adjuvant in the treatment of rubeosis iridis and neovascular glaucoma--results from a prospective interventional case series. Graefes Arch Clin Exp Ophthalmol. 2013; 251(10): 2403–13. PubMed Abstract \| Publisher Full Text\n\nOlmos LC, Sayed MS, Moraczewski AL, et al.: Long-term outcomes of neovascular glaucoma treated with and without intravitreal bevacizumab. Eye (Lond). 2016; 30(3): 463–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBartz-Schmidt KU, Thumann G, Psichias A, et al.: Pars plana vitrectomy, endolaser coagulation of the retina and the ciliary body combined with silicone oil endotamponade in the treatment of uncontrolled neovascular glaucoma. Graefes Arch Clin Exp Ophthalmol. 1999; 237(12): 969–75. PubMed Abstract \| Publisher Full Text\n\nKinoshita N, Ota A, Toyoda F, et al.: Surgical results of pars plana vitrectomy combined with pars plana lensectomy with anterior capsule preservation, endophotocoagulation, and silicon oil tamponade for neovascular glaucoma. Clin Ophthalmol. 2011; 5: 1777–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeiss DI, Gold D: Neofibrovascularization of iris and anterior chamber angle: a clinical classification. Ann Ophthalmol. 1978; 10(4): 488–91. PubMed Abstract\n\nAbbas A, Agrawal P, King AJ: Exploring literature-based definitions of hypotony following glaucoma filtration surgery and the impact on clinical outcomes. Acta Ophthalmol. 2018; 96(3): e285–9. PubMed Abstract \| Publisher Full Text\n\nTan LT, Isa H, Lightman S, et al.: Prevalence and causes of phthisis bulbi in a uveitis clinic. Acta Ophthalmol. 2012; 90(5): e417–8. PubMed Abstract \| Publisher Full Text\n\nShaarawy TM, Sherwood MB, Grehn F: WGA Guidelines on Design and Reporting of Glaucoma Surgical Trials. Kugler Publications; 2009; 83. Reference Source\n\nBach M, Schulze-Bonsel K, Feltgen N, et al.: Author response: numerical imputation for low vision states [electronic letter]. Invest Ophthalmol Vis Sci. 2007; 26.\n\nStrzalkowski P, Strzalkowska A, Loewen N: Combined vitrectomy, near-confluent panretinal endolaser, bevacizumab and cyclophotocoagulation for neovascular glaucoma — a retrospective interventional case series. 2020. http://www.doi.org/10.17605/OSF.IO/QTCGV\n\nCalculate Your BMI - Standard BMI Calculator [Internet]. [cited 2020 Sep 13]. Reference Source\n\nPagenstecher H: Mittheilungen aus der Augenheilanstalt zu Wiesbaden: Beiträge zur Lehre vom hämorrhagischen Glaukom. Albrecht von Graefes Archiv für Ophthalmologie. 1871; 17(2): 98–130.\n\nWeiss DI, Shaffer RN, Nehrenberg TR: Neovascular Glaucoma complicating carotid-cavernous fistula. Arch Ophthalmol. 1963; 69(3): 304–7. PubMed Abstract \| Publisher Full Text\n\nStefánsson E: Ocular oxygenation and the treatment of diabetic retinopathy. Surv Ophthalmol. 2006; 51(4): 364–80. PubMed Abstract \| Publisher Full Text\n\nStefansson E, Landers MB 3rd, Wolbarsht ML: Increased retinal oxygen supply following pan-retinal photocoagulation and vitrectomy and lensectomy. Trans Am Ophthalmol Soc. 1981; 79: 307–34. PubMed Abstract \| Free Full Text\n\nSimpson ARH, Dowell NG, Jackson TL, et al.: Measuring the effect of pars plana vitrectomy on vitreous oxygenation using magnetic resonance imaging. Invest Ophthalmol Vis Sci. 2013; 54(3): 2028–34. PubMed Abstract \| Publisher Full Text\n\nStefansson E, Landers MB 3rd, Wolbarsht ML: Vitrectomy, lensectomy, and ocular oxygenation. Retina. 1982; 2(3): 159–66. PubMed Abstract \| Publisher Full Text\n\nRice TA, Michels RG, Maguire MG, et al.: The effect of lensectomy on the incidence of iris neovascularization and neovascular glaucoma after vitrectomy for diabetic retinopathy. Am J Ophthalmol. 1983; 95(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nde Juan E Jr, Hardy M, Hatchell DL, et al.: The effect of intraocular silicone oil on anterior chamber oxygen pressure in cats. Arch Ophthalmol. 1986; 104(7): 1063–4. PubMed Abstract \| Publisher Full Text\n\nRinkoff JS, de Juan E Jr, McCuen BW: Silicone oil for retinal detachment with advanced proliferative vitreoretinopathy following failed vitrectomy for proliferative diabetic retinopathy. Am J Ophthalmol. 1986; 101(2): 181–6. PubMed Abstract \| Publisher Full Text\n\nYalvac IS, Eksioglu U, Satana B, et al.: Long-term results of Ahmed glaucoma valve and Molteno implant in neovascular glaucoma. Eye(Lond). 2007; 21(1): 65–70. PubMed Abstract \| Publisher Full Text\n\nEvery SG, Molteno ACB, Bevin TH, et al.: Long-term results of Molteno implant insertion in cases of neovascular glaucoma. Arch Ophthalmol. 2006; 124(3): 355–60. PubMed Abstract \| Publisher Full Text\n\nXie Z, Liu H, Du M, et al.: Efficacy of Ahmed Glaucoma Valve Implantation on Neovascular Glaucoma. Int J Med Sci. 2019; 16(10): 1371–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNetland PA, Ishida K, Boyle JW: The Ahmed Glaucoma Valve in patients with and without neovascular glaucoma. J Glaucoma. 2010; 19(9): 581–6. PubMed Abstract \| Publisher Full Text\n\nYazdani S, Hendi K, Pakravan M, et al.: Intravitreal Bevacizumab for Neovascular Glaucoma: A Randomized Controlled Trial. J Glaucoma. 2009; 18(8): 632–7. PubMed Abstract \| Publisher Full Text\n\nMarra KV, Wagley S, Omar A, et al.: Case-matched comparison of vitrectomy, peripheral retinal endolaser, and endocyclophotocoagulation versus standard care in neovascular glaucoma. Retina. 2015; 35(6): 1072–83. PubMed Abstract \| Publisher Full Text" }	[ { "id": "73906", "date": "20 Nov 2020", "name": "Andrew W. Eller", "expertise": [ "Reviewer Expertise Ophthalmology", "Vitreo-Retinal Diseases and Surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study Dr Strzalkowski et al. provide a retrospective observational analysis of a single center’s experience with aggressive upfront treatment of neovascular glaucoma (NVG) with combined vitrectomy, near confluent pan retinal endolaser, bevacizumab and transscleral cyclophotocoagulation.\nThe authors hypothesize that upfront combined treatment would improve outcomes in terms of IOP, antiglaucoma medications, pain, necessity of further surgeries and help reduce follow-up visits. They report baseline characteristics and one-year outcomes for 77 patients with neovascular glaucoma that underwent the combined procedure. We congratulate them on implementing a success strategy in treating these complex cases. Often patients with NVG have disastrous outcomes with poorly controlled IOP and significant loss of vision. In this series, a number of patients were able to maintain reasonable vision. It can often be difficult to “titrate” treatment of IOP, often resulting in hypotony. There were three patients with phthisis bulbi and another six with hypotony for a total of nine or 11.7%. Three patients developed retinal detachment. Perhaps these were the same three patients who developed phthisis bulbi.\n\nThe major drawback of this study is the lack of alternate treatments for randomization. This limits our ability to draw any conclusions about optimal management strategies and only hypotheses can be generated. This is appropriately acknowledged by the authors who are careful not to overstate their conclusions.\nThe authors seemed to have corelated surgical success with the stage of iris neovascularization. In other words, there were more surgical interventions with stage 4, then stages 2 or 3. It is the experience of this reviewer that one can corelate treatment success of NVG with presenting IOP and the immediate response of IOP to medical therapy. In clinic, if the initial IOP is less than 40 mmHg and medical treatment can reduce the IOP into the low 20’s, these patients can often be treated with an initial injection of an anti-VEGF medication, followed by PRP laser. In the long term, any residual glaucoma can usually be treated with medical therapy alone. Those patients presenting with an IOP greater than 40 mmHg, and not responding to medical therapy will require more aggressive treatment. If the visual potential is reasonable, this treatment may include anti-VEGF, PRP laser (with vitrectomy if visualization of the retina is poor), and glaucoma surgery. If the visual potential is very poor, then not invasive glaucoma treatment such as transscleral CPC) is advised. Using their data, are the authors able to confirm our observations regarding treatment of NVG.\nAs the authors have stated, there are a number of different underlying causes of neovascular glaucoma and they have been combined in this series. This is a fairly large series and we question whether the severity of the NVG and response to treatment may have differed according to the underlying pathology. For example, the iris neovascularization and glaucoma in central retinal vein occlusions (CRVO) tends to progress more rapidly then seen with diabetic retinopathy. Once the retinal ischemia has been addressed in CRVO, it seems these eyes are more likely to develop hypotony. What was the experience of the authors in this study?\nThe authors cite two studies from the mid-1980’s when the use of silicone oil was very much in its early stages of clinical usage. At that time, it was felt that silicone oil reduced or perhaps prevented the flow of VEGF into the anterior chamber and therefore decreased the risk of iris neovascularization and glaucoma. We believe this theory has been discounted as we know that there is a flow of aqueous humor from the posterior segment into the anterior chamber. Retinal detachment alone increases VEGF production and it occurs in cases of proliferative diabetic retinopathy there can be an even more profound angiogenic effect. An alternative explanation for this observation is that silicone oil improves the outcome in these eyes by simply improving the chances for retinal re-attachment.\nIn the Introduction, the authors hypothesize that the combined approach would help decrease outpatient visits. This is again emphasized in the Discussion where authors report that patients with neovascular glaucoma have significant comorbidities and lower life expectancy and would therefore benefit from decreased outpatient visits. The counter-argument to be made is that combined aggressive surgical intervention would increase risk of complications and require closer monitoring. No data is provided on the number of follow up visits. Consider adding to results if available. The authors could consider performing a regression analysis to determine which baseline characteristics predicted success with this combined technique. This analysis will help identify patients in whom this technique would be most helpful. It could also show when this technique may be more aggressive than necessary in some cases, and could be more aggressive in others when the addition of a glaucoma drainage device may be beneficial.\nSpecific comments for revision:\n\nIn the Introduction, second paragraph, suggest following change. “In 1994, Miller et al. showed that laser occlusion of all branch retinal veins in a primate could lead to CRVO and subsequent retinal ischemia.” In the Results, consider rephrasing “arterial hypertension” to hypertension as this is the currently accepted terminology. In Table 1 – consider rephrasing “R stadium” to read “NVI Stage” or Weiss and Gold Rubeosis grading to ensure clarity. In the Discussion, it would be beneficial to see outcomes with staged surgical approach (Incisional surgical management with trabeculectomy/tubes, followed by cryotherapy if poor response) from existing literature to enable some comparison with the combined approach reported in this manuscript. In the Discussion, the authors state “Patients with stage 4 anterior chamber neovascularization needed significantly more major interventions than stage 3 or stage 2, but there was a difference between stage 2 and stage 3”. The data reports NO significant difference between stage 2 and 3. Please clarify.\n\nOverall, a well-organized manuscript with a practical approach to the treatment of neovascular glaucoma with novel 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?\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": "6297", "date": "02 Mar 2021", "name": "Piotr Strzalkowski", "role": "Author Response", "response": "Dear Reviewers, We are grateful for the opportunity to improve our manuscript. We appreciate the helpful comments and have provided our replies below. We trust that we were able to address the concerns that were raised. Thank you, Piotr Strzalkowski Reviewers' comments: Reviewer #1 (“R1” in the following): There were three patients with phthisis bulbi and another six with hypotony for a total of nine or 11.7%. Three patients developed retinal detachment. Perhaps these were the same three patients who developed phthisis bulbi. Authors: That is exactly correct. We appreciate the thoughtful analysis. All three patients with retinal detachment also developed phthisis bulbi. We added this important information to our manuscript. R1, 1: As the authors have stated, there are a number of different underlying causes of neovascular glaucoma and they have been combined in this series. This is a fairly large series and we question whether the severity of the NVG and response to treatment may have differed according to the underlying pathology. For example, the iris neovascularization and glaucoma in central retinal vein occlusions (CRVO) tends to progress more rapidly than seen with diabetic retinopathy. Once the retinal ischemia has been addressed in CRVO, it seems these eyes are more likely to develop hypotony. What was the experience of the authors in this study? Authors: Thank you for sharing your clinical experience. Based on our data we could not find a statistical significant difference in postoperative IOP or hypotony between CRVO and PDR patients. R1, 2: No data is provided on the number of follow up visits. Consider adding to results if available. Authors: Information about the number of follow up visits is provided in section “study design”: All patients were treated at the University Eye Hospital in Würzburg, Germany. The combined intervention was part of routine patient care at our clinic and all patients with decompensated neovascular glaucoma and preserved visual function received this intervention. BCVA, intraocular pressure (IOP, mmHg), the number of glaucoma medication, visual analog pain scale (VAPS, 0-10) was recorded at baseline and at follow-up visits at one, three, six, and 12 months as part of routine care. R1, 3: The authors could consider performing a regression analysis to determine which baseline characteristics predicted success with this combined technique. This analysis will help identify patients in whom this technique would be most helpful. Authors: Regression analysis was not included in this study on purpose because this analysis is part of our ongoing study. R1, Specific Comments: In the Introduction, second paragraph, suggest following change. “In 1994, Miller et al. showed that laser occlusion of all branch retinal veins in a primate could lead to CRVO and subsequent retinal ischemia.” In the Results, consider rephrasing “arterial hypertension” to hypertension as this is the currently accepted terminology. In Table 1 – consider rephrasing “R stadium” to read “NVI Stage” or Weiss and Gold Rubeosis grading to ensure clarity. In the Discussion, it would be beneficial to see outcomes with staged surgical approach (Incisional surgical management with trabeculectomy/tubes, followed by cryotherapy if poor response) from existing literature to enable some comparison with the combined approach reported in this manuscript. In the Discussion, the authors state “Patients with stage 4 anterior chamber neovascularization needed significantly more major interventions than stage 3 or stage 2, but there was a difference between stage 2 and stage 3”. The data reports NO significant difference between stage 2 and 3. Please clarify. Authors: We have changed the manuscript to include these suggestions." } ] }, { "id": "75197", "date": "01 Dec 2020", "name": "Stefan Dithmar", "expertise": [ "Reviewer Expertise Retina 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\nThe authors present a very interesting observational study on the therapy of neovascularization glaucoma. The treatment of this clinical entity is difficult and often unsatisfactory. The authors report on a convincing surgical approach and present retrospective results in a relatively large patient group. The presentation of the results is good and the manuscript is well written.\nTaking into account the reviewer report by Andrew W. Eller, I have no further comments to make.\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/9-1236
https://f1000research.com/articles/9-1185/v1	30 Sep 20	{ "type": "Clinical Practice Article", "title": "Minimally invasive (flapless) crown lengthening by erbium:YAG laser in aesthetic zone", "authors": [ "Saverio Capodiferro", "Angela Tempesta", "Luisa Limongelli", "Giuseppe Barile", "Daniela Di Venere", "Massimo Corsalini", "Angela Tempesta", "Luisa Limongelli", "Giuseppe Barile", "Daniela Di Venere", "Massimo Corsalini" ], "abstract": "Crown lengthening is a surgical procedure aimed at exposure of a larger tooth surface by gingivectomy alone or with cortical bone remodelling for aesthetic purposes in the anterior zone of the maxilla or for reconstruction of teeth affected by subgingival caries. We report two cases of crown lengthening in the anterior maxilla for aesthetic purposes by gingival and bone re-contouring performed by erbium-doped yttrium aluminium garnet (erbium:YAG) laser. As highlighted in this report, the erbium:YAG laser-assisted crown lengthening is less invasive and also leads to faster clinical outcomes in contrast to the conventional surgical technique by scalpel incision, flap elevation and osteoplastic.", "keywords": [ "flap-less crown lengthening", "Erbium:YAG laser", "smile line" ], "content": "Introduction\n\nSeveral clinical situations may require dental crown lengthening (CL) such as irregular smile line, gummy smile, decayed or fractured teeth, worn out teeth by parafunction habits (e.g. bruxism)1,2. Regardless of aesthetic or functional purpose, the conventional technique of CL involves scalpel incision, flap elevation and bone remodeling by burns, with or without adjunctive gingivectomy, the latter essentially related to the gingival biotype3,4. Despite the excellent clinical outcome, the conventional surgical technique may be more invasive depending on the severity of the clinical situation as well patient’s general health condition (e.g. medically compromised patients or in therapy with anticoagulant drugs). Many alternatives techniques for CL have been reported in literature but it is generally accepted that the least invasive are the laser-assisted techniques5,6. Of these, the erbium:YAG laser has the advantage to work on both hard (bone) and soft tissues (gingiva)7. We report on 2 cases treated by a mini-invasive erbium:YAG laser-assisted procedure (including gingiva and bone re-contouring) for CL in the anterior maxilla.\n\n\nCases presentation\n\nThe patient was a 53 y.o. Caucasian woman with an no relevant medical history who was unemployed at the time of presentation (March, 2015). She presented an abundant gingiva covering tooth 1.2 which she wished to remove for aesthetical purposes (Figure 1a,b). Gingival remodeling and bone re-contouring by erbium:YAG laser was suggested. A small amount of anesthesia was injected locally (0.9 ml of mepivacaine cloridrate 2%, 1:100,000 epinephrine) after which the gingiva was remodeled by laser (Key Laser 3-Kavo s.r.l.) in de-focalized modality (not in contact tip, 180 MJ/10 Hz, poor water emission) until the dental crown was sufficiently exposed according to the patient smile line (Figure 1c,d). After one week (Figure 2a), a second procedure was performed to re-contour the marginal bone by the same laser, using a surgical tip (small scalpel-like tip, 120 MJ/10 Hz, abundant water emission) in contact modality and through the gingival sulcus (flap-less); a light bleeding occurred during the procedure (Figure 2b). The gingival margin was completely healed, and the smile line appeared significantly improved 12 days after surgery (Figure 2c).\n\nAlteration of the smile line related to the abundant gingiva of tooth 1.2 (a,b); gingival remodelling by erbium-doped yttrium aluminium garnet (erbium:YAG) laser and its immediate clinical appearance (c,d).\n\nSecond step after seven days (a); flapless (through the gingival sulcus) bone re-contouring by erbium-doped yttrium aluminium garnet (erbium:YAG) laser (b) and its clinical appearance after 12 days (c).\n\nThis 47 y.o. Caucasian housewife who presented in April 2016 with severe abrasion of the anterior teeth related to bruxism over a long duration (Figure 3a). Her medical history was un-remarkable. No pain and/or teeth hyper-sensibility were indicated by the patient, however, she was unhappy with her smile. A laser-assisted CL of the lateral and central incisors was planned to re-define a new marginal gingiva profile. After local injection of anesthesia, (1,8 ml of mepivacaine cloridrate 2%, 1:100,000 epinephrine), the marginal gingiva was careful recontoured by erbium:YAG laser (Key Laser 3-Kavo s.r.l.) (not in contact tip, 180 MJ/10 Hz, poor water emission) till an adequate teeth exposure (Figure 3b,c); subsequently, the cortical bone was-remodeled by a surgical tip (small scalpel-like tip, 160 MJ/10 Hz, abundant water emission) on both aspects of the maxilla through the gingival sulcus without flap elevation (Figure 3d). After 14 days, gingival tissues appeared healed and teeth prepared for the following prosthetic restoration by cemented metal-ceramic crowns. (Figure 3e,f).\n\nSevere abrasion of incisors due to bruxism (a); erbium-doped yttrium aluminium garnet (erbium:YAG) laser-assisted gingivectomy (b) and contextual flapless bone remodelling (c); the clinical appearance after 14 days (d), the teeth preparation as appearing on computer-aided design and the following prosthetic rehabilitation (e,f).\n\n\nDiscussion\n\nSeveral medical devices have been proposed to make CL less invasive, including piezosurgery4,8. Several lasers such as diode, neodymium-doped yttrium aluminum garnet (Nd:YAG), potassium titanyl phosphate (KTP), CO2, Erbium, chromium-doped yttrium, scandium, gallium and garnet (Er,Cr:YSGG) and erbium:YAG are widely used for CL1,2,6,9. However, the main difference between these is their capability to work exclusively on soft or hard or both tissues3,9,10. Diode, Nd:YAG, KTP and CO2 lasers may be useful when only gingival remodeling alone is necessary and this is essentially related to their surgical capabilities, especially contextual cuts and coagulation2,9,11,12. In fact, they are generally suggested for many surgical and non-surgical procedures in the oral cavity (frenectomy/frenulotomy, vestibuloplasty, mucosal biopsy, treatment of tooth hyper-sensibility, benign, potentially malignant and malignant lesions removal, surgical and not-surgical periodontal treatments including drug-related gingival overgrowth, photocoagulation of venous malformations, etc), but not for bone treatments12–19. When both gingival and bone remodeling is required, instead, the choice necessarily must fall on Er;Cr:YSGG or erbium:YAG lasers thanks to their selectivity for water, resulting in the capability to work by ablation on hard tissues as tooth and bone10,11,20,21. Therefore, such lasers can be used for dental cavity preparation, periodontal treatments and bone remodeling or cutting7,9,10,20. In the reported cases, authors used an erbium:YAG laser both for soft and hard tissue treatment but with different tips and output energy parameters. The excellent clinical outcomes we described in terms of minimal invasiveness, lack of intra- and post-operative complications and pain, fast and predictable healing, are essentially related to the intrinsic proprieties of the erbium:YAG laser light and to the generally recognized gentle laser-oral tissues interaction10,11,20–22.\n\n\nConclusion\n\nThe overall clinical benefits of the erbium:YAG laser allows flapless CL to be simplified, even in difficult cases. The total absence of laser-related thermal injuries to the oral hard and soft tissues leads to highly predictable clinical results, and this is important in the treatment of the anterior teeth for aesthetic purposes. However, a good knowledge of laser-tissue interaction principles, sufficient experience on laser use and, obviously, familiarity with the general and basic guidelines of oral/periodontal surgery are mandatory to achieve desirable clinical results.\n\n\nConsent\n\nWritten informed consent for publication of their 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\nHempton TJ, Dominici JT: Contemporary crown-lengthening therapy: a review. J Am Dent Assoc. 2010; 141(6): 647–655. PubMed Abstract \| Publisher Full Text\n\nNarayanan M, Laju S, Erali SM, et al.: Gummy Smile Correction with Diode Laser: Two Case Reports. J Int Oral Health. 2015; 7(Suppl 2): 89–91. PubMed Abstract \| Free Full Text\n\nFarista S, Kalakonda B, Koppolu P, et al.: Comparing Laser and Scalpel for Soft Tissue Crown Lengthening: A Clinical Study. Glob J Health Sci. 2016; 8(10): 55795. PubMed Abstract \| Publisher Full Text\n\nMcGuire MK, Scheyer ET: Laser-assisted flapless crown lengthening: a case series. Int J Periodontics Restorative Dent. 2011; 31(4): 357–364. PubMed Abstract\n\nKazakova RT, Tomov GT, Kissov CK, et al.: Histological Gingival Assessment after Conventional and Laser Gingivectomy. Folia Med (Plovdiv). 2018; 60(4): 610–616. PubMed Abstract \| Publisher Full Text\n\nFekrazad R, Moharrami M, Chiniforush N: The Esthetic Crown Lengthening by Er;Cr:YSGG laser: A Case Series. J Lasers Med Sci. 2018; 9(4): 283–287. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiaci J, Gaspirc B: Review Comparison of Er: YAG and Er Cr: YSGG lasers used in dentistry. J Laser Health Acad. 2012; 2012(1): 1–13. Reference Source\n\nLavu V, Arumugam C, Venkatesan N, et al.: A Present Day Approach to Crown Lengthening - Piezosurgery. Cureus. 2019; 11(11): e6241. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTachmatzidis T, Dabarakis N: Technology of Lasers and Their Applications in Oral Surgery: Literature Review. Balkan Journal of Dental Medicine. 2016; 20(3): 131–137. Publisher Full Text\n\nStubinger S: Advances in bone surgery: the Er:YAG laser in oral surgery and implant dentistry. Clin Cosmet Investig Dent. 2010; 2: 47–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker S: Verifiable CPD paper: laser-tissue interaction. Br Dent J. 2007; 202(2): 73–81. PubMed Abstract \| Publisher Full Text\n\nDerikvand N, Chinipardaz Z, Ghasemi S, et al.: The Versatility of 980 nm Diode Laser in Dentistry: A Case Series. J Lasers Med Sci. 2016; 7(3): 205–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLimongelli L, Tempesta A, De Caro A, et al.: Diode Laser Photocoagulation of Intraoral and Perioral Venous Malformations After Tridimensional Staging by High Definition Ultrasonography. Photobiomodul Photomed Laser Surg. 2019; 37(11): 722–728. PubMed Abstract \| Publisher Full Text\n\nCapodiferro S, Limongelli L, Tempesta A, et al.: Diode laser photocoagulation of sublingual varices in 706 patients on antithrombotic therapy without drug discontinuation. Ann Ital Chir. 2020; 91: 100–104. PubMed Abstract\n\nCapodiferro S, Limongelli L, Tempesta A, et al.: Diode laser treatment of venous lake of the lip. Clin Case Rep. 2018; 6(9): 1923–1924. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapodiferro S, Tempesta A, Limongelli L, et al.: Nonsurgical Periodontal Treatment by Erbium:YAG Laser Promotes Regression of Gingival Overgrowth in Patient Taking Cyclosporine A: A Case Report. Photomed Laser Surg. 2019; 37(1): 53–56. PubMed Abstract \| Publisher Full Text\n\nLimongelli L, Capodiferro S, Tempesta A, et al.: Early tongue carcinomas (clinical stage I and II): echo-guided three-dimensional diode laser mini-invasive surgery with evaluation of histological prognostic parameters. A study of 85 cases with prolonged follow-up. Lasers Med Sci. 2020; 35(3): 751–758. PubMed Abstract \| Publisher Full Text\n\nFavia G, Capodiferro S, Limongelli L, et al.: Malignant transformation of oral proliferative verrucous leukoplakia: a series of 48 patients with suggestions for management. Int J Oral Maxillofac Surg. 2020; S0901-5027(20)30206-X. PubMed Abstract \| Publisher Full Text\n\nLuke AM, Mathew S, Altawash MM, et al.: Lasers: a review with their applications in oral medicine. J Lasers Med Sci. 2019; 10(4): 324–329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarashima T, Kinoshita J, Kimura Y, et al.: Morphological comparative study on ablation of dental hard tissues at cavity preparation by Er:YAG and Er,Cr:YSGG lasers. Photomed Laser Surg. 2005; 23(1): 52–55. PubMed Abstract \| Publisher Full Text\n\nChen CK, Wu YT, Chang NJ, et al.: Er:YAG Laser for Surgical Crown Lengthening: A 6-Month Clinical Study. Int J Periodontics Restorative Dent. 2017; 37(2): e149–e153. PubMed Abstract \| Publisher Full Text\n\nKao RT, Dault S, Frangadakis K, et al.: Esthetic crown lengthening: appropriate diagnosis for achieving gingival balance. J Calif Dent Assoc. 2008; 36(3): 187–91. PubMed Abstract" }	[ { "id": "72302", "date": "17 Nov 2020", "name": "Rada T. Kazakova", "expertise": [ "Reviewer Expertise Laser dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Saverio Capodiferro et al.’s article reports two cases of a crown lengthening procedure with an Er:YAG laser. This procedure aims at exposure of a larger tooth surface usually on the anterior maxillary region by gingivectomy alone (soft tissue crown lengthening), or gingivectomy and cortical bone remodeling (hard tissue crown lengthening). The advantages of the Er:YAG method, compared to the classical scalpel one, is that it leads to a predictable outcome and a faster healing The background of the cases’ history and progression described is in sufficient detail. The authors have introduced us to the patients, their age, history of the disease and the reason why they sought for the crown lengthening procedure in particular. There is enough data provided of the examinations and diagnostic tests, treatment given and outcomes. The authors described in detail what the gingival level was, and illustrated the gingival overgrowth, the steps and the clinical outcome with excellent photos, including a CAD/CAM scan. They also added photos after the procedures and the results after the final restorations. The discussion is sufficient and it accentuates on the importance of the outcome and the better and faster healing of the Er:YAG laser crown lengthening, compared to the classical method with the scalpel. It gives much details on the wide areas of use of the different lasers, as well as a comparison between them – their advantages and disadvantages, as well as their indications and contraindications. The conclusion is balanced and summarizes the findings. One of the most important advantage of the Er:YAG laser method described is the better healing and the predictable results, which is mentioned in the conclusion. My only small recommendations for them, which are not obligatory, are the following: to change ‘a small anesthesia’ to ‘a little bit of anesthesia’ and to describe what type of anesthesia they used (e.g. terminal, intra-ligamental, etc.); ‘light bleeding’ – should be written without ‘a’; it would be useful if they could provide the readers with some more information and the precise specification of the laser, laser mode and scalpel tip used; ‘her medical history was un-remarkable’ is a statement that I could not quite understand, maybe the readers need some more clarity, as well. I congratulate the team with the good work, the efforts and the wonderful outcomes and wish them to more and more success in the future. The article is interesting and well-described. I will definitely vote ‘approved’ for this article.\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": [] }, { "id": "72300", "date": "03 Dec 2020", "name": "Nasim Chiniforush", "expertise": [ "Reviewer Expertise photodynamic therapy", "laser surgery", "photobiomodulation", "hard tissue surgery", "soft tissue surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn two cases please add pulse duration of laser device you used for each purpose and laser tip diameter and length.\n\nYou can add some more information about the advantages of erbium lasers.\n\nIt's better to add case report in title of your manuscript.\n\nThis article after minor revision can be acceptable for indexing.\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": "6275", "date": "22 Jan 2021", "name": "saverio capodiferro", "role": "Author Response", "response": "Thank you very much. Changes have been made accordingly." } ] } ]	1	https://f1000research.com/articles/9-1185
https://f1000research.com/articles/10-164/v1	01 Mar 21	{ "type": "Research Article", "title": "The importance of mentorship and collaboration for scientific capacity-building and capacity-sharing: perspectives of African scientists", "authors": [ "Heather E. Burgess", "Joanna Chataway", "Joanna Chataway" ], "abstract": "Long-term goals for capacity-building in Africa centres around building a self-sufficient scientific community, however there is a lack of research on the interactions that are needed to make up a thriving academic community or the steps needed to realise such a goal. Through interviews with researchers supported by a capacity-building initiative, we have characterised their interactions with other scientists and the impact that these have on capacity-building. This has revealed a wide range of interactions that have not been captured by traditional bibliometric studies of collaboration and shown that a substantial amount of intra-African collaboration is taking place. This collaboration allowed the researchers to share capacity with their colleagues and this could provide an alternative to, or supplement, traditional North-South capacity-building. We have shown that this capacity-sharing can enable capacity to spill over from capacity-building programmes to the broader scientific community. Furthermore, researchers are deliberately hastening this capacity-sharing through training or mentoring others outside of their capacity-building initiative, including those from more resource-poor groups. To understand how capacity-building initiatives can harness the power of these interactions, we investigated how interactions between researchers originated, and found that collaborations tended to be formed around pre-existing networks, with researchers collaborating with previous colleagues, or contacts formed through their mentors or consortium activities. Capacity-building organisations could capitalise on this through actions such as expanding mentorship schemes but should also ensure that researchers are not limited to pre-established networks but have exposure to a changing and growing pool of expertise. As interactions continue to move online since the appearance of COVID-19 this will present opportunities for new interaction patterns to develop. This study highlights the need to develop new metrics for collaboration that will take into account these new modes of interaction and the full range of interactions that make up a scientific community.", "keywords": [ "Collaboration", "Health research", "Capacity-building", "Mentorship" ], "content": "Introduction\n\nBuilding scientific capacity in Africa is a key aspect of development plans for the continent, and there are a large number of international and African organisations working towards this goal (Chataway & Ochieng, 2017). Traditionally this capacity-building has been carried out by pairing African research groups with groups in more scientifically-advanced countries, termed ‘North-South’ collaborations. These collaborations can allow the African research group to access capacity in the form of resources, equipment, and expertise (Cochrane et al., 2017). However, there is a growing recognition that such collaborations can be imbalanced and misaligned with African needs (Walsh et al., 2016). Capacity-building organisations are beginning to encourage more intra-African collaboration. For example, the African Academy of Sciences’ Coalition for African Research and Innovation (CARI) (The African Academy of Sciences, n.d.) aims to build systematic scientific collaboration on the continent. Through intra-African collaboration, organisations hope to build a more self-sufficient scientific community, working to solve local challenges through more equitable collaborations (Marjanovic et al., 2017). This involves not only formal collaborations, but building a scientific community that can share ideas, hold meetings and be a part of the broader international research community. However, there is a lack of research on the interactions that are needed to make up a thriving academic community.\n\nMost work on collaborations has used just one metric for collaboration, shared publications, however this only captures a small proportion of scientific interactions, those formal collaborations that result in a publication and has a long lag with many projects taking years until they reach publication, if at all. It fails to capture a whole range of informal and pre-publication collaborations and does not reveal insights into how the collaborations were formed (Tijssen and Kraemer-Mbula, 2017). Previous attempts to characterise these broader interactions have included two large-scale surveys of researchers in the US (Boardman & Corley, 2008; Bozeman & Corley, 2004) and UK (Abreu et al., 2009). The UK study showed that 50% of scientists engage in collaborative research with researchers from other organisations and that many engage in activities that allow them to share ideas and expertise; 90% attend conferences and 65% give lectures at other institutions and participate in networks (Abreu et al., 2009). The US study, The Research Value Mapping Project, used a survey to measure the amount of time scientists spent interacting with other researchers (Boardman & Corley, 2008; Bozeman & Corley, 2004). This identified that researchers spent the bulk of their time working by themselves or with researchers at their university and around 6% with international researchers.\n\nHowever, more work is needed to fully characterise the collaborations and interactions that researchers engage in, particularly in the context of capacity-building where collaborations may be most valuable. In a developed country context, it has been shown that research collaborations are associated with higher productivity (Abramo et al., 2009; Lee & Bozeman, 2005), but collaborations may be even more important as part of capacity-building projects, where they can allow researchers to build their capacity by accessing the expertise and equipment of others. There have been a number of studies of collaborations in Africa, but these have largely used bibliometrics measures. These studies have revealed that most co-authored papers in Africa are the result of North-South collaborations, with less than 10% being produced by collaborations between authors in more than one African country (De Vré, RM Rial Verde, E & Santos da Silva, 2010; E Fonesca et al., 2016; Owusu-Nimo & Boshoff, 2017). Studies that have looked at the impact of these collaborations on capacity building have focused predominantly on formal North-South collaborations (Burgess, unpublished report) at the expense of informal and broader research interactions, particularly intra-African ones.\n\nOnly a small proportion of these studies have sought the perspectives of African researchers on collaborations. These found that researchers particularly value international collaborations for the visibility and funding provided (Franzen et al., 2013, 2017a; Zdravkovic et al., 2016). However local and, in particular, institutional (Zdravkovic et al., 2016) collaboration was also valued, particularly as it provides more opportunity to work on problems of local relevance (Zdravkovic et al., 2016). Researchers also placed a high importance on other research interactions such as mentoring (Franzen et al., 2013, 2017a; Tijssen & Kraemer-Mbula, 2017, 2018; Zdravkovic et al., 2016), with researchers stating that involvement in mentoring and training the next generation of researchers is a vital part of research excellence (Tijssen & Kraemer-Mbula, 2017). There were also found to be many barriers to intra-African interactions, and like intra-African co-authorship, informal collaborations and networking were found to be rare (Franzen et al., 2013, 2017a), with researchers often feeling isolated from the regional research community (Franzen et al., 2013). Researchers attributed this to a lack of networking opportunities (Franzen et al., 2013, 2017b), poor communications infrastructure (Marjanovic et al., 2013), the need to prioritise international partners and their institution’s focus on teaching (Franzen et al., 2013, 2017a; Muriithi et al., 2018; Owusu-Nimo & Boshoff, 2017; Zdravkovic et al., 2016). Three recent studies examined whether capacity-building initiatives could ease these barriers. A study of an effort to increase local research interactions in the field of reproductive health by bringing together five research centres into a network, found that the network instead ended up being dominated by North-South interactions (van der Veken et al., 2017). However, the other studies, evaluations of major health capacity-building initiatives, found that they did facilitate interactions, such as conferences, between members of an initiative (Marjanovic et al., 2017) or shared publications (Uwizeye et al., 2020), including after researchers have left the programme. However as highlighted by a recent review of evaluations of capacity-building initiatives (Pulford et al., 2020), in order to effectively study the impact of these initiatives on informal collaborations requires better quality metrics for collaboration in capacity building.\n\nThrough interviews with African researchers supported by a capacity-building initiative, this study aims to characterise the collaborations of these researchers, in particular the interactions that have not been effectively captured in previous studies: intra-African collaborations, and informal and pre-publication collaborations, and other interactions such as mentoring and attending meetings. Through this, we gain insight into the researchers’ patterns of collaborations and how they are formed. This allows us firstly to identify areas in which it is important that better quality metrics are developed, secondly to determine the impact that collaborations have on capacity-building, and thirdly to highlight the ways in which capacity-building organisations can harness the power of these interactions.\n\n\nMethods\n\nThis work arose out of two literature reviews we conducted in 2018 and 2020 (Burgess, unpublished report) on perspectives to South-South collaboration within Africa in the literature on capacity-building and collaboration in health research. This found that references to South-South intra-African collaboration were relatively rare, with most of the literature focusing on North-South collaborations. Furthermore, these reviews showed that only a very small portion of the literature took into account the views of the scientists themselves on these collaborations. Consequently, we set out to carry out interviews with African researchers with the aim of characterising their collaborations, in particular intra-African ones, and gaining their perspectives on these. We were particularly interested to study these collaborations in the context of a capacity-building initiative to determine the impact of these collaborations on capacity building, so we contacted researchers working under the DELTAS1 capacity-building initiative, one of the largest capacity-building programmes operating on the continent and one that has as one of its stated aims to encourage scientific mentorship and collaboration. DELTAS funds 11 programmes across the continent and we interviewed researchers from six of these: MARCAD, Afrique-One Aspire, Kemri-IDeAl, WACCBIP, MUII plus and Santhe2, providing perspectives from a range of different contexts. Researchers from the programmes who focused primarily on laboratory rather than clinical research were identified from the programme websites and contacted via email. 15 scientists were interviewed, comprising four principal investigators, five postdoctoral researchers and six PhD students. This was to some extent a selective sample, as those who agreed to participate may be those who are more likely to have an interest in collaboration, thus we might expect that they might experience more collaboration than average.\n\nPrior to the interview, researchers were asked to complete a short questionnaire on the frequency with which they experience different interactions such as meetings, training, mentorship and collaborations. This was used to structure the interview, allowing us to ask the researchers follow-ups on these interaction patterns, such as how the collaborations were formed, any barriers and their perspectives on these interactions. 13 of the 15 researchers completed this survey. In the interview, researchers occasionally recalled collaborations that they had not at the time of the questionnaire, thus the interview appeared to give more complete information. Interviews were conducted via Skype and lasted between 32 and 70 minutes. The study was approved through the University of Sussex Business School ethical review process (ER/HEB35/1) and consent was obtained via email and verbally at the start of the interview. Interviews were recorded and transcribed. Qualitative analysis of the interview data was carried out using a deductive approach. Reponses were split by type of interaction and information collated on the different modes, attitudes, facilitators, and barriers for each type of interaction. This is a small exploratory study aiming to highlight areas where new metrics should be developed and identify areas where capacity-building organisations might wish to focus their efforts in building a scientific community. As the number of participants was low, we have not attempted to provide full quantitative analysis of the data but have presented summary statistics of the survey data and the number of responses in a particular category, where appropriate. It should be born in mind that these proportions cannot be extrapolated to the general population of researchers due to the small and selective sample, but give an overview of the interactions experienced by this particular group of researchers.\n\n\nResults\n\nFrom our questionnaire we find that researchers spend a substantial amount of time on collaborative research, spending on average 33% (range 7-60%) of their research-related work time working with researchers outside their own work group. As discussed, previous bibliometric studies have found that most of African scientists’ collaborations are North-South (De Vré, RM Rial Verde, E & Santos da Silva, 2010; E Fonseca et al., 2016; Ettarh, 2016; Ogachi, 2018; Onyancha & Maluleka, 2011; Owusu-Nimo & Boshoff, 2017). However, our interviews reveal that researchers are participating in a broad array of intra-African collaborations and according to the responses to the questionnaire, the average proportion of a researcher’s time spent on intra-African collaboration outstrips collaboration outside the continent by 4:1. Six of the 13 researchers reported having no current collaborations at all outside of Africa (although on follow up in the interview two of these had at least some current international collaboration), whereas all of the researchers experienced intra-African collaboration at a minimum level of 7% of total research time.\n\nThe large disparity between the amount of intra-African collaboration from bibliometric studies and the amount captured in this study, highlights the need to build more comprehensive and real-time measures of collaboration. This study captures informal and pre-publication collaborations that are not captured in publication metrics, both in the questionnaire and in examples detailed in the interviews. These include collaborations where a publication is currently in preparation, collaborations with students where a publication may not be an endpoint and informal assistance between researchers.\n\nResearchers mentioned a wide range of benefits of both intra-African and international collaborations, including building more complex projects, involvement in the publications and grants of other researchers and personal benefits to their careers such as finding a new position or career mentorship. A major benefit cited by all researchers was capacity-building; developing collaborations to gain expertise or access resources, funding or equipment. Traditionally capacity-building has been focused on building North-South collaborations and indeed researchers described in the interviews many examples of this, including training placements in laboratories outside of Africa (8/15 researchers) and training from visiting researchers (mentioned by three researchers). However, as we have seen from the questionnaire data there appears to be a large amount of intra-African collaboration taking place which raised the question of whether this is also playing a role in capacity-development.\n\nIndeed, the researchers all gave examples of intra-African collaborations that had provided them with access to the expertise, equipment or resources of other groups within Africa or in which they provided this to others. For example, one researcher in a new research group carried out part of her project in a more established laboratory that provided access to expertise, equipment and reagents. All scientists had also given or received training or mentoring from groups of researchers inside Africa. One particularly common way to share capacity between two groups appeared to be via the exchange or co-supervision of students (all four principal investigators experienced this).\n\nThus, intra-African collaborations can allow African researchers to develop new expertise and access other resources through sharing of capacities within the continent rather than by import. This ‘capacity-sharing’ was valued by researchers. All of the researchers were either in the process of, planning to or hoping to build more intra-African collaborations and more than half of the researchers (eight) specified specifically that these would play a capacity-building role. Thus “capacity-sharing” could provide an alternative mode of capacity-building that allows researchers to develop their capacities in ways that are more equitable and more self-sustaining than external capacity-building. We will next describe how these intra-African collaborations can also work with traditional capacity building schemes to amplify their effects by spreading the imported capacity to an expanding circle of other institutes and researchers.\n\nResearchers interviewed were current or recent members of one of six different consortia under the DELTAS umbrella. Researchers spoke positively about the effect being a part of this network had on their research capacity. They also described a wide range of interactions they participate in that have the potential to diffuse this capacity. These interactions can be classed into two groups. Firstly, the sharing of capacity in the course of mutually beneficial intra-African project collaborations as we have described above. Secondly, deliberate sharing of the capacity to resource-poor groups through collaboration, training and mentorship. Researchers were questioned on the interactions that they have with more resource-poor groups and this revealed that many researchers (five) were part of research groups or institutes that welcomed visiting researchers from more resource poor groups for training or access to their better equipment and facilities. Five researchers also shared their expertise with less research-intensive universities and institutes though providing training, teaching or co-supervising of students. This includes a researcher who had an unpaid placement to provide training at a local university and another who provided training to lecturers at a lower-capacity university. Another two fellows were also actively involved in training the non-DELTAS members at their institute. As well as these ad hoc interactions, capacity-sharing also occurred officially through the DELTAS programmes; for example researchers on the MUII plus programme described how some meetings and equipment were open to non-MUII members of the local scientific community.\n\nA further important way in which researchers are deliberately trying to share the capacity is by mentoring researchers outside their programme. All researchers were involved in supervising students, and all but one also mentored researchers that were not directly under their supervision. For five of these researchers, this was as part of official mentoring schemes that had been established by themselves or by researchers in their programmes. This included a scheme at one consortium to match mentors with students and a programme at another to provide career development training to postdoctoral researchers, which were both open to researchers outside of DELTAS. Researchers involved in supervising and mentoring mentioned the positives for their own work, but also the motivation to give back, the satisfaction of helping others and the need to pass on and build on the mentoring they themselves had received. By mentoring others they could help to pass on the capacity they have gained from the schemes and this is something that appears to be recognised by senior researchers in these capacity-building programmes as members from two consortia described how they had participated in or organised training programmes on how to mentor others and members of two others described how the mentoring schemes established by researchers were then supported by their consortia. Thus, it appears that the organisations recognise the importance of good mentorship in building up research capacity. Further, by opening up mentorship schemes to external researchers, this ensures that the training and experience built up through capacity-building programmes can be amplified by passing it to the broader scientific community.\n\nWe have outlined how the extensive collaboration, training and mentorship activities that these researchers take part in can allow both traditional capacity-building and capacity-sharing, developing and amplifying capacity. However, for capacity-building organisations to be able to harness this, requires an understanding of how these collaborations are formed and what can be done to facilitate or to remove barriers to these interactions.\n\nWithin their institute researchers describe one of the facilitating factors for forming collaborations being a positive culture that facilitates interactions, with an informal structure where other researchers and supervisors are approachable and where students and fellows help each other. For all but three researchers these interactions were facilitated by researchers getting together at an active schedule of meetings, including departmental or institute meetings at least once a month.\n\nFor collaborations beyond the researchers’ own institutes the most common modes by which collaborations were established were through consortia or through their current or past colleagues, supervisors and mentors. All researchers described collaborations established through consortia activities and all but two, through their current or previous supervisors, mentors or colleagues. This is compared to just four through non-consortia meetings and workshops and two by getting in contact through phone or email.\n\nResearchers outlined a number of avenues by which consortia such as DELTAS, or others such as H3ABioNet, were able to facilitate collaborations. Firstly, they provide networking opportunities, for example three quarters of researchers were attending the AGMs of DELTAS or their sub-programme. These bring together scientists from different institutes and enable researchers to meet and identify potential collaborators and to raise their profiles. Secondly, consortia would bring in external speakers (this was described for three different consortia), particularly from abroad, through which the researchers describing this, had identified potential collaborators. Thirdly, it created a ready-made community of peers who could provide mentoring to each other. Fellows also interacted with each other at consortia training sessions and fellows from three consortia were interacting with each other via email and instant messaging and at workshops. Finally, consortia also provide travel opportunities enabling travel to conferences or training workshops which allow researchers to expand their networks, gain exposure to more research, raise their profile and identify more collaborators. Furthermore, the PhD training schemes of consortia such as Kemri-IDeAL enable researchers to spend a six-month training placement abroad which facilitates long-term collaborations.\n\nMentorship also appeared to play a crucial role in shaping collaboration. Seven of the nine postdoctoral researchers and Principal Investigators interviewed continued to have ongoing collaboration with prior supervisors or mentors. In addition, researchers (12/15) were also often introduced to new collaborators via their supervisors or mentors or through co-supervising or mentoring students themselves. Also particular to extra-continental collaborations, researchers with the highest levels of international collaboration (10-30%) had previously worked abroad with the international researchers in previous positions or training placements.\n\nSeveral researchers (four) mentioned the importance of having a profile and establishing trust in forming collaborations. Consortia or links with mentors likely provide the researchers with credibility and establish trust, reducing the risk to potential collaborators. Furthermore, as mentioned by one researcher, potential collaborators are likely to place more trust in a potential collaborator once they have met face to face, for example at consortia meetings. Seven researchers mentioned that they experienced some difficulty forming collaborations without a profile, network or contacts in a particular region or field. Two researchers mentioned that their consortia maintaining relationships with alumni would enhance their networks. Thus, membership of a consortium and introductions through previous collaborators and mentors might enhance the network of researchers, unlocking access to project collaborations through which they can enhance their capacities and build bigger projects. However, these facilitators may also act to constrain collaborations by restricting them to pre-existing patterns based around the networks of their previous collaborators and networks. This lock-in could limit their exposure to new collaborations and ideas and also act as a barrier to researchers who lack these contacts. Indeed, there is some evidence that researchers felt constrained in their collaborations; a francophone researcher mentioned that the default is to look to France for collaborations, another that the UK is the default for DELTAS interactions and a third that they were experiencing a poor response to their enquiries about potential collaborations in the EU due to their mentor not having contacts there.\n\nWe have described how consortia and mentorship can act as facilitators to collaborations, we will next describe the factors that researchers cited as barriers to collaboration.\n\nResearchers indicated that they had experienced barriers to forming an even higher level of intra-African collaboration. Whilst collaboration was high in the researchers’ own institute at 12% of total working time, it was low in their local area and country at a total of 5% compared to elsewhere in Africa at 10%. Over half (eight) of the researchers cited the lack of capacity within Africa. This was expressed as the lack of African researchers in their field or that it was difficult to identify them, or that the labs that do exist face the same challenges in their lack of facilities or resources. Researchers mentioned that it was easier to find the funding and capacities needed outside of the continent. Six researchers also cite a lack of openness from other researchers, that individuals or consortia are not open to new collaborations or that people will sign up for collaborations but lack the time or interest to fully engage with them. Two of these researchers mentioned that due to a lack of local meetings and collaborations they would often only meet local researchers at international conferences.\n\nAlthough many areas seemed to lack local meetings, there were positive examples described, such as the local area around where the MUII consortium is based at the Uganda Virus Institute in Entebbe, Uganda. All of the MUII researchers we interviewed mentioned the good links between MUII and the other programmes based at UVRI as well as with other research institutes and private universities in the area. Particularly strong links were formed with the University of Makerere.\n\nResearchers believed more meetings could help overcome the barriers to local collaborations including a lack of engagement (four researchers) and difficulties in identifying potential collaborators (five researchers). From our questionnaire data and interviews, 10/13 researchers were attending both frequent lab and institutional meetings and all but one were attending international conferences, inside or outside of Africa, although they experienced some barriers to these such as insufficient funding and difficulties getting visas. However, researchers identified a lack of local and regional African meetings as a problem and all but one researcher attended local and regional meetings six or fewer times a year. However, there is a lack of evidence from our interviews that increased meetings would lead to increased collaborations because, as we described earlier, there were few collaborations described that were formed at meetings without the further facilitator of consortium activities or introduction by mentors. More work is needed to determine whether meetings are an important factor in collaborations, particularly at the intermediate geographic levels between institute and international collaborations.\n\nA further area for urgent study is the impact of new technologies on research collaborations. We found that researchers are increasingly using Zoom and other platforms to keep in touch with collaborators. A particularly striking new opportunity for researchers to interact has arisen through WhatsApp, with six researchers using this to keep in touch with other fellows, providing peer-to-peer mentoring and sharing opportunities. One project collaboration described was also taking place over WhatsApp, allowing video meetings and sharing of documents more instantly and conveniently via phone, than via email and other video-calling platforms. Use of these technologies by the researchers has increased since the beginning of the COVID-19 pandemic, with even whole conferences being conducted virtually and researchers attending online training workshops and webinars. There is an opportunity for these to open up new opportunities to researchers from Africa, allowing capacity-building via webinars and removing the barriers of limited travel funding, travel time and visa issues thereby creating a more level playing field for participation in meetings and conferences. However, researchers also cited the importance of meetings in providing face-to-face interactions in forming collaborations and it is worthy of investigation whether this same benefit can be derived from virtual meetings.\n\n\nDiscussion\n\nIn this study we aimed to characterise the interactions of researchers being supported by a capacity-building initiative, how these interactions are formed and how they can impact upon capacity-building. We hoped to capture the full range of interactions that make up a scientific community, from formal and informal collaborations to meetings, training and mentorship. By capturing a wide-range of interactions, including those that might not be covered by publication metrics, such as collaborations with students and training, and by asking researchers about both their previous and current interactions, we are able to reveal extensive interactions taking place between researchers within the continent that have been hidden from previous bibliometric studies. Some of this increased intra-African collaboration that we have shown may reflect an increase in these collaborations over time that has not yet come through in the publication metrics which are subject to a large lag-time between research and publication.\n\nThe researchers valued these intra-African collaborations and detailed many ways in which these are allowing them to share capacity. This capacity-sharing could be an alternative to, or supplement traditional capacity-building, allowing positive spillovers to the broader scientific community. Strikingly researchers are also deliberately spreading the capacity they are provided with through being part of a capacity building initiative, to less well-resourced researchers. Through collaborations and interactions, capacity could become amplified, spreading out and contributing to the development of a self-sustaining scientific community.\n\nTo understand more about how these collaborations can be harnessed by capacity-building initiatives we sought to examine how they are formed and what might facilitate or hinder them. We identified that collaborations are most commonly formed along the lines of prior networks and relationships. Researchers were introduced to potential collaborators through consortia networks or worked with former colleagues and mentors, or the collaborators of those former colleagues. These introductions likely facilitated interactions by increasing the profile and network of the researchers, but also by providing the credibility and trust necessary for a collaboration to begin. Capacity-building organizations may be able to capitalize on this to encourage collaboration by further stepping up consortia activities that enable networking such as meetings between members and training placements, and by forming mentorship and alumni schemes, which would allow researchers to access the networks of other researchers.\n\nAs well as mentors providing networks and contacts to their mentees so that they can form collaborations that could help capacity-building, mentorship and training also allows direct capacity-sharing from mentor to mentee. A recent survey that asked researchers what they thought were the most important facets of being an excellent researcher showed that respondents placed the highest value on ‘training and supporting future generations of researchers (Tijssen & Kraemer-Mbula, 2017, 2018). This is reinforced by our study, which shows that researchers greatly value the mentorship they have received, whilst also feeling a duty to pass on their knowledge and experience. Researchers are taking part in a wide array of mentorship activities, taking the initiative in setting up mentorship schemes and training people that do not have access to the same support that they do. The potential power of these interactions in increasing the impact of capacity-building appears to be recognised by capacity-building programmes who provide support for mentorship schemes and provide training in mentoring others.\n\nThus, this study highlights ways in which collaborations can impact on capacity building and ways in which capacity-building organisations can capitalize on this. However, it also reiterates the point made in a recent review of metrics for capacity-building (Pulford et al., 2020) that better metrics are needed to capture the full range of interactions that make up a scientific community, from formal and informal collaborations, to mentorship, training and meetings. Capacity-building organisations may also benefit from being aware of the constraints on collaborations that have been revealed by this study. The ready-established networks that they are providing to researchers may constrain collaborations to these pre-set patterns and an awareness of this may help to make sure that these do not become too entrenched. Capacity-building organisations could ensure to seek out new interactions, encouraging more local and regional meetings, increasing the range of countries they collaborate with and perhaps developing interactions with other consortia, increasing the pool of ideas and expertise to which their researchers are exposed. Researchers stated that developing their profile is essential and that identification of potential collaborators within the continent is not always easy; building a platform for linking potential collaborators and displaying the research that is being done across the continent could assist with this. The move towards virtual interactions through Zoom and WhatsApp, especially in a post-COVID world, is likely to further change the nature of collaboration. It presents new opportunities such as the removal of travel barriers from conferences and the ease of peer-to-peer mentoring via messaging apps, and new metrics should be considered to capture this collaboration as it develops.\n\nCapacity-building organisations are increasingly recognising the power of collaborations in developing research capacity and we have shown that they can further harness this by developing intra-African collaborations to enable the sharing of capacity without external input and to amplify the capacity introduced through traditional-capacity building activities. To do this effectively will involve recognizing, understanding and being able to evaluate the full range of interactions that make up a scientific community.", "appendix": "Acknowledgements\n\nWe would like to thank the researchers who participated in the interviews for sharing their experiences and insight with us, and Frédérique Bone for help in designing the interview protocol.\n\n\nConsent\n\nWritten informed consent for participation in the study was obtained from the participants. Consent was reaffirmed verbally before interviews. Prior to consent, participants were informed of possible uses of the findings, including publication of a report using anonymised, amalgamated responses.\n\n\nReferences\n\nAbramo G, D’Angelo CA, di Costa F: Research collaboration and productivity: is there correlation? High Edu 2009; 57(2): 155–171. Publisher Full Text\n\nAbreu M, Grinevich V, Hughes A, et al.: Knowledge Exchange between Academics and the Business, Public and Third Sectors. 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Sci Public Policy 2018; 45(3): 392–403. Publisher Full Text\n\nUwizeye D, Karimi F, Otukpa E, et al.: Increasing collaborative research output between early-career health researchers in Africa: lessons from the CARTA fellowship program. Glob Health Action 2020; 13(1): 1768795. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Veken K, Belaid L, Delvaux T, et al.: Research capacity building through North-South-South networking: Towards true partnership? An exploratory study of a network for scientific support in the field of sexual and reproductive health. Health Res Policy Syst 2017; 15(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalsh A, Brugha R, Byrne E: The way the country has been carved up by researchers: ethics and power in north–south public health research. Int J Equity Health 2016; 15(1): 204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZdravkovic M, Chiwona-Karltun L, Zink E: Experiences and perceptions of South–South and North–South scientific collaboration of mathematicians, physicists and chemists from five southern African universities. Scientometrics 2016; 108(2): 717–743. Publisher Full Text\n\n\nFootnotes\n\n1 DELTAS: Developing Excellence in Leadership, Training and Science Initiative.\n\n2 MARCAD: Malaria Research Capacity Development, IDeAL: The Initiative to Develop African Research Leaders at the KEMRI-Wellcome Trust Research Programme, Afrique One-Aspire African Science Partnership for Intervention Research Excellence, WACCBIP: West African Centre for Cell Biology of Infectious Pathogens, MUII plus: Makerere University/UVRI Infection and Immunity Research Training Programme, SANTHE: Sub-Saharan African Network for TB/HIV Research Excellence." }	[ { "id": "80811", "date": "17 Mar 2021", "name": "Elaine Byrne", "expertise": [ "Reviewer Expertise Health services research", "research for development", "qualitative methodology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall: An interesting article in terms of perspectives of African researchers on benefits and some challenges of research collaborations. Interesting concepts of intra-African mentorship and capacity sharing raised which should be highlighted more as the main contribution of the article. Some areas for further development of this article are provided below.\n\nDefine what is meant by scientific - the term in this article is quite specific and seems to refer only to laboratory sciences in terms of inclusion criteria used (‘Researchers from the programmes who focused primarily on laboratory rather than clinical research were identified from the programme websites and contacted via email’). It would be best to refer in the title and throughout the article that the authors are referring to laboratory research capacity. As a social scientist I would have included all research conducted in a scientific manner, so some rationale for this narrower focus should also be given.\n\nDefine what is meant by capacity-building - there are many references to ‘traditional capacity building’ but this is not described and needs to be. Capacity development would be a preferable term as it goes beyond training and beyond the implicit assumptions of capacity building (where it is assumed that the community do not have any capacity at all to begin with and that the outsider is starting from scratch). As Suchman et al. (19881) (1988 - so we are going a long time back!) explains, we need to move beyond the “fallacy of the empty vessel”. Nchinda (20022) goes further and refers to both capacity and capability strengthening as often changes in the institution/organisation and environment such as national level policies are needed for large scale sustainable research capacity. Why the term capacity building is used and what this means needs to be explained.\n\nIndicate that it is specifically African lab research capacity that is the focus.\n\nPosition article in the debate of approaches to capacity development and highlight contributions in terms of views from the south and capacity development through mentorship and capacity sharing. Once the argument has been made in terms of the need for research capacity development then the authors should explain why within research capacity development the focus is on interactions. A way to do this is to look at the models/frameworks on capacity development (such as Bergeron et al., 20173 review of capacity development models) and argue what the added value is of your article. As Bergeron et al. note in their review knowledge networks and professional coaching were identified as common approaches to capacity development, but the authors could argue in this article that this debate is often in relation to research capacity straightening in north-south partnerships rather than south-south.\n\nThe arguments for (i) how to assess collaborations, and (ii) the approach to research capacity development are conflated - these are two different, though interlinked, arguments. You rightly argue that most collaborative research partnerships use publications as one of their metrics (but avoid generalised statements such as ‘Most work on collaborations has used just one metric for collaboration, shared publications’ - though measuring collaborations through publications is the predominant measure it is not the only one used and is often used with others - see Tigges et al. (20194) review on measuring quality and outcomes of research collaborations.). Success or not of a partnership is often determined and driven by publishing. However, alternative metrics are not proposed in this article, so either include metrics that would enable measurement of interactions, mentorship and capacity sharing or leave this argument out. The second argument is how capacity is developed and this is often from a north-south view and this is where this article is different.\n\nAdditionally there is the need to separate out the benefits of the collaboration in general and then focus on the south-south capacity development that is facilitated through capacity sharing and mentoring. In the current version the discussion mixes these together. Not quite sure of the relevance of having a profile has to this debate - not very clear whether the collaboration helps create this profile.\n\nThe methodology is unusual. I am from a social science background and would have commenced this research from a qualitative lens as little is understood about this area. Possibly then after a better understanding is obtained a questionnaire might have been developed to see how widespread the issues raised in the interviews were. The authors should explain why a questionnaire was used initially and then interviews followed by deductive analysis. It would seem better to start with open ended interviews with inductive analysis as ‘This is a small exploratory study aiming to highlight areas where new metrics should be developed and identify areas where capacity-building organisations might wish to focus their efforts in building a scientific community’. How can the themes to be analysed be predetermined?\n\nThe last section on results is more an interpretation than a finding ‘A move towards online modes of interaction may remove barriers to intra-African collaboration’ - the findings seems to suggest that there are limited interactions currently. The situation with COVID does mean many are using technology to interact and could possibly with increased confidence and acceptability of interacting in this manner expand these interactions with other African partners. However, this overlooks the other barriers to these interactions raised by the authors. There is possibly an argument to be made but would put this in the discussion section as a suggestion on how this could happen.as it doesn't come out of the findings. Additionally little numerical analysis and no direct quotes are included to highlight the interviews held.\n\nSome copy editing needed ‘In the interview, researchers occasionally recalled collaborations that they had not at the time of the questionnaire, thus the interview appeared to give more complete information.’’ “For collaborations beyond the researchers’ own institutes …” Some sentences are unclear: ‘Consortia or links with mentors likely provide the researchers with credibility and establish trust, reducing the risk to potential collaborators’ ‘As well as mentors providing networks and contacts to their mentees so that they can form collaborations that could help capacity-building, mentorship and training also allows direct capacity-sharing from mentor to mentee’.\nOverall though, a useful contribution and hopefully the authors can address some of my comments above.to develop the article further.\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/10-164
https://f1000research.com/articles/9-366/v1	14 May 20	{ "type": "Research Article", "title": "Use of the journal impact factor for assessing individual articles need not be statistically wrong", "authors": [ "Ludo Waltman", "Vincent A. Traag", "Vincent A. Traag" ], "abstract": "Most scientometricians reject the use of the journal impact factor for assessing individual articles and their authors. The well-known San Francisco Declaration on Research Assessment also strongly objects against this way of using the impact factor. Arguments against the use of the impact factor at the level of individual articles are often based on statistical considerations. The skewness of journal citation distributions typically plays a central role in these arguments. We present a theoretical analysis of statistical arguments against the use of the impact factor at the level of individual articles. Our analysis shows that these arguments do not support the conclusion that the impact factor should not be used for assessing individual articles. In fact, our computer simulations demonstrate the possibility that the impact factor is a more accurate indicator of the value of an article than the number of citations the article has received. It is important to critically discuss the dominant role of the impact factor in research evaluations, but the discussion should not be based on misplaced statistical arguments. Instead, the primary focus should be on the socio-technical implications of the use of the impact factor.", "keywords": [ "Journal impact factor", "citation", "skewness", "research evaluation", "research assessment" ], "content": "1. Introduction\n\nThe journal impact factor (IF) is the most commonly used indicator for assessing scientific journals. IFs are calculated based on the Web of Science database. They are reported each year in the Journal Citation Reports published by Clarivate Analytics. Essentially, for a certain year y, the IF of a journal equals the average number of citations received in year y by articles published in the journal in years y–1 and y–2. Although the IF is an indicator at the level of journals, it is used not only for assessing journals as a whole, but also for assessing individual articles in a journal. IF-based assessments of individual articles are usually used to evaluate the researchers or the institutions by which the articles have been authored.\n\nThere is a lot of criticism on the IF and its use in research evaluations (e.g., DORA, 2013; Seglen, 1997; Vanclay, 2012). One of the most common concerns relates to the use of the IF for assessing individual articles. It is often argued that from a statistical point of view it is incorrect, or at least highly problematic, to use the IF in the assessment of individual articles (e.g., Garfield, 2006; Gingras, 2016; Larivière et al., 2016; Leydesdorff et al., 2016; Seglen, 1992; Seglen, 1997; Zhang et al., 2017). This point is also made in the well-known San Francisco Declaration on Research Assessment (DORA, 2013). The argument is that the IF is a journal-level indicator and that it therefore tells us something about a journal as a whole, but not about an individual article in a journal. Typically the argument is supported by pointing out that the distribution of citations over the articles in a journal is highly skewed, with a small share of the articles in a journal receiving a large share of the citations (Seglen, 1992; Seglen 1997). The IF of a journal therefore is not representative of the number of citations of an individual article in the journal. According to Curry (2012), a prominent critic of the IF, the use of the IF at the level of individual articles reflects ‘statistical illiteracy’.\n\nIn this paper, we analyze in detail the above statistical argument against the use of the IF for assessing individual articles. We point out that the argument does not logically lead to the conclusion that the IF should not be used at the level of individual articles. This conclusion can be reached only when additional assumptions are made. Whether the use of the IF for assessing individual articles should be rejected depends on whether one considers these assumptions to be reasonable or not. In fact, depending on the assumptions that one makes, it can be argued that the use of the IF for assessing individual articles is preferable over the use of indicators defined at the level of individual articles, such as the number of citations of an article.\n\nThe aim of this paper is not to argue in favor of or against either the IF in general or the specific use of the IF for assessing individual articles. The analysis that we present does not enable us to draw a general conclusion on the appropriateness of IF-based assessment of individual articles. Rather, our aim is to criticize the statistical objections typically raised against the use of the IF at the level of individual articles. We argue that these objections are misguided. In our view, it is important to critically discuss the dominant role of the impact factor in research evaluations, but the discussion should not be based on misplaced statistical arguments. Instead, the primary focus should be on the socio-technical implications of the use of the impact factor.\n\nAlthough the discussion in this paper focuses on the IF, we emphasize that the discussion also applies to other citation-based indicators for journals. Indicators such as Eigenfactor Score and Article Influence Score (West et al., 2010), Source Normalized Impact per Paper (SNIP; Moed, 2010; Waltman et al., 2013), Scimago Journal Rank (SJR; González-Pereira et al., 2010; Guerrero-Bote & Moya-Anegón, 2012), and CiteScore (James et al., 2019) differ from the IF in various ways. However, like the IF, these indicators are all defined at the level of journals. The discussion on the use of journal-level indicators at the level of individual articles is therefore equally relevant for these indicators as it is for the IF. Our focus in this paper is on the IF simply because the IF is the most commonly used journal-level indicator, and consequently also the indicator that is debated most heavily. We refer to Waltman (2016, Section 8) for an overview of the literature on citation-based indicators for journals.\n\nThis paper is organized as follows. Section 2 gives an overview of the discussion on the use of the IF for assessing individual articles. Section 3 provides an illustrative example analyzing the use of the IF at the level of individual articles. This is followed in Section 4 by a more general conceptual discussion on the use of the IF for assessing individual articles. The illustrative example in Section 3 and the conceptual discussion in Section 4 aim to make clear that from a statistical point of view the use of the IF at the level of individual articles does not need to be wrong. Section 5 presents computer simulations to further illustrate this point. Finally, in Section 6, we discuss our findings and summarize our conclusions.\n\n\n2. Background\n\nThere is a sizeable literature discussing the IF and its use in research evaluations (for a recent overview, see Larivière & Sugimoto, 2019). The discussion has partly focused on technical and statistical issues in the calculation of the IF (e.g., Glänzel & Moed, 2002; Seglen, 1997), such as the definition of so-called ‘citable items’ in the denominator of the IF (e.g., Moed & Van Leeuwen, 1995; Moed & Van Leeuwen, 1996) and the time window based on which the IF is calculated (e.g., Glänzel & Schoepflin, 1995; Moed et al., 1998). In addition, there has also been discussion about the transparency of the IF (e.g., PLoS Medicine Editors, 2006; Rossner et al., 2007; Vanclay, 2012), the vulnerability of the IF to manipulation (e.g., Chorus & Waltman, 2016; Martin, 2016; Wilhite & Fong, 2012), and the dominant role of the IF in research evaluations (e.g., McKiernan et al., 2019; Quan et al., 2017; Rushforth & De Rijcke, 2015). An extensive discussion about the IF has taken place in a special issue of Scientometrics (Braun, 2012). This discussion was triggered by a critical paper about the IF by Vanclay (2012). The producers of the IF have also repeatedly contributed to discussions about the IF (e.g., Garfield, 1996; Garfield, 2006; Pendlebury, 2009; Pendlebury & Adams, 2012; Wouters et al., 2019).\n\nIn this paper, we restrict our attention to statistical objections against the use of the IF for assessing individual articles. Below we first review some literature that argues against IF-based assessment of individual articles. We then discuss a few sources that suggest that there are some limited opportunities for IF-based assessment of individual articles.\n\nStatistical objections against IF-based assessment of individual articles go back at least to classical papers by Seglen (1992); Seglen (1997). Seglen shows that the distribution of citations over the articles in a journal is highly skewed. He then draws the following conclusion (Seglen, 1992, p. 631):\n\nThe great variability in citedness within a journal has important implications for the significance attached to the journal impact factor. In several countries, this easily available factor has been used in academic evaluations of individual scientists, on the implicit premise that the impact factor of the journal is representative of its constituent articles, and hence, of the article authors. The skewness of the journal article distributions shows that this premise does not hold true: only a minor fraction of the articles are cited anywhere near the journal mean ... Assigning the same value to all articles in a journal will overestimate the less influential and underestimate the more influential articles, thus effectively leveling out the very differences that evaluation procedures should seek to identify.\n\nEugene Garfield, who created the IF in the early days of the Science Citation Index, draws a similar conclusion (Garfield, 2006, p. 92):\n\nTypically, when the author’s work is examined, the impact factors of the journals involved are substituted for the actual citation count. Thus, the journal impact factor is used to estimate the expected count of individual papers, which is rather dubious considering the known skewness observed for most journals.\n\nIn 2013, the San Francisco Declaration on Research Assessment (DORA) was published. It has attracted a lot of attention and support. DORA strongly rejects the use of the IF for assessing individual articles. A number of arguments are given, one of them being that “citation distributions within journals are highly skewed”, leading to the recommendation not to “use journal-based metrics, such as Journal Impact Factors, as a surrogate measure of the quality of individual research articles (or) to assess an individual scientist’s contributions” (DORA, 2013). DORA also recommends journal publishers to “make available a range of article-level metrics to encourage a shift toward assessment based on the scientific content of an article rather than publication metrics of the journal in which it was published”.\n\nIn line with DORA, in his monograph on bibliometrics and research evaluation, Gingras (2016) also uses the skewness of citation distributions to argue against the use of the IF for assessing individual articles (p. 47–48):\n\nThe IF remains a measure related to the journal, not to the articles it contains. The fundamental reason that makes it a flawed indicator of the value of individual articles is that the distribution of actual citations received by the articles published in a given journal follows a power law similar to that of Alfred Lotka for productivity, which means that most articles are in fact cited very little. Only a few are very highly cited, and they inflate the value of the IF ... If one wants to measure the quality or visibility of a particular item, one must look at the citations actually received in the years following its publication. But that of course takes time, and those who prefer ‘quick and dirty’ evaluation do not want to wait three to five years. So they use the IF of the journal in which the papers are published as a proxy of their quality and impact, even though such a measure is totally inappropriate.\n\nA high-profile paper by Larivière et al. (2016) again draws attention to the skewness of the distribution of citations over the articles in a journal. The authors recommend that, when a journal publishes its IF, it should also publish the underlying citation distribution. In this way, awareness will be drawn to the skewness of the citation distribution, and this skewness can then be taken into account in the interpretation of the IF. Like the sources discussed above, Larivière et al. regard the skewness of the citation distribution of a journal as an argument against the use of the IF for assessing individual articles. They observe that “for all journals there are large numbers of papers with few citations and relatively few papers with many citations”, which they argue “underscores the need to examine each paper on its own merits and serves as a caution against over-simplistic interpretations of the JIF” (p. 5).\n\nIn a recent paper by Zhang et al. (2017), the work by Seglen (1992); Seglen (1997) is revisited. Seglen’s empirical findings are confirmed based on a much larger amount of data, leading to the following conclusion (p. 14–15):\n\nAlthough some journals are certainly more prestigious, attractive and selective than others, one should not infer the quality of the individual article from the status of the journal. Moreover, even if citations are taken as an indication of quality, the citation impact of a journal remains a weak predictor of the citation impact of each of its articles. Consequently, individual contributions should not be evaluated by where they are published.\n\nLeydesdorff et al. (2016) also present a statistical objection against the use of the IF for assessing individual articles. Their objection does not relate to the skewness of citation distributions. Instead, it is based on the concept of ecological fallacy (p. 2140):\n\nThe use of the JIF for the evaluation of individual papers provides an example of the so-called “ecological fallacy” ...: inferences about the nature of single records (here: papers) are deduced from inferences about the group to which these records belong (here: the journals where the papers were published). However, an individual child can be weak in math in a school class which is the best in a school district. Citizen bibliometricians ... may nevertheless wish to continue to use the JIF in research evaluations for pragmatic reasons, but this practice is ill-advised from the technical perspective of professional bibliometrics.\n\nPaulus et al. (2018) also use the concept of ecological fallacy to criticize the use of the IF at the level of individual articles.\n\nUsing statistical arguments similar to the ones presented above, most scientometricians reject the use of the IF for assessing individual articles. However, some scientometricians argue that there is some room for assessing individual articles using the IF or some other journal-level indicator.\n\nAccording to Abramo et al. (2010, p. 832), “there is an agreement among scholars on the superiority of citations over impact factor as proxy of quality of publications for ‘old’ articles”. However, for recent articles, Abramo et al. argue that the situation is different:\n\nCitations observed at a moment too close to the date of publication will not necessarily offer a proxy of quality that is preferable to impact factor. Yet bibliometric evaluation exercises ... should be based on observations of the most recent possible past. For evaluations over periods that are very close in time to the date of conducting the exercise, and especially in certain disciplines, the impact factor can thus be a predictor of the real impact of an article, and possibly a better one than citations.\n\nA similar argument is made by Levitt & Thelwall (2011). Rather than choosing between the number of citations of an article and the IF of the journal in which an article has appeared, Levitt and Thelwall suggest combining the number of citations and the IF into a hybrid indicator. In the context of providing indicators to peer review panels in the UK Research Excellence Framework, Levitt and Thelwall reach the following conclusion (p. 307):\n\nParticularly for very recently published articles, an indicator based on the average of the standard indicator of citation and the IF of the journal ... could form the basis of a useful indicator for peer review panels.\n\nAncaiani et al. (2015) discuss how the Italian research evaluation exercise takes into account both the number of citations of an article and the IF of the journal in which an article has appeared. In line with the ideas of Abramo et al. (2010) and Levitt & Thelwall (2011), the IF plays a prominent role especially in the assessment of recent articles. When the number of citations and the IF provide conflicting information, the IF is given more weight in the case of recent articles, while the number of citations has more weight in the case of older articles.\n\nAnother perspective is provided by Moed (2005) in his monograph on citation analysis and research evaluation. According to Moed, assessing articles using journal-level indicators is acceptable, but the assessment should focus on the entire oeuvre of a research group rather than on individual articles. Moreover, Moed emphasizes that journal-level indicators reflect a different aspect of the performance of a research group than article-level indicators (p. 84–85):\n\nJournal impact is a performance aspect in its own right, but cannot be used to predict actual citation rates. The extent to which groups of scientists publish their output in the more prestigious, or even the ‘top’ journals in their fields, is often viewed as an important aspect of scientific research performance. (An) indicator of the impact of a group’s journal packet ... can be validly used to assess this aspect.\n\n\n3. Illustrative example\n\nIn this section, we present a simple illustrative example analyzing the use of the IF for assessing individual articles. The example introduces some of the key ideas that will play an important role in the conceptual discussion in Section 4 and in the computer simulations in Section 5. Before presenting the example, we first discuss the difference between observable and non-observable concepts in citation analysis.\n\nIn order to have a careful and precise discussion on the use of the IF for assessing individual articles, it is essential to distinguish between observable and non-observable concepts in citation analysis (for a similar idea in a somewhat different context, see Waltman et al., 2013). Important observable concepts are the number of citations of an article and the IF of a journal. These observable concepts are important not so much because they are of interest themselves, but mainly because they may tell us something about certain non-observable concepts that we are interested in. In the context of the assessment of scientific articles, examples of these non-observable concepts could be the quality, the impact, and the influence of an article. The general idea of citation analysis is that an observable concept, such as the number of citations of an article, provides an approximate representation of a non-observable concept, such as the impact of an article. The observable concept is then regarded as an indicator of the non-observable concept. The number of citations of an article for instance is often regarded as an indicator of the impact of the article. Likewise, the IF of a journal is sometimes seen as an indicator of the quality of the journal.\n\nThe use of a certain observable concept as an indicator of a certain non-observable concept often causes debate. There usually is disagreement on whether the observable concept provides a sufficiently close approximation of the non-observable concept. For instance, some may consider the number of citations of an article to be a suitable indicator of the impact of the article, but others may disagree and may argue that citations do not provide a sufficiently close approximation of impact. At a more fundamental level, the difficulty is that non-observable concepts typically lack a clear and unambiguous definition. The concepts of quality, impact, and influence for instance are understood differently by different people, making it challenging to agree on the use of citations as an indicator of any of these concepts.\n\nIn this paper, we do not want to enter the debate about which non-observable concepts may or may not be represented by the number of citations of an article. Instead, we start from the idea that assessing an article is equivalent to determining the value of the article, where we use value as a general non-observable concept that, depending on the precise criterion based on which one wants articles to be assessed, may for instance be understood as quality, impact, influence, importance, or usefulness. The main point that we want to make in this paper does not depend on the specific understanding that one has of the concept of value, and therefore there is no need to provide a precise definition of this concept. However, in Subsection 4.2, we will say a bit more about the implications of different ways in which the concept of value can be understood.\n\nFor further discussion on the conceptual foundation of citation analysis, we refer to Bornmann & Daniel (2008); De Bellis (2009, Chapter 7); Moed (2005, Chapters 15–17); Nicolaisen (2007) & Tahamtan & Bornmann (2019).\n\nWe now provide a simple example comparing the assessment of articles based on either the IF of the journal in which they have appeared or the number of citations they have received. The example introduces some of the key ideas that will be further elaborated in Sections 4 and 5. It also illustrates the importance of making a careful distinction between observable and non-observable concepts.\n\nThe situation that we analyze in our example is an extreme simplification of reality (for a somewhat similar type of analysis, see Waltman et al., 2013). Some may regard this as a weakness of the example. However, we regard it as a strength, because the extreme simplification enables us to focus on the most essential issues, without being distracted by irrelevant details.\n\nWe consider a situation in which the value of an article is either low or high and in which an article is either lowly cited or highly cited. There are 200 articles. Of these articles, 100 are of low value and 100 are of high value. Likewise, 100 are lowly cited and 100 are highly cited. Furthermore, there are just two journals, journal A and journal B. Each journal has published 100 articles.\n\nOur aim is to identify as accurately as possible the articles that are of high value. As pointed out in Subsection 3.1, the value of an article is a non-observable concept. This means that high-value articles cannot be directly identified. We therefore compare two approaches that try to identify these articles in an indirect way. One approach is to select all articles that are highly cited. The other approach is to select all articles that have appeared in the journal with the higher IF. In the situation in which each article is either lowly cited or highly cited, the journal with the higher IF is the journal with the larger share of highly cited articles. We want to find out which of the above two approaches for identifying high-value articles is more accurate.\n\nThe number of citations of an article may provide an approximate representation of the value of an article. Because the representation is approximate, being highly cited does not need to coincide with being of high value. In the first scenario that we consider (i.e., scenario 1), 90% of the articles that are of high value are highly cited. The other 10% are lowly cited. Conversely, 90% of the articles that are of low value are lowly cited. The other 10% are highly cited. This information is summarized in Table 1.\n\nSuppose that 80 articles in journal A are of high value, while only 20 articles in journal B are of high value. The remaining articles in both journals are of low value. This yields the situation presented in Table 2. As can be seen in the table, the number of highly cited articles in journal A equals 90% x 80 + 10% x 20 = 74. On the other hand, journal B has published 90% × 20 + 10% × 80 = 26 highly cited articles. Consequently, journal A has published a larger share of highly cited articles than journal B, and therefore journal A has a higher IF than journal B.\n\nIf we choose to identify high-value articles based on the IF, we select all 100 articles in journal A, which yields 80 high-value articles. The other approach is to identify high-value articles based on an article’s number of citations. If we choose this approach, we select all 100 highly cited articles. 90% of these articles are of high value, so this results in 90 high-value articles. Hence, in scenario 1, it is more accurate to identify high-value articles based on an article’s number of citations than based on the IF. This is in agreement with commonly used statistical arguments against the use of the IF for assessing individual articles.\n\nWe now consider a second scenario (i.e., scenario 2). In this scenario, instead of 90% only 70% of the high-value articles are highly cited. The other 30% are lowly cited. Of the low-value articles, 70% are lowly cited and 30% are highly cited. Like in scenario 1, 80 articles in journal A are of high value, while only 20 articles in journal B are of high value. All other articles are of low value. Scenario 2 is summarized in Table 3 and Table 4.\n\nTo what extent does scenario 2 lead to different outcomes than scenario 1? In scenario 2, journals A and B have published respectively 70%×80+30%×20=62 and 70%×20+30%×80=38 highly cited articles. Hence, like in scenario 1, journal A has a higher IF than journal B. If we choose to identify high-value articles based on the IF, we select all 100 articles in journal A. This yields 80 high-value articles, which is identical to the outcome obtained in scenario 1. On the other hand, if we choose to identify high-value articles based on an article’s number of citations, we select all 100 highly cited articles. In scenario 2, only 70% of these articles are of high value, and therefore we obtain only 70 high-value articles.\n\nImportantly, the conclusion that we reach in scenario 2 is the opposite of the conclusion drawn in scenario 1. In scenario 2, identifying high-value articles based on an article’s number of citations is less accurate than identifying high-value articles based on the IF. The accuracy of citations as an indicator of the value of an article is lower in scenario 2 than in scenario 1, but this difference in the accuracy of citations does not affect the accuracy of the IF. This explains why the two scenarios yield opposite conclusions and why in scenario 2 the IF is a more accurate indicator of the value of an article than the number of citations of the article.\n\nOf course, the situation analyzed in the above example is an extreme simplification of reality. Nevertheless, the example shows that the number of citations of an article is not necessarily a more accurate indicator of the value of the article than the IF of the journal in which the article has appeared. Which of the two indicators is more accurate depends on the degree to which citations provide an accurate representation of the value of an article. In the next two sections, we will study this in more detail, first by providing a conceptual discussion and then by presenting computer simulations.\n\n\n4. Conceptual discussion\n\nIn the previous section, we provided an illustrative example of a situation in which it is possible that the IF of the journal in which an article has appeared is a more accurate indicator of the value of the article than the number of citations of the article. The situation analyzed in the example in the previous section is an extreme simplification of reality. As we have seen in Section 2, in discussions on the use of the IF for assessing individual articles, the skewness of the distribution of citations over the articles in a journal usually plays a crucial role. The skewness of journal citation distributions was not taken into account in the simple example presented in the previous section. In this section, we provide a more general conceptual discussion on the use of the IF for assessing individual articles. The skewness of journal citation distributions is a key element in this discussion1.\n\nLike in the example presented in the previous section, the distinction between the value of an article and the number of citations of an article is essential. We again consider two scenarios. In scenario 1, the number of citations of an article is a more accurate indicator of the value of the article than the IF of the journal in which the article has appeared. Scenario 2 represents the opposite situation. In both scenarios, journal citation distributions are highly skewed.\n\nScenario 1 can be summarized in the following three points:\n\n1. The number of citations of an article is a relatively accurate indicator of the value of the article.\n\n2. Journals are rather heterogeneous in terms of the values of the articles they publish.\n\n3. The skewness of journal citation distributions results mainly from point 2.\n\nCompared with scenario 1, scenario 2 offers an opposite explanation of the skewness of journal citation distributions:\n\n1. The number of citations of an article is a relatively inaccurate indicator of the value of the article.\n\n2. Journals are fairly homogeneous in terms of the values of the articles they publish.\n\n3. The skewness of journal citation distributions results mainly from point 1.\n\nIn scenario 1, the number of citations of an article and the value of an article are strongly correlated. The skewness of a journal citation distribution therefore reflects the skewness of the distribution of the values of the articles in a journal. The IF is not representative of the number of citations of an individual article in a journal, and in scenario 1 this directly implies that the IF is not an accurate indicator of the value of an individual article.\n\nThe situation is very different in scenario 2. In this scenario, the articles in a journal all have a relatively similar value. The skewness of a journal citation distribution therefore does not result from large differences in the values of the articles in a journal. Instead, it results from the inaccuracy of citations as an indicator of the value of an article. As a consequence of this inaccuracy, articles that have a similar value may have very different numbers of citations. In line with the literature on cumulative advantage (De Solla Price, 1976) or preferential attachment (Barabási & Albert, 1999) processes, this causes the citation distribution of a journal to be skewed even though the articles in the journal all have a relatively similar value.\n\nLike in scenario 1, in scenario 2 the IF is not representative of the number of citations of an individual article in a journal. However, this is not a problem in scenario 2. If a journal has published a sufficiently large number of articles, the IF may be expected to be a quite accurate indicator of the average value of the articles in the journal. This is the case despite the fact that in scenario 2 the number of citations of an individual article is a relatively inaccurate indicator of the value of the article. To understand this, it is essential to recognize that the IF is calculated at the level of an entire journal rather than at the individual article level. At the journal level, ‘errors’ in citations may be expected to largely cancel out. This is in agreement with what Nicolaisen (2007) refers to as the standard account of citation analysis (e.g., Van Raan, 1998). If ‘errors’ in citations largely cancel out at the journal level, the IF is a quite accurate indicator of the average value of the articles in a journal. Since the articles in a journal all have a relatively similar value in scenario 2, this implies that the IF is also a quite accurate indicator of the value of an individual article.\n\nCritics of the use of the IF for assessing individual articles implicitly appear to assume that reality is like scenario 1. Critics do not seem to be aware of the possibility of reality being more like scenario 2, or alternatively, they may consider scenario 2 to be highly unrealistic and may therefore not take it seriously. In our view, there is no easy way to determine whether scenario 1 or scenario 2 is closer to reality. Nevertheless, we can make some comments on the degree to which scenarios 1 and 2 may be realistic.\n\nWe first consider the accuracy of citations as an indicator of the value of an article. In scenario 1 citations are a relatively accurate indicator of the value of an article, while in scenario 2 they are a relatively inaccurate indicator. There are two reasons why it is difficult to say which of the two scenarios is more realistic.\n\nFirst, there are conflicting viewpoints on the accuracy of citations as an indicator of the value of an article. For instance, following the well-known distinction between the normative and the social constructivist perspectives on citations (Nicolaisen, 2007), it is clear that those who adopt the normative perspective will have more confidence in the accuracy of citations than those who adopt the social constructivist perspective. Hence, followers of the normative perspective will be more likely to accept the viewpoint of scenario 1 on the accuracy of citations, while followers of the social constructivist perspective will reject this viewpoint and may find the viewpoint of scenario 2 more acceptable (although they may also disagree with this viewpoint).\n\nThere is a second reason why it is difficult to say which of the two scenarios provides a more realistic perspective on the accuracy of citations. As discussed in Subsection 3.1, we have chosen not to provide a precise definition of the concept of the value of an article. However, depending on how this concept is understood, one may prefer either scenario 1 or scenario 2. For instance, if the value of an article is understood as the extent to which the article is used in other articles, citations may perhaps be considered a relatively accurate indicator of the value of an article. From this point of view, scenario 1 may then be regarded as more realistic than scenario 2. On the other hand, if the value of an article is understood as the quality of the article according to the judgment of scientific peers, citations may be considered a relatively inaccurate indicator of the value of an article. Scenario 2 may then be regarded as more realistic than scenario 1.\n\nWe now consider the homogeneity or heterogeneity of journals in terms of the values of the articles they publish. In scenario 1 there are large differences in the values of the articles published in a journal, while in scenario 2 the articles published in a journal all have a relatively similar value.\n\nThe homogeneity of journals in scenario 2 can be motivated based on two ideas. One idea is that the peer review system of a journal will ensure that all or almost all articles in a journal have a value above a certain journal-specific minimum threshold. The other idea is that researchers will generally try to publish their work in a journal that is as ‘prestigious’ as possible, which means that they will try to avoid publishing their work in a journal that also publishes work of much lower value. Together, these two ideas may cause journals to be relatively homogeneous in terms of the values of the articles they publish.\n\nThe above motivation for the homogeneity of journals in scenario 2 requires a relatively high level of confidence in the accuracy of the journal peer review system. However, the accuracy of the journal peer review system has been questioned (for an overview of the literature, see Bornmann, 2011), which provides support for the heterogeneity of journals in scenario 1. There are also other arguments that may be used to support the heterogeneity of journals. For instance, when a journal publishes lots of articles, it seems unlikely that these articles are all of similar value. In general, the larger a journal, the more the journal can be expected to be heterogeneous in terms of the values of its articles. In addition, in a small field with only a limited number of journals (such as the field of scientometrics), even a relatively small journal may need to publish articles that are of quite different value. This also results in journals being heterogeneous.\n\nWe have now made a number of comments on the degree to which scenarios 1 and 2 may be realistic. Based on these comments, which of the two scenarios is closer to reality? In our opinion, there is no easy answer to this question. The answer is likely to be field- and journal-dependent. It is also likely to be time-dependent. In line with some of the literature discussed in Subsection 2.2 (Abramo et al., 2010; Ancaiani et al., 2015; Levitt & Thelwall, 2011), shortly after an article has been published, scenario 1 seems unrealistic, since there has not been much time for the article to be cited. Scenario 1 may be more realistic in the longer term. Scenario 2, on the other hand, may be realistic both in the short term and in the longer term. Furthermore, as we have already pointed out, the answer to the above question also depends on the precise understanding that one has of the concept of the value of an article. In other words, the appropriateness of the use of the IF for assessing individual articles is dependent on the precise criterion based on which one wants articles to be assessed.\n\nImportantly, whether the IF can or cannot be used for assessing individual articles is perhaps not even the most relevant question to ask. Any method for assessing articles has weaknesses. This applies not only to the IF but also to the number of citations of an article and to assessment based on peer review. The question whether the use of a specific method for assessing articles is appropriate or not therefore seems to be of limited relevance. Instead, a more relevant question seems to be which of the various methods available for assessing articles is most appropriate relative to the others. For instance, critics of IF-based assessment of individual articles typically seem to believe that for assessing an article it is more appropriate to use the number of citations of the article than the IF of the journal in which the article has appeared. This seems to be the case for the critics quoted in Subsection 2.1, although some of them are more explicit about this than others. Gingras (2016, p. 48) is very explicit: “If one wants to measure the quality or visibility of a particular item, one must look at the citations actually received in the years following its publication.” In our opinion, determining the relative appropriateness of different methods for assessing articles is a much more intricate problem than critics of IF-based assessment seem to believe. We reject a simple binary perspective in which some methods are valid and others are invalid. Instead, it is a matter of degree. Depending on the assumptions that one makes, one method may be more appropriate than another, but the difference need not be large. Also, the situation may reverse when the assumptions are changed. In the next section, we will use computer simulations to further elaborate our viewpoint.\n\n\n5. Computer simulations\n\nWe now use computer simulations to further illustrate the ideas introduced in the previous two sections. We start by presenting our simulation model and by discussing how we analyze the accuracy of an indicator for assessing individual articles. We then report the results of our computer simulations2.\n\nWe consider a scientific field in which there are m journals. In a certain time period, n articles are published in these journals. Each journal is of the same size, so each journal publishes n/m articles.\n\nFor each article i (i = 1,2, ... , n), the value of the article, denoted by vi, is drawn from a lognormal distribution, that is,\n\n\n\nWe use logN(σ2) to denote a lognormal distribution for which the mean and the variance of the underlying normal distribution are equal to –σ2/2 and σ2, respectively. In this way, the mean of the lognormal distribution always equals 1, regardless of the value of σ2. A lognormal distribution is used in (1) because in reality there are probably many more articles that have a low or moderate value than articles that have a high value. This is captured by the skewness of the lognormal distribution. The degree to which the distribution is skewed is determined by the parameter σv2 in (1).\n\nJournal 1 is regarded as the most prestigious journal in the field, journal 2 is regarded as the second most prestigious journal in the field, and so on. Journal m is seen as the least prestigious journal. Our model does not specify why one journal is regarded as more prestigious than another journal. However, one could imagine that this is based on the IFs of the journals in earlier time periods or on the value of the articles published in the journals in earlier time periods. Our model assumes that the authors of an article first try to publish their article in journal 1. If their article is rejected by this journal, they try to publish it in journal 2, and so on. This goes on until there is a journal that accepts the article.\n\nTo decide which articles to accept and which ones to reject, a journal k estimates the value of each article it receives. To do so, the journal sends each article to reviewers. Based on the comments reviewers provide on an article, the journal obtains an estimate of the value of the article. The value of article i estimated by journal k, denoted by eik, equals the value of the article multiplied by a value drawn from a lognormal distribution. More precisely, eik is given by\n\n\n\nThe parameter σr2 determines the accuracy of the journal peer review system. The smaller the value of this parameter, the more accurate the journal peer review system. If σr2 = 0, the journal peer review system provides a perfectly accurate estimate of the value of an article. Of all articles received by journal k, the journal accepts the n/m articles that have the highest estimated value. All other articles are rejected. Hence, journal 1 receives n articles and rejects n – n/m of them, journal 2 receives n – n/m articles and rejects n – 2(n/m) of them, and so on. Journal m, the least prestigious journal, receives n/m articles, which it all accepts.\n\nAfter all n articles have been published, they accumulate citations. Our model assumes that the number of citations of an article correlates with the value of the article. On average, articles that have a higher value receive more citations. For each article i, the number of citations of the article, denoted by ci, equals the value of the article multiplied by a value drawn from a lognormal distribution, that is,\n\n\n\nThe parameter σc2 determines the accuracy of citations as an indicator of the value of an article. The smaller the value of this parameter, the higher the accuracy of citations. If σc2 = 0, citations are a perfectly accurate indicator of the value of an article. In reality, the number of citations of an article is an integer. For simplicity, we do not require the number of citations of an article to be an integer in our model. As we discuss in Subsection 5.3, citations may be interpreted as rescaled citations (Radicchi et al., 2008) in our model.\n\nIt follows from (1) and (3) that the distribution of citations over articles is also lognormal. More precisely, the distribution of citations over articles is logN(σv2+σc2). The lognormal distribution of citations over articles is in line with empirical studies that show that the distribution of citations over articles is highly skewed and approximately lognormal (Evans et al., 2012; Radicchi et al., 2008; Stringer et al., 2008; Thelwall, 2016a; Thelwall 2016b).\n\nFinally, for each journal k, the IF of the journal, denoted by IFk, is calculated. In our model, the IF of a journal is defined as the average number of citations of the articles published in the journal. Hence, IFk is given by\n\n\n\nwhere pik equals 1 if article i has been published in journal k and 0 otherwise. In our model, each journal publishes n/m articles, and therefore the denominator in (4) always equals n/m.\n\nThe model introduced above of course provides a simplified representation of reality. For instance, in reality journals are not all of the same size and researchers do not all have the same perception of the prestige of the journals in their field. Also, when researchers want to publish an article, they do not always start by submitting their article to the most prestigious journal. Based on their knowledge of the journals in their field, researchers may know the journal in which their article can best be published, and they may immediately submit their article to this journal rather than first submitting it to other more prestigious journals.\n\nImportantly, we consider the simplicity of our model to be a strength, not a weakness. We could develop a more realistic model. However, such a model would also be more complex, making it more difficult to obtain clear insights from the model. A good model captures the essential elements that need to be taken into account to get a proper understanding of the phenomenon of interest, while it leaves out the non-essential elements. We believe that our model indeed captures the elements that are essential for our analysis. At the same time, non-essential elements are left out, so that unnecessary complexity is avoided and clear insights can be obtained.\n\nWe focus on two indicators for assessing individual articles. One indicator is the IF of the journal in which an article has appeared3. The other indicator is the number of citations of an article. Our aim is to analyze and compare the accuracy of these two indicators. This of course requires a precise definition of the accuracy of an indicator.\n\nOur definition of the accuracy of an indicator relies on a binary classification of articles based on their value. Like in Subsection 3.2, we distinguish between low-value and high-value articles. To make this distinction, we introduce the parameter α. This parameter specifies the share of articles that are considered to be of high value. Of the n articles in our simulation model, the αn articles with the highest values are classified as high-value articles, while the remaining articles are classified as low-value articles.\n\nTo obtain the accuracy of an indicator, we select the αn articles that are most highly ranked by the indicator (i.e., the αn articles with the highest IF or the largest number of citations) and we calculate the percentage of the selected articles that are of high value. The accuracy of an indicator can be anywhere between 0% and 100%. An indicator has an accuracy of 100% if the αn articles that are most highly ranked by the indicator coincide with the αn high-value articles. An indicator has an accuracy of 0% if the αn most highly ranked articles are all of low value.\n\nWe now present the results of our computer simulations. We consider a situation in which n = 2000 articles are published in a certain scientific field and in a certain time period. These articles appear in m = 20 journals, which means that each journal publishes n/m = 2000/20 = 100 articles.\n\nTo choose suitable values of σv2 and σc2, we rely on empirical work carried out by Radicchi et al. (2008). Radicchi et al. rescale citations in such a way that in each field the average number of citations per article equals 1. This is in agreement with our simulations, in which we also have an average number of citations per article of 1. Radicchi et al. report that the distribution of rescaled citations over the articles in a field is lognormal, with the variance of the underlying normal distribution being equal to 1.34. In order to obtain citation distributions that are in line with the findings of Radicchi et al., we require that σv2+σc2=1.3 in our simulations. In the presentation of the simulation results, we report the value of σc2. Values of σc2 between 0 and 1.3 are considered. The value of σv2 is not reported, but this value equals 1.3 – σc2. Suitable values of σr2 cannot be easily derived from empirical analyses. We therefore simply consider a number of different values of σr2 in our simulations.\n\nIn the calculation of the accuracy of an indicator, we set the parameter α equal to 0.1. Hence, we determine the accuracy of an indicator based on the capability of the indicator to identify the 10% highest-value articles. The choice of α = 0.1 is somewhat arbitrary. However, we also tested other values of α, and our results do not change in an essential way when a different value of α is chosen. Our simulation results are based on 1000 simulation runs. The accuracy of an indicator is calculated as the average accuracy over all simulation runs.\n\nFigure 1 shows for different values of σr2 and σc2 the accuracy of both the IF of the journal in which an article has appeared and the number of citations of an article. Four different values of σr2 are considered. In our simulation model, the value of σr2 has no influence on the accuracy of citations, but it does influence the accuracy of the IF. The higher the value of σr2 (i.e., the lower the accuracy of the journal peer review system), the less accurate the IF. As can be expected, the accuracy of both the IF and citations decreases as the value of σc2 increases. However, the value of σc2 has more influence on the accuracy of citations than on the accuracy of the IF. As discussed in Subsection 4.1, this is because ‘errors’ in citations tend to cancel out in the IF, making the IF relatively insensitive to these ‘errors’.\n\nThe most important observation based on Figure 1 is that for a range of values of σr2 and σc2 the IF is more accurate than citations. This is the case when the value of σr2 is not too high (i.e., the journal peer review system is at least moderately accurate) and the value of σc2 is not too low (i.e., citations are at least moderately inaccurate). For these values of σr2 and σc2, the IF benefits from its limited sensitivity to ‘errors’ in citations while it does not suffer too much from heterogeneity in the values of the articles published in a journal. As shown in the top-left panel in Figure 1, when σr2 = 0 (i.e., the journal peer review system is perfectly accurate), the IF outperforms citations for all values of σc2. On the other hand, the bottom-right panel in Figure 1 shows that for high values of σr2 (i.e., the journal peer review system is highly inaccurate) the IF is always outperformed by citations, regardless of the value of σc2. In this case, journals are highly heterogeneous and publish a mix of high-value and low-value articles, making the IF a very weak indicator of the value of an article.\n\nThe results presented in Figure 1 are based on a situation in which there are m = 20 journals. Figure 2 shows the effect of increasing or decreasing the number of journals, while keeping the total number of articles fixed at n = 2000. In the left panel of Figure 2, the number of journals has been halved (and the number of articles per journal has been doubled), which means that we have m = 10 journals with n/m = 200 articles per journal. In the right panel, the number of journals has been doubled (and the number of articles per journal has been halved), resulting in m = 40 journals with n/m = 50 articles per journal. In both panels, σr2 has a value of 0.4. Hence, we consider an intermediate level of accuracy of the journal peer review system.\n\nIncreasing the number of journals from 10 (left panel of Figure 2), to 20 (top-right panel of Figure 1), to 40 (right panel of Figure 2) yields a modest improvement in the accuracy of the IF. Of course, it does not affect the accuracy of citations. The increase in the number of journals therefore broadens the range of values of σc2 in which the IF outperforms citations. When the IF is used as an indicator of the value of an article, it is clear that the number of journals should not be too small. In the extreme case in which there is only one journal (i.e., m = 1), the IF is completely useless as an indicator of the value of an article. However, the number of journals should not be too large either. Having a large number of journals is fine as long as the number of articles per journal does not become too small. When the number of articles per journal is very small, the IF will be highly sensitive to ‘errors’ in citations. The smaller the number of articles in a journal, the less one can expect ‘errors’ in citations to cancel out. In the extreme case in which each journal publishes only one article (i.e., m = n), the IF and citations have exactly the same accuracy. However, this is an artefact that results from our choice not to take time into account in our simulation model (see footnote 3).\n\nWe have seen that, depending on the values of σr2, σc2, and m, the accuracy of the IF may be either higher or lower than the accuracy of citations. A natural question to ask is whether the IF and citations can be combined into a hybrid indicator that is more accurate than both the IF and citations separately. This possibility was already suggested by Anfossi et al. (2016); Levitt & Thelwall (2011). To explore this possibility, we obtain hybrid indicators by calculating a weighted average of the IF of the journal in which an article has appeared and the number of citations of the article5. We give a weight of 0%, 25%, 50%, 75%, or 100% to the IF. The remaining weight is given to citations. Of course, when the IF has a weight of 0%, the hybrid indicator coincides with the citations indicator. Likewise, using a weight of 100% for the IF, the hybrid indicator coincides with the IF indicator. We focus on the situation in which σr2 = 0.4 and m = 20.\n\nThe results are presented in Figure 3. The figure confirms that hybrid indicators indeed perform well. Except for very low values of σc2, citations are consistently outperformed by a hybrid indicator that gives a weight of 25% to the IF and a weight of 75% to citations. The other way around, for any value of σc2, the IF is outperformed by a hybrid indicator that gives a weight of 75% to the IF and a weight of 25% to citations. These results show that one does not necessarily need to make an absolute choice between the IF and citations. Instead, the two indicators can be combined into a hybrid indicator that is likely to be more accurate than each of the two indicators separately.\n\n\n6. Discussion and conclusion\n\nAccording to Van Raan (quoted by Van Noorden, 2010, p. 864–865), “if there is one thing every bibliometrician agrees, it is that you should never use the journal impact factor to evaluate research performance for an article or for an individual — that is a mortal sin”. As discussed in Section 2, many scientometricians indeed reject the use of the IF for assessing individual articles. Moreover, the widespread support for DORA (2013) shows that the same applies to the scientific community more generally. However, the use of the IF at the level of individual articles is often rejected based on incorrect statistical arguments. We believe it is time to develop a more nuanced perspective on the IF and on journal-level indicators more generally.\n\nAs we have shown using an illustrative example in Section 3, a conceptual discussion in Section 4, and computer simulations in Section 5, commonly used statistical arguments against the use of the IF for assessing individual articles are not convincing. This applies to arguments based on the skewness of citation distributions, and it also applies to other related types of arguments, such as the ecological fallacy argument (Leydesdorff et al., 2016; Paulus et al., 2018). Although these arguments may appear compelling at first sight, a more careful analysis reveals that the arguments do not logically lead to the conclusion that the IF should not be used at the level of individual articles. This conclusion can be reached only when additional assumptions are made, for instance the assumption that citations accurately reflect the value of an article or the assumption that journals are very heterogeneous in terms of the values of the articles they publish. Our analysis not only shows that statistical objections against the use of the IF at the level of individual articles are not convincing. It also shows that, depending on the assumptions that are made, the IF can even be a more accurate indicator of the value of an article than the number of citations of the article.\n\nOur analysis is of a theoretical nature, and it therefore does not make clear whether in practice it can be recommended to use the IF for assessing individual articles and whether in practice the IF is more or less accurate than citations. These questions require empirical follow-up research. One could for instance compare the accuracy of the IF and of citations by correlating both of them with peer review assessments of articles. Such an analysis is presented by HEFCE (2015) (for similar analyses at a smaller scale, see Allen et al., 2009; Eyre-Walker & Stoletzki, 2013). The analysis is based on the outcomes of the Research Excellence Framework in the United Kingdom. It shows that two field-normalized journal-level indicators, SNIP and SJR, and field-normalized citations all correlate more or less to the same degree with peer review assessments. However, in this analysis, peer review took place after articles had been published, and therefore peer review assessments may have been influenced by the fact that reviewers knew in which journal an article had appeared and how often an article had been cited. Ideally, when using peer review assessments to compare the accuracy of the IF and of citations, one would like the peer review assessments to be completely independent of this type of information.\n\nFollow-up research may also focus on developing more advanced simulation models for analyzing the use of the IF in research evaluations. The model presented in Section 5 is static and involves only a single time period. In a dynamic model with multiple time periods, the IF of a journal can be calculated in a more realistic way (by using appropriate publication and citation windows) and may evolve over time. Moreover, in a dynamic model, the citations of the articles published in a journal may not only determine the IF of the journal but may also be influenced by the IF of the journal in earlier time periods, creating a kind of Matthew effect of the IF (for further discussion on this possibility, see Kim et al., in press; Larivière & Gingras, 2010; Traag, 2019). A more advanced simulation model may also consider that the peer review carried out by journals takes time and that researchers may not want to risk delaying publication of their work by submitting it to a journal by which it will most likely be rejected. Hence, researchers may make their own assessment of the value of their work, and based on this they may choose a suitable journal to which they submit their work. Another idea that can be considered in a more advanced simulation model is that even within a single field of science journals may differ significantly in their topical focus. This influences how researchers choose the journal to which they submit their work. The situation becomes especially complex when some topics attract more citations than others. The IF may then create an incentive both for journals and for researchers to shift their attention to specific topics (e.g., Müller & De Rijcke, 2017). A final possibility for a more advanced simulation model is to regard the IF and citations as proxies of different aspects of the value of an article, leading to a situation in which the IF and citations may be seen as two complementary indicators that each provide useful information.\n\nImportantly, the simplicity of the simulation model presented in Section 5 does not weaken our claim that commonly used statistical arguments against the use of the IF for assessing individual articles are incorrect. We received a lot of feedback on an earlier version of this paper, which we published as a preprint in 2017 (Waltman & Traag, 2017). Many of the critical comments that we received were about unrealistic assumptions in our simulation model. We agree that the model is unrealistic in many ways, and we are open to the possibility that more realistic models may provide stronger arguments against the use of the IF at the level of individual articles. However, this does not invalidate our claim that commonly used statistical arguments against the use of the IF at the article level are incorrect. In our view, the question whether article-level use of the IF is justified or not cannot be settled based only on statistical arguments. For instance, in our simulation model, the appropriateness of article-level use of the IF depends on the assumptions that one makes, and whether certain assumptions are realistic or not is an empirical rather than a statistical matter. This illustrates that arguments against article-level use of the IF should not be based exclusively on statistical considerations. These arguments need to be supported by careful empirical analyses. The idea that the use of the IF at the article level simply reflects ‘statistical illiteracy’ (Curry, 2012) is misguided.\n\nBased on their study of the use of the IF in university medical centers in the Netherlands, Rushforth & De Rijcke (2015) state that they “feel ambivalent about statements coming from scientometricians that the JIF ‘misleads.’ By limiting indicator uses to questions of validity, movements like DORA also assume displacing the JIF for ‘better’ (i.e. more valid) indicators would necessarily give rise to better evaluation practices” (p. 136). In a similar spirit, Cronin & Sugimoto (2015) consider the use of the IF for assessing individual articles to be “as much a socio-technical as a statistical issue: growing adoption of the IF is changing scientists’ behavior and causing displacement activity”. While the use of the IF for assessing individual articles may be statistically justifiable, we acknowledge that it seems highly problematic from a socio-technical perspective. We have not considered the socio-technical perspective in this paper, but we recognize that there is ample evidence of undesirable consequences of the dominant role of the IF in research evaluations (e.g., Chorus & Waltman, 2016; Larivière & Sugimoto, 2019; Martin, 2016; Wilhite & Fong, 2012).\n\nWe are in full support of initiatives aimed at improving research evaluation, and we believe that critical discussions about the use of the IF should be an important element of such initiatives. However, it is not clear whether replacing the IF by article-level indicators will improve research evaluation. If article-level indicators become as dominant as the IF, this will probably also have undesirable consequences. The use of the IF and other journal-level indicators (Wouters et al., 2019) in research evaluations needs to be critically discussed, but the discussion should not be based on misplaced statistical arguments.\n\n\nData availability\n\nNo data is associated with this article.\n\n\nNotes\n\n1Our use of the term ‘skewness’ in this paper follows the literature discussed in Subsection 2.1. However, we note that it would actually be more appropriate to consider the variance rather than the skewness of journal citation distributions. If the citation distribution of a journal is perfectly symmetrical (and therefore completely non-skewed) but has a high variance, the IF would still not be representative of the number of citations of an individual article in the journal. Presumably, many scientometricians would then still have statistical objections against the use of the IF at the level of individual articles.\n\n2Our use of computer simulations is somewhat related to work by Kapeller & Steinerberger (2016). Kapeller and Steinerberger use computer simulations to study the journal publishing system. Their focus is on analyzing the efficiency of the system, not on analyzing the accuracy of indicators for assessing individual articles.\n\n3In reality, the way in which the IF is used for assessing individual articles is slightly different from the way in which this is done in our simulation model. In reality, when an article published in year y is assessed using the IF, the IF is calculated based on citations received by articles published in the same journal in years y – 1 and y – 2. To keep our simulation model as simple as possible, time is not taken into account in the model. Essentially, in our model, the IF is calculated based on citations received by articles published in year y rather than in years y – 1 and y – 2. Although our model provides a simplified representation of reality, this does not affect our analysis in an essential way. The key element in the discussion on the use of the IF for assessing individual articles is the skewness of citation distributions, and this skewness is properly reproduced in our model.\n\n4The findings of Radicchi et al. are criticized by Waltman et al. (2012), who show that rescaled citation distributions do not have exactly the same shape in different fields. Nevertheless, the findings of Radicchi et al. provide a reasonable approximation of the true shape of citation distributions, and we therefore use these findings to inform the choice of the values of σv2 and σc2 in our simulations.\n\n5In our simulation model, the IF and citations have the same scale (i.e., they both have an average value of 1) and therefore it makes sense to combine them in a straightforward way by calculating a weighted average. In practice, the IF and citations are likely to have different scales. This needs to be accounted for when combining them into a hybrid indicator.", "appendix": "Acknowledgements\n\nWe have received feedback on our work from a large number of colleagues. We are grateful for all feedback. 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Publisher Full Text\n\nTraag VA: Inferring the causal effect of journals on citations.2019; arXiv: 1912.08648. Reference Source\n\nVanclay JK: Impact factor: Outdated artefact or stepping-stone to journal certification? Scientometrics. 2012; 92(2): 211–238. Publisher Full Text\n\nVan Noorden R: Metrics: A profusion of measures. Nature. 2010; 465(7300): 864–866. PubMed Abstract \| Publisher Full Text\n\nVan Raan AF: In matters of quantitative studies of science the fault of theorists is offering too little and asking too much. Scientometrics. 1998; 43(1): 129–139. Publisher Full Text\n\nWaltman L: A review of the literature on citation impact indicators. J Informetr. 2016; 10(2): 365–391. Publisher Full Text\n\nWaltman L, Traag VA: Use of the journal impact factor for assessing individual articles need not be wrong.2017; arXiv: 1703.02334. Reference Source\n\nWaltman L, Van Eck NJ, Van Leeuwen TN, et al.: Some modifications to the SNIP journal impact indicator. J Informetr. 2013; 7(2): 272–285. Publisher Full Text\n\nWaltman L, Van Eck NJ, Van Raan AF: Universality of citation distributions revisited. J Am Soc Inf Sci Technol. 2012; 63(1): 72–77. Publisher Full Text\n\nWest JD, Bergstrom TC, Bergstrom CT: The Eigenfactor metricsTM: A network approach to assessing scholarly journals. College & Research Libraries. 2010; 71(3): 236–244. Publisher Full Text\n\nWilhite AW, Fong EA: Scientific publications. Coercive citation in academic publishing. Science. 2012; 335(6068): 542–543. PubMed Abstract \| Publisher Full Text\n\nWouters P, Sugimoto CR, Larivière V, et al.: Rethinking impact factors: better ways to judge a journal. Nature. 2019; 569(7758): 621–623. PubMed Abstract \| Publisher Full Text\n\nZhang L, Rousseau R, Sivertsen G: Science deserves to be judged by its contents, not by its wrapping: Revisiting Seglen's work on journal impact and research evaluation. PLoS One. 2017; 12(3): e0174205. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63446", "date": "15 May 2020", "name": "Mike Thelwall", "expertise": [ "Reviewer Expertise Scientometrics" ], "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 uses a mix of conceptual analysis, simplified examples, and simulations to argue that using journal Impact Factors (IFs) is not from a statistical perspective inferior to using citation counts for the task of identifying high-value articles. The argument revolves around the key point that neither the citation count of the article nor the IF of its journal measures the value of the article: both are (approximate) indicators of its value, however the concept of value is interpreted. Once this point is accepted, which I think it must be, then this article proves that on a purely statistical basis it is impossible to say whether citation counts or IFs are the best indicators of value for individual articles. The point that the authors make that both IFs and citations are indicators of something that we cannot measure is a key one that needs to be understood by anyone using citation-based indicators for evaluations. I suspect that this will make sense to many authors that value some journals above others but recognize that even these occasionally publish poor articles. As the authors make clear, they are not arguing in favour of or against the use of IFs for article-level evaluations, they are only showing that one of the arguments against the use of IFs is not correct. I think that this paper makes a positive contribution by shifting the debate to non-statistical issues when considering the value of IFs (but see below). I have one minor quibble: since the IF is calculated using methods that do not take into account the skewing of citation counts, I think it is reasonable to call the IF statistically illiterate, even though the authors have demonstrated that it is not statistically illiterate to use the IF for identifying high value articles.\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": "6371", "date": "01 Mar 2021", "name": "Ludo Waltman", "role": "Author Response", "response": "Thank you for reviewing our paper. Please find below a point-by-point response. This paper uses a mix of conceptual analysis, simplified examples, and simulations to argue that using journal Impact Factors (IFs) is not from a statistical perspective inferior to using citation counts for the task of identifying high-value articles. The argument revolves around the key point that neither the citation count of the article nor the IF of its journal measures the value of the article: both are (approximate) indicators of its value, however the concept of value is interpreted. Once this point is accepted, which I think it must be, then this article proves that on a purely statistical basis it is impossible to say whether citation counts or IFs are the best indicators of value for individual articles. The point that the authors make that both IFs and citations are indicators of something that we cannot measure is a key one that needs to be understood by anyone using citation-based indicators for evaluations. I suspect that this will make sense to many authors that value some journals above others but recognize that even these occasionally publish poor articles. As the authors make clear, they are not arguing in favour of or against the use of IFs for article-level evaluations, they are only showing that one of the arguments against the use of IFs is not correct. I think that this paper makes a positive contribution by shifting the debate to non-statistical issues when considering the value of IFs (but see below). I have one minor quibble: since the IF is calculated using methods that do not take into account the skewing of citation counts, I think it is reasonable to call the IF statistically illiterate, even though the authors have demonstrated that it is not statistically illiterate to use the IF for identifying high value articles. In our view, the use of a journal-level indicator, such as the IF, at the level of individual articles is not statically illiterate. We do concede that there could be reasons for questioning some of the statistical properties of the IF. To better indicate our position on this issue, we have rephrased the sentence on statistical illiteracy in the concluding section of our paper." } ] }, { "id": "63448", "date": "29 May 2020", "name": "Stephen Curry", "expertise": [ "Reviewer Expertise Structural biology", "research assessment", "research culture" ], "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 interests of full disclosure, I want to declare that I am the chair of the steering group of DORA and the author of the blogpost ‘Sick of Impact Factors’, both of which are subject to critique by the authors of this manuscript. These potential conflicts of interest should be borne in mind when reading this review.\n\nThis manuscript presents a theoretical model to contest the view that statistical considerations, primarily the inappropriateness of a single indicator – the journal impact factor (JIF) – to characterise the skewed citation distributions found in all journals, should not be grounds for denying it a role in the assessment of individual articles. The rationale for this work is presented carefully and the theoretical model that forms the centre-piece of the manuscript is clearly laid out. The authors take some pains to acknowledge the simplified nature of this exercise and the wider (and in my view more substantive) debate surrounding the problematic nature of undue reliance on journal impact factors in research assessment. On the whole, this manuscript is an interesting and thoughtful contribution to an important debate and I enjoyed reading it. However, I think there are still some serious problems, both technical and rhetorical, that the authors should address.\n\nFirst, the problem being addressed is extremely narrowly framed. To a degree, the authors have been admirably precise in outlining the very particular objection that they seek to address. This is centred on the particular question of whether the skewness of journal citation distributions means that use of the JIF or the paper citation count is a better indicator of the value of an individual article.\n\nHowever, this very narrow focus comes at the cost of discounting many additional arguments against the use of aggregate indicators like the JIF to assess individual papers. These include problems – many of which are statistical in nature – with the definitions of citable items, the JIF’s short time window, the lack of transparency of the underlying data, the possible manipulation of the JIF, and the performative impact of explicit reliance on JIFs in assessment criteria. Although these problems are discussed briefly at the beginning of section 2 of the paper, they are downplayed in the authors’ subsequent survey of the literature on statistical objections to the JIF. To take one example, the authors quote a single phrase from the DORA declaration (https://sfdora.org/read/) but make no mention of the richer argument that is presented there describing the problematic nature of the JIF. I accept that the skewness of this discussion is partially justified by the authors’ desire to focus their critique, but that focus needs to be maintained throughout. In the discussion, the authors claim that their analysis shows that “commonly used statistical arguments” against the use of the JIF are incorrect, but in fact, they have only addressed one particular statistical (or technical) argument. I think more precise phrasing is warranted. A reminder to the reader in the discussion of the unaddressed technical problems with the JIF (which are not taken into account in the model) would be also helpful.\n\nSecond, there is to my mind a weakness in the argument constructed from the simple example presented in section 3.2. This theoretical model shows (for the synthetic data relating to 200 papers presented in Tables 2 and 3) that it is possible to imagine scenarios where either reliance on the JIF or paper citation counts could give more accurate selections of groups of high-value papers. While this may be the case, this type of selection (of groups of papers) does not map onto any real-world exercises in research assessment. Further, the claim following from this argument that “the number of citations of an article is not necessarily a more accurate indicator of the value of the article than the IF of the journal in which the article has appeared,” is correct only insofar as the method allows the calculation of a probability that one or the other selection method will identify a high-value article, a probability that can only be determined for synthetic data. The probabilistic nature of the authors’ claim should be given more emphasis.\n\nThis weakness of this argument is not resolved in the more sophisticated mathematical modelling presented in section 5. Although the authors are at pains to point out the theoretical nature of their argument and the fact that it presents a simplified view of reality, as they themselves concede, there is no visible route to testing their hypothetical model with real data. As an academic exercise, there is some merit in using purely theoretical approaches to think through a problem, but ultimately theoretical models should give rise to real-world predictions or applications. It is somewhat telling that, although a previous version of this manuscript was posted to the arXiv in 2017, in the intervening three years the authors have made no further progress in testing their ideas with real data. This is likely an indication of the immense difficulty in producing accurate estimates for the key parameters of the model, sigma-r-squared (the accuracy of journal peer review) and sigma-c-squared (the accuracy of citations as an indicator of value). The manuscript discusses how “empirical follow-up research” might be conducted but the ideas presented are speculative or confine themselves to the pursuit of more sophisticated simulations. In the absence of a test on real data – or a clear pathway to such a test – the conclusions drawn must remain hypothetical and unconvincing.\n\nThere is some implicit acknowledgement of these limitations in the authors’ careful use of language, especially perhaps in the “need not” of their title and the double-negative construction in the abstract: “Our analysis show that these arguments do not support the conclusion that the impact factor should not be used for assessing individual articles.”\n\nHowever, I don’t think these go far enough. I have found myself asking whether the authors have fully considered the rhetorical impact of their title and summary, which may well be the only sections of this paper read by university research managers. Obviously, it is the responsibility of readers to read in full, but the Leiden Manifesto (https://www.nature.com/news/bibliometrics-the-leiden-manifesto-for-research-metrics-1.17351), of which Waltman is a co-author, urges us all to act responsibly in thinking about and using metrics. In the spirit of the manifesto, I would strongly suggest that the authors modify their title and abstract to make it clear that they have no way to apply the theoretical analysis presented in their paper to decisions that may impact the careers of real people and the conduct of research.\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": "6372", "date": "01 Mar 2021", "name": "Ludo Waltman", "role": "Author Response", "response": "Thank you for reviewing our paper. We appreciate your in-depth feedback. Please find below a point-by-point response. In the interests of full disclosure, I want to declare that I am the chair of the steering group of DORA and the author of the blogpost ‘Sick of Impact Factors’, both of which are subject to critique by the authors of this manuscript. These potential conflicts of interest should be borne in mind when reading this review. This manuscript presents a theoretical model to contest the view that statistical considerations, primarily the inappropriateness of a single indicator – the journal impact factor (JIF) – to characterise the skewed citation distributions found in all journals, should not be grounds for denying it a role in the assessment of individual articles. The rationale for this work is presented carefully and the theoretical model that forms the centre-piece of the manuscript is clearly laid out. The authors take some pains to acknowledge the simplified nature of this exercise and the wider (and in my view more substantive) debate surrounding the problematic nature of undue reliance on journal impact factors in research assessment. On the whole, this manuscript is an interesting and thoughtful contribution to an important debate and I enjoyed reading it. However, I think there are still some serious problems, both technical and rhetorical, that the authors should address. First, the problem being addressed is extremely narrowly framed. To a degree, the authors have been admirably precise in outlining the very particular objection that they seek to address. This is centred on the particular question of whether the skewness of journal citation distributions means that use of the JIF or the paper citation count is a better indicator of the value of an individual article. However, this very narrow focus comes at the cost of discounting many additional arguments against the use of aggregate indicators like the JIF to assess individual papers. These include problems – many of which are statistical in nature – with the definitions of citable items, the JIF’s short time window, the lack of transparency of the underlying data, the possible manipulation of the JIF, and the performative impact of explicit reliance on JIFs in assessment criteria. Although these problems are discussed briefly at the beginning of section 2 of the paper, they are downplayed in the authors’ subsequent survey of the literature on statistical objections to the JIF. To take one example, the authors quote a single phrase from the DORA declaration (https://sfdora.org/read/) but make no mention of the richer argument that is presented there describing the problematic nature of the JIF. I accept that the skewness of this discussion is partially justified by the authors’ desire to focus their critique, but that focus needs to be maintained throughout. In the discussion, the authors claim that their analysis shows that “commonly used statistical arguments” against the use of the JIF are incorrect, but in fact, they have only addressed one particular statistical (or technical) argument. I think more precise phrasing is warranted. A reminder to the reader in the discussion of the unaddressed technical problems with the JIF (which are not taken into account in the model) would be also helpful. In the context of the IF, there are two types of statistical objections that are commonly raised. First, there are statistical objections that apply to the IF in general, irrespective of the level at which the IF is used (i.e., at the journal level or at the article level). Second, there are statistical objections that are specifically about the use of the IF, and of journal-level indicators more generally, at the article level. Our paper is about the latter objections, not about the former ones. As our literature review shows, the latter objections play a prominent role in the debate about the IF, and they therefore deserve close attention. The latter objections are also more fundamental than the former ones, since the former objections could be addressed by modifying the way in which the IF is calculated, which is not possible for the latter objections. Throughout our paper, we point out that our focus is on statistical objections against the use of the IF at the level of individual articles, not on other statistical objections against the IF. In our revised paper, we have made a number of changes in the introductory and concluding sections to emphasize this point even more. In addition, as suggested by the reviewer, in our revised paper we are more explicit about the fact that there are also other statistical objections against the IF, which we do not consider in our paper. Second, there is to my mind a weakness in the argument constructed from the simple example presented in section 3.2. This theoretical model shows (for the synthetic data relating to 200 papers presented in Tables 2 and 3) that it is possible to imagine scenarios where either reliance on the JIF or paper citation counts could give more accurate selections of groups of high-value papers. While this may be the case, this type of selection (of groups of papers) does not map onto any real-world exercises in research assessment. Further, the claim following from this argument that “the number of citations of an article is not necessarily a more accurate indicator of the value of the article than the IF of the journal in which the article has appeared,” is correct only insofar as the method allows the calculation of a probability that one or the other selection method will identify a high-value article, a probability that can only be determined for synthetic data. The probabilistic nature of the authors’ claim should be given more emphasis. In our revised paper, we have added a sentence in which we explicitly indicate the probabilistic nature of our example. However, we do not see this probabilistic nature as a weakness. Indicators provide only approximate information (which is why we call them indicators rather than metrics). Sometimes the information provided by an indicator is accurate, while in other cases it is not. This applies both to journal-level indicators and to article-level indicators. The probabilistic nature of our example is a reflection of the probabilistic nature of indicators. This weakness of this argument is not resolved in the more sophisticated mathematical modelling presented in section 5. Although the authors are at pains to point out the theoretical nature of their argument and the fact that it presents a simplified view of reality, as they themselves concede, there is no visible route to testing their hypothetical model with real data. As an academic exercise, there is some merit in using purely theoretical approaches to think through a problem, but ultimately theoretical models should give rise to real-world predictions or applications. It is somewhat telling that, although a previous version of this manuscript was posted to the arXiv in 2017, in the intervening three years the authors have made no further progress in testing their ideas with real data. This is likely an indication of the immense difficulty in producing accurate estimates for the key parameters of the model, sigma-r-squared (the accuracy of journal peer review) and sigma-c-squared (the accuracy of citations as an indicator of value). The manuscript discusses how “empirical follow-up research” might be conducted but the ideas presented are speculative or confine themselves to the pursuit of more sophisticated simulations. In the absence of a test on real data – or a clear pathway to such a test – the conclusions drawn must remain hypothetical and unconvincing. We do not agree that our conclusions are unconvincing. We conclude that the use of journal-level indicators such as the IF at the level of individual articles need not be statistically wrong. This conclusion follows logically from our analysis. If the reviewer is not convinced that this conclusion is justified, the reviewer should point to a logical flaw in our analysis. Importantly, we do not conclude that journal-level indicators are preferable over article-level indicators. Our analysis does not support such a conclusion. Those who want to argue that journal-level indicators are preferable over article-level indicators indeed need to provide additional empirical evidence. Likewise, those who want to argue that article-level indicators are preferable over journal-level indicators also need to provide additional empirical evidence. The burden of proof is not on us, but on those who want to argue in favor of either journal-level or article-level indicators. Regarding the choice between the two types of indicators, we consider ourselves to be agnostic. In fact, depending on the context, we think it may not even be desirable to choose between the two types of indicators and it may in fact be better to combine the use of the different types of indicators, so that a more complete picture is obtained. There is some implicit acknowledgement of these limitations in the authors’ careful use of language, especially perhaps in the “need not” of their title and the double-negative construction in the abstract: “Our analysis show that these arguments do not support the conclusion that the impact factor should not be used for assessing individual articles.” However, I don’t think these go far enough. I have found myself asking whether the authors have fully considered the rhetorical impact of their title and summary, which may well be the only sections of this paper read by university research managers. Obviously, it is the responsibility of readers to read in full, but the Leiden Manifesto (https://www.nature.com/news/bibliometrics-the-leiden-manifesto-for-research-metrics-1.17351), of which Waltman is a co-author, urges us all to act responsibly in thinking about and using metrics. In the spirit of the manifesto, I would strongly suggest that the authors modify their title and abstract to make it clear that they have no way to apply the theoretical analysis presented in their paper to decisions that may impact the careers of real people and the conduct of research. We do not agree with the reviewer that we “have no way to apply the theoretical analysis presented in (our) paper to decisions that may impact the careers of real people and the conduct of research”. Our analysis shows that article-level indicators are not necessarily preferable over journal-level indicators, and this point has important real-world consequences. Recent developments at the Medical Faculty of the University of Bern illustrate this. After signing DORA, this institution decided to replace the IF by the relative citation ratio (RCR; Steck, Stalder, & Egger, 2020) to support decision making about promotions. The IF is a relatively transparent indicator, enabling anyone who uses the indicator to reflect on its pros and cons and to discuss what kinds of conclusions can and cannot be drawn from the indicator. The RCR is a highly complex black-box indicator, making it much more difficult for the typical user of the indicator to reflect on its pros and cons and to discuss how the indicator can best be interpreted. In addition, just like the IF calculation, the calculation of the RCR has been subject to substantial criticism. From the viewpoint of responsible research assessment, we believe that the use of a relatively transparent journal-level indicator may be preferable over the use of a non-transparent article-level indicator. This example, which we briefly discuss in footnote 7 in our revised paper, illustrates the danger of dogmatically insisting that article-level indicators are preferable over journal-level indicators. Despite our disagreement with the reviewer, we have decided to make a (small) change to the title of our paper. The title has been changed from ‘Use of the journal impact factor for assessing individual articles need not be statistically wrong’ to ‘Use of the journal impact factor for assessing individual articles: Statistically flawed or not?’. This will hopefully stimulate readers to carefully reflect on the use of the IF for assessing individual articles and to neither reject nor accept this practice in an uncritical manner, but to engage in a more meaningful discussion on pros and cons of the use of journal-level indicators. We have also made some (small) changes to the abstract of our paper." } ] } ]	1	https://f1000research.com/articles/9-366
https://f1000research.com/articles/9-780/v1	28 Jul 20	{ "type": "Brief Report", "title": "Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes", "authors": [ "Andrew W. Liu", "Yongkai Tan", "Aki Masunaga", "Charles Plessy", "Nicholas M. Luscombe", "Aki Masunaga", "Charles Plessy", "Nicholas M. Luscombe" ], "abstract": "Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.", "keywords": [ "karyotype", "chromosome", "centromere", "histone H3", "Oikopleura", "oocyte", "embryo", "H3S28P" ], "content": "Introduction\n\nKaryotyping is a long-established histochemical method to visualize chromosomes of eukaryotes (Hsu & Benirschke, 1967; Tjio & Levan, 1950). A multi-dye reagent developed at the turn of the 20th century for the diagnosis of infections in human histological preparations (Giemsa, 1902; Giemsa, 1904) was later used to stain chromosomes themselves in order to study their numbers, translocations, and other aberrations. This rapid technique, involving the use of stains including methylene blue, eosin, and azure B allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis.\n\nAlthough individual chromosomes have been resolved by histochemical techniques in O. dioica, the reported results differ in numbers from n=3 (Körner, 1952) to n=8 (Colombera & Fernaux, 1973). More recently, metaphase-specific histone 3 (H3) markers have been used to determine the structure and the segregation of genetic material during oogenesis in situ (Ganot et al., 2006; Schulmeister et al., 2007) while providing greater detail and resolution. One such marker is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003); although it is typically used to identify centromeres during metaphase (Kurihara et al., 2006), we observed in data presented in previous studies that signals were not confined to centromeres. More importantly, the localization of the H3S28P signal depends on the phase of the cell cycle: spatially punctate signals were found evenly spread within the nuclear envelope during prophase, while condensed chromatin gave an outlined staining of the sister chromatids during metaphase in a manner consistent with alignment along the metaphase plate (Table 1; Campsteijn et al., 2012; Feng & Thompson, 2018; Feng et al., 2019; Olsen et al., 2018). Moreover, a structure in which genetic material is sequestered in a ∏-shaped conformation has been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008). However, these results were all obtained from the same laboratory strain originating from the Atlantic Ocean. Considering the discrepancy of past findings, and the fact that our laboratory strain originates from a geographically distinct ocean, we applied H3S28P staining on intact embryos and oocytes to confirm the chromosome count and validate our genome sequencing assemblies of Okinawan O. dioica marine populations among the Ryukyu Islands of southern Japan.\n\n\nMethods\n\nHistochemical staining. Live specimens were collected from Ishikawa Harbor (26 °25'39.3 \"N, 127 °49'56.6 \"E) by a hand-held plankton net and cultured in the lab (Masunaga et al., 2020). Mature females were collected prior to spawning, individually washed with filtered autoclaved seawater (FASW) 3 times for 10 minutes and placed in separate 1.5 ml tubes containing 500 µl of FASW. Nearly mature males, full of sperm, were also washed 3 times in FASW. Mature males that successfully made it through the washes intact were placed in 100 µl of fresh FASW and allowed to spawn naturally. As soon as females spawned, each individual clutch of 100-200 eggs was washed three times for 10 minutes by moving eggs along with a pulled capillary micropipette from well to well in a 6-well dish, each containing 5 ml of FASW, and left in a fresh well of 5 ml FASW in the same dish. These were stored at 17 °C and set aside for fertilization. Staged embryos were initiated by gently mixing 10 µl of the spawned male sperm with the awaiting eggs in FASW at 23 °C. Developing embryos were staged and collected by observation under a Leica M165C dissecting microscope. These embryos were quickly dechorionated using 0.1% sodium thioglycolate and 0.01% actinase in FASW for 2–3 minutes, then promptly washed with 2 washes with filtered autoclaved seawater prior to fixation and staining. Unfertilized eggs were treated similarly.\n\nEmbryos were Giemsa stained as previously described in Shoguchi et al., 2005. Briefly, approximately 20–30 dechorionated embryos were treated with 0.04% colchicine in FASW for 30 minutes and then treated with decreasing amounts of KCl (50 mM and 25 mM) for five minutes each. Fixation was quickly performed with cold methanol:glacial acetic acid (3:1). The fixative was changed three times in the span of 18 hours while at -30 °C. The next morning, the fixed cells were quickly resuspended in 60% Acetic acid and methodically dropped from a height of 7 – 8cm onto a 48°C pre-warmed slide (Matsunami Glass, S2441). The slide was incubated for an additional 2 hours at 48°C; then stained with 6% Gimesa in 67mM sodium phosphate pH 7.0 for 2 hours at room temperature and rinsed with ddH2O. These were dried for two hours at room temperature, mounted with DPX Mountant (Sigma, 06522) and covered with No.1 35 x 50 mm glass coverslips (Matsunami Glass, C035551).\n\nImmunostaining. Washed eggs and embryos were immediately fixed in 4% w/v paraformaldehyde, 100 mM MOPS pH 7.5, 0.5 M NaCl, 0.1% triton-X100 at 23 °C ON (Campsteijn et al., 2012). The samples were then washed for 10 minutes once with PBSTE (PBS supplemented with 1 mM EDTA) and 3 times for 10 min with PBSTEG (PBS supplemented with 1 mM EDTA and 0.1 M glycine). The samples were blocked using PBSTE supplemented with 3% bovine serum albumin at 4 °C overnight. Rabbit polyclonal (Thermo Fisher Scientific Cat# 720099, RRID:AB_2532807) or rat monoclonal (Abcam Cat# ab10543, RRID:AB_2295065) primaries directed against H3S28P were diluted 1:100 in PBSTE 3% BSA and incubated at 4 °C for 3 days. The next morning, these were washed in PBSTE for 10 minutes 3 times and incubated with anti-rabbit (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217) or anti-rat (Molecular Probes Cat# A-11006, RRID:AB_141373) Alexa488 conjugated secondary antibodies diluted 1:500 with PBSTE 3% BSA at 4 °C ON. The following morning, samples were washed 3 times for 10 min with PBSTE. The samples were mounted on cleaned glass slides (Matsunami Glass, S2441) with fluorescence preserving mounting medium (ProLong. Fluoromount G Mounting Medium, RRID:SCR_015961) covered with No.1 35 x 50 mm glass coverslips (Matsunami Glass, C035551) and sealed with nail polish.\n\nBoth a Nikon Ni-E epifluorescent and a Zeiss LSM 510 Meta confocal microscopes were used to acquire Z-stack images of eggs and embryos. Brightfield images were obtained using a 20x/0.75 CFI Plan Apo λ objective (Nikon, MRD00205) for histochemical staining. Epifluorescent immunofluorescent images were obtained with both 20x/0.75 and 40x/0.95 CFI Plan Apo λ air objectives (Nikon, MRD00405); each sample acquisition was Z-stacked with each plane set at an interval of 1 µm. Confocal images were acquired using a 40x/0.75 EC Plan-Neofluar M27 (Zeiss, 420360-9900-000) and 63x/1.4 Plan-Apochromat M27 oil immersion (Zeiss, 420782-9900-79) objectives; each sample acquisition was Z-stacked, line averaged twice with each plane set at an interval of 0.6 and 0.27 µm, respectively.\n\nImages acquired from a Nikon Ni-E epifluorescent were deconvoluted with Nikon Elements-AR v5.0 software. Images for both epifluorescent and confocal acquisitions were analyzed using Imaris software SPOT DETECTION tool (Imaris, RRID:SCR_007370) for embryos and unfertilized eggs, parameters set at 0.5 and 0.43 µm spot detection size, respectively, and software preset to QUALITY auto signal threshold for each individual cell within a sample. Alternatively, ImageJ v1.51 3D Objects Counter may be employed to count signals. Epifluorescent and confocal acquisitions of embryos and their subsequent analysis were performed independently by different researchers to exclude bias.\n\nConfidence intervals were calculated with Prism 8 (GraphPad) and histograms plotted with R (v3.6.3).\n\n\nResults\n\nInitial attempts at visualizing individual chromosomes were done with developing embryos and Giemsa staining. The spreads from two time points, 32- and 64-cell developmental stages, gave results with counts ranging between 11–27 stains per cell (BioImage Archive, S-BIAD21, Experiment A). Although hypotonic-induced cell spreads were confined as a result of incomplete dechorionation and digestion with the enzymatic dissociation cocktail, groups of chromosomes were easily associated to a single cell. However, individual chromosomes were difficult to resolve due to the low resolution of images. In order to eliminate possible miscounts and other Giemsa staining artifacts, immunostaining was used to count individual chromosomes using a centromere-specific primary antibody directed against H3S28P and a secondary antibody conjugated to Alexa488 directed against the primary antibody.\n\nSignal-based thresholding was employed to determine the number of distinct 515 nm emission signals present in acquired images from epifluorescent and laser confocal microscopes (BioImage Archive, S-BIAD21, Experiment B & D). The data was analyzed using the Imaris SPOT DETECTION tool (Oxford Instruments). Two types of nuclei were apparent within each embryo: nuclei containing evenly distributed, clearly separated spots that were interpreted as being in prophase (Figure 1A and 1B, blue circles) and nuclei with intense clusters of signals in the center, considered to be in metaphase (Figure 1A and 1B, red squares). Counts from these two classes of nuclei fall into separate distributions (Figure 1C and 1D). Both epifluorescent and confocal acquisitions were in near agreement, epifluorescence n = 20, mean 6.2 , 95% CI 5.6 – 6.8; confocal n = 13, mean 6.4, 95% CI 5.7 – 7.1 and epifluorescence n = 20, mean 12, 95% CI 11.0 – 13.0; confocal, n = 14, mean 14.1, 95% CI 12.9 – 15.3. We interpret the results as a count of 12 distinct centromeres in prophase cells and a count of 6 larger spots identifying pairs of centromeres in metaphase (Figure 1B).\n\nAnti-H3S28P rabbit-derived polyclonal stained 64-cell whole-embryo chromosomal imaging data analyzed by Imaris software SPOT DETECTION tool using different microscopy techniques. A Maximum projection of confocal image of an embryo demonstrating the differences in signal localization appearance and signal count, which was inferred to represent distinct cell cycle phases. Red box, metaphase; blue circle, prophase. B Schematic interpretation of signals with respect to chromatin structure during prophase and metaphase cell cycle states. As a simplification, all chromosomes have been drawn at an equal length although they actually vary in O. dioica. C Distribution of signal counts within individual cells using epifluorescent (n = 40) and D confocal (n = 27) microscopes. Two distinct populations were observed in a bimodal distribution, which corresponded with cell cycle stage. Red, metaphase; blue, prophase.\n\nTo confirm our observations on germ cells and therefore rule out polyploidy, which is frequent in O. dioica’s somatic cells (Ganot & Thompson, 2002), we also analyzed oocytes in prometaphase I before fertilization (Schulmeister et al., 2007). We identified confined groupings of signals in unfertilized eggs (Figure 2A; BioImage Archive, S-BIAD21, Experiment E). Images were analyzed using the Imaris SPOT DETECTION tool to determine chromosome counts and their distributions (Figure 2B). Counts from the compact rosette-shaped genetic material averaged near 6 (n = 23, mean 5.70, 95% CI 5.2 – 6.2). Visual inspection of individual Z-sections (Figure 2C) confirm agreement with the Imaris count analysis and annotation (Figure 2D). We interpret these results as each spot corresponding to a pair of centromeres from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).\n\nA Maximum signal projection of a representative confocal Z-stack acquisition of anti-H3S28P rat monoclonal stained oocyte used for the count analysis. B Distribution of signal counts from centromere-stained oocyte genetic material, analyzed by Imaris software SPOT DETECTION tool (n = 23). C Individual Z-sections from same image acquisition showing the 3D structure of the genetic material, each plane is 0.54 µm apart. D Imaris spot analysis and annotation of signal positions from Z-stack acquisition. E Schematic representation of our interpretation that each signal is a pair of closely associated centromeres from a pair of sister chromatids. As a simplification, all chromosomes have been drawn at an equal length although they actually vary in O. dioica.\n\n\nDiscussion\n\nDespite the variation in signal counts across different image acquisitions settings, a haploid chromosomal count of three provides the most parsimonious explanation of the collected data and agrees with previously published assemblies (Denoeud et al., 2010).\n\nOocyte staining with rat anti-H3S28P and a conjugated secondary fluorophore gave rise to a compact area in which signals appear to stack on top of one another (Figure 2A). Previously, DNA stains at this stage have been interpreted as a structure resembling the Greek character ∏ (Ganot et al., 2007), representing condensed chromosomes seen in mature oocytes arrested in meiosis I. Our data does not include DNA stains and therefore our illustration (Figure 2E) should not be interpreted as precluding the previously reported ∏-structure.\n\nCurrently, the sequence of the centromeres is not known, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information. However, our whole embryo staining data and the previous literature (Table 1) show that non-centromeric signal present outside metaphase stages may introduce noise. Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable. Availability of centromeric sequences would open the possibility of confirming our results with fluorescence in situ hybridization.\n\nIn summary, we conclude that the Okinawan Oikopleura dioica genome consists of three pairs of chromosomes in diploid cells. We believe that the images may be useful for examining cell cycle specific changes to chromosome structure and encourage the reuse and reanalysis of our data located in the EBI BioImage Archive (Ellenberg et al., 2018).\n\n\nData availability\n\nImage acquisitions: Image data are available in the BioImage Archive\n\nCentromere specific antibody mediated karyotyping of Okinawan Oikopleura dioica. Accession number S-BIAD21. https://www.ebi.ac.uk/biostudies/preview/studies/S-BIAD21", "appendix": "Acknowledgements\n\nWe thank Drs. Daniel Chourrout, Hiroki Nishida & Eiichi Shoguchi for discussions and suggestions regarding the subject matter. Additionally, great appreciation is given the staff (Drs. Toshiaki Mochizuki, Shinya Komoro & Paolo Barzaghi) in the Imaging Section of the Research Support Division at OIST for providing technical assistance. Finally, we are grateful for Dr. Michael Mansfield and Charlotte West comments on the manuscript’s draft and appearance.\n\n\nReferences\n\nCampsteijn C, Øvrebø JI, Karlsen BO: Expansion of Cyclin D and CDK1 Paralogs in Oikopleura dioica, a Chordate Employing Diverse Cell Cycle Variants. Mol Biol Evol. 2012; 29(2): 487–502. PubMed Abstract \| Publisher Full Text\n\nColombera D, Fenaux R: Chromosome form and number in the larvacea. Boll Zoll. 1973; 40: 347–353. Reference Source\n\nDenoeud F, Henriet S, Mungpakdee S, et al.: Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate. Science. 2010; 330(6009): 1381–1385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEllenberg J, Swedlow JR, Barlow M, et al.: A call for public archives for biological image data. Nat Methods. 2018; 15(11): 849–854. 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Centralblatt für Bakteriologie. 1904; 32: 308–311. Reference Source\n\nHsu TC, Benirschke K: An atlas of mammalian chromosomes. Springer Verlag. 1967; 10. Publisher Full Text\n\nKawajiri A, Yasui Y, Goto H, et al.: Functional Significance of the Specific Sites Phosphorylated in Desmin at Cleavage Furrow: Aurora-B May Phosphorylate and Regulate Type III Intermediate Filaments during Cytokinesis Coordinatedly with Rho-kinase. Mol Biol Cell. 2003; 14(4): 1489–1500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurihara D, Matsunaga S, Kawabe A, et al.: Aurora kinase is required for chromosome segregation in tobacco BY-2 cells. Plant J. 2006; 48(4): 572–580. PubMed Abstract \| Publisher Full Text\n\nKörner WF: Untersuchungen über die gehäusebildung bei appendicularien (Oikopleura dioica FOL). Z Morph u Ökol Tiere. 1952; 41(1): 1–53. 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Publisher Full Text" }	[ { "id": "68260", "date": "05 Aug 2020", "name": "Haiyang Feng", "expertise": [ "Reviewer Expertise cell cycle", "oogenesis" ], "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’s interesting to know, though not surprising, that Japanese O. dioica has the same number of centromeres and chromosomes as that in Norwegian species. This piece of work can boost broad interests in using O. dioica as a new model in epigenetics and cell cycle studies. However, some results are a bit confusing to me and may be misinterpreted. In Fig 1, centromere counts at prophase are 12, and at metaphase are 6, which are inconsistent. H3S28p signals locate at inner centromeric regions, flanked by CenpA signals that mark kinetochores at metaphase in embryonic mitosis in Norwegian O. dioica. The counts of H3S28p signals should be the same at prophase and metaphase, which are 6. In addition, centromere is a piece of DNA sequence that holds a pair of sister chromatids in mitotic phase before they separate at anaphase. We can say that a chromosome has one centromere and a pair of sister chromatids at prophase. Thus, the schema representing prophase in Fig 1B should be a pair of sister chromatids is linked by one red dot at centromere. H3S28p signals in female meiosis of Norwegian O. dioica are a bit different from those in mitosis. It localizes on entire chromosomes in prophase, moves towards centromeric regions during prometaphase, and is enriched at centromeric regions (or accurately speaking, midline of a bivalent) at metaphase I. Since chromosomes are more condensed in meiosis, and the midline of a bivalent should be crossover site between homologous chromosomes, we don’t know how far away it is between centromere and crossover in meiotic chromosomes of O. dioica, and how many crossovers a bivalent has. I would say centromeric region of H3S28p signals in female meiosis with caution. Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 cell cycle. The stages of meiosis depend on when the oocytes are collected. Just after spawning, the oocytes are before prometaphase I. Within 10 to 15 min after spawning, it is prometaphase I. Later, it should be at metaphase I. The timing of sampling is not indicated, which makes it even harder to interpret the data. But again, it should one red dot between a pair of sister chromatids in Fig 2E.\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? I 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": "6318", "date": "01 Mar 2021", "name": "Andrew W Liu", "role": "Author Response", "response": "We thank Dr Feng’s helpful feedback on our manuscript. We have done our best to address all the comments, which are listed below. An aspect of the data that puzzles us is the evenly distributed, clearly separated H3S28P signal observed in non-mitotic cells, which we incorrectly referred to as prophase in the previous version of the manuscript (please see responses 2.1 and 3.1). We have consulted cell cycle experts who were unable to explain the results. Given Dr Feng’s extensive experience with H3S28P staining, we would like to ask if these patterns have been observed in his laboratory? We’d be grateful to organize a videoconference with Dr Feng to discuss the data archived at the EBI BioArchive to share our observations in detail. _____________________ Reviewer 1 comment 1.1 – Comparison of chromosome numbers between Japanese and Norwegian O. dioica Reviewer comment It’s interesting to know, though not surprising, that Japanese O. dioica has the same number of centromeres and chromosomes as that in Norwegian species. Author response We thank the reviewer for this comment. 1. After submission of this manuscript to F1000Research, two additional O. dioica genomes were published for (i) samples acquired in mainland Japan and (i) an individual from the Okinawa coastline. Preliminary comparison of the three O. dioica genomes have revealed very divergent sequences at single nucleotide and kilo/megabase scales (unpublished results). Given that mainland and Okinawan O. dioica are both “Japanese”, we avoid the term “Japanese O. dioica” in the present manuscript. 2. Historical studies reported between 3 and 8 chromosomes for O. dioica. 3. For these reasons, it was not obvious to us that the Okinawan O. dioica would have the same number of chromosomes as the Norwegian and mainland Japanese O. dioica. 4. Therefore, we wished to confirm independently the chromosome number of the Okinawan O. dioica. Manuscript changes We rewrote the Abstract and Introduction to strengthen the justification for this study. Abstract. Oikopleura dioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome. It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems. The genome of north Atlantic O. dioica comprises three chromosomes. However, comparisons with the genomes of O. dioica sampled from mainland and southern Japan revealed extensive sequence differences. Moreover, historical studies have reported widely varying chromosome counts. We recently initiated a project to study the genomes of O. dioicaindividuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan. Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145). The data lead us to conclude that diploid cells from Okinawan O. dioicacontain three pairs of chromosomes, in line with the north Atlantic populations. The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and give insight into the genomic diversity of O. dioica from different geographical locations. The data deposited in the EBI BioImage Archive provide representative images of the antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy. Introduction (paragraphs 1-3, complete). The larvacean, Oikopleura dioica, possesses a fascinating genome: it has reduced to a mere 70Mbp and exhibits unique characteristics such as non-canonical splicing and the scattering of Hox genes (Seo et al., 2001; Edvardsen et al., 2005; Marz et al., 2008; Denoeud et al., 2010). It is thought that a combination of large effective population size and high mutation rate per generation have led to fast evolution (Berná et al., 2014). The recently published genome sequence of a “Japanese O. dioica” from mainland Japan highlighted large sequence variations between the Pacific and Atlantic populations (Wang et al., 2020). In addition, we recently released a telomere-to-telomere genome sequence of an O. dioica individual collected from the Okinawan coastline in southern Japan (Bliznina et al., 2020), which, to our surprise, revealed large differences in synteny to the mainland Japanese genome despite the geographical proximity. The genetic map of the north Atlantic O. dioica is reported to contain three chromosomes (two autosomes, X and Y sex chromosomes; Denoeud et al., 2010); however, prior studies based on histochemical techniques reported three (Körner, 1952) and eight chromosomes (Colombera & Fernaux, 1973). Given the large sequence and synteny differences between the assembled O. dioicagenomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population. Karyotyping is a long-established histochemical method to visualize eukaryotic chromosomes (Hsu & Benirschke, 1967; Tjio & Levan, 1950). This rapid technique, involving the use of stains including methylene blue, eosin, and azure B, allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis. However, we were unable to determine an accurate count for the Okinawan O. dioica by this method due to variability which ranged from 11-27 chromosomes per nucleus. As an alternative approach, we decided to immunostain the centromere as a means of quantifying the number of chromosomes. Metaphase-specific histone 3 (H3) markers have been used to determine the structure and the segregation of genetic material during oogenesis in situ (Ganot et al., 2006; Schulmeister et al., 2007). One such marker that has been successfully visualized in O. dioica is histone H3 phosphorylated at Ser-28 (Kawajiri et al., 2003; Kurihara et al., 2006), whose localization depends on the phase of the cell cycle: during metaphase, sister chromatids were stained in a manner consistent with alignment along the metaphase plate, whereas in non-mitotic cells, spatially punctate signals were found evenly spread within the nuclear envelope (Campsteijn et al., 2012; Feng & Thompson, 2018; Feng et al., 2019; Olsen et al., 2018). A structure in which chromosomes are sequestered in a ∏-shaped conformation has also been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al., 2008). In Table 1, we list the publications in which the H3S28P marker was applied to O. dioica: the studies were all performed using cultured strains originating from the north Atlantic Ocean. Here, we visualized anti-H3S28P stained embryos from two commercially available antibody sources and unfertilized oocytes to determine the chromosome count of the local Okinawan O. dioica. _____________________ Reviewer 1 comment 1.2 – Misinterpretation of data Reviewer comment This piece of work can boost broad interests in using O. dioica as a new model in epigenetics and cell cycle studies. However, some results are a bit confusing to me and may be misinterpreted. Author response We thank the referee for the detailed comments below. We agree that we misinterpreted some of the results and we have now revised the manuscript to correct this. Manuscript changes Specific instances of misinterpretations (response to comments 2.1 & 3.1) and changes in schematics have been addressed below (response to comments 2.2 & 3.3). Clarification of timing of oocyte collection has explained in more detail (response to comment 3.2). _____________________ Reviewer 1 comment 2.1 – Cell cycle state of cells containing 12 spots Reviewer comment In Fig 1, centromere counts at prophase are 12, and at metaphase are 6, which are inconsistent. H3S28p signals locate at inner centromeric regions, flanked by CenpA signals that mark kinetochores at metaphase in embryonic mitosis in Norwegian O. dioica. The counts of H3S28p signals should be the same at prophase and metaphase, which are 6. Author response We thank the reviewer for this comment. We agree that the cells containing ~12 spots cannot be in prophase. In fact, we cannot explain the cell cycle state of these cells, so we now refer to them as “non-mitotic”. Manuscript changes Paragraph 12. “Cells were manually classified into two types depending on the staining pattern visible in the nucleus: (i) those with intense clusters of signals in the center, considered to be in metaphase and (ii) those containing evenly distributed, clearly separated spots within a faint background of signal defining a region encompassed by the nuclear envelope, interpreted as non-mitotic (Figure 1A and 1B, blue circles; Figure 1A and 1B, red squares). Counts from these two classes of nuclei fall into separate distributions (Figure 1C and 1D), with both epifluorescence and confocal acquisitions in agreement with each other. We interpreted the nuclei with an average of six large, clustered signals as centromeric regions in metaphase (Figure 1B), however, we cannot explain the cell cycle state of those containing the average of 12 spatially distinct punctate signals.” _____________________ Reviewer 1 comment 2.2 – Schematic representation of chromosomes in embryos in Figure 1B Reviewer comment In addition, centromere is a piece of DNA sequence that holds a pair of sister chromatids in mitotic phase before they separate at anaphase. We can say that a chromosome has one centromere and a pair of sister chromatids at prophase. Thus, the schema representing prophase in Fig 1B should be a pair of sister chromatids is linked by one red dot at centromere. Author response We thank the referee for this comment. We have corrected our use of “centromere” and redrawn Figure 1B. Manuscript changes 1. We have changed all instances of “a pair of centromeres” to “centromere”. 2. Figure 1B. We have corrected the schematic representation of metaphase and non-mitotic nuclei in Figure 1B. 3. We have updated our manuscript to replace “centromere” with “centromeric region” when referring to the DNA sequence regardless of the state of assembly of the centromere, and removed mentions of “a pair of” from the remaining occurences of “centromere”. _____________________ Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations Reviewer comment H3S28P signals in female meiosis of Norwegian O. dioica are a bit different from those in mitosis. It localizes on entire chromosomes in prophase, moves towards centromeric regions during prometaphase, and is enriched at centromeric regions (or accurately speaking, midline of a bivalent) at metaphase I. Since chromosomes are more condensed in meiosis, and the midline of a bivalent should be crossover site between homologous chromosomes, we don’t know how far away it is between centromere and crossover in meiotic chromosomes of O. dioica, and how many crossovers a bivalent has. I would say centromeric region of H3S28p signals in female meiosis with caution. Author response We thank the referee for this comment. To make a clearer distinction between observation and interpretation, we now refer to the spots in the imaging data as “H3S28P signal” and only equate them to the centromeric region in specific instances. We have also included caveats to the interpretation of the oocyte data in the discussion section. Manuscript changes 1. We now refer to the image spots as “H3S28P” signal, and only equate them to centromere in specific, appropriate contexts. 2. Paragraph 12. “.... We interpreted the nuclei with an average of six large, clustered signals as centromeric regions in metaphase (Figure 1B), however, we cannot explain the cell cycle state of those containing the average of 12 spatially distinct punctate signals. ” 3. Paragraph 13. “.... We interpreted each spot as representing a centromere from paired chromatids forming a synapsis in unfertilized eggs (Figure 2E).” 4. Updated Paragraph 16-17. Paragraph 16. “An important consideration is what the H3S28P signal represents. It has been used to visualize centromeric regions in O. dioica (Table 1), but the signal is not confined to the centromere and its localization depends on the cellular state (Figure 1; Hake  et al., 2005; Feng and Thompson, 2018). However, we are confident that the signals seen in Figure 1 labelled as metaphase and Figure 2 represent centromeres and their associated chromosome. Further, DNA-staining images of mature oocyte have previously been interpreted as chromosomes condensed in a structure resembling the Greek character ∏ (Ganot et al., 2007). Since we did not perform DNA stains, our interpretation of the H3S28P signal in the oocyte does not preclude the previously reported ∏-structure. Additionally, the positions and numbers of crossovers between homologous pairs are unresolvable in this highly condensed state and the signal positions are not definitive of centromeric-regions.” Paragraph17. “Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information. However, our whole embryo staining data (Figure 1) and the previous literature (Table 1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis. Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable. Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situhybridization.” 5. Figure 2 legend, last sentence. “… The positions of centromeric regions cannot be determined as chiasmata(s) are present along the homologous pairs of chromosomes in a highly condensed state.” _____________________ Reviewer 1 comment 3.2 – Timing of oocyte collection Reviewer comment Actually, H3S28p shows several spots (more than 6) during prometaphase I, as can be seen Fig S4 in Feng and Thompson, 2018 Cell Cycle publication. The stages of meiosis depend on when the oocytes are collected. Just after spawning, the oocytes are before prometaphase I. Within 10 to 15 min after spawning, it is prometaphase I. Later, it should be at metaphase I. The timing of sampling is not indicated, which makes it even harder to interpret the data. Author response We thank the referee for this comment. The process of rinsing the eggs took more than 15 min and so the oocytes were metaphase I. Changes were made in the methods section. Manuscript changes Paragraph 4. “Unfertilized eggs were treated similarly with three successive 10-minute washes.” Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…” _____________________ Reviewer 1 comment 3.3 – Schematic representation of chromosomes in embryos in Figure 2E Reviewer comment But again, it should one red dot between a pair of sister chromatids in Fig 2E. Author response We thank the referee for this comment and we have corrected Figure 2E. Manuscript changes 1. Figure 2E. Schematic corrected so there is one red spot between each pair of sister chromatid." } ] }, { "id": "68262", "date": "12 Aug 2020", "name": "Shigeki Fujiwara", "expertise": [ "Reviewer Expertise Tunicate embryogenesis", "asexual reproduction", "and evolutionary developmental biology. Transcriptional regulation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a new method for karyotyping using the antibody raised against Ser28-phosphorylated Histone H3 (H3S28P). Using this method, the authors obtained the results suggesting that Okinawan Oikopleura dioica somatic cells contain three sets of chromosomes. Specific detection of O. dioica’s phosphorylated H3 by the antibody has been proven in other papers, shown in Table 1. The data presented in this article are therefore reliable, and the conclusion seems appropriate. However, after I read to the end of the article, I did not really understand what the main aim and novelty of this article were. Which is the main aim, development of a new karyotyping method or determination of the number of chromosomes in diploid O. dioica somatic cells? Although the article type is “BRIEF REPORT”, clearer statements and more detailed explanations are required. I hope that the following comments are useful for the authors. All of my comments are for presentation and description.\n\nMajor concerns:\nThe Introduction section starts with the history of karyotyping. This implies that the development of a new karyotyping technique appears to be the main aim of this study. The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres). Shoguchi et al. (2005) (cited in this article) clearly showed 14 pairs of chromosomes of the Ciona intestinalis genome by means of Giemsa staining and FISH. While the size of the genome in O. dioica is a half of that in C. intestinalis, the number of chromosomes in O. dioica is about one-fifth of that in C. intestinalis. Therefore, readers may feel that the average size of the O. dioica chromosomes is large enough to be examined by the standard methods. If the development of the new method is really the main aim of this study, I would like the authors to describe merits of this new method in further detail. Without sufficiently convincing explanations, the authors’ method appears to be a less sophisticated alternative to the standard karyotyping methods. Particularly, discussion is required for the observation of seven or eight signals within a single nucleus. It will help if the authors explain why the standard methods are not applicable to O. dioica.\n\nIf the authors’ main aim is to determine the number of chromosomes in Okinawan O. dioica, they should explain more about particularity of this species. Is there a hypothesis that Pacific and Atlantic O. dioica are different species? If not, is there the possibility that different populations (Pacific and Atlantic) have different numbers of chromosomes within the same species? The number of chromosomes is highly variable even between closely related species. However, to my knowledge, the number of chromosomes is essentially invariant within a species. Uncommon exceptions are chromosome reorganization in Ascaris embryos and Paramecium macronuclei. Although the authors discuss the discrepancy in the number of the O. dioica chromosomes (n = 3, or n = 8), I felt that the argument has already been settled (on n = 3) by the extensive genome sequencing (Denoeud et al., 2010). If the authors want to insist that the number of chromosomes in Pacific O. dioica may not be three, more detailed biological information (rationale) is necessary.\n\nMinor points:\nIn Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”.\n\nI guess that “ddH2O” (page 4 line 17) is double-distilled H2O. Anyway, “ddH2O” is a laboratory-specific jargon. Similarly, I guess that “ON” (page 4 line 23) means “overnight”? These abbreviations cannot be recommended to be used in articles.\n\nSince the authors have knowledge that some somatic cells are polyploid in O. dioica. Therefore, they had better clearly state that the cells shown in Figure 1 are not the case. Although the authors state that 32~64-cell embryos were used for Giemsa staining, they did not tell the developmental stages they used for the antibody staining (in the second paragraph of the Results section). Are they also the early embryos? And do they consist exclusively of diploid cells?\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? I 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": "6319", "date": "01 Mar 2021", "name": "Andrew W Liu", "role": "Author Response", "response": "We thank Dr Fujiwara’s helpful feedback and critique on our manuscript. We have done our best to address all the concerns and minor points he has brought to our attention, which are listed below. Reviewer 2 synopsis Reviewer comment This manuscript describes a new method for karyotyping using the antibody raised against Ser28-phosphorylated Histone H3 (H3S28P). Using this method, the authors obtained the results suggesting that Okinawan Oikopleura dioica somatic cells contain three sets of chromosomes. Specific detection of O. dioica’s phosphorylated H3 by the antibody has been proven in other papers, shown in Table 1. The data presented in this article are therefore reliable, and the conclusion seems appropriate. However, after I read to the end of the article, I did not really understand what the main aim and novelty of this article were. Which is the main aim, development of a new karyotyping method or determination of the number of chromosomes in diploid O. dioica somatic cells? Although the article type is “BRIEF REPORT”, clearer statements and more detailed explanations are required. I hope that the following comments are useful for the authors. All of my comments are for presentation and description. Author response We thank the referee for the feedback, which have helped improve the clarity and quality of the manuscript. To clarify, the aim of this paper is to determine the number of chromosomes for the Okinawan O. dioica genome. We have detailed the reasons for this in our response to Reviewer Comment 1.1 above. Manuscript changes We clarified the main aim of the paper and strengthened the justification for this in the Abstract and Introduction (please see authors response to Reviewer Comment 1.1). _____________________ Major concerns Reviewer 2 comment 1.1 – Clarification of study aim Reviewer comment The Introduction section starts with the history of karyotyping. This implies that the development of a new karyotyping technique appears to be the main aim of this study. Author response We thank the referee for this comment. We have now substantially revised the Introduction to clarify that the main aim of the study is to determine the chromosome count. We have retained the description of the histochemical and immunostaining methods as two contrasting approaches, in order to explain why we chose the latter approach here; however, we hope that it is now clear that we are not implying the publication of a new karyotyping technique. Manuscript changes 1. Abstract. “Oikopleura dioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome. It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems. The genome of north Atlantic O. dioica comprises three chromosomes. However, comparisons with the genomes of O. dioica sampled frommainland and southern Japan revealed extensive sequence differences. Moreover, historical studies have reported widely varying chromosome counts. We recently initiated a project to study the genomes of O. dioica individuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan. Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145). The data lead us to conclude that diploid cells from Okinawan O. dioica contain three pairs of chromosomes, in line with the north Atlantic populations. The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and give insight into the genomic diversity of O. dioica from different geographical locations. The data deposited in the EBI BioImage Archive provide representative images of the antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.” 2. Paragraph 1 (Introduction). “… Given the large sequence and synteny differences between the assembled O. dioica genomes, as well as the discrepancies among previous studies, we wished to assess the karyotype for the local Okinawan O. dioica population.” 3. Paragraph 2 (Introduction). “However, we were unable to resolve individual O. dioica chromosomes by this method [Giemsa staining]...” 4. Paragraph 3 (Introduction). “As an alternative approach, we decided to immunostain the centromere as a means of quantifying the numbers of chromosomes… Here, we visualized anti-H3S28P stained embryos from two commercially available antibody sources and unfertilized oocytes to determine the chromosome count of the local Okinawan O. dioica.” _____________________ Reviewer 2 comment 1.2 – Variability of data Reviewer comment The authors intend to argue the advantage of the karyotyping method using H3S28P-specific antibody. However, the authors observed fairly large variation in the number of H3S28P signals (number of centromeres). Particularly, discussion is required for the observation of seven or eight signals within a single nucleus. Author response We thank the referee for this comment. 1. Despite the apparent certainty in chromosome numbers, variability in signal counts does not appear to be unusual. For instance, Fenaux and Colombera noted (1973) reported “In another five anaphase plates, presumably because of chromosome losses during the squashing, a lower number was found.” However, most karyological papers generally present a very small number of representative images; therefore, we cannot comment on whether the variation we observe is unusually large compared with other studies. It is worth noting that in our hands, Giemsa-staining yielded even larger variability than immunostaining in our hands. 2. It is because of this variability that we decided to use a statistical approach: calculating confidence intervals allows us to quantify the uncertainty in the conclusions that we draw from each set of experiments, fully accounting for the variability. 3. We have added a discussion of the possible sources of variation in the number of H3S28P signals. Specifically, we believe that nuclei containing 7-8 counts, arise from non-uniform spots being split into multiple counts; for individual nuclei, this could be resolved by adjusting the signal threshold, but this is not possible if a uniform threshold is applied across all nuclei. 4. From a statistical perspective, the intervals are all narrow and centre on a mean of 6 across three different experimental set ups (Figure 1: mean 6.2, 95% CI 5.6 – 6, mean 6.4, 95% CI 5.7 – 7.1; Figure 2: mean 5.70, 95% CI 5.2 – 6.2). From a biological perspective, the observation is consistent with our genome sequence assembly. Together, these give us reasonable confidence that we have reached the correct conclusion that there are three chromosomes. Manuscript changes Paragraph 15 (Discussion). “Most karyotyping studies display a representative image to support the conclusion; however, given the variability in signal counts between nuclei, we decided to take a statistical approach that quantifies the uncertainty in the estimated chromosome count. Despite testing many different image acquisition settings, we were unable to eliminate the variability; we believe there are several possible reasons that explain them. (i) We applied uniform signal thresholds to all cells, so any spots below the threshold would have been missed. (ii) Spots displayed non-uniform signals, and individual centromeres may have occasionally contributed multiple counts. (iii) The H3S28P signal is not always confined to centromeres, and so may have caused multiple counts (see below). (iv) Finally, the three-dimensional rosette structures in oocytes might not have always been captured reliably in the focal plane. It is worth noting that for O. dioica, immunostaining showed much smaller variabilities than Giemsa-staining.” _____________________ Reviewer 2 comment 1.3 – Use of immunostaining over histochemical methods Reviewer comment Shoguchi et al. (2005) (cited in this article) clearly showed 14 pairs of chromosomes of the Ciona intestinalis genome by means of Giemsa staining and FISH. While the size of the genome in O. dioica is a half of that in C. intestinalis, the number of chromosomes in O. dioica is about one-fifth of that in C. Intestinalis. Therefore, readers may feel that the average size of the O. dioica chromosomes is large enough to be examined by the standard methods. If the development of the new method is really the main aim of this study, I would like the authors to describe merits of this new method in further detail. Without sufficiently convincing explanations, the authors’ method appears to be a less sophisticated alternative to the standard karyotyping methods. It will help if the authors explain why the standard methods are not applicable to O. dioica. Author response We thank the referee for this comment. We were equally frustrated by the difficulties in performing Giemsa staining, which gave even larger variations in signal counts. Anecdotally, this appears to be a similar experience in other laboratories studying O. dioica. FISH is an attractive future possibility for further validation of the immunostaining and genome assembly results. Manuscript changes Please also see authors response to Reviewer 1 comment 3.1 – Interpretation of H3S28P signal locations above. 1. Paragraph 14 (Discussion). “Our initial attempts at karyotyping by traditional Giemsa staining gave us wildly varying counts which we unable to overcome with or without mitotic arrest. Giemsa-staining has been applied successfully to other organisms with small chromosomes such as the tunicate Ciona intestinalis (Shoguchi et al., 2005). The difference in outcome might be explained by the higher AT content of those genomes compared with O. dioica, since Giema preferentially stains AT-rich sequences. Although we do rule out Giema-staining as an effective method for studying O. dioica chromosomes, in our hands, immunostaining yielded more consistent results.” 2. Paragraph 17 (Discussion). “Currently, the nucleotide sequence of the centromeric region is unknown for O. dioica, although chromatin immunoprecipitation with a H3S28P antibody followed by long-read sequencing might be able to provide this information. However, our whole embryo staining data (Figure 1) and the previous literature (Table 1) show that the H3S28P antibody produces non-centromeric signals which may confound such analysis. Thus, alternative targets such as other centromeric histone 3 variants (Moosmann et al., 2011) might be preferable. Knowledge of centromeric sequences would also open the possibility of confirming these results with fluorescence in situ hybridization.” _____________________ Reviewer 2 comment 2.1 – Rationale of study Reviewer comment If the authors’ main aim is to determine the number of chromosomes in Okinawan O. dioica, they should explain more about particularity of this species. Is there a hypothesis that Pacific and Atlantic O. dioica are different species? If not, is there the possibility that different populations (Pacific and Atlantic) have different numbers of chromosomes within the same species? The number of chromosomes is highly variable even between closely related species. However, to my knowledge, the number of chromosomes is essentially invariant within a species. Uncommon exceptions are chromosome reorganization in Ascaris embryos and Paramecium macronuclei. Although the authors discuss the discrepancy in the number of the O. dioica chromosomes (n = 3, or n = 8), I felt that the argument has already been settled (on n = 3) by the extensive genome sequencing (Denoeud et al., 2010). If the authors want to insist that the number of chromosomes in Pacific O. dioica may not be three, more detailed biological information (rationale) is necessary. Author response We thank the referee for this comment. Please also see authors response to Reviewer 1 Comment 1.1. Briefly, we observe large genome sequence variations between north Atlantic, mainland Japanese and Okinawan O. dioica samples, both at nucleotide level and kilo-megabase scale. This is why we decided to check the number of chromosomes in the Okinawan O. dioica. We feel it’s too early to conclude whether they represent distinct species. Regarding the earlier literature, since Colombera and Fenaux (1973) reported 8 chromosomes, it is possible that they examined a different species of Oikopleura. Manuscript changes Please see authors response to Reviewer 1 Comment 1.1 _____________________ Minor points Reviewer 2 minor point 1 Reviewer comment In Table 1, “Ganot et al.” should be “Ganot & Thompson”. Similarly, “Feng et al. (2018)” should be “Feng & Thompson (2018)”. Author response We have made the changes to the citations in the manuscript as suggested. Manuscript changes 1. Feng et al. (2018) was changed to “Feng & Thompson”. 2. Ganot et al. (2008) was left unchanged as it refers to Ganot P, Schulmeister A, Thompson EM, (2008). ___________________ Reviewer 2 minor point 2 Reviewer comment I guess that “ddH2O” (page 4 line 17) is double-distilled H2O. Anyway, “ddH2O” is a laboratory-specific jargon. Similarly, I guess that “ON” (page 4 line 23) means “overnight”? These abbreviations cannot be recommended to be used in articles. Author response We have replaced jargon and abbreviations with full terminology in the methods sections. Manuscript changes Changes made in paragraph 5 and 6. _____________________ Reviewer 2 minor point 3 Reviewer comment Since the authors have knowledge that some somatic cells are polyploid in O. dioica. Therefore, they had better clearly state that the cells shown in Figure 1 are not the case. Although the authors state that 32~64-cell embryos were used for Giemsa staining, they did not tell the developmental stages they used for the antibody staining (in the second paragraph of the Results section). Are they also the early embryos? And do they consist exclusively of diploid cells? Author response We updated the methods section to indicate the developmental stage of the stained embryos (“32 and 64-cell embryos”) and underlined that the same stage was used for Giemsa and antibody staining by adding the words “similarly staged embryos” in the first paragraph of the results section. It is our understanding that the polyploid cells outlined in Ganot & Thompson, 2002 which are responsible for the extrusion of the mucosal house are present in the later stages of development. Manuscript changes 1. Paragraph 6. “Washed eggs, 32 and 64 cell embryos (described above) were immediately fixed…” 2. Paragraph 11. “Consequently, we performed immunostaining of similarly staged embryos…” 3. Paragraph 13. “To rule out polyploidy, which occurs in O. dioica somatic cells that give rise to the mucosal house (Ganot & Thompson, 2002), we also analyzed oocytes in metaphase I before fertilization”" } ] } ]	1	https://f1000research.com/articles/9-780
https://f1000research.com/articles/9-1204/v1	06 Oct 20	{ "type": "Case Report", "title": "Case Report: Unusual association of gubernacular canal, supernumerary tooth and odontoma with an impacted canine on cone beam computed tomography", "authors": [ "Lubna K. Elsayed", "Sara M. El Khateeb", "Suzan A. Alzahrani", "Shatha Subhi ALHarthi", "Raidan Ba-Hattab", "Sara M. El Khateeb", "Suzan A. Alzahrani", "Shatha Subhi ALHarthi", "Raidan Ba-Hattab" ], "abstract": "This report describes a clinical case of asymptomatic compound odontoma in the anterior left side of the maxilla associated with an impacted canine and supernumerary tooth with a gubernacular canal of a 47- year-old female with no relevant medical history. Cone-beam computed tomography (CBCT) was performed for precise three-dimensional localization of each structure and assessment of their spatial relationship with the associated structures before surgery. The treatment protocol involved surgical enucleation of the odontoma and open extraction of both impacted and supernumerary teeth. Patient had uneventful healing and proceeded with the prosthodontic treatment plan. An experienced clinician can accurately diagnose a compound odontoma, as it has distinctive clinical and radiographic features. Thus, early detection and management of odontomas can help correction of any dental irregularity and avoid further complications.", "keywords": [ "Gubernacular canal", "Compound Odontoma", "Supernumerary teeth", "Cone Beam CT", "Impacted Canine" ], "content": "Introduction\n\nOdontoma is defined as a benign odontogenic tumor containing enamel, dentin and cementum, and are classified by the World Health Organization into two main types: compound and mixed1,2. Compound odontoma consists of a tumorlike malformation (hamartoma) with varying numbers of tooth-like elements (odontoids). The complex odontoma consists of a tumour-like malformation (hamartoma) in which the enamel and dentin, and sometimes cementum, are present3. Pathogenesis of odontoma is still unclear, although some etiologic factors have been suggested such as trauma during primary dentition, genetic factors, and chronic inflammation4. They are usually small, asymptomatic and discovered through radiographic examination when patients present with a missing permanent tooth5,6.\n\nOdontomas may cause disturbances in the eruption of teeth such as impaction, delayed eruption, or retention of deciduous or permanent teeth, despite these critical disturbances only a few patients have been described7\n\nAn impacted tooth is one that fails to fully erupt into the dental arch within the usual range of expected time. The tooth becomes impacted because abnormal tooth orientation, adjacent teeth, dense overlying bone, excessive soft tissue, or a genetic abnormality prevents eruption8. Exceptionally, it can be associated with the supernumerary teeth or an odontoma1.\n\nThe gubernacular cord is a structure composed of conjunctive tissue that connects the tooth follicle to the overlying gingiva, this cord guides or directs the course of the tooth eruption. A canal is formed within the bone by the osteoblastic activity to contain the gubernacular cord which is named gubernacular canal9–11\n\nWe present a case of an impacted upper canine with compound odontoma and a supernumerary tooth accompanied by gubernacular canal, where we utilised cone-beam computed tomography (CBCT) to diagnose the tumor prior to surgical treatment.\n\n\nCase report\n\nA 47-year old house wife – of African origin - presented to the dental clinic to treat multiple carious teeth, and to replace multiple missing teeth. On Intraoral clinical examination, both the maxillary canines were missing as well as the third molars, and there was a bulge on the buccal cortex of the missing maxillary left canine, which was asymptomatic (Figure 1a). The patient's medical history showed no previous incidence of dental/maxillofacial trauma or infections. She was referred to the Oral and Maxillofacial Surgery clinic for management. Panoramic radiograph revealed the presence of an impacted maxillary left canine and two small radiopaque masses distal to the impacted canine (Figure 1b) and the periapical radiograph showed that the apical mass is a supernumerary tooth and the coronal mass is a rounded denticle like mass (Figure 1c).\n\na) preoperative clinical intraoral photo showing buccal bulging opposite to the area of impacted left maxillary canine. b) preoperative panoramic radiograph showing impacted left maxillary canines and two radiopaque masses distal to it. c) preoperative periapical radiograph showing impacted canine, supernumerary tooth and radiopaque mass.\n\nCBCT showed that tooth #23 was palatally impacted between teeth #21 and #22. There was a very small well defined rounded denticle like mass positioned distal to tooth #22 and coronal to the impacted supernumerary tooth (crown only), causing a small clinical buccal bulging and preventing its eruption. (Figure 2a, b, c).\n\na) Coronal cone-beam computed tomography (CBCT) scan showing impacted left maxillary canine, supernumerary tooth and odontoma with continuous follicular space b) Three-dimensional CBCT reconstruction showing palatal impacted maxillary left canine together with supernumerary tooth and odontoma c) Sagittal CBCT scan showing odontoma occlusal to supernumerary tooth d) Sagittal CBCT scan showing the proposed gubernacular canal (white arrow).\n\nThe sagittal CBCT slice showed radiographic evidence of continuity of the follicular space of the supernumerary tooth along the bone up to its most inferior part at the alveolar ridge. This was determined to most likely be the gubernacular canal as it followed its possible eruption pathway through the bone. However, the denticle like structure was located inside this pathway and prevented the eruption of the supernumerary tooth. (Figure 2d)\n\nThe most probable differential diagnosis would be a compound odontoma because of its denticle like density, organization, and coronal position.\n\nIntraoral surgery was planned under local anaesthesia, based on the clinical, radiographic findings and multidisciplinary consultation diagnosis of the radiopaque mass as a compound odontoma.\n\nLocal infiltration of the area was performed, a full-thickness mucoperiosteal buccal flap was raised to expose both the mass and supernumerary tooth and soft tissue in between, a palatal flap was raised to expose the impacted canine. The cortical bone was removed utilizing rotary instruments accompanied by normal saline irrigation to minimize heat generation. The calcified mass was identified and ditched all around and then elevated with a Coupland elevator. The incompletely formed supernumerary tooth along with soft tissue attached to it was extracted using straight forceps, and for identification, the soft tissue was tagged with a black silk suture. By access from the palatal region, the impacted canine was sectioned horizontally and extracted (Figure 3 a, b, c), then the whole area was irrigated using normal saline and the flap was stitched back to its original position. A pre-surgical fabricated acrylic stent was placed to prevent falling of the palatal flap and to promote healing. Post-operative analgesia was prescribed in the form of acetaminophen 500mg every 6 hours. Clinical examination was performed 3 months post-surgical; patient was symptoms free and had uneventful recovery.\n\nIntra-operative clinical picture showing surgical procedures to extract impacted maxillary left canine, supernumerary and odontoma a) Labial approach to expose the odontoma. b) Exposing the supernumerary tooth c) Palatal approach to extract the impacted maxillary left canine d&e) Measuring size of odontoma, supernumerary tooth and follicular tissue.\n\nMacroscopically, the calcified mass measured 0.7 × 0.5 × 0.3 cm (Figure 3 d), while the soft tissue mass measured 1.9 × 0.4 × 0.3 cm (Figure 3e). Both specimens were placed in 10% buffered formalin for histopathological/histological examination and final diagnosis.\n\nHistopathologic examination of the excised mass showed a tooth-like structure with dentin, dentinoid tissue, and some enamel matrix confirming the diagnosis of compound odontoma (Figure 4 a,b). Histological examination for the soft tissue mass showed epithelial lamina surrounded by collagenous connective tissue which confirms the diagnosis of gubernacular tissue (Figure 4 c).\n\nHistopathologic picture showing a&b) tooth-like structure with dentin, dentinoid tissue and some enamel matrix. c) epithelial lamina surrounded by collagenous connective tissue.\n\n\nDiscussion\n\nOdontoma is a benign tumour that is usually asymptomatic, diagnosed clinically through routine radiographic examinations, or when investigating other events such as the delayed eruption of permanent teeth, or ectopic position of teeth12–14, as is the case in our findings.\n\nOdontomas are mostly located at the anterior maxilla and associated primarily with permanent teeth, although they can also be associated with deciduous teeth15–18. Due to its rare recurrence, conservative surgical excision is the treatment of choice for odontomas8,19\n\nSupernumerary teeth occur most frequently at the anterior midline, causing malposition of neighboring teeth or preventing their eruption1,6,20. In the present case, the supernumerary tooth was incompletely formed, and it was extracted along with the impacted canine and odontoma.\n\nPanoramic and periapical radiographs are conventional two-dimensional radiographic techniques which are widely available and are frequently used for multiple diagnostic purposes like detection of impaction, odontoma, bone loss and various intraosseous lesions. Nevertheless, these radiographs have various limitations in revealing the buccolingual relationship, and presence of superimposition, also having geometric inaccuracy21. In the current case, we primarily detect the presence of impaction of a supernumerary tooth and odontoma through panoramic and periapical radiograph.\n\nCBCT is an advanced 3D imaging modality that offers precise localization and detection of the spatial relationships of any dental structure with the surroundings, also it lessens the radiation dose in comparison with conventional CT and delivers high spatial resolution22. In our case, we used CBCT for 3D localization of the odontoma, impacted canine and supernumerary tooth before surgical removal22,23.\n\nThe role of gubernacular cord (GC) and the canal in tooth eruption is not clear, although it has been suggested that it may have a central role in inducing normal tooth eruption as it constitutes a pathway from the dental follicle to the gingiva for the eruption of permanent teeth11. Oda M. et al.24 suggested that the presence of and contact with the gubernacular tract (GT) should be added as a characteristic CT finding of some types odontogenic masses including dentigerous cysts, calcifying odontogenic cysts (Gorlin Cyst), odontomas, Adenomatoid Odontogenic Tumor and perhaps others. Moreover, they concluded that dentists should pay more attention to the association between the GT and odontogenic masses24,25. Our case demonstrated an association between the GT and the odontoma where the odontoma was inside the GT of the supernumerary tooth with the same spatial relationship in agreement with Oda M. et al.24 which reports that the majority of odontoma cases (about 70%) detected with CBCT were inside the GT of the unerupted teeth (Figure 2d). Furthermore, during surgery, it was apparent that the GT was contiguous with the supernumerary follicle, suggesting that the GT was guiding the eruption of the supernumerary tooth, but the development of the odontoma prevented its eruption. Gaˆeta-Araujo H et al. reported that the most communal attachment site of GC was to the occlusal side of the dental sac of the unerupted tooth (93.2%) and was classified as a usual attachment. These findings were in agreement with our case, where we also found the GT to be attached to the occlusal aspect of the dental sac of the supernumerary tooth25.\n\n\nConclusion\n\nThe anterior maxilla is the most common site for the development of a compound odontoma, causing various disturbances in eruption and teeth position. Having distinctive clinical and radiographic features; an experienced clinician can accurately diagnose a compound odontoma. Thus, early detection and management of odontomas can help the correction of any dental irregularity and avoid further complications. The dentist should also be aware of the probability of a close relationship between the development of odontoma and presence of the gubernacular tract, which in future could be used as a radiographic diagnostic criterion of an odontoma.\n\n\nConsent\n\nWritten informed consent for publication of their 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\nKrichen G, Hentati H, Hadhri R, et al.: Odontoma associated with supernumerary and impacted teeth. 2013; 1(4): 6. Reference Source\n\nSoluk-Tekkesin M, Wright JM: The world health organization classification of odontogenic lesions: a summary of the changes of the 2017 (4th) edition. Turk Patoloji Derg. 2018; 34(1). PubMed Abstract \| Publisher Full Text\n\nBarnes L, Eveson JW, Reichart P, et al.: Pathology and Genetics of Head and Neck Tumours. Accessed January 8, 2020. Reference Source\n\nSilva Gomes Ferreira PH, Ferreira S, Faverani LP, et al.: Compound Odontoma in a Pediatric Patient With Aspects Similar to Complex Odontoma. J Craniofac Surg. 2015; 26(4): 1429–1431. PubMed Abstract \| Publisher Full Text\n\nKaneko M, Fukuda M, Sano T, et al.: Microradiographic and microscopic investigation of a rare case of complex odontoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998; 86(1): 131–134. PubMed Abstract \| Publisher Full Text\n\nRajendran R: Shafer’S Textbook Of Oral Pathology (6th Edition). Elsevier India; 2009. Reference Source\n\nIsola G, Cicciù M, Fiorillo L, et al.: Association Between Odontoma and Impacted Teeth. J Craniofac Surg. 2017; 28(3): 755–758. PubMed Abstract \| Publisher Full Text\n\nHupp JR, Tucker MR, Ellis E: Contemporary Oral and Maxillofacial Surgery. 7th ed. Elsevier; 2018. Reference Source\n\nHodson JJ: The gubernaculum dentis. Dent Pract Dent Rec. 1971; 21(12): 423–428. PubMed Abstract\n\nCahill DR, Marks SC Jr: Tooth eruption: evidence for the central role of the dental follicle. J Oral Pathol. 1980; 9(4): 189–200. PubMed Abstract \| Publisher Full Text\n\nFerreira DCA, Fumes AC, Consolaro A, et al.: Gubernacular cord and canal – does these anatomical structures play a role in dental eruption? 5. Reference Source\n\nChoudhary PJ, Gharote HP, Hegde K, et al.: Compound Odontoma Associated with Impacted Teeth: A Case Report. 2014; 1(3): 12. Reference Source\n\nAlam MK: Management of bilateral impacted maxillary canines (BIMC): open surgical exposure and orthodontic traction. Bangladesh J Med Sci. 2019; 19(1): 169–173. Publisher Full Text\n\nSilva Dos Santos SR, Brando Mendes F, Da Silva TE, et al.: Odontoma Compound Preventing Dental Irruption and Start of Orthodontic Treatment: Case Report. Braz J Surg Clin Res. 2016; 17(3): 18–23. Reference Source\n\nBereket C, Çakır-Özkan N, Şener İ, et al.: Complex and compound odontomas: Analysis of 69 cases and a rare case of erupted compound odontoma. Niger J Clin Pract. 2015; 18(6): 726–730. PubMed Abstract \| Publisher Full Text\n\nHisatomi M, Asaumi JI, Konouchi H, et al.: A case of complex odontoma associated with an impacted lower deciduous second molar and analysis of the 107 odontomas. Oral Dis. 2002; 8(2): 100–105. PubMed Abstract \| Publisher Full Text\n\nActon CH, Savage NW: Odontomes and their behaviour: a review. Aust Dent J. 1987; 32(6): 430–435. PubMed Abstract \| Publisher Full Text\n\nSinger SR, Mupparapu M, Milles M, et al.: Unusually large complex odontoma in maxillary sinus associated with unerupted tooth. Report of case and review of literature. N Y State Dent J. 2007; 73(4): 51–53. PubMed Abstract\n\nOral and Maxillofacial Pathology - 4th Edition. Accessed March 2, 2020. Reference Source\n\nNagaraj K, Upadhyay M, Yadav S: Impacted maxillary central incisor, canine, and second molar with 2 supernumerary teeth and an odontoma. Am J Orthod Dentofacial Orthop. 2009; 135(3): 390–399. PubMed Abstract \| Publisher Full Text\n\nAlqerban A, Jacobs R, Fieuws S, et al.: Comparison of two cone beam computed tomographic systems versus panoramic imaging for localization of impacted maxillary canines and detection of root resorption. Eur J Orthod. 2011; 33(1): 93–102. PubMed Abstract \| Publisher Full Text\n\nGürler G, Delilbasi C, Aydin K: Evaluation of Unerupted Odontomas With Cone Beam Computed Tomography (CBCT). J Int Dent Sci. 2017; 2(1): 36–42. Publisher Full Text\n\nKapila SD, Nervina JM: CBCT in orthodontics: assessment of treatment outcomes and indications for its use. Dentomaxillofacial Radiol. 2015; 44(1): 20140282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOda M, Miyamoto I, Nishida I, et al.: A spatial association between odontomas and the gubernaculum tracts. Oral Surg Oral Med Oral Pathol Oral Radiol. 2016; 121(1): 91–95. PubMed Abstract \| Publisher Full Text\n\nGaêta-Araujo H, da Silva MB, Tirapelli C, et al.: Detection of the gubernacular canal and its attachment to the dental follicle may indicate an abnormal eruption status. Angle Orthod. 2019; 89(5): 781–787. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72618", "date": "17 Nov 2020", "name": "Hatem W. Amer", "expertise": [ "Reviewer Expertise Oral and maxillofacial pathology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction is well written. The methodology and approach to diagnosis was systematic and the discussion was fruitful. It would have been more fulfilling to provide more past literature about the case and to discuss how the gubernacular canal could affect differential diagnosis both radiographically and histologically. All in all I found it very interesting.\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": "6360", "date": "26 Feb 2021", "name": "Lubna Elsayed", "role": "Author Response", "response": "Thank you for your valuable comments and we appreciate your recommendations, and they were considered, a brief description about how the gubernacular canal could affect differential diagnosis both radiographically and histologically was added to the discussion section." }, { "c_id": "6379", "date": "26 Feb 2021", "name": "Lubna Elsayed", "role": "Author Response", "response": "Thank you for your valuable comments, and your recommendations were considered as we added more literature about similar cases and a discussion about how the gubernacular canal could affect differential diagnosis both radiographically and histologically was added in the discussion section." } ] }, { "id": "78510", "date": "02 Feb 2021", "name": "Prashanth Panta", "expertise": [ "Reviewer Expertise Oral medicine and radiology." ], "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\nOdontoma is a common clinical entity and is often spotted on dental radiographs taken to inspect missing permanent teeth. However, this report's merit lies in the CBCT demonstration of an odontoma within the 'gubernacular canal' (GC)-a finding less frequently spotted on radiographs. Although vital in tooth eruption, GC goes unnoticed in dental radiographs due to a lack of focus on this crucial anatomical landmark. As rightly suggested by few previous authors and the authors of this report, this typical association could aid the radiographic diagnosis of odontoma. Because odontomas are closely associated with GC, they cause delayed/failure of eruption of the underlying permanent tooth. Since many clinicians may still be unaware of some of these aspects, more technical reports are necessary for this direction. The current report by Lubna K. Elsayed et al. fills this gap.\n\nHere are a few recommendations which could improve the manuscript:\nThe 'A 47-year old housewife – of African origin presented' can be modified as 'A 47-year old female – of African origin presented'\n\nSupernumerary tooth and odontoma are also associated with syndromes. If the patient is non-syndromic, the phrase, 'an otherwise healthy and non-syndromic patient,' can be added to the case history section. A brief discussion on the syndromes associated with odontomas and supernumerary teeth may be a valuable addition to this report.\n\nIn the conclusion section, the sentence \"Thus, early detection and management of odontomas can help correct any dental irregularity and avoid further complications\" has been included. When odontomas are asymptomatic entities and discovered incidentally during radiographic surveys taken for evaluation of missing permanent teeth, how is early detection achievable? Consider adding some info regarding the same and mention which are the further complications.\n\nSince the report mainly circles around GC, the conclusion paragraph could straightaway focus on the GC-odontoma connection. Consider avoiding conventional odontoma literature in conclusion. The below paragraph seems to be fitting:\n'The dentist should be aware of the probability of a close relationship between the development of odontoma and presence of the gubernacular tract, which could be used as a radiographic diagnostic criterion of an odontoma.'\n\nThe discussion section can also include a brief review of odontoma-GC literature and appropriate differential diagnosis.\n\nTo avoid hype, the word 'unusual' can be removed from the title. The title 'Association of the gubernacular canal, supernumerary tooth, and odontoma with an impacted canine on cone-beam computed tomography' seems fitting.\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": "6361", "date": "26 Feb 2021", "name": "Lubna Elsayed", "role": "Author Response", "response": "Recommendation 1: The 'A 47-year old housewife – of African origin presented' can be modified as 'A 47-year old female – of African origin presented' Answer: Thanks for the recommendation and the change is accepted, although it was a request of the editor to mention “occupation”. Recommendation 2: Supernumerary tooth and odontoma are also associated with syndromes. If the patient is non-syndromic, the phrase, 'an otherwise healthy and non-syndromic patient,' can be added to the case history section. A brief discussion on the syndromes associated with odontomas and supernumerary teeth may be a valuable addition to this report. Answer: Thanks for your opinion, it was considered. Recommendation 3: In the conclusion section, the sentence \"Thus, early detection and management of odontomas can help correct any dental irregularity and avoid further complications\" have been included. When odontomas are asymptomatic entities and discovered incidentally during radiographic surveys taken for evaluation of missing permanent teeth, how is early detection achievable? Consider adding some info regarding the same and mention which are the further complications. Answer: Thanks for the suggestion and further complications were added to the discussion section Recommendation 4: Since the report mainly circles around GC, the conclusion paragraph could straightaway focus on the GC-odontoma connection. Consider avoiding conventional odontoma literature in conclusion. Answer: Thanks for the valuable comment and the recommended phrase was added to the conclusion section. Recommendation 5: The discussion section can also include a brief review of odontoma-GC literature and appropriate differential diagnosis. Answer: Thanks for the comment and the suggestion was considered and added to the discussion section. Recommendation 6: To avoid hype, the word 'unusual' can be removed from the title. The title 'Association of the gubernacular canal, supernumerary tooth, and odontoma with an impacted canine on cone-beam computed tomography' seems fitting. Answer: Thanks for the suggestion and it was considered." } ] }, { "id": "77029", "date": "04 Feb 2021", "name": "Ronell Bologna-Molina", "expertise": [ "Reviewer Expertise Oral Pathology", "Histopathology", "Oral Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a clear, simple and interesting article, easily understood by the reader. The authors describe a rare case of an odontoma in the anterior left side of the maxilla associated with an impacted canine and supernumerary tooth with a governmental canal.\nThere are some points that could improve the quality of the article:\na) The x-ray image is not clearly representative of a composite odontoma, it could also be a complex odontoma. Due to the aforementioned, I suggest that in the histopathological image 4a and 4b) the authors mark and mark each dental tissue that makes up the odontoma (dentin, enamel, cementum). In image 4c, the authors should put the following description: It is observed odontogenic epithelium arranged in islands and cords surrounded by mature connective tissue, suggesting the presence of epithelial lamina surrounded by collagenous. Please indicate the magnification of the image (for example 50X, 100X or 400X).\n\nb) The reviewer suggests (in the discussion section) to go deeper into the imaging of the gubernacular tract with CBCT and the presence of odontomas or odontogenic tumors, I suggest reading in this article: Chaudhry and Sobti (20201).\n\nc) I believe that the conclusion paragraph that says \"The dentist should also be aware of the probability of a close relationship between the development of odontoma and the presence of the governmental tract, which in future could be used as a radiographic diagnostic criterion of an odontoma\" is very ambitious. I suggest that the authors clearly explain why they believe that it could become a radiographic criterion for an odontoma and/or not for another odontogenic tumor that has the presence of mineralized tissue.\n\nMinor points:\nIn the case report section, the authors describe the patient in the following way: \"A 47-year old house wife - of African origin -\" I think that this data is not important for the clinical case, I suggest describing only; \"A 47-year old female ...\".\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6362", "date": "26 Feb 2021", "name": "Lubna Elsayed", "role": "Author Response", "response": "a) The x-ray image is not clearly representative of a composite odontoma, it could also be a complex odontoma. Due to the aforementioned, I suggest that in the histopathological image 4a and 4b) the authors mark and mark each dental tissue that makes up the odontoma (dentin, enamel, cementum). In image 4c, the authors should put the following description: It is observed odontogenic epithelium arranged in islands and cords surrounded by mature connective tissue, suggesting the presence of epithelial lamina surrounded by collagenous. Please indicate the magnification of the image (for example 50X, 100X or 400X). Response a: thanks for your opinion, it was considered and added (changes were added to the newly submitted version of manuscript). b) The reviewer suggests (in the discussion section) to go deeper into the imaging of the gubernacular tract with CBCT and the presence of odontomas or odontogenic tumors, I suggest reading in this article: Chaudhry and Sobti (2021). Response b: Thanks for your recommendation and the suggested paper was very valuable. c) I believe that the conclusion paragraph that says \"The dentist should also be aware of the probability of a close relationship between the development of odontoma and the presence of the governmental tract, which in future could be used as a radiographic diagnostic criterion of an odontoma\" is very ambitious. I suggest that the authors clearly explain why they believe that it could become a radiographic criterion for an odontoma and/or not for another odontogenic tumor that has the presence of mineralized tissue. Response c: Thanks and a paragraph was added in the discussion to explain the role of GT in differentiating between odontogenic and non-odontogenic lesions based on previous studies. Minor points: In the case report section, the authors describe the patient in the following way: \"A 47-year old housewife - of African origin -\" I think that this data is not important for the clinical case, I suggest describing only; \"A 47-year old female ...\". Response for minor point: The recommendation was considered and changes were made, although it was a request by the editor." } ] } ]	1	https://f1000research.com/articles/9-1204
https://f1000research.com/articles/9-826/v1	30 Jul 20	{ "type": "Study Protocol", "title": "Stage 1 Registered Report: Anomalous perception in a Ganzfeld condition - A meta-analysis of more than 40 years investigation", "authors": [ "Patrizio E. Tressoldi", "Lance Storm", "Lance Storm" ], "abstract": "This meta-analysis is an investigation into anomalous perception (i.e., conscious identification of information without any conventional sensorial means). The technique used for eliciting an effect is the ganzfeld condition (a form of sensory homogenization that eliminates distracting peripheral noise). The database consists of peer-reviewed studies published between January 1974 and June 2020 inclusive. The overall effect size will be estimated using a frequentist model and a Bayesian random model. Moderator analysis will be used to examine the influence of level of experience of participants and the type of task. Publication bias will be estimated by using three different tests. Trend analysis will be conducted on the cumulative database.", "keywords": [ "meta-analysis", "ganzfeld", "anomalous cognition", "publication bias", "consciousness" ], "content": "Introduction\n\nThe possibility of identifying pictures or video clips without conventional (sensorial) means, in a ganzfeld environment, is a decades old controversy, dating back to the pioneering investigation of Charles Honorton, William Braud and Adrian Parker between 1974 and 1975 (Parker, 2017).\n\nIn the ganzfeld, a German term meaning ‘whole field’, participants are immersed in an homogeneous sensorial field were peripheral visual information is masked out by red light diffused by translucent hemispheres (often split halves of ping-pong balls or special glasses) placed over the eyes, while a relaxing rhythmic sound, or white or pink noise, is fed through headphones to shield out peripheral auditory information. Once participants are sensorially isolated from external visual and auditory stimulation, they are in a favourable condition for producing inner mental contents about a randomly-selected target hidden amongst decoys. The mentation they produce can either be used by the participant to guide his/her target selection, or it can be used to assist in an independent judging process.\n\nIn the prototypical procedure, participants are tested in a room isolated from external sounds and visual information. After they made themselves comfortable in a reclining armchair, they receive the instructions related their task during the ganzfeld condition. Even if there are different verbatim versions, the instructions describe what they should do mentally in order to detect the information related to the target and how to filter out the mental contents not related to it. This information will be described aloud and recorded for playback before or during the target identification phase. After the relaxation phase, they are exposed to the ganzfeld condition for a period ranging from 15 to 30 minutes. During this phase, participants describe verbally all images, feelings, emotions, they deem related to the target usually a picture or a short videoclip of real objects or events.\n\nOnce the ganzfeld phase is completed, participants are presented with our choices (the target plus three decoys) of the same format, e.g. picture or videoclip, and they must choose which one is the target (binary decision). Alternatively, they may be asked to rate all four (e.g., from 0 to 100), to indicate the strength of relationship between the information detected during the ganzfeld phase and the images or video clips contents.\n\nA variant of the judgment phase is to send the recording of the information retrieved during the ganzfeld phase to an external judge for independent ratings of the target. In order to prevent voluntary or involuntary leakage of information about the target by the experimenters, the research assistant who interact with the participants must be blind to the target identity until the participants’ rating task is over. The choice of the target and the decoys is usually made using automatic random procedures, and scores are automatically fed onto a scoring sheet.\n\nThere are three different ganzfeld conditions:\n\n-Type 1: the target is chosen after the judgment phase;\n\n-Type 2: the target is chosen before the ganzfeld phase;\n\n-Type 3: the target is chosen before the ganzfeld phase and presented to a partner of the participant isolated in a separate and distant room.\n\nThese differences are related to some theoretical and perceptual concepts we will discuss later. It is important to note that type of task makes no difference to the participant who only engages in target identification after the ganzfeld phase.\n\n\nReview of the Ganzfeld Meta-Analyses\n\nIt is interesting to note that most of the cumulative findings (meta-analyses) of this line of investigation were periodically published in the mainstream journal Psychological Bulletin.\n\nHonorton (1985) undertook one of the first meta-analyses of the many ganzfeld studies completed by the mid-1980s. In total, 28 studies yielded a collective hit rate of 38%, where mean chance expectation (MCE) was 25%. Various flaws in his approach were pointed out by Hyman (1985), but in their joint-communiqué they agree that “there is an overall significant effect in this database that cannot reasonably be explained by selective reporting or multiple analysis” (Hyman & Honorton, 1986, p. 351).\n\nA second major meta-analysis on a set of ‘autoganzfeld’ studies was performed by Bem & Honorton (1994). These studies followed the guidelines laid down by Hyman & Honorton (1986). Moreover the autoganzfeld procedure avoids methodological flaws by using a computer-controlled target randomization, selection, and judging technique. They overall reported hit rate of 32.2% exceeded again the mean chance expectation.\n\nMilton & Wiseman (1999) meta-analysed further 30 studies collected for the period 1987 to 1997; reporting an overall nonsignificant standardized effect size of 0.013. However, Jessica Utts (personal communication, December 11, 2009) using the exact binomial test on trial counts only (N = 1198; Hits = 327), found a significant hit rate of 27% (p = 0.036).\n\nStorm & Ertel (2001) comparing Milton & Wiseman’s (1999) database with Bem & Honorton’s (1994) one, found the two did not differ significantly. Furthermore Storm and Ertel went on to compile a 79-study database, which had a statistically significant mean standardized effect size of 0.138.\n\nStorm et al. (2010), meta-analysed a database of 29 ganzfeld studies published during the period 1997 to 2008, yielding a standardized effect size of 0.14. Rouder et al. (2013) reassessing Storm et al.’s (2010) meta-analysis, with a Bayesian meta-analysis found evidence for the existence of an anomalous perception in the original dataset observing a Bayes Factor of 330 in support to the alternative hypothesis (p. 241). However they contended the effect could be due to “difficulties in randomization” (p. 241), arguing that ganzfeld studies with computerized randomization had smaller effects than those with manual randomization. The reanalysis by Storm et al.’s (2013) showed that this conclusion was unconvincing as it was based on Rouder et al.’s faulty inclusion of different categories of study.\n\nIn the last meta-analysis by Storm & Tressoldi (2020), related to the studies published from 2008 to 2018, the overall standardized effect size was 0.133; 95%CI: 0.06 - 0.18.\n\n\nThis study\n\nThe main aim of this study is to meta-analyse all available ganzfeld studies dating from 1974 up to June 2020 in order to assess the overall effect size of the database and determine whether there are moderator variables that affect task performance; in particular, we hypothesize that participant type and type of task are two major moderators of effect size (see Methods section).\n\n\nMethods\n\nThis study will follow the guidelines of the APA Meta-Analysis Reporting Standard (Appelbaum et al., 2018) and the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P, Moher et al., 2015).\n\nRetrieval of studies related to anomalous perception in a Ganzfeld environment is simplified, firstly by the fact that most of these studies have already been retrieved for previous meta-analyses, as cited in the introduction. Secondly, this line of investigation is carried out by a small community of researchers. Thirdly, most of the studies of interest to us are published in specialized journals that adopted the editorial policy of accepting paper with results that are statistically non-significant (according to the frequentist approach). This last condition is particularly relevant because it reduces the publication bias due to the non-rejection of the statistical null hypothesis often consequent to reduced statistical power. However, in order to integrate the previous retrieval method, we will carry-out an online search with Google Scholar, PubMed and Scopus databases of all papers from 1974 to 2020 including in the title and/or the abstract the word “ganzfeld” (e.g. for PubMed: Search: ganzfeld[Title/Abstract] Filters: from 1974 – 2020).\n\nThe following inclusion criteria will be adopted:\n\n- Studies related to anomalous perception in a ganzfeld environment;\n\n- Studies must use human participants only (not animals);\n\n- Number of participants must be in excess of two to avoid the inherent problems that are typical in case studies;\n\n- Target selection must be randomized by using a Random Number Generator (RNG) in a computer or similar electronic device, or a table of random numbers. Randomization procedures must not be manipulated by the experimenter or participant;\n\n- Studies must provide sufficient information (e.g., number of trials and outcomes) for the authors to calculate the direct hit-rates and effect size values, so that appropriate statistical tests can be conducted.\n\n- Peer reviewed studies even if published in proceedings;\n\nFor each included study, one of the authors, expert in meta-analyses, will code the following variables:\n\n- Authors;\n\n- Year of publication;\n\n- Number of trials;\n\n- Number of hits;\n\n- Number of choices of each trial;\n\n- Task type (Type 1,2 or 3);\n\n- Participants type (selected vs. unselected). The authors of the study will score as selected all participants that were screened for one or more particular characteristic deemed favourable for the performance in this type of task.\n\nThe second author will randomly check 10% of all studies, and the data will be compared with those extracted by the other author. Discrepancies will be corrected by inspecting the original papers.\n\nThe complete database will be made available through open access posting within the dedicated project in the Open Science Framework (https://osf.io/t7sya/) platform.\n\nAs standardized measure of effect size, we will use the following formula: Binomial Z score/√number of trials. The Binomial Z score will be obtained applying the formula implemented online at http://vassarstats.net/binomialX.html\n\nIn order to take in account the effect size overestimation bias in small samples, the effect size will be transformed in the Hedge’s g effect size, obtaining the corresponding variance by applying the formula presented in Borenstein et al. (2009, pp. 27–28).\n\nThe overall effect size estimation of the whole database will be calculated by applying both a frequentist and a Bayesian random model for testing its robustness.\n\nFollowing the recommendations of Langan et al. (2019), we will use the restricted maximum likelihood (REML) approach to estimate the heterogeneity variance with the Knapp and Hartung method for adjustment to the standard errors of the estimated coefficients (Rubio-Aparicio et al., 2018).\n\nFurthermore, in order to control for possible influence of outliers, we will calculate the median and mode of the overall effect size applying the method suggested by Hartwig et al. (2020).\n\nThese calculations will be implemented in the R statistical environment with the metafor package v. 2.4 (Viechtbauer, 2017). See syntax provided as extended data (Tressoldi & Storm, 2020).\n\nAs priors for the overall effect size we will use a normal distribution with Mean = 0.01; SD =0.03, constrained positive, lower bound = 0 (Rouder et al., 2019), given our expectation of a positive value. This Bayesian meta-analysis will be implemented with the MetaBMA package v. 0.6.3 (Heck et al., 2017). See Syntax in the Supporting Information.\n\nFollowing the suggestions of Carter et al. (2019), we will apply three tests to assess publication bias:\n\n- the 3-parameter selection model (3PSM), as implemented by Coburn & Vevea (2019) with the package ‘weightr’ v.2.0.2;\n\n- the p-uniform* (star) v. 0.2.2 test as described by van Aert & van Assen (2019), and\n\n- the sensitivity analysis using the Mathur & VanderWeele (2020) package PublicationBias v.2.2.0.\n\nThe three parameters model represent the average true underlying effect, δ; the heterogeneity of the random effect sizes, τ2; and the probability that there is a nonsignificant effect in the pool of effect sizes. The probability parameter is modeled by a step function with a single cut point at p = 0.025 (one-tailed), which corresponds to a two-tailed p value of 0.05. This cut point divides the range of possible p values into two bins: significant and nonsignificant. The three parameters are estimated using maximum likelihood (Carter et al., 2019, p. 124).\n\nThe p-uniform* test, is an extension and improvement of the p-uniform method. P-uniform* improves upon p-uniform giving a more efficient estimator avoiding the overestimation of effect size in case of between-study variance in true effect sizes, thus enabling estimation and testing for the presence of between-study variance in true effect sizes.\n\nSensitivity analysis as implemented by Mathur & VanderWeele (2020), assumes a publication process such that “statistically significant” results are more likely to be published than negative or “nonsigniﬁcant” results by an unknown ratio, η (eta). Using inverse-probability weighting and robust estimation that accommodates non-normal true eﬀects, small meta-analyses, and clustering, it enables statements such as: “For publication bias to shift the observed point estimate to the null, ‘signiﬁcant’ results would need to be at least 30-fold more likely to be published than negative or ‘nonsigniﬁcant’ results” (p. 1). Comparable statements can be made regarding shifting to a chosen non-null value or shifting the conﬁdence interval. See Syntax in the Supporting Information\n\nIn order to study the overall trend of the cumulative evidence and in particular for testing the presence of a decline or incline effect, we will perform a cumulative effect size estimation (see Syntax in the Supporting Information)\n\nWe will compare the influence of the following two moderators: (i) Type of participant, and (ii) Type of task.\n\nAs described in the Variable Coding paragraph, the variable Type of participant, will be coded in a binary way: selected vs unselected. Type of task will be coded as Type 1, Type 2, and Type 3, as described in the Introduction. See Syntax in the Supporting Information.\n\nOnce the overall effect size and its precision are estimated, we will calculate the number of trials necessary to achieve a statistical power of at least .80 with an α = .05. With this estimation we can examine how many studies in the database reached this threshold.\n\nThe search and selection of the studies will be presented by using a PRISMA flowchart.\n\nDescriptive statistics will be produced related to the variables, trials, hits, participant type, and task types.\n\nWe will present the estimated average effect size along with the corresponding 95% Confidence Intervals or Credible Intervals of both the Frequentist and Random Models as described in the Methods section. We will calculate the values of τ2 and I2 (Higgins & Thompson, 2002), and their confidence intervals, as measures of between-study variance.\n\nWe will present the results of the three publication bias tests described in the Methods section.\n\nThe results of the cumulative meta-analysis will be represented with a cumulative forest plot.\n\nWe will present and compare the average effect size along with the corresponding 95% Confidence Intervals of the two types the participant, and of the three task types.\n\nApart the Registered Report, all information related to this study will be made available open access at Open Science Framework.\n\nThe study has not started yet.\n\n\nDiscussion\n\nWe will discuss the robustness of the overall results in order to determine a degree of confidence in the evidence for anomalous perception. In case of an insufficient degree of confidence in the evidence, we will consider whether it is worthwhile pursuing such a line of investigation and offer solutions to improve the evidence.\n\nHowever, even if the overall results show a sufficient degree of evidence, we will discuss how this line of investigation can instil greater confidence by using a preregistration registry as proposed by Watt & Kennedy (2016) in order to reduce so-called questionable research practices (John et al., 2012), and provide more transparent procedures during data collection and analysis (see for example, the Transparent Psi Project; Kekecs et al., 2019).\n\n\nData availability\n\nNo data are associated with this article\n\nFigshare: Anomalous perception in a gazfeld condition: a meta-analysis of more than 40 years of investigation. https://doi.org/10.6084/m9.figshare.12674618.v1 (Tressoldi & Storm, 2020)\n\n- Syntax Details.docx (Syntax related to all statistical analyses)\n\nFigshare: PRISMA-P checklist for ‘Stage 1 Registered Report: Anomalous perception in a Ganzfeld condition - A meta-analysis of more than 40 years investigation’ https://doi.org/10.6084/m9.figshare.12674618.v1 (Tressoldi & Storm, 2020)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAppelbaum M, Cooper H, Kline RB, et al.: Journal article reporting standards for quantitative research in psychology: The APA Publications and Communications Board task force report. Am Psychol. 2018; 73(1): 3–25. PubMed Abstract \| Publisher Full Text\n\nBem DJ, Honorton C: Does psi exist? Replicable evidence for an anomalous process of information transfer. Psychol Bull. 1994; 115(1): 4–18. Publisher Full Text\n\nBorenstein M, Hedges LV, Higgins JPT, et al.: Introduction to Meta-Analysis. Chichester, UK: John Wiley & Sons, Ltd. 2009. Publisher Full Text\n\nCarter E, Schönbrodt F, Gervais W, et al.: Correcting-bias-in-psychology. Adv Methods Pract Psychol Sci. 2019; 2(2): 115–144. Publisher Full Text\n\nCoburn KM, Vevea JL: Package ‘weightr’. Estimating Weight-Function Models for Publication Bias. 2019. Reference Source\n\nHartwig FP, Smith GD, Schmidt AF, et al.: The median and the mode as robust meta-analysis estimators in the presence of small-study effects and outliers. Res Synth Methods. 2020; 11(3): 397–412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeck DW, Gronau QF, Wagenmakers E: metaBMA: Bayesian model averaging for random and fixed effects meta-analysis. 2017. Publisher Full Text\n\nHiggins JPT, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med. 2002; 21(11): 1539–1558. PubMed Abstract \| Publisher Full Text\n\nHyman R: The ganzfeld psi experiment: A critical appraisal. J Parapsychol. 1985; 49(1): 3–49. Reference Source\n\nHyman R, Honorton C: Joint communiqué: The psi ganzfeld controversy. J Parapsychol. 1986; 50(4): 351–364. Reference Source\n\nHonorton C: Meta-analysis of psi ganzfeld research: A response to Hyman. J Parapsychol. 1985; 49(1): 51–91. Reference Source\n\nJohn LK, Loewenstein G, Prelec D: Measuring the Prevalence of Questionable Research Practices With Incentives for Truth Telling. Psychol Sci. 2012; 23(5): 524–532. PubMed Abstract \| Publisher Full Text\n\nKekecs Z, Aczel B, Palfi B, et al.: Raising the value of research studies in psychological science by increasing the credibility of research reports: The Transparent Psi Project - Preprint. 2019. Publisher Full Text\n\nLangan D, Higgins JPT, Jackson D, et al.: A comparison of heterogeneity variance estimators in simulated random-effects meta-analyses. Res Synth Methods. 2019; 10(1): 83–98. PubMed Abstract \| Publisher Full Text\n\nMathur MB, VanderWeele TJ: Sensitivity analysis for publication bias in meta-analyses [preprint]. 2020. Reference Source\n\nMilton J, Wiseman R: Does psi exist? Lack of replication of an anomalous process of information transfer. Psychol Bull. 1999; 125(4): 387–391. PubMed Abstract \| Publisher Full Text\n\nMoher D, Stewart L, Shekelle P, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015; 4(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParker A: ‘Ganzfeld’. Psi Encyclopedia. London: The Society for Psychical Research. 2017. Reference Source\n\nRouder JN, Morey RD, Province JM: A Bayes factor meta-analysis of recent extrasensory perception experiments: Comment on Storm, Tressoldi, and Di Risio (2010). Psychol Bull. 2013; 139(1): 241–247. PubMed Abstract \| Publisher Full Text\n\nRouder JN, Haal JM, Davis-Stober CP, et al.: Beyond overall effects: A Bayesian approach to finding constraints in meta-analysis. Psychol Methods. 2019; 24(5): 606–621. PubMed Abstract \| Publisher Full Text\n\nRubio-Aparicio M, López-López JA, Sánchez-Meca J, et al.: Estimation of an overall standardized mean difference in random-effects meta-analysis if the distribution of random effects departs from normal. Res Synth Methods. 2018; 9(3): 489–503. PubMed Abstract \| Publisher Full Text\n\nStorm L, Ertel S: Does psi exist? Comments on Milton and Wiseman’s (1999) meta-analysis of ganzfeld research. Psychol Bull. 2001; 127(3): 424–433, discussion 434-8. PubMed Abstract \| Publisher Full Text\n\nStorm L, Tressoldi PE, Di Risio L: Meta-analyses of free-response studies, 1992–2008: Assessing the noise reduction model in parapsychology. Psychol Bull. 2010; 136(4): 471–485. PubMed Abstract \| Publisher Full Text\n\nStorm L, Tressoldi PE, Utts J: Testing the Storm et al. (2010) meta-analysis using Bayesian and frequentist approaches: Reply to Rouder et al. (2013). Psychol Bull. 2013; 139(1): 248–254. PubMed Abstract \| Publisher Full Text\n\nStorm L, Tressoldi P: Meta-Analysis of Free-Response Studies 2009–2018: Assessing the Noise-Reduction Model Ten Years On. PsyArxiv. 2020. Publisher Full Text\n\nTressoldi P, Storm L: Anomalous perception in a Ganzfeld condition: A meta-analysis of more than 40 years investigation. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12674618.v1\n\nvan Aert RCM, van Assen MALM: Correcting for publication bias in a Meta-Analysis with the P-Uniform* method. 2019. Publisher Full Text\n\nViechtbauer W: The metafor Package. 2017. Reference Source\n\nWatt CA, Kennedy JE: Options for Prospective Meta-Analysis and Introduction of Registration-Based Prospective Meta-Analysis. Front Psychol. 2016; 7: 2030. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68427", "date": "09 Sep 2020", "name": "Jessica Utts", "expertise": [ "Reviewer Expertise Statistical analysis and methods", "with applications to various disciplines including parapsychology", "statistics education." ], "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 outlines a meta-analysis the authors plan to conduct of all studies meeting certain criteria for the experimental realm in parapsychology called “ganzfeld.” The first ganzfeld experiments were conducted in the early 1970s. There have been multiple meta-analyses of ganzfeld studies over the years, but none have covered the entire research period, as the authors plan to do with this one. In addition to estimating an overall effect size, the proposed meta-analysis will examine two additional questions. One is whether the timing and/or participation of a “sender” makes a difference. This question will be examined by classifying the sessions into one of 3 types – target selected after the session, target selected before the session but with no sender, target selected before the session and a sender used. The other question of interest is whether there is a difference in effect size when the participant in the session was selected for characteristics thought to enhance performance.\nThe authors are to be commended for addressing so many different issues that arise in meta-analysis, and for planning to use both frequentist and Bayesian methods. However, there are some details missing from the report that led to my answer of “partially provided” for the question about sufficient details to allow replication by others. Here are some details that are not provided as completely as needed in the paper if someone were to try to replicate their analyses. (It’s possible that they are provided in one of the references on protocols and reporting of meta-analysis, but not in the paper.)\nWill effect sizes be weighted by the size of the study? Obviously they should be. It makes no sense to give equal weight to a study with n = 20 and n = 100. But it isn’t clear in the methodology part of the report how the effect sizes will be combined.\n\nWill any measure of study quality be used? Some of the earlier studies were criticized for possible methodological problems. Or will studies that don’t meet certain quality criteria be omitted? Or is the plan to omit the studies that didn’t use proper randomization methods sufficient?\n\nWill studies that did not use standard targets (photographs, videos, locations) be excluded? For instance, at least one study used music instead of photographs or videos. Those probably should be excluded, because they represent possibly testing a different ability.\n\nThe reference to using Hedges g to reduce bias for small studies is not clear. Hedge’s g is usually used for comparing means.\n\nIt is not clear exactly what effect size measure will be used, but if I understand it correctly, it will be z/√n where z is found using the normal approximation to the binomial with continuity correction. Although that method gives results very close to using an exact binomial probability for sample sizes of perhaps 20 or more, it may not work well for small sample sizes. In fact the computation website mentioned in the report (http://vassarstats.net/binomialX.html) won’t even compute z if either np or nq is less than 5. In such cases, an effect size could be found by using the exact binomial p-value, then finding the inverse normal z that gives that area in the upper tail. There is an effect size measure specially intended for proportions (Cohen’s h) but it may not be applicable if a study uses ratings instead of direct hits.\n\nIt isn’t clear how the three types of studies will be compared. Will analysis of variance be used? Or, as mentioned, only looking at 95% confidence intervals for each type?\n\nHave the authors pre-specified sufficient outcome-neutral tests for ensuring that the results obtained can test the stated hypotheses, including positive controls and quality checks? Yes\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6024", "date": "21 Oct 2020", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for your comments and suggestions. Here follows our replies: Will effect sizes be weighted by the size of the study? Obviously they should be. It makes no sense to give equal weight to a study with n = 20 and n = 100. But it isn’t clear in the methodology part of the report how the effect sizes will be combined. Reply: the random-effect model explained in the “Frequentist random-effect model” paragraph. weights the studies by using the inverse of their variance plus an estimate of the heterogeneity of the studies τ2 ; wi=1/(τ2+vi) Will any measure of study quality be used? Some of the earlier studies were criticized for possible methodological problems. Or will studies that don’t meet certain quality criteria be omitted? Or is the plan to omit the studies that didn’t use proper randomization methods sufficient? Reply: In our 2010 and 2020 meta-analyses we assessed study quality using two judges whose ratings were highly correlated. We did not find a statistically significant correlation between study quality and ES. As to the proper randomization methods, our oldest studies applied the proper randomisation according to the Honorton-Hyman’s joint communiqué.As a new way to test the correlation between study quality and ES we added a comparison between the studies published in journals with a full peer-review and the studies published in conference proceedings that usually have a less complete peer-review. Will studies that did not use standard targets (photographs, videos, locations) be excluded? For instance, at least one study used music instead of photographs or videos. Those probably should be excluded, because they represent possibly testing a different ability. Reply: There are no theoretical reasons why targets different from images, pictures or video clips, cannot be used. We could assess whether their use will generate ES outliers.We have assessed dynamic vs. static vs. objects/music in our 2020 study (no statitstical differences in ES). Objects/music category is a heterogeneous group, but ES for the single musical target study is not statistical different to the ES for objects. The reference to using Hedges g to reduce bias for small studies is not clear. Hedge’s g is usually used for comparing means. Reply: Hedges’ g can be applied to all continuous effect size like Cohen’s d independently from the experimental design (e.g. one and two-groups) see Borenstein et al (2009) pag. 30 It is not clear exactly what effect size measure will be used, but if I understand it correctly, it will be z/√n where z is found using the normal approximation to the binomial with continuity correction. Although that method gives results very close to using an exact binomial probability for sample sizes of perhaps 20 or more, it may not work well for small sample sizes. In fact the computation website mentioned in the report (http://vassarstats.net/binomialX.html) won’t even compute z if either np or nq is less than 5. In such cases, an effect size could be found by using the exact binomial p-value, then finding the inverse normal z that gives that area in the upper tail. There is an effect size measure specially intended for proportions (Cohen’s h) but it may not be applicable if a study uses ratings instead of direct hits. Reply: In the effect size measures paragraph, we added where that is the case, we will use wolframalpha calculator available online: https://www.wolframalpha.com/widgets/gallery/view.jsp?id=540d8e149b5e7de92553fdd7b1093f6d It isn’t clear how the three types of studies will be compared. Will analysis of variance be used? Or, as mentioned, only looking at 95% confidence intervals for each type? Reply: I the “Moderator effects” paragraph we added we will compare the moderators effect by comparing the overlap of their 95% CIs and with a focused hypothesis testing statistic e.g. ANOVA." } ] }, { "id": "70878", "date": "05 Oct 2020", "name": "Julia Haaf", "expertise": [ "Reviewer Expertise Quantitative and mathematical psychology", "expertise in Bayesian statistics", "multilevel modeling and meta-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\nIn this stage 1 registered report the authors describe a planned meta-analysis to target the Ganzfeld effect as is found in the parapsychological literature. Summarizing all conducted studies on the topic seemingly is a relevant research objective, though I might add that I do wonder about the quality of the conducted studies and their reporting. While the authors plan to conduct publication bias correction, to this point it is virtually impossible to fully account and correct for all the biases baked into the literature, let alone the parapsychological literature. I have a few comments on the statistical analysis, but some of these require some additional work by the authors.\n\nEffect size measure of interest. The authors plan to report effect size measures based on the Binomial test. The binomial z-score seems like an appropriate choice given the models they plan to use. I wonder, however, why the authors decided to divide the binomial z-score by the square root of n, the sample size, given that the binomial z is calculated from the binomial mean and standard deviation, both dependent on n. In addition, if the binomial z corresponds to Fisher’s z then we know the standard error is 1/sqrt(n – 3). What is the standard error for z/sqrt(n), and how do we transform it to the standard error of Hedge’s g, as planned by the authors? Both frequentist and Bayesian meta-analysis requires the calculation of standard errors to weigh the study effects which is how meta-analysis accounts for sample size/precision. This point must be addressed to make the article scientifically sound.\n\nRandom model or random-effects model. The authors plan to use a model to account for between-study heterogeneity. In the meta-analytic literature, these models are called random-effects models (not random models). The wording of fixed-effect model vs. random-effects model from this literature is a bit unfortunate because it does not correspond to what is typically considered fixed vs. random effects in the statistical literature (Gelman, 2004, p. 20)1. It might be better to describe the so-called random-effects model simply as a model accounting for between-study heterogeneity.\n\nBayesian model with ordinal constraints. The authors reference Rouder et al. (2019). I think this reference is perhaps misplaced. It does not really correspond to the sentence. Rouder et al. propose instead of interpreting the mean effect size across studies to focus on the distribution of true effect sizes. Therefore, the ordinal constraint is placed on each study’s true effect simultaneously. The way the authors describe it they plan to (only) apply an ordinal constraint on the overall effect. If the authors are interested in the question of whether all studies show an effect in the same direction, and I think this would be an interesting question for this application, I might shamelessly refer them to some of my recent work in this area (Haaf & Rouder, preprint)2.\n\nPriors. If the authors want to use a model that accounts for between-study heterogeneity (aka a random-effects model) they need to specify an additional prior distribution on that heterogeneity parameter. I would suggest adding this prior to this stage of the registered report. In the Haaf & Rouder preprint I mentioned above there are suggestions for priors on Fisher’s z that might be useful here. This point must be addressed to make the article scientifically sound.\n\nWhich publication bias correction method is the best? The authors plan to implement three ways of correcting for publication bias. If the three methods diverge in results, how will they interpret the results? Is there an ordering of method quality, or a way of combining them? Additionally, there have been newer development on publication bias corrections for Bayesian meta-analysis (Maier et al., preprint)3. Maybe this is also an option.\n\nI really like the idea of a cumulative meta-analysis for this application! In Jasp (JASP Team, 2020) there is also an option to apply a cumulative Bayesian meta-analysis, maybe as a nice addition.\n\nHave the authors pre-specified sufficient outcome-neutral tests for ensuring that the results obtained can test the stated hypotheses, including positive controls and quality checks? Partly\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6025", "date": "21 Oct 2020", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for your comments and suggestions. Here follows our replies; Effect size measure of interest. The authors plan to report effect size measures based on the Binomial test. The binomial z-score seems like an appropriate choice given the models they plan to use. I wonder, however, why the authors decided to divide the binomial z-score by the square root of n, the sample size, given that the binomial z is calculated from the binomial mean and standard deviation, both dependent on n. In addition, if the binomial z corresponds to Fisher’s z then we know the standard error is 1/sqrt(n – 3). What is the standard error for z/sqrt(n), and how do we transform it to the standard error of Hedge’s g, as planned by the authors? Both frequentist and Bayesian meta-analysis requires the calculation of standard errors to weigh the study effects which is how meta-analysis accounts for sample size/precision. This point must be addressed to make the article scientifically sound. Reply: In the paragraph “Effect size measures” we have added how the effect sizes standard errors will be computed and how both will be transformed with the Hedges’s formulas. Random model or random-effects model. The authors plan to use a model to account for between-study heterogeneity. In the meta-analytic literature, these models are called random-effects models (not random models). The wording of fixed-effect model vs. random-effects model from this literature is a bit unfortunate because it does not correspond to what is typically considered fixed vs. random effects in the statistical literature (Gelman, 2004, p. 20)1. It might be better to describe the so-called random-effects model simply as a model accounting for between-study heterogeneity. Reply: In the “Overall effect size estimation”, we have added this clarification Bayesian model with ordinal constraints. The authors reference Rouder et al. (2019). I think this reference is perhaps misplaced. It does not really correspond to the sentence. Rouder et al. propose instead of interpreting the mean effect size across studies to focus on the distribution of true effect sizes. Therefore, the ordinal constraint is placed on each study’s true effect simultaneously. The way the authors describe it they plan to (only) apply an ordinal constraint on the overall effect. If the authors are interested in the question of whether all studies show an effect in the same direction, and I think this would be an interesting question for this application, I might shamelessly refer them to some of my recent work in this area (Haaf & Rouder, preprint)2. Reply: In the “Bayesian random-effect model” paragraph we have corrected this reference. Priors. If the authors want to use a model that accounts for between-study heterogeneity (aka a random-effects model) they need to specify an additional prior distribution on that heterogeneity parameter. I would suggest adding this prior to this stage of the registered report. In the Haaf & Rouder preprint I mentioned above there are suggestions for priors on Fisher’s z that might be useful here. This point must be addressed to make the article scientifically sound. Reply: In the “Bayesian random-effect model” paragraph we have added the tau parameter prior distribution (already available in the Syntax details file). Which publication bias correction method is the best? The authors plan to implement three ways of correcting for publication bias. If the three methods diverge in results, how will they interpret the results? Is there an ordering of method quality, or a way of combining them? Additionally, there have been newer development on publication bias corrections for Bayesian meta-analysis (Maier et al., preprint)3. Maybe this is also an option. Reply: The available literature hasn’t found yet the “best” publication bias for all conditions. We will analyse the robustness of our findings comparing the results of all the publication bias tests. As a fourth test, we will add the RoBMA test as suggested. I really like the idea of a cumulative meta-analysis for this application! In Jasp (JASP Team, 2020) there is also an option to apply a cumulative Bayesian meta-analysis, maybe as a nice addition. Reply: as a further test of the decline effect we will perform a meta-regression analysis using “Year of publication” as covariate (see “ Cumulative meta-analysis” paragraph)." } ] } ]	1	https://f1000research.com/articles/9-826
https://f1000research.com/articles/9-1201/v1	06 Oct 20	{ "type": "Research Article", "title": "Increase in public interest concerning alternative medicine during the COVID-19 pandemic in Indonesia: a Google Trends study", "authors": [ "Dewi Rokhmah", "Khaidar Ali", "Serius Miliyani Dwi Putri", "Khoiron Khoiron", "Khaidar Ali", "Serius Miliyani Dwi Putri", "Khoiron Khoiron" ], "abstract": "Background: The COVID-19 pandemic has triggered individuals to increase their healthy behaviour in order to prevent transmission, including improving their immunity potentially through the use of alternative medicines. This study aimed to examine public interest on alternative medicine during the COVID-19 pandemic using Google Trends in Indonesia. Methods: Employing a quantitative study, the Spearman rank test was used to analyze the correlation between Google Relative Search Volume (RSV) of various search terms, within the categories of alternative medicine, herbal medicine and practical activity, with COVID-19 cases. In addition, time lag correlation was also investigated. Results: Public interest toward alternative medicine during COVID-19 pandemic in Indonesia is dramatically escalating. All search term categories (alternative medicine, medical herbal, and alternative medicine activities) were positively associated with COVID-19 cases (p<0.05). The terms ‘ginger’ (r=0.6376), ‘curcumin’ (r=0.6550) and ‘planting ginger’ (0.6713) had the strongest correlation. Furthermore, time lag correlation between COVID-19 and Google RSV was also positively significant (p<0.05). Conclusion: Public interest concerning alternative medicine related terms dramatically increased after the first COVID-19 confirmed case was reported in Indonesia. Time lag correlation showed good performance using weekly data. The Indonesian Government will play an important role to provide and monitor information related to alternative medicine in order for the population to receive the maximum benefit.", "keywords": [ "COVID-19", "alternative medicine", "pandemic", "search activity" ], "content": "Introduction\n\nThe COVID-19 pandemic is a massive health crisis worldwide. Within seven months, it has affected 216 countries, and more than 11 million population have been infected by the SARS-COV-2 virus, which causes COVID-191. In Indonesia, COVID-19 transmission has been reported in all provinces, with 68,226 confirmed cases recorded by July 8th 20202. The World Health Organization (WHO) noted that Indonesia is the third country with largest number of cases in South East Asia3. Therefore, appropriate action is urgently needed to halt COVID-19 transmission among the public.\n\nEffenberger et al.4 noted that the high virulence of SARS-COV-2 contributes to the super-spread of COVID-19. In addition, the large number of asymptomatic cases catalyze the intensity of the transmission among population. Currently, no vaccine has been developed for COVID-19. The pandemic has triggered a large-scale behavior change among the global population to protect their health5. This may include an increase of public interest concerning alternative medicine.\n\nAlternative medicine in Indonesia is called Jamu and is well-known. It is commonly composed by herbal medicines, such as ginger and curcumin, which are extracted and added to water to be drinkable. Both ingredients and other methods of Jamu are accessible and available to the general population of Indonesia. Jamu is commonly used to preserve immunity. Therefore, this study aimed to examine public interest concerning alternative medicines in Indonesia during the COVID-19 pandemic. Time lag scenarios were also investigated.\n\n\nMethods\n\nThis was a quantitative study using secondary data from Indonesia. The data was obtained from Google Trends using Google Relative Search Volume (RSV) and COVID-19 case data. Google RSV presents information on how many terms have been searched at a particular time using the Google search engine, i.e. the data provides information about public interest towards a particular term6. A high RSV (maximum 100 points) indicates high public interest; while the lowest (0 points) shows an absence of public interest7. In this study, COVID-19 cases were defined as laboratory-confirmed cases positive for SARS-COV-2 virus as reported by the Indonesian Government. The data were retrieved from January 1st 2019 to June 6th 2020 weekly (total of 74 weeks; 2019: weeks 1–52, 2020: weeks 53–74).\n\nData for confirmed cases of COVID-19 nationwide were collected from the Indonesian Ministry of Health (MoH), where COVID-19 cases are reported daily (https://www.kemkes.go.id/article/view/20031900002/Dashboard-Data-KasusCOVID-19-di-Indonesia.html).\n\nGoogle RSV data for Indonesia were collected from Google Trends (www.trends.google.com) with web search as default option8. Search terms were divided into three categories with subterms in each of the categories as follows: 1) alternative medicine (‘Jamu’ [alternative medicine]; 2) herbal medicine (‘tanaman obat’ [herbal medicine], ‘jahe’ [ginger], ‘kunyit’ [curcumin]); and 3) alternative medicine activities (‘cara membuat jamu’ [how to make jamu], ‘membuat jamu’ [make jamu], ‘menanam tanaman obat’ [planting herbal medicines], ‘menanam jahe’ [planting ginger], ‘menanam kunyit’ [planting curcumin]).\n\nThe first category ‘Jamu’ was employed to recognize public interest toward alternative medicine during the pandemic in Indonesia; as stated before ‘Jamu’ is traditional alternative medicine in Indonesia used for maintaining and improving immunity. The second category (herbal medicine) was used to understand public interest on the types of medical plants being used. According to Salim and Munadi9, the production of ginger and curcumin in Indonesia was the highest compared to other medicinal plants, where the consumption trend during 2011–2015 increased by 21.95% and 5.92%, respectively. Moreover, the Statistics Office of Indonesia recorded that the total harvest of ginger and curcumin on 2018 is the largest in Indonesia10. Therefore, search terms of ‘jahe’ [ginger] and ‘kunyit’ [curcumin] was selected in the second category. The third category (alternative medicine activities) collected information about public interest toward performing Jamu and planting herbal medicines.\n\nThis study followed the methodology of previous studies7,11. After checking and cleaning the data, there was no missing data noted. The data was stored in Microsoft Excel 2010, and then transferred to STATA v13 (College Station, TX, USA) for analysis. Google RSV data was available weekly, and therefore COVID-19 case data was also analyzed weekly.\n\nThe data was not normally distributed, so Spearman rank test was used to examine the correlation between Google RSV and COVID-19 cases. Time lag correlation between Google RSV and COVID-19 was also analyzed, where the procedure referred to Husnayain et al.11 and Torres-Reyne12. The significance level was set at 0.05.\n\n\nResults\n\nThe pattern of COVID-19 case and Google RSV in Indonesia is visualized in Figure 1. Since the first confirmed COVID-19 case was reported in Indonesia on March 2nd 2020 (week 61 of this study), COVID-19 cases have been increasing in Indonesia. According to the MoH, 30,514 confirmed cases of COVID-19 were reported during 14 weeks (March 2nd–June 6th 2020); mean weekly cases were recorded as ~315 cases.\n\nCOVID-19 cases compared with (A) ‘Jamu’ [alternative medicine] search term; (B) herbal medicine search terms (‘tanaman obat’ [herbal medicine], ‘jahe’ [ginger], ‘kunyit’ [curcumin]); (C) alternative medicine activities search terms (‘cara membuat jamu’ [how to make jamu], ‘membuat jamu’ [make jamu], ‘menanam tanaman obat’ [planting herbal medicines], ‘menanam jahe’ [planting ginger], and ‘menanam kunyit’ [planting curcumin]). Letters: A, January 30th 2020: COVID-19 declared as Public Health Emergency of International Concern; B, March 2nd 2020: first imported case was reported in Indonesia; C, March 16th 2020: social distancing declared by Indonesian Government.\n\nRSV of ‘Jamu’ [alternative medicine] from week 1 until week 60 was 40–60 points, with search activity increasing from week 61 (March 1st-7th 2020). The highest RSV score for this search term was in week 63 with 100 points (Figure 1A). The RSV of ‘tanaman obat’ [herbal medicine], ‘jahe’ [ginger], and ‘kunyit’ [curcumin] before the pandemic (week 1–60) was 19–49 points, with the RSV dramatically increasing from week 61 (42–79 points). The peak for all herbal medicine search terms was found in week 64 (100 points) (Figure 1B).\n\nA similar trend is shown for alternative medicine activities search terms (Figure 1C). Before the pandemic (week 1–60) these terms had an RSV of 0–36 points. In week 61, the RSV increases ~2 fold higher. The term ‘cara membuat jamu’ [how to create jamu] and ‘membuat jamu’ [create jamu] reached their peak on week 63 (100 points) and 64 (100 points), respectively. Meanwhile, the peak for ‘menanam jahe’ [planting ginger] and ‘menanam kunyit’ [planting curcumin] was recorded on week 65 and week 63, respectively, with 100 points. The peak for ‘menanam tanaman obat’ [planting herbal medicines] reached its peak on week 63 (similar to ‘cara membuat jamu’ [how to create jamu]) with the highest score of 48 points.\n\nTable 1 displays the correlation between COVID-19 cases and Google RSV in Indonesia. All search term categories (alternative medicine, herbal medicine, and alternative medicine activities) are positively correlated with COVID-19 cases (p<0.05). The terms ‘jahe’ [ginger] (r=0.6376), ‘kunyit’ [curcumin] (r=0.6550) and ‘menanam jahe’ [planting ginger] (r=0.6713) have the strongest correlation towards COVID-19 new cases in Indonesia. Based on a time lag scenario, the correlation between COVID-19 cases and Google RSV showed good performance with weekly data, where all search terms are significant (p<0.05). In the time lag scenario, a strong correlation is also found for the terms ‘jahe’ [ginger], ‘kunyit’ [curcumin], and ‘menanam jahe’ [planting ginger] (r>0.6; p<0.05).\n\nsignificant (p<0.05); *significant (p<0.01)\n\n\nDiscussion\n\nSince the first COVID-19 confirmed case was reported on March 2nd 2020 (week 61), there have been a dramatic increases in cases in Indonesia. The mean weekly cases of COVID-19 is ~315 case (Figure 1), and we noted the highest case load reported on week 74 (4741 cases). We also show in our data that COVID-19 cases in Indonesia have increased by ~305% within 14 weeks (30,514 cases; Figure 1). This indicates a super-spread of COVID-19 in Indonesia. The high population and population mobility may take an essential role in intense COVID-19 transmission13,14.\n\nAlternative medicine is one option for individuals to maintain and increase their immunity during the COVID-19 pandemic. In our study, we found that the search activity of alternative medicine-related terms, including herbal medicine and activities surrounding alternative medicine, was low and steady before the pandemic (weeks 1–60). This was even though a Public Health Emergency of International Concern had been declared by the WHO on January 30th 2020 (week 56). Interestingly, only after the first COVID-19 confirmed case in Indonesia was announced on week 61 did the search activity dramatically increased. Most of the search terms looked at in this study reached their peak on week 63–64, after which social distancing issue has been established in Indonesia (on March 16th 2020)15. The alternative medicine issue also appeared among the public around March 13th – 16th (week 63) during the pandemic. In this period, the President of Indonesia claimed that herbs can fight COVID-19, which may have increased public interest toward alternative medicine16.\n\nIn this study, all search terms were associated positively with COVID-19 cases in Indonesia (p<0.05). This indicated that increasing COVID-19 cases elevated the public interest concerning alternative medicine. A similar result was also shown with the time lag scenario, where all search terms were positively associated with COVID-19 cases (p<0.05). This finding shows that there was an increase in search activities 1–3 weeks after and before the increase of COVID-19 cases in Indonesia. However, a strong correlation is detected at the present time (lag 0), particularly for the herbal medicine category. This study found that correlation analysis using weekly data of Google RSV compared to COVID-19 new cases in Indonesia showed good performance, which is collaborated by previous studies7,17–19.\n\nThe trend of Google RSV for all search terms was higher during the pandemic. This indicates increasing public interest toward alternative medicine during the pandemic in Indonesia. Wise et al.20 noted that awareness of the public related to the COVID-19 pandemic is elevated due to the risk posed by the virus, and the large number of available information sources serves to reinforce their protective behavior. Galankis21 also reported that the public tend to search for information related to health either short- or long-term during the pandemic.\n\nThe Indonesian Government plays an important role in the high public interest toward alternative medicine during the pandemic. Actions concerning monitoring and providing valid information regarding alternative medicine to the public are urgently needed. These actions should prevent misuse of medical herbal among the public. In addition, information could be used to empower communities to provide self-remedial source at a household level, such as planting herbal medicines.\n\n\nConclusion\n\nPublic interest on alternative medicine related-terms has dramatically increased during the COVID-19 pandemic in Indonesia. Search terms relating to alternative medicine, herbal medicines and activities surrounding alternative medicines correlate positively with an increase of COVID-19 cases in Indonesia. This study recommends that the Indonesian Government take an active role in informing the public about alternative medicines, and monitoring and providing valid information. This may empower households to produce medical herbs independently.\n\n\nData availability\n\nCOVID-19 case data available from: https://www.kemkes.go.id/article/view/20031900002/Dashboard-Data-KasusCOVID-19-di-Indonesia.html\n\nGoogle Trend data available from: https://trends.google.com/. Search terms and other parameters are provided in the text.\n\nMendeley: Public interest on alternative medicine during pandemic in Indonesia, http://dx.doi.org/10.17632/fv7tprb24j.122.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nWorld Health Organization: Coronavirus disease (COVID-19) Pandemic. 2020; (Accessed on 10 July 2020). Reference Source\n\nKemenkes: Dashboard data kasus COVID-19 di Indonesia. 2020; (Accessed on 10 July 2020). Reference Source\n\nWorld Health Organization: Coronavirus disease (COVID-19) situation report-170. Geneva: World Health Organization. 2020. Reference Source\n\nEffenberger M, Kronbichler A, Shin JI, et al.: Association of the COVID-19 pandemic with Internet Search Volumes: A Google TrendsTM Analysis. Int J Infect Dis. 2020; 95: 192–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Bavel JJ, Baicker K, Boggio PS, et al.: Using social and behavioural science to support COVID-19 pandemic response. Nat Hum Behav. 2020; 4(5): 460–471. PubMed Abstract \| Publisher Full Text\n\nHeerfordt C, Heefordt LM: Has there been an increased interest in smoking cessation during the first months of the COVID-19 pandemic? A Google Trends study. Public Health. 2020; 183: 6–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHusnayain A, Fuad A, Su ECY: Applications of Google Search Trends for risk communication in infectious disease management: A case study of the COVID-19 outbreak in Taiwan. Int J Infect Dis. 2020; 95: 221–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMavragani A, Ochoa G: Google trends in infodemiology and infoveillance: methodology framework. JMIR Public Health Surveill. 2020; 5(2): e13439. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalim Z, Munadi E: Info komoditi tanaman obat (Information of herbal medicine commodity). Jakarta: Kementerian Perdagangan Republik Indonesia. 2017. Reference Source\n\nBadan Pusat Statistik: Statistics of medicinal plants of Indonesia. Jakarta: Badan Pusat Statistik Indonesia. 2019. Reference Source\n\nHusnayain A, Fuad A, Lazuardi L: Correlation between Google Trends on dengue fever and national surveillance report in Indonesia. Glob Health Action. 2019; 12(1): 1552652. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTorres-Reyne O: Time series. Data and statistical services. Princeton university. 2013; (Accessed on 5th August 2020). Reference Source\n\nKhairat S, Meng C, Xu Y, et al.: Interpreting COVID-19 and Virtual Care Trends: Cohort Study. JMIR Public Health Surveill. 2020; 6(2): e18811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocklöv J, Sjödin H: High population densities catalyse the spread of COVID-19. J Travel Med. 2020; 27(3): taaa038. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCahya GM: Stay home, President says. The Jakarta Post. 2020; (Accessed on 10 July 2020). Reference Source\n\nThe Star: Indonesia president Jokowi claims herbs can fight COVID-19. 2020; (Accessed on 10 July 2020). Reference Source\n\nLin YH, Liu CH, Chiu YC: Google searches for the keywords of “wash hands” predict the speed of national spread of COVID-19 outbreak among 21 countries. Brain Behav Immun. 2020; 87: 30–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMavragani A: Tracking COVID-19 in Europe: Infodemiology Approach. JMIR Public Health Surveill. 2020; 6(2): e18941. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyyoubzadeh SM, Ayyoybzadeh SM, Zahedi H, et al.: Predicting COVID-19 Incidence through analysis of google trends data in Iran: data mining and deep learning pilot study. JMIR Public Health Surveill. 2020; 6(2): e18828. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWise T, Zbozinek T, Michelini G, et al.: Changes in risk perception and protective behavior during the first week of the COVID-19 pandemic in the United States. PsyArXiv. 2020. Publisher Full Text\n\nGalanakis CM: The food systems in the era of the coronavirus (COVID-19) pandemic Crisis. Foods. 2020; 9(4): 523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli K: Public interest on alternative medicine during pandemic in Indonesia. Mendeley Data, v1. 2020. http://www.doi.org/10.17632/fv7tprb24j.1" }	[ { "id": "73844", "date": "11 Nov 2020", "name": "Lanjing Zhang", "expertise": [ "Reviewer Expertise clinical epidemiology", "statistical methodology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article focused on the links between google search trend and daily incidence of COVID-19 in Indonesia. The findings appear novel since my search of the literature shows no similar works in the Pubmed. However, I have the following concerns:\nMajor:\n\nThe correlation coefficients appeared moderate (about 0.5-0.6). This low degree of correlation should be addressed. One of the approaches is to correlate the keyword with the google trend. If such a correlation is moderate (in Indonesia), the correlation coefficient become acceptable. Of course, some discussions are needed even so.\n\nLiterature review is less comprehensive. It could be improved by citing related articles 1,2,3 and others.\n\nThe authors may compare the trends of search interest in these alternative medicine terms, whose change may have a better correlation with the COVID-19 incidence trends.\n\nDo the symptoms correct with COVID-19 daily incidence? They may correlate better than these alternative medicine terms.\n\nNot sure why Spearman ranked test was used. In my view, Pearson's rho may be a better choice.\n\nMinor:\nIt will be more helpful if the authors could discuss more on how direct use of these trends could improve disease surveillance and prevention.\n\nDetailed p values should be provided even they are smaller than .05.\n\nThe case number is probably meant daily new cases or daily incidence. Please use the more precise terms.\n\nThe total case number should be updated since the one in the text is published 4 months. The number may have been doubled.\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": [ { "c_id": "6355", "date": "25 Feb 2021", "name": "Dewi Rokhmah", "role": "Author Response", "response": "Dear Dr. Lanjing Zhang, Thank you for your constructive review. This is our response: Major: 1. The correlation coefficients appeared moderate (about 0.5-0.6). This low degree of correlation should be addressed. One of the approaches is to correlate the keyword with the google trend. If such a correlation is moderate (in Indonesia), the correlation coefficient become acceptable. Of course, some discussions are needed even so. Response: Sure. In the latest version, we informed this condition in discussion section. 2. Literature review is less comprehensive. It could be improved by citing related articles 1,2,3 and others. Response: Sure. Thank you for your suggestion. 3. The authors may compare the trends of search interest in these alternative medicine terms, whose change may have a better correlation with the COVID-19 incidence trends. Response: Sure, it is. In this article, we classified the terms of alternative medicine into three categories in order to obtain proper information about public interest on alternative medicine during pandemic, namely: 1) alternative medicine, 2) herbal medicine, and 3) alternative medicine activities. Group 1 (alternative medicine) represent the term of alternative medicine itself in Bahasa or synonym (“jamu” [alternative medicine]). Group 2 (herbal medicine) represent the term of the type of herbal medicine that commonly used in public (tanaman obat’ [herbal medicine], ‘jahe’ [ginger], ‘kunyit’ [curcumin]). Group 3 (alternative medicine activities) represent the term of the public activities to provide or create alternative medicine ((‘cara membuat jamu’ [how to make jamu], ‘membuat jamu’ [make jamu], ‘menanam tanaman obat’ [planting herbal medicines], ‘menanam jahe’ [planting ginger], ‘menanam kunyit’ [planting curcumin]). The author performed not only statistical test, but also graphical analysis in this study. The statistical test was used to assess the correlation between alternative medicine terms to COVID-19 daily case. On the other hand, graphical analysis was also performed to analyse the trend of public search/ public interest on alternative medicine terms to COVID-19 daily case. In addition, graphical analysis could also compare the trend of search interest in the alternative medicine term. 4. Do the symptoms correct with COVID-19 daily incidence? They may correlate better than these alternative medicine terms. Response: As we mentioned in the article, the objective of the study is to analyze the public interest on alternative medicine during COVID-19 pandemic, in which alternative medicine is well-known in Indonesia. The hypothesis of this study is the public interest should be rising during pandemic condition, where this condition occur related to health seeking behaviour among community during pandemic. In addition, Google Trend study is used to obtain information of public interest. This study is necessary, where the study supply information about public interest on alternative medicine to Indonesian Government during pandemic, where Indonesian Government may play important role to provide and monitor appropriate information related to the use of alternative medicine during COVID-19 pandemic. 5. Not sure why Spearman ranked test was used. In my view, Pearson's rho may be a better choice. Response: In this article, we used spearman ranked test due to the result of normality test of the data, where the data is not normally distributed. Therefore, spearman rank test is fit to assess the correlation between dependent variable to independent variable1. In addition, if we referred to Mavragani et al (2018)2 in her systematic review of Google Trend study, the spearman rank test is also the second most used to examine the correlation in Google Trends study. 1 Sarmento, D. no year. Chapter 22: Correlation Types and When to Use Them. Available: https://ademos.people.uic.edu/Chapter22.html#22_spearman_correlation (accessed on 10-02-2021) 2 Mavragani, A., Ochoa, G., Tsagarakis, K.P. 2018. Assessing the methods, tools, and statistical approaches in google trends research: systematic review. Journal of Medical Internet. 20(11):e270 Minor: 1. It will be more helpful if the authors could discuss more on how direct use of these trends could improve disease surveillance and prevention. Response: Sure, this recommendation has been applied in this study, where we added related information in discussion section 2. Detailed p values should be provided even they are smaller than .05. Response: Sure. According to statistical test, the range of p-value was between 0.0194 – 0.0000. But, mostly the p-value score is 0.0000. Therefore, we create 4 groups/classifications in order to present the specific p-value: * : p<0.05 : p<0.01 * : p<0.001 **** : p <0.0001 3. The case number is probably meant daily new cases or daily incidence. Please use the more precise terms. Response: The case number refers to total daily case of COVID-19 in Indonesia. In the latest version of our article, we insert this information in methodology. 4. The total case number should be updated since the one in the text is published 4 months. The number may have been doubled. Response: This issue become limitation of our study, and we had declared this limitation on discussion section in which the author also mentioned recommendation for further study." } ] }, { "id": "78462", "date": "05 Feb 2021", "name": "Seyed Mohammad Ayyoubzadeh", "expertise": [ "Reviewer Expertise Medical Informatics", "eHealth", "mHealth", "IoT", "smartphone apps", "data mining", "CDSS" ], "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 analyzed if the public interest in alternative medicine had changed during the COVID-19 pandemic in Indonesia using Google Search data provided by Google Trends. The authors used the Spearman rank test for performing the statistical test. The result showed that the public interest in alternative medicine has increased during COVID-19.\nThe study has cited related literature analyzing Google Trends in COVID-19. However, It will be great if the authors could add some references about alternative medicine in the introduction section.\nThe study design seems appropriate, choosing a reasonable time range for the analysis and performing a suitable statistical test for the analysis.\nPlease remove the sentence \"Currently, no vaccine has been developed for COVID-19\".\nThe URL for google trends is \"https://trends.google.com/\" not \"www.trends.google.com\" mentioned in the data source.\nI couldn't find the data from the mentioned URL: https://www.kemkes.go.id/article/view/20031900002/Dashboard-Data-KasusCOVID-19-di-Indonesia.html\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": "6354", "date": "25 Feb 2021", "name": "Dewi Rokhmah", "role": "Author Response", "response": "Dear Dr. Seyed Mohammad Ayyoubzadeh, Thank you for your constructive review. This is our feedback for your review: 1. We had added some references about alternative medicine in the introduction section. 2. The sentence “Currently, no vaccine has been developed for COVID-19” has been remove in the article. 3. We updated the data source of google trend in the article to \"https://trends.google.com/\" 4. The following website (https://www.kemkes.go.id/article/view/20031900002/Dashboard-Data-KasusCOVID-19-di-Indonesia.html) is the oldest version of surveillance COVID-19 website from Indonesian Ministry of Health (MoH). The data of COVID-19 case in Indonesia is also found in https://covid19.go.id/peta-sebaran, which is the data is integrated with the Indonesian Ministry of Health (MoH). The website (https://covid19.go.id/peta-sebaran) itself is published by COVID-19 Response Acceleration Task Force of Indonesia (RATF) that is directly created by Indonesian President to combat COVID-19 in Indonesia. Therefore, in order to create accessible data source in this article, we consider to use the data from RATF which is the data is integrate with previous website from MoH." } ] }, { "id": "78465", "date": "11 Feb 2021", "name": "Sinan Kardeş", "expertise": [ "Reviewer Expertise Balneotherapy", "Rheumatic and Musculoskeletal Diseases", "Google Trends" ], "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 important study. Congratulations. I have few points:\nPlease add a paragraph mentioning Google Trends is used in COVID-19 studies to highlight its usefulness in health studies. You may refer following papers 1,2,3.\n\nIn Methods: Please add when the Google Trends database searched: E.g. On 11 February 2021, Google Trends data were retrieved from...\n\nIn Methods: Google Trends search: The time period was mentioned; but please mention the other filters: country (Indonesia?), categories (All categories or Health?).\n\nr > 0.7 indicates strong correlation. Please mention this and revise thoroughly the manuscript particularly for the word 'strong correlation'.\n\nIn the Results: Please change the name of subheading \"Statistical analysis\". It was generally used in the Methods section. You may change \"Correlation analysis results\", or something appropriate.\n\nFor your further studies, I suggest to consider performing time series analysis.\n\nPlease discuss limitations of the study. The authors may refer following papers for limitations 4,5.\n\nConclusion: Please add \"early\": \"during the early COVID-19 pandemic\".\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": "6353", "date": "25 Feb 2021", "name": "Dewi Rokhmah", "role": "Author Response", "response": "Dear Dr. Sinan Kardeş, Thanks for your constructive review. This is our feedback for your review. 1. Please add a paragraph mentioning Google Trends is used in COVID-19 studies to highlight its usefulness in health studies. You may refer following papers 1,2,3. Response: Sure, we have added the following literature as our references (discussion section). Thank you for your suggestion 2. In Methods: Please add when the Google Trends database searched: E.g. On 11 February 2021, Google Trends data were retrieved from... Response: Sure, we applied this recommendation. 3. In Methods: Google Trends search: The time period was mentioned; but please mention the other filters: country (Indonesia?), categories (All categories or Health?). Response: Sure, we applied this recommendation 4. r > 0.7 indicates strong correlation. Please mention this and revise thoroughly the manuscript particularly for the word 'strong correlation'. Response: Sure. 5. In the Results: Please change the name of subheading \"Statistical analysis\". It was generally used in the Methods section. You may change \"Correlation analysis results\", or something appropriate. Response: Sure. 6. For your further studies, I suggest to consider performing time series analysis. Response: Sure, it become our recommendation for further study. Thank you for your suggestion. 7. Please discuss limitations of the study. The authors may refer following papers for limitations 4,5. Response: Thank you. We had added this literature as our reference in order to describe limitation on our study. 8. Conclusion: Please add \"early\": \"during the early COVID-19 pandemic\". Response: Sure. In the latest version of our manuscript is revised based on your recommendation. Thank you for your review." } ] } ]	1	https://f1000research.com/articles/9-1201
https://f1000research.com/articles/10-149/v1	25 Feb 21	{ "type": "Research Article", "title": "Measure of unevenness in human genomes, described as a self-affine phase transition in a 'spin-chain’ model", "authors": [ "Sergey Feranchuk" ], "abstract": "Background: Non-Gaussian distribution of polymorphic positions across a genome can substantially influence the results of any approach to molecular evolution based on a 'classical' probability model. The infinite dispersion of non-Gaussian perturbations is a challenge in an attempt to accept it in a probability-based model of evolution. Methods: Here a model is proposed where non-Gaussian distribution is introduced to an exact solution of the 'Ising model'; it describes a behavior of one-dimensional chain of spins in an approaching to a phase transition. The distribution of fragments which are identical between two genomes is similar to distribution of islands of spins with the same orientation, in the model where non-integer dimension is introduced. Results: Application of this model allows us to compare the relative contributions of non-Gaussian perturbations for pairs of human genomes from different ethnic groups. An evolution of the three human races in a most compact presentation is considered, rates of development on the separated stages of the evolution are assumed to be proportional to a value of relative unevenness between the appropriate groups of genomes. In the resolved model, the meaningful details of the separation between Asian and European races are clarified, in a period around ten thousand years ago; a particular viewpoint to the separation of the African race is also presented. Conclusion: The proposed approximation of non-Gaussian perturbations in human genomes allows to support the statements which are otherwise missed in the scientific investigations of the early history of modern humans.", "keywords": [ "mutation rate", "self-affine development", "human evolution", "phase transition", "genome coverage", "Landau-Zener transition" ], "content": "Introduction\n\nThe issues about an unevenness of a genome arose in particular in a distribution of coverage of sequencing reads mapped to a genome1. There, the deviations of a reads’ coverage from a 'classical' Lange-Waterman model, which was constructed following a Poisson distribution for short genome fragments, reflects some features of self-affinity for most frequent genome fragments in addition to the previously observed over-dispersion of a ''Poisson'' peak. There, the effect was observed stably in several analyzed genomes and is to be treated as a robust enough phenomenon to be discovered further and in depth.\n\nThe features of self-affinity in DNA sequences were detected at the very early days of genomics, in a classical work of Peng et al.2; a definition of the fractal dimension for the one-dimensional series was proposed there, and DNA sequences were a model of ''fractal''-like series.\n\nThe self-affinity features in a phenomenon imply the influence of perturbations with infinite dispersion, or the presence of a so-called 'fat-tail' in their probability distribution, and these features are difficult to detect and describe.\n\nHere, an approach is proposed to detect and apply a measure to these 'perturbations' focusing on the phenomenon mentioned, which was observed in coverage distributions. The relevance of a proposed research project is demonstrated on a model of evolution of humans restored from some of the present-day human genomes; confusions which are accumulated in solving of this scientific problem were in fact a motif to drive out the research.\n\n\nMethods\n\nSelf-affinity features are a property of 'transitional' period, and a description of these features is borrowed from approaches to a so-called 'Ising model', the model where a phase transition in a mutual orientation of spins in a crystal is explained. The heating of magnetic crystal leads to abrupt disappearance of magnetic momentum, and an approximation of this phenomenon is simulated in the Ising model. In a very simplified form, the interactions in crystal are presented there as an increase of energy if two spins in a linear chain are oriented in the same direction, and a critical temperature of phase transition (Tc) is derived from a strength of these interactions ('coupling constant').\n\nThe cooling led in turn to a sudden appearance of magnetic momentum, and a decrease of a temperature close to a critical temperature leads to accumulation of 'islands', long enough fragments where spins are oriented in the same order. Ordered fragments in a one-dimensional spin chain can be compared to identical fragments of genome sequences, and a distribution of these fragments allows to describe there the features of self-affinity.\n\nIn the frames of statistical physics, an expression for the probability of island of length k was obtained by Dziamagra3, as an applied case of the so-called 'Landau-Zener' transition'':\n\n\n\nThis is a point where a distribution with infinite dispersion can be introduced, assuming that a power coefficient 2 in this Gaussian-like distribution is substituted to some floating power coefficient D < 2, a dimension of 'intrinsic' self-affinity of the underlaid process. The model constructed above depends on the two flexible parameters; intrinsic dimension D and a parameter τ, a rate of cooling, or a rate of approaching a transitional phase. This model allows us to explain over-dispersion of the genome coverage distributions mentioned above (Figure 1) and to fit the parameters to a measure of unevenness of human genomes, trying to reconstruct their evolution most precisely . The distribution of 'islands' for human genomes can be obtained as distribution of lengths of completely identical fragments in the genomes; lists of polymorphic positions (SNP) from ''1000 genomes'' project4 were used as a representation of genomes. A similar distribution of fragment sizes is observed for this data (Figure 2A; Table 1). The clusters for the three races are clearly seen for both genetic distance and for tails of 'island' sizes in genome-genome comparison (Figure 2B). To interpret this, higher unevenness relative to same genetic distance means higher 'equilibration rate', higher mutation rates, and a lesser slope of a fitting line.\n\n(A) Simulation of over-dispersion effect in a genome following modified Ising model of islands in a chain of spins. Here, D = 1.9, τ = 0.0005, number of spins N = 200. The dashed line emulates an ordinary Poisson distribution, τ is the same. (B) Dependency between estimated fitting coefficient, the average of distribution, and underlying closeness to 'phase transition'.\n\n(A) Distribution of fragments’ sizes in human genomes in a pairwise comparison. Measures of closeness between genomes: genetic distance (B) and unevenness of fragment sizes (C). Here races are, from left to right, from bottom to top: Asia, Mexico, Europe, India, Africa.\n\n\nResults\n\nFor the two independent populations, the distance does not depend on heterogeneity in populations; a simple model of exponential development can provide a dependency of average genetic distance within population.\n\n\n\nThe simplified model of evolution of human ethnic groups is shown in Figure 3, and for further consideration it was reduced to just clarify a separation between three human races. In this case, the model can be further reduced to a system of equations (2). The events which are assumed here as events of separation between races are (a) separation between modern Asians and modern Europeans, which happened nearly just after an expansion to America, about ten thousand years ago; and (b) expansion of modern humans to Eurasia, about fifty thousand years ago.\n\npa, pe, ... - heterogeneity of genomes within a communicating group; dae, ... - distance between genomes of separated groups; be, ba, ... relative heterogeneity of a group in the events of separation between groups. ka, ke, ... - rates of 'exponential' development.\n\n\n\nThe mutation rate 10-9 per nucleotide per year can be transformed as 3 per genome per year = 3000 per genome per thousand years, 3000 mutations per 700000 SNP, so that r in the equations above should be about 0.004.\n\nHaving a requirement that p0 ≥ 0, the r should be less than 0.0029. Rates of development are assumed to be unknown, what is only known is a dependency between a rate of development and a linear coefficient m. Values of ba, be, bA, pae, kae which are attributes of a passed history are also assumed to be unknown.\n\nFor a marginal but the most confident assumption, p0 = 0, pae = 0.21, kA = 1.65 and kae = 1.81. Unevenness in a comparison between groups is a weighted average of evolutionary paths from a time of separation, so that if kA ~ mA, ke ~ me, then kae ~ (mAeT + mA(T + t) + me t)/2(T + t); kae ≈ 0.0141. Following a log-linear approximation, ka and ke, for modern Asians and Europeans, should be about 1.74 and 1.76.\n\nThe genetic diversity of Asians in a time of separation is than estimated as 0.15, much lower than a pool of genotypes just before the separation (pae = 0.21). For Europeans, the pool of genotypes was wider, about 0.20.\n\nWhat is known is that Eurasia is a continent with good communications, and that it was populated mostly by ancestors of present-day Asian race before the time of last separation, or 'crash', in better words. It is also known that ancestors of modern Europeans began to expand to Europe mostly after that 'crash'; the founders of the latest expansion originated from tribes which developed slowly for a long time somewhere in an area of central Asia mountains. The wide expansion to Eurasia for the ancient pre-Asian race was characterized, instead, by a substantive increase of genome unevenness. Some of it now is lost, some are kept in native Americans, and, for modern Asians, the instabilities in diverged genomes were neutralized by a long enough period of stable slow development after the crash.\n\n\nConclusions\n\nSelection of individuals was almost the same as in Schiffels and Durbin5, the difference with that model is that non-Gaussian features in genomes are considered here explicitly. This has a substantial influence on a reconstructed history of the three human races. Dealing with self-affine phenomena is difficult and risky, but it by no way can be ignored in any of valuable challenges to a present-day science.\n\n\nData availability\n\nZenodo: Measure of unevenness in human genomes: supplement software utilities and intermediate data files, http://doi.org/10.5281/zenodo.44954446.\n\nThis project contains the utilities to convert, pre-process and compare genotypes.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nThe International Genome Sample Resource: 1000 Genomes phase 3 release, https://www.internationalgenome.org/data-portal/data-collection/phase-3", "appendix": "References\n\nKuzmin D, Feranchuk SI, Sharov VV, et al.: Stepwise large genome assembly approach: a case of Siberian larch (Larix sibirica Ledeb). BMC Bioinformatics. 2019; 20(Suppl 1): 37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeng CK, Buldyrev SV, Havlin S, et al.: Mosaic organization of DNA nucleotides. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1994; 49(2): 1685–9. PubMed Abstract \| Publisher Full Text\n\nDziarmaga J: Dynamics of quantum phase transition: exact solution in quantum Ising model. arxiv.org. 2005; 95(24). Publisher Full Text\n\n1000 Genomes Project Consortium, Abecasis GR, Auton A, et al.: An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422): 56–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiffels S, Durbin R: Inferring human population size and separation history from multiple genome sequences. Nat Genet. 2014; 46(8): 919–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSergey F: \"Measure of unevenness in human genomes\": supplement software utilities and intermediate data files (Version 1.5) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4495444" }	[ { "id": "127304", "date": "04 Apr 2022", "name": "Ying Ji", "expertise": [ "Reviewer Expertise statistical genetics" ], "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 author presented a model to approximate non-Gaussian perturbations in human genomes, and provided reconstructed history of Europe, Asia, and Africa based on the model results.\nHuman evolution history is very interesting and important problem to work, and I appreciate the authors for the efforts to tackle this problem. However, I found it challenging to follow the study design and analysis from this article.\nIntroduction\nI found it challenging to figure out what scientific gap this article address. For example, the specific shortcoming of \"without explicit consider the non-Gaussian features\" are not clearly stated, and the definition of \"self-affinity\", 'Ising model' are not clearly described.\n\nResults\nIt's unclear to me how the models are designed, and what underlying assumptions or approximations were made. An example is that the model equations on page 3 were presented without stating what each parameter mean. And I'd recommend adding more details on captions for tables and figures, it'll help the readers understand the data analysis process and interpretation of results.\n\nConclusions\nThe authors mentioned dealing with the problem can not be ignored, it'll be helpful if the authors could elaborate more on the importance of the discovery.\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": [ { "c_id": "8061", "date": "07 Apr 2022", "name": "Sergey Feranchuk", "role": "Author Response", "response": "a foreword. I appreciate the efforts of a reviewer who is a specialist in a statistical genetics. In my reply, first, I would like to point out the meaning and a definition of a term \"statistics\". A lot of efforts, including the contribution of the reviewer, were put into a development of a statistics in a \"classical\" meaning. This meaning is, historically, started from the subjects like \"probability theory\", rules of gambling and Gaussian distribution. These methods are good to answer to some challenges and are inadequate in some other challenges. A man can not know the everything in the mathematics, so the incompetence in the areas beyond the \"classical statistics\" is not a \"sin\". Anyway, complex developing systems, and genetic codes in particular, are beyond the applicability of a classical statistics. I will not prove this statement here, as it can be treated as a provocation. But, having education in a classical statistics, I had a need to \"migrate\" towards the \"fractal\" statistics. It is more complex and challenging. But the methods there are suitable for the object under study. I became to feel myself a fool when I was trying to apply the statistical methods to a system which is not suitable to the basic assumption of the statistical theory. \"I found it challenging to figure out what scientific gap this article address. For example, the specific shortcoming of \"without explicit consider the non-Gaussian features\" are not clearly stated, and the definition of \"self-affinity\", 'Ising model' are not clearly described.\" Speaking about a \"fractal methods\", there is a choice, either to explain everything, or to assume that the reader is know what I'm speaking about. In a format of a submitted publication, I choose the second. I'm unable to explain what I mean speaking \"non-Gaussian\" without reminding the whole context around the \"fat-tailed distributions\". \"Ising model\" is a term known for physicists, it is a most complex of physical problems which can be solved exactly. The model explain a \"collective behavior\", mathematicians are not too familiar with it. I intended to put an attention to power of this model in a case of fractal mathematics. That is, to bring a gap to physics, but not to explain physics. \"It's unclear to me how the models are designed, and what underlying assumptions or approximations were made. An example is that the model equations on page 3 were presented without stating what each parameter mean. And I'd recommend adding more details on captions for tables and figures, it'll help the readers understand the data analysis process and interpretation of results.\" A man wrote a text by different reasons. And a scientific text can be motivated by different needs. The science is an universal way to prove falsehood or truth of the statement. Anyway, there are scholar texts, grant proposals, popular explanations, and the reporting of the results. In a present-day flow of scientific information, I choose to separate a style of the text from the \"popular-style\" explanation for \"newbies\" with precise definitions and detailed captions. \"The authors mentioned dealing with the problem can not be ignored, it'll be helpful if the authors could elaborate more on the importance of the discovery.\" My bid is to the changed times. The results are anyway contradictory and difficult to disseminate. Either the importance of the discovery would be revealed anyway, or all the efforts to elaborate these results will be anyway in vain." } ] } ]	1	https://f1000research.com/articles/10-149
https://f1000research.com/articles/10-147/v1	25 Feb 21	{ "type": "Opinion Article", "title": "Personalisation of treatment pathways – analysis of chances and barriers by the implementation of digital technologies under the conditions of the German Health System", "authors": [ "Armin Töpfer", "Georg Brabänder", "Georg Brabänder" ], "abstract": "Background: The potential of digital technologies is far from being exhausted for patients. The regulatory framework becomes a brake on innovation due to digitalisation, but also due to the trend towards individualisation. Strategies, corporate culture and processes, which are necessary for the design of high-quality and cost-effective healthcare services, are still lacking in many healthcare providing organisations. Health Services 4.0 and patient integration as leverage: With Health Services 4.0 it is possible to improve the outcome of the individual healthcare service and meet the regulatory requirements. This requires the capabilities of the provider to dynamically balance exploitation and exploration. The challenges are to develop innovations in a continuously changing working environment and/or to adapt (medical) technical innovations into their own service processes. Conclusion: This article is focused on hypotheses of cause-and-effect analyses formulated as scenarios, related to the implementation of digital technologies in order to improve efficiency and effectiveness for a high medical expertise as well as for a higher level of service quality. The output is a more detailed analysis of key value drivers, success factors as well as internal and external value generators for the design of Health Services 4.0. Up to now many issues regarding the use of digital technologies are still only partly analysed and not yet proved for a more efficient care on high-quality level. The company's capacity for ambidexterity is becoming an important dynamic capability, with on one hand flexibility for new developments and on the other hand stability for hard factors in physical value chains and soft factors in value-oriented attitudes and behaviour based on empathy. This article was previously published in German in \"Monitor Versorgungsforschung\" under the original title \" Personalisierung von Behandlungspfaden – Das Potenzial digitaler Technologien\". This translated version faithfully reflects the authors, data, and interpretations of the original.", "keywords": [ "Personalisation of treatment pathways", "Health Services 4.0", "cyber-physical systems", "translation of innovations", "customer journey", "ambidexterity" ], "content": "Introduction\n\nThe German Healthcare system shows a wide variety of specific features compared to other healthcare systems. Examples include the delegation of supply contracts to associations of service providers, while simultaneously regulating the content of service, as well as the third payer principle of financing. Thus, suppliers at the provider level see themselves confronted with a multitude of legal regulations concerning the integration of the patient with joint decision-making, data protection and quality assurance. Simultaneously, there exists demand for a more economic provision of services as well as high- quality medical care with rapid availability. Technological innovations, such as big data analytics applications, sensor technology and the Internet of Things, analyses of OMICS technologies, augmented reality and robotics have the potential to change the previously existing – largely analogue – value chains through integration of the patient with the combination of conventional healthcare with digital technologies, or Healthcare Services 4.0. The patient’s needs are tailored to individual needs in the form of personalised treatment pathways, increasing the effectiveness and the efficiency of health services.\n\nExisting literature focuses mainly on individual applications of digital technologies in regard to specific disease patterns. Increasing digitalisation is accompanied by a change in work and organisational structures as well as a change in leadership culture. The purpose of this article is to explain how, and under which (organisational) conditions within the requirements of the German Healthcare system, a high level of patient orientation, a more economic provision of services with digital technologies, and improved ambidexterity in leadership and organisational structure can be accomplished.\n\nThe article has been published in a German Journal (Monitor Versorgungsforschung)51. Feedback showed that the topic and content should also be accessible to other disciplines for further discussions.\n\n\nHealth Services 4.0 for personalised treatment pathways\n\nAlthough the trends in the German healthcare system point to a higher significance of health as a benefit for the insured and for patients, healthcare in Germany is still not sufficiently oriented to the needs of the individual patient. Studies have shown that it has not been possible to meet expectations for concepts such as ‘shared decision making’ to a sufficient extent. In some disciplines, the medical profession is therefore still a long way from adopting the basic principles of professional discussion to involve the patient1. This means that significant potential for the improvement of outcomes, and thus the level of effectiveness of a healthcare service, cannot be realised, and that the legal requirements in the Patient Rights Act are also not being fulfilled.\n\nThe potential for patient integration in the form of personalised treatment pathways can be exploited through the convergence of information technology and biotechnology, so-called ‘super-convergence’2, and the resulting combination of (medical) technical innovations and their application trends. By combining suitable components of the respective technologies, Health Services 4.0 can be developed to increase the quality and value of health services (see Figure 1), and the provider can react more quickly to market developments and competition (see Figure 2).\n\nIn this way, Health Services 4.0 refers to the integration of previously standard health services, which in the German health system are financed by statutory and private health insurance funds and provided by inpatient and outpatient service providers, using digital technologies. In the resulting cyber-physical processes, central components of digitalisation are the consistent use of sensors for monitoring biomarkers, the expansion of the interaction between humans and computers, the use of mobile devices (mHealth), big-data technologies and the use of machine learning. Technologies are used here to close the gap between the virtual and the real world and are capable of interacting with people and things3. In conjunction with cloud computing (i.e. where information technology resources are provided via networks), cyber-physical systems represent a cross-sectional technology for real diagnosis, therapy and care services to provide reality-based services in real time, in a virtualised and omnipresent manner with multimedia control and communication4. In this way, processes that were previously analogue can be digitalised and new processes that are only available digitally – cyber-physical processes in the narrower sense – and data-driven decision support systems can be used for the real treatment of patients. They allow the linking, in compliance with data protection regulations, of standardised and personalised partial services that increase benefit in the form of Health Services 4.0. The intensive integration of the patient in this joint service creation represents a decisive lever in this process.\n\nThe more thorough integration of patients and relatives in the treatment process in this way means, in principle, that healthcare services are personalised in a general, conceptual approach to focus on individuals, who are comparable according to certain criteria, in order to overcome non-specific standardisation. This usually amounts to the creation of standards for individual groups. Here, personalisation means even greater differentiation and detailing on specific implementation for the individual person or the individual patient with his or her specific clinical profile and personal circumstances5.\n\nIn general, the added value of a service is generated in the direct interaction of the provider system with the contributions of the customer (see Figure 1). Therefore, the treatment processes that create value directly represent the most important lever to achieve competitive advantages. The personalisation of treatment processes - ideally in the form of treatment pathways - thus forms the linchpin for the strategy, organisation and implementation, and for the effectiveness of treatment programmes and sequences6. Consistent process management on the basis of an ethical and moral foundation with respect to the importance of a health service and 'data literacy', i.e. the ability to handle data in a competent manner7, creates the prerequisites for increasing patient benefit, productivity, and outcome, and is thus the most important value driver, ensuring that focus on the patient is maintained in all diagnostic, treatment and care services.\n\n\nThe potential of digital technologies for the personalisation of treatment processes in various medical fields\n\nIn general, digital technologies have considerable potential to provide higher quality and simultaneously more economical inpatient, outpatient and cross-sectoral care. This is achieved both by accelerating the communication and coordination processes between patients and service providers as well as between the service providers themselves and by developing innovative diagnosis and treatment procedures in the individual medical fields. The following are examples of technologies that can be used by healthcare providers to personalise a treatment pathway (see Figure 1).\n\nThe electronic networking of patients with service providers and between these providers offers many opportunities to make the service processes in prevention, diagnostics, therapy and aftercare more efficient and effective and to involve patients more closely in the treatment of chronic diseases by using apps and sensors to measure biomarkers. Through electronic networking, patients gain easier access to their own medical data and to medical knowledge and can thus become customers in the 'health market'8. One example of the possibilities of networking at the system level in Germany is the 'Healthy Kinzigtal Valley'9 value-added network, a cooperation of various general practitioners, specialists and hospital doctors, psychotherapists, nursing and rehabilitation facilities. Within the framework of a contract for integrated care, it plans and coordinates the treatment of insured members of certain health insurance funds. The focus of the business model here is on innovations that create new product features in conjunction with new business models and on the use of innovative information, communication and big data technologies to achieve results, i.e. concrete effects rather than an increase in the volume of services.\n\nThe provision of tele-medical services was approved in 2018 by an amendment of the model professional code of conduct for physicians and further developed at the 121st Ärztetag Medical Congress in Frankfurt in 2018. This defined the relevant benefits - from the medical perspective - for the provision of telemedical services. In the broadest sense, this is understood as the overcoming of temporal and/or spatial distances in the context of medical issues. This mainly includes the measurement, recording and transmission of information or the application of medical procedures with the help of information and communication technology between doctors and between doctors and patients, if necessary with the involvement of non-physician specialists10. There are now already a number of examples of applications, such as a remote application for the care and continuous aftercare of patients with cardiac insufficiency (telecoaching) or the ambulant aftercare of stroke patients (telestroke). Telemedicine services have great potential to adapt to the preferences of the patient, but there are fewer benefits for the physician providing these services. The physician must provide the time for the service both online and offline, and the set-up time for the telemedical infrastructure must be added to this.\n\nFrom the analysis of gene sequences (genomics), epigenetic modifications (epigenomics), RNA transcripts (transcriptomics), proteins (proteomics) and metabolic products (metabolomics)11, the so-called OMICS technologies, it is possible to identify biological and psycho-social subtypes of diseases that are incongruent with traditional diagnostic categories by using machine learning techniques from large amounts of data. Based on the classification, predictive therapy recommendations can be given and treatment effects can be predicted with high accuracy12.\n\nAn example of the significance of genetic research and the importance of translational orientation for future targeted treatments is, for example, the discovery of 30 new genetic variants associated with depression. More effective drugs with fewer side effects can be expected for certain patient groups on the basis of further research13.\n\nAnother example of advances in this area is the treatment of acute lymphatic leukaemia in children, a specific form of lymphoma that can be carried out individually for each patient using CAR-T, or the development of 'chimeric antigen receptors'. This enables leukaemia cells to be detected and killed. First results show complete remission in some cases and the absence of visible signs of tumour in the computer tomogram14. Another advantage of this treatment method is that this form of cancer therapy could replace stem cell transplantation in the future15.\n\nVirtual reality applications, i.e. the imitation of physical reality with all its physical properties in a computer-generated, interactive, virtual environment16, can be used in the training of medical students and the testing of personalised treatment approaches. For example, heart function can be modelled with a computational method, supported by artificial intelligence (digital twin)17, and tailor-made cyber-physical treatments can be tested with the interdisciplinary integration of clinical patient data before the actual implementation. This technology can also be used in the treatment of anxiety disorders18 and addictions19.\n\nNanotechnology can be used to produce tiny devices and particles that are used in the human body for in vivo diagnostics and for the transport of active substances in drugs20.\n\nRobotics are controlled by computers with artificial intelligence programmes. Depending on the software used, they can be used, for example, as support for physically arduous work in nursing and rehabilitation, and as support systems in the operating theatre.\n\nResearchers have used an interdisciplinary approach involving methods from neurotechnology, clinical neurology, robotics and computer simulation to develop prostheses with which patients can move more naturally. Electrical impulses are simulated so that the brain interprets them as if they came from a natural arm, enabling patients to grip more precisely21. Individual operation and support processes can be structured, digitalised and automated with Brainlab. The operation steps are announced, processed and simultaneously documented. This achieves a better quality of results and greater efficiency22.\n\nThe MicroSurge robot system was developed at the German Aerospace Centre (DLR) for use in minimally-invasive procedures. The devices are held by robot arms and remotely controlled by the surgeon. The system not only provides a visual insight into the operating area, but even enables haptic feedback23. In combination with Augmented Reality, with which the user experiences the real world supplemented by a virtual component16, it is possible to practise complex operations and plan them in detail or to use the method for intra-operative support. With SpectoVive, real-time computed tomography data can be converted into a three-dimensional virtual environment that provides the surgeon with a realistic representation of the environment24.\n\nThe extraction of new information from all this data, which arises or is generated from the use of these technologies and may also have been collected for other purposes, is a core idea in big data25. In a structured treatment pathway, these analyses can also contribute to improving treatment directly to the patient. This is done in such a way that predictive analytics26 can be used in an established data warehouse to derive prognostic statements from the large quantities of information from various sources and stored in so-called big data lakes26 (see Figure 1).\n\n\nPrevious obstacles and barriers to digital solutions in the German healthcare system\n\nIn the German healthcare system, however, there are still considerable obstacles and barriers that make the adoption and application of innovative and digital services difficult or impossible.\n\nImplementation is hampered by the extensive incompatibility of information and communication systems, which make it difficult to integrate data and use it across locations because of the lack of structural equality. In the medical informatics initiative DIFUTURE, universities of excellence and clinical partners have therefore joined forces to bring together the necessary data volumes from various research areas and digital products so that every doctor, researcher and patient has access to the information they need27. In addition to the obstacles relating to data protection and data security, common standards are also lacking, and not all health professionals are convinced by digital techniques or trained in their use28.\n\nA further difficulty for the translation of innovations is that the costs are only covered by the health insurance funds once they have proven their clinical benefit, which is difficult given the relatively small number of cases. A change with regard to reimbursement is currently being initiated with NOVARTIS, which has agreed a reimbursement model for CART-T cell therapy using company health insurance funds. In this model, the insurance company is returned a portion of the cost of the treatment drug (Kymriah®) if the patient dies within a predefined period of time29.\n\nWhile hospitals are subject to the so-called proviso of prohibition regarding the use of innovative methods, the outpatient sector, in contrast, is subject to the proviso of permission30. New examination and treatment methods (NUB) may be provided in the inpatient sector until the Federal Joint Committee (G-BA) has carried out an evaluation. In the outpatient sector, the proviso that permission be obtained means that services can only be provided after the Federal Joint Committee has examined, evaluated and permitted them in advance. In the current health policy discussion, it is becoming apparent that the Ministry of Health is also criticising the long decision-making processes of self-administration and is planning changes31.\n\nIn general, the close and cross-sectoral cooperation of clinicians with innovative partners, namely research institutions and the medical industry, represents an important value driver for the translation of innovative services (see Figure 2). As an important success factor for patients, it offers innovative service variants and choice in terms of place, time, participants and methods at a highly medical quality and state-of-the-art research.\n\n\nExpansion of the value-added chain ‘treatment pathways’ – Dissolution of organisational divisions\n\nIn the future, healthcare will take place less in the waiting rooms of registered doctors and hospitals and more often on a smartphone32. The physical dependence of many service processes on the presence of the patient can be eliminated under the conditions of digital infrastructure and interconnectivity. Sensor technology and smart diagnostic devices at the point of care provide comprehensive diagnostics independently of the respective supplier and, ideally, compatibly with the relevant systems. Telemedical service processes allow value-added chains in the form of treatment pathways to be developed further and extended, so that previously unknown or unfeasible methods and new business models can emerge. This means that there are considerably more options available to map healthcare services across all three sectors (outpatient, inpatient, home) in a continuous end-to-end treatment pathway and to provide personalised treatment (see below in Figure 1). Healthcare services can thus be personalised - oriented to the optimum course of action for the patient5.\n\nIf the data from the above-mentioned technologies is used, it will be possible to better understand how diseases arise and how processes that cause illness can be detected at an early stage in the bodies of healthy people (i.e. people without symptoms of illness). In this way, earlier and earlier diagnoses and effective interventions can be used to intervene at an early stage instead of only providing treatment once the disease has already spread33. Alzheimer's dementia, for example, appears to manifest itself in some forms long before the actual onset of the disease. This gives us time to prevent the disease, possibly by as long as 20 years34. In another example, following investigation of the genetic risk, effective substances can be used before the onset of a related disease caused by underlying type one diabetes33.\n\nThe early approach to and integration of the patient thus makes it possible to reduce the information asymmetry between doctor and patient and, with early detection, to swiftly focus on primary prevention35.\n\nAccordingly, medicine is developing from reactive medicine with a one-size-fits-all approach to proactive and anticipatory preventive medicine, which can plan treatment more precisely with combinations of therapies instead of often having to rely on trial and error as has been the case in the past. However this requires patients to reflect on their own position with regard to the handling of their data and the risk regarding knowing and not knowing about their current health status and treatment (Figure 1: A), and a debate as to where disease begins and what is therefore included in the service catalogue of the health insurance companies is needed36.\n\nFor healthcare providers, this means that the chain of contact with potential patients and relatives (customer journey) must begin much earlier in order to identify significant touchpoints and proactively point out personalised solutions that can be integrated into the patient's everyday life without major friction losses.\n\n\nJoint value creation with patients and relatives\n\nOne central element of successful treatment is the integration of the patient into the diagnosis and treatment process, which is also codified in the German Patient Rights Act. The aim is to achieve more knowledge and control over the disease or health service as well as greater compliance/adherence.\n\nIntegration gives the provider access to implicit patient knowledge and to feedback from which improvement processes and innovations can be developed37. But patients and relatives can also act as innovators in their function as users. Their knowledge about the disease represents a great potential for process and product innovation, especially in the field of rare diseases38. This requires the willingness of the patient to take responsibility for the recovery process and to give feedback to the practitioner (Figure 1: B).\n\nSmartphones and wearables, i.e. computer systems that are worn on the body and/or integrated into clothing, make patient integration possible independent of time and place. These systems can be used with digital technologies such as telemedical health services and open the possibility of using powerful sensors in conjunction with cloud computing (see Figure 1). They thus represent a considerable increase in benefit to the patient, who must be willing and able to use the technologies, in the context of prevention, diagnosis and treatment (Figure 1: C). In the context of modelling a treatment pathway on the basis of a treatment concept, such personalisation promotes the relationship between provider and patient in the sense of a specific customer orientation and creates interactive added value in the sense of a co-design process39.\n\nBlockchain technology (Figure 1: D) can be used to create a system that collects all data that can only be released by the owner of the data to certain users40. This would allow significant problems such as the dispersed nature and the security of medical and social data to be resolved. Blockchain data is more complete, accurate, trustworthy and widely available20. It would then no longer be possible for data to be used for other purposes without the knowledge of patients. This would give the patient more security and autonomy in dealing with healthcare providers.\n\nThis development will fundamentally change the role of the doctor. By making information and knowledge accessible and easy for all to access, the physician will no longer be responsible for the transfer of knowledge as an indispensable knowledge carrier, but rather operate in the role of a consultant and coach. Doctors will instead configure a personalised preventive and/or treatment path with the patient using Health Services 4.0 and control the entire process with empathy and ethical clarity, so that the patient does not feel like a ‘test specimen’.\n\n\nMaking new knowledge effective\n\nDigitalisation will also change the future of work in the health sector, albeit not (yet) as radically as is the case for Industry 4.0 with its framework conditions of Volatility, Uncertainty, Complexity and Ambiguity (VUCA)41. Even if the challenges of Google, Apple, Facebook, Amazon (GAFA) and many small health apps acting as drivers, the persistent forces in the individual sectors of the German healthcare industry still appear very strong. In this sector, which is characterised by complexity due to regulations, different incentive and billing systems, quality assurance measures, security routines and data protection regulations, the more difficult framework conditions as in Industry 4.0 do not yet apply. Nevertheless, digitalisation and the focus on translation of concepts from industry frameworks in the German healthcare system are accompanied by changes in work and organisational structures and a different management culture.\n\nThe challenges for providers are to enable the organisation and employees to develop their own innovations and to take over innovative diagnosis, treatment and care services from pharmaceutical and medical research institutions. This makes the management of automated and non-automated treatment processes and their interconnection to quality and risk management with zero-defect quality into the most important value drivers and into success-critical prerequisites for entrepreneurial action as a provider of healthcare services (Figure 2).\n\nThe healthcare provider must constantly rebalance the conflicting goals of exploitation and exploration. New knowledge is created by specialising and refining existing knowledge in a continuous improvement process during exploitation as use and utilisation; new knowledge is developed during exploration by creating radically new application and solution possibilities in the interests of users42. Both approaches can be pursued by realising ambidexterity in organisational structure and management behaviour - an approach that is also relevant for expert organisations in the healthcare sector in a time of digital transformation. This dynamic ability represents a key value driver for a health organisation to become or remain competitive in the long term.\n\nAmbidexterity can be implemented in different organisational models depending on size, available resources and the chosen innovation strategy:\n\n• In a cooperative organisational model, exploration is carried out in certain domains while exploitation is carried out in others (domain ambidexterity), for example in the concept of a dual-operating system43.\n\n• In the autonomous organisational model, a company carries out exploration and exploitation in separate organisational units, each of which is managed by autonomous units (structural ambidexterity).\n\n• In the case of temporal ambidexterity, a company periodically alternates between the phases of exploration and exploitation.\n\n• In the integrated organisational model, the business units conduct exploration and exploitation in parallel, i.e. the responsibility for innovations is with a single party (contextual ambidexterity)44.\n\nAll four forms of organisation have their justifications and chances of success. In the case of Health Services 4.0, the fourth organisational model represents the greatest potential for success, alongside the third. Organisations in the German healthcare system are subject to a multitude of regulations and budgets, which usually do not allow scope for additional units, so that contextual ambidexterity in an integrated organisational model is the predominant option. Ambidexterity has also been examined closely in networks45 and is therefore also relevant for transposition to physician networks, which are playing an increasingly large role in supply within the German healthcare system.\n\nDigitalisation creates new forms of cooperation with one another, but also with machines and cyber systems, so that we are already speaking of Work 4.041. Due to the fusion of software, hardware and new diagnostic and treatment services, established patterns of thought and routines will have to be questioned in general in order to compete for more demanding and informed patients. The focus on translational orientation with the consistent use of innovative diagnosis and treatment methods and their mapping in personalised treatment pathways requires a framework that enables innovative thinking and action, consistently processes knowledge and makes it available to everyone - including patients. It is not the knowledge itself that is in the foreground, but rather the methodological competence to apply this knowledge effectively for the individual health service6. In principle, this requires a managerial, value-based attitude and an ethical, value-based attitude in the focus of several groups of stakeholders, in which patient orientation and ‘clinical credibility’46 are just as firmly anchored as a consistently business-oriented focus. This will require a corporate culture that is oriented towards both. Self-organisation and participation, development of teams away from hierarchical control and towards self-control, self-organisation to the point of independence47 and to the entrepreneur within the company are key elements of design and levels of maturity48.\n\nWhat is needed is an approach to leadership that masters the ‘slider' of transactional and transformational leadership, that can accompany the change in the range of activities and role models as well as the increasing demands on the technical and emotional skills of employees. This adds ‘both-handedness’ of management to the organisational ambidexterity49, a qualification that is directed firstly towards efficiency and excellent (the ‘left hand’) and secondly more strongly towards speed and innovation (the ‘right hand’) and can also withstand and communicate this balancing act between great openness and tight management50.\n\n\nConclusion\n\nDigital technologies already exist in many sectors and are gradually being adopted in medicine, although - particularly in the German healthcare system - many (professional) legal, health policy and ethical issues are still completely unresolved and the debate on them can hardly keep pace with the development of digitisation technologies. There are many opportunities and justified prospects for high-quality, precise diagnosis and treatment services with fewer side effects, which can be used proactively and in a personalised manner in various business models. It remains to be seen whether the hopes for a more economical supply can be realised.\n\nIn addition to legal and technical issues, the main competitive challenges for providers are to develop process and product innovations in a continuously changing working environment and/or to incorporate and adapt technical medical and pharmacological innovations from collaborations with research and development into their own service processes.\n\nIn times of digital change, a company's ability to achieve an ambidexterity of organisation and management is a dynamic ability that provides sufficient flexibility for new developments and sufficient stability for a value- and values-oriented attitude.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nOpen Access Funding by the Publication Fund of the TU Dresden\n\nThe original version of the article has been published in German language in „Monitor Versorgungsforschung“, printed (MVF 05/2019, Volume 12, October 07, 2019, p. 54-61). Permission to reprint in English in F1000Research was granted.\n\n\nReferences\n\nSchmacke N: Der Schlüssel zur Partizipation. Monitor Versorgungsforschung 2018; 3: 40–42.\n\nHahn H, Schreiber A: Potenziale der Digitalen Transformation in der Medizin. In: Neugebauer R Editor. Digitalisierung – Schlüsseltechnologien für Wirtschaft & Gesellschaft Berlin Heidelberg: Fraunhofer Forschungsfokus; 2018. p. 323–345.\n\nBruhn M, Hadwich K: Relevanz von Dienstleistungen 4.0 in Wissenschaft und Praxis. In: Bruhn M, Hadwich K Editor. Dienstleistungen 4.0 – Konzepte – Methoden – Instrumente. Band I. Wiesbaden 2017. p. 5–40.\n\nacatech: Integrierte Forschungsagenda Cyber-Physical System. München 2012.\n\nTöpfer A, Brabänder G: Personalisierung von Gesundheitsleistungen – Gestaltungsempfehlungen zur Umsetzung. ZEFQ 2018; 130: 27–34. Publisher Full Text\n\nStöger R: Prozessmanagement. In: 4th Edition Stuttgart 2018; p. IX.\n\nKuhn S: Transformation durch Bildung. Deutsches Ärzteblatt Volume 2018; 115, 14: A633–A638.\n\nWilhelm A: Der Patient im deutschen Gesundheitssystem: Einstellungen, Präferenzen und Erwartungen. In: Gellner W, Wilhelm A Editor. Vom klassischen Patienten zum Entrepreneur. Baden Baden; 2006, p. 18–57.\n\nHildebrandt H: Sprunginnovationen in der Organisation der Versorgung. Versorgungsforschung 2014; 7: 29–33. Publisher Full Text\n\nKassenärztliche Bundesvereinigung: Telemedizin.Reference Source Last accessed on 18.03.2019.\n\nLeopoldina –Nationale Akademie der Wissenschaften: Zukunftsreport Wissenschaft.Reference Source Last accessed on 14.03.2019.\n\nBzdok D, Karrer TM, Habel U, et al.: Big-Data-Ansätze in der Psychiatrie: Beispiele aus der Depressionsforschung. Der Nervenarzt 2018; 89:869–874.\n\nWray NR, Ripke S, Mattheisen M, et al.: Genome-wide association analyses identify 44 risk variants and refine the genetic architecture of major depression.Reference Source Last accessed on 21.03.2019.\n\nChustecka Z: Leukämie und Lymphome: Euphorie über CAR-modifizierte T-Zellen als neue Therapieoption.Reference Source Last accessed on 21.03.2019.\n\nReisinger K: Zielgerichtetes Instrument.Reference Source Last accessed on 21.03.2019.\n\nSpecht P: Die 50 wichtigsten Themen der Digitalisierung, München.2018.\n\nWallenfels W: Digitale Zwillinge – ein Gipfelhighlight.Reference Source Last accessed on 21.03.2019.\n\nÄrztezeitung: Heilen mit künstlicher Wirklichkeit.Reference Source Last accessed on 14.03.2019.\n\nÄrztezeitung: VR-Brille statt Zigarette.Reference Source Last accessed on 21.03.2019.\n\nBogdan B: MedRevolution – Neue Technologien am Puls der Patienten Berlin2018.\n\nÄrztezeitung: Armprothesen mit Feingefühl.Reference Source Last accessed on 21.03.2019.\n\nBrainlab: Reference Source Last accessed on 21.03.2019.\n\nDLR – Institut für Robotik und Mechatronik: Reference Source Last accessed on 21.03.2019.\n\nUniversity of Basel: Virtual Reality in Medicine: New Opportunities for Diagnostics and Surgical Planning.Reference Source Last accessed on 21.03.2019.\n\nLandrock H, Gadatsch A: Big Data im Gesundheitswesen kompakt. Wiesbaden 2018.\n\nSeiter M: Business Analytics. München 2017.\n\nMedizininformatik Initiative Difuture: Reference Source Last accessed on 21.03.2019.\n\nReference Source Last accessed on 21.03.2019.\n\nWallenfels M: Künstliche Intelligenz – bald Rückgrat des Versorgungsalltags?Reference Source Last accessed on 12.03.2019.\n\nGemeinsamer Bundesausschuss: Methodenbewertung.Reference Source Last accessed on 21.03.2019.\n\nFrankfurter Allgemeine Zeitung: Spahn macht Fettabsaugen zur Kassenleistung.11.01.19, p. 17.\n\nRytina S: Hauptstadt-Kongress erörtert Künstliche Intelligenz: “Das Gesundheitssystem der Zukunft findet auf dem Smartphone statt.„Reference Source Last accessed on 24.11.2020.\n\nHofmann S, Telgheder M: “Wir erleben eine neue Ära der Medizin.„Reference Source Last accessed on 21.03.2019.\n\nMüller T: Alzeimer-Anzeichen schon 25 Jahre vor Ausbruch.Reference Source Last accessed on 24.11.2010.\n\nArbeitskreis Ökonomie im Gesundheitswesen der Schmalen-Gesellschaft für Betriebswirtschaft e.V. Digitalisierung im Krankenhaus: Technische Entwicklungen und deren Implikationen für Behandlungsprozesse. In: Krause S, Pellens B. Editor. Wiesbaden: Betriebswirtschaftliche Implikationen der digitalen Transformation; 2018, p. 203–219.\n\nStreek H: Das sagen Experten zu dem neuen Forschungsgebiet.Reference Source Last accessed on 24.11.2020.\n\nLucius-Hoene G, Palant A: Zwei verschiedene Welten – Wie medizinisches Personal von Patientenerfahrungen lernen kann. KU Gesundheitsmanagement 2015; 84: 69–71.\n\nHabicht H, Oliveira P, Shcherbatiuk V: User Innovators: When Patients Set Out to Help Themselves and End Up Helping Many. Die Unternehmung 2013; 66 (3): 277–294.\n\nReichwald R, Piller F.T: Interaktive Wertschöpfung. In: 2nd Edition Wiesbaden 2009.\n\nPerez S: Proof Work aims to decentralize medical data by using the blockchain.Reference Source Last accessed on 24.11.2020.\n\nBruckner L, Werther S: Allgemeiner Überblick über Arbeit 4.0. In: Werther S, Bruckner L Editor. Arbeit 4.0 aktiv gestalten Berlin2018, p. 15–21.\n\nMarch JG: Exploration and Exploitation in organizational Learning. Org Sci 1991; 2 (1), p. 71–87.\n\nKotter JB: Accelerate. In: München 2015.\n\nEbers M: Organisationsmodelle für Innovation. ZfbF 2017; 69: 81–109.\n\nWessel L, Gersch M, Goeke C: Netzwerk-Ambidextrie: Ist eine Balance explorativen und exploitativen Lernens auch in Netzwerken möglich? In: Stephan M, Kerber W Editor. Jahrbuch Strategisches Kompetenz-Management, Volume 4: „Ambidextrie“: Der unternehmerische Drahtseilakt zwischen Ressourcenexploration und –exploitationMünchen Mering2010, p. 121–147.\n\nKoch T, Töpfer A, Heller A.R: Team Management im Krankenhaus für eine Lernkultur. In: Albrecht DM, Töpfer A (Editor). Handbuch Changemanagement im Krankenhaus, 2nd EditionBerlin2017, p. 587–608.\n\nKaltenecker S: Selbstorganisierte Unternehmen – Management und Coaching in der agilen Welt. Heidelberg 2017.\n\nTöpfer A, Duchmann C: Motivationseffekte bei den Mitarbeitern durch die Krankenhaus-Kultur. In: Albrecht DM, Töpfer A Editor. Handbuch Changemanagement im Krankenhaus 2nd EditionBerlin2017, p. 529–537.\n\nBurkhart S, Grabmeier S: Der Einfluss von Digital Leadership auf Organisationen im Gesundheitswesen. In: Matusiewicz D, Pittelkau C, Elmer A Editor. Die Digitale Transformation im Gesundheitswesen Berlin2017, p. 255–261.\n\nDuwe J: Beidhändige Führung. Stuttgart 2018.\n\nTöpfer A, Brabänder G: Personalisierung von Behandlungspfaden – Das Potenzial digitaler Technologien. Monitor Versorgungsforschung 2019; 7 (05)." }	[ { "id": "80263", "date": "12 Mar 2021", "name": "Aline Gottlieb", "expertise": [ "Reviewer Expertise gastroenterology", "hepatology", "digitization processes and developments in health/care", "medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the topic discussed accurately? In context of current literature?\nThe topic is very accurately discussed from a German perspective. The provided literature sources are accurate and deliver a good overview from older citations (to depict the beginning of developments) to recent sources and updates.\n\nOne point to mention (which is based on the nature of what the paper is describing): almost all cited sources are in German. This makes it more difficult for an international audience to follow up/ go deeper in the topic. I would advise the authors to try to provide more English sources where possible.\nAre all factual statements correct and adequately supported by citations? A couple of times, statements need to be supported with additional citations or need clarification, for example:\np 3: sources for funding “health systems are financed by statutory and private health insurance funds and provided by inpatient and outpatient service providers, using digital technologies”\n\nsame page: “more thorough interpretation: is written in italic and it is unclear to me why ( is this a term introduced by the authors? – needs more clarification)\n\nI would recommend that the authors critically look at especially sections on page 3 and 4 to ensure proper citing of all statements that are not their own.\nAre arguments sufficiently supported by evidence from published literature?\nEspecially the section in the paper highlighting ongoing examples of how developments in technology and medicine can be linked, are very well cited and explained for a broader audience.\nAre the conclusions drawn balanced and justified on the basis of the presented arguments?\nOverall, drawn conclusions are clear and well-presented from the economic point of view. As a suggestion, I would recommend trying to link more clearly how ambidextry influences/affects the intertwining of digitalization and medical knowledge (and patient diagnostics and treatment)\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? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "95623", "date": "13 Oct 2021", "name": "Maurice Mars", "expertise": [ "Reviewer Expertise Telemedicine and eHealth", "implementation", "and related legal and ethical issues." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the topic of the opinion article discussed accurately in the context of the current literature?\nThe issue of providing personalised healthcare through the judicious use of digital technologies is of global interest. The paper is from the German perspective of its legislative, funding, health system and service provision milieu. Germany is in the interesting position of having a Patients’ Rights Act, promulgated in 2013, a Digital Health Act passed in 2019 and being a late adopter of aspects of telemedicine in 2018. As the paper is a translation of a 2019 paper in German it would be interesting to know whether subsequent developments, especially the Digital Health Act and technology advances have altered any of the points made.\nIt would be helpful for the average reader to have the concept of ambidexterity explained in the introductory section.\nAre all factual statements correct and adequately supported by citations?\nMost of the cited papers are in German and so have not been checked. There are no obvious errors in the information provided.\n\nAre arguments sufficiently supported by evidence from the published literature?\nSee the comment above.\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? The matter of the 'completely unresolved' legal, health policy and ethical issues raised in the conclusion is not addressed in the body of the paper.\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? Partly\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": [] } ]	1	https://f1000research.com/articles/10-147
https://f1000research.com/articles/9-32/v1	22 Jan 20	{ "type": "Opinion Article", "title": "Respiratory concerns of gabapentin and pregabalin: What does it mean to the pharmacovigilance systems in developing countries?", "authors": [ "Sunil Shrestha", "Subish Palaian", "Subish Palaian" ], "abstract": "Gabapentin and pregabalin, commonly known as gabapentinoids, have been widely used globally. This paper highlights the serious breathing problems due to using gabapentin and pregabalin which was warned by the United States Food and Drug Administration on December, 2019. In this article, we tried to recommend suggestions for controlling these adverse drug reactions (ADRs). Safety reports of gabapentin and pregabalin should be obtained from concerned manufacturers and reviewed for respiratory depression effects. There should be strict prescription monitoring and drug use evaluation studies. Concurrent use of gabapentin and pregabalin with other respiratory depressants should be strictly monitored. Educating patients can help in the early detection of ADRs due to gabapentin and pregabalin. Anecdotal reports on these medications should be encouraged.", "keywords": [ "Gabapentin", "Pregabalin", "pharmacovigilance", "Developing countries" ], "content": "Introduction\n\nGabapentin and pregabalin, commonly known as gabapentinoids, have been widely used globally. Gabapentin is an anticonvulsant agent used in treating various illnesses such as amyotrophic lateral sclerosis, analgesia, anxiety, neuralgia, restless legs syndrome and bipolar disorder1. Pregabalin is commonly used for the treatment of painful diabetic neuropathy, fibromyalgia, diabetic neuropathy, cancer chemotherapy-induced neuropathic pain, post-herpetic neuralgia, trigeminal neuralgia, and post-operative pain2,3. Pregabalin also acts to be an effective treatment therapy in refractory partial-onset seizures and the existing data recommends that pregabalin may be favorable as adjunctive therapy in adults with generalized or social anxiety disorder3.\n\nAs per the available safety data, the use of gabapentin and pregabalin may cause neuropsychiatric related adverse drug reactions (ADRs) followed by hepatic, cutaneous and hematological reactions4. Suicidal ideation, cognitive impairment, motor incoordination, dizziness are also severe forms of ADRs associated with gabapentinoids5. The use of pregabalin is associated with hematological ADRs and gabapentin is also associated with liver toxicity.\n\n\nRespiratory concerns with gabapentin and pregabalin\n\nRespiratory depression, a highly mortal condition, due to gabapentin and pregabalin has been emerging for the past few years even in patients who were not on opioids, though post-marketing studies showed similar effects among patients taking these medications concurrently along with other respiratory suppressants6. In December 2019, the United States Food and Drug Administration (US FDA) issued a drug safety alert on serious breathing problems with gabapentin and pregabalin noticed when used along with central nervous system (CNS) depressants or in patients with lung problems. US FDA reviewed data from the FDA Adverse Event Reporting System (FAERS)7 database of almost five years, i.e. from January 1, 2012 – October 26, 2017, which revealed 49 cases of gabapentinoid-induced respiratory depression. Out of 49, the majority of cases (n=34; 69.3%) were reported with pregabalin and 30.6% (n=15) cases were reported with gabapentin. Of these cases, 92% reported either a respiratory risk factor, including age-related loss of lung function, or the use of a CNS depressant. This report also revealed that 24% percent (n=12) of the 49 patients with respiratory depression died due to respiratory depression and were taking gabapentinoids.\n\n\nWhat do the recent respiratory problems of gabapentin and pregabalin mean for pharmacovigilance in the developing world?\n\nSince these ADR reports are from developed countries, it is difficult for regulators in developing countries to take decisions on these two medications that are also abundantly used in the developing world. For example, in India, there are escalating sales of gabapentin and pregabalin and while comparing the sales in 2017 to those in 2019, it was found that sales of gabapentin and pregabalin increased by 25% and 16%, respectively8.\n\nAs is well documented in the literature9–11, pharmacovigilance programs in developing countries lack robust reporting of ADRs and underreporting is a common issue. In order to bring any regulatory changes such as labeling changes or even ultimately banning the medications, one needs evidence, which largely is lacking at this point in time among developing countries12.\n\nThere have often been issues like this in the past where safety concerns emerge, largely from the developed world, with drugs like selective cyclooxygenase-2 inhibitors (coxibs)13, cerivastatin14 and glitazones15, and the developing world, due to lack of stringent pharmacovigilance mechanisms, is left with little or no choice but to follow the actions taken by the developed world. In addition, developing countries lack options for communicating pharmacovigilance information16 among key stakeholders including consumers, which is another concern. It is astonishing that many developing countries where large quantities of medicines are used still lack strong mechanisms to monitor the safety of their products.\n\n\nRecommendations\n\nIn the current scenario, safety reports of gabapentinoids should be obtained from concerned manufacturers and reviewed for respiratory depression effects. As understood, manufacturers are an important partner in the pharmacovigilance process with the unique advantage of formulation related information. The periodic safety update reviews (PSURs) submitted by the manufacturers of gabapentanoids can be an important source for new signal detection17. In addition, the pharmaceutical manufacturer also has an obligation to report serious ADRs to the regulatory authorities18.\n\nHealthcare professionals should be watchful and report ADRs associated with gabapentinoids. Spontaneous reporting of suspected ADRs in the past have been crucial in detecting ADRs at an early stage19.\n\nLabeling changes should be made and a drug can be banned if needed20. New warnings are necessary to incorporate into prescribing information including package inserts about possible respiratory depression8. Concurrent use of these drugs with other respiratory depressants should be strictly monitored. Anecdotal reports on these medications should be encouraged as they can be crucial in the detection of ADRs21. If noticed, causality and severity assessments should be made for the suspected ADRs.\n\nThe patients on these two medications (and the ones closely chemically related to them) should be provided with proper counseling. Educating patients can help in the early detection of ADRs and active participation of patients can help in the identification of adverse events and ADRs, description of ADRs, and ultimately prevention of drug-related harm22. Hospital drug and therapeutics committees have a crucial role to play in situations like this by disseminating information within the hospital23. Social media may also play a crucial role in the signal generation of suspected ADRs in these situations24. Hospitals using these medications should develop risk management plans associated with gabapentoinoids usage and must disseminate any safety issues to concerned authorities.\n\n\nConclusions\n\nDrug safety is a constantly evolving process and one must be constantly vigilant on the use of these medications. Healthcare professionals, especially prescribing physicians, nurses and pharmacists should be more cautious about using these medications in vulnerable people. Patient education and prescription restrictions of gabapentin and pregabalin may be needed until more data are available.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nQuintero GC: Review about gabapentin misuse, interactions, contraindications and side effects. J Exp Pharmacol. 2017; 9: 13–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInoue K, Takano H: Gabapentin for refractory chronic cough. Lancet. 2013; 381(9867): 623. PubMed Abstract \| Publisher Full Text\n\nTassone DM, Boyce E, Guyer J, et al.: Pregabalin: a novel gamma-aminobutyric acid analogue in the treatment of neuropathic pain, partial-onset seizures, and anxiety disorders. Clin Ther. 2007; 29(1): 26–48. PubMed Abstract \| Publisher Full Text\n\nFuzier R, Serres I, Guitton E, et al.: Adverse drug reactions to gabapentin and pregabalin: a review of the French pharmacovigilance database. Drug Saf. 2013; 36(1): 55–62. PubMed Abstract \| Publisher Full Text\n\nToth C: Pregabalin: latest safety evidence and clinical implications for the management of neuropathic pain. Ther Adv Drug Saf. 2014; 5(1): 38–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGabapentin and the risk of respiratory depression without concomitant opioids. 2017. Reference Source\n\nFDA: Potential Signals of Serious Risks/New Safety Information Identified from the FDA Adverse Event Reporting System (FAERS). 2019. Reference Source\n\nThePrint: US flags two top neurological drugs used in India, warns of serious breathing difficulties. 2019. Reference Source\n\nElshafie S, Zaghloul I, Roberti AM: Pharmacovigilance in developing countries (part I): importance and challenges. Int J Clin Pharm. 2018; 40(4): 758–763. PubMed Abstract \| Publisher Full Text\n\nBiswas P: Pharmacovigilance in Asia. J Pharmacol Pharmacother. 2013; 4(Suppl 1): S7–S19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmin MN, Khan TM, Dewan SM, et al.: Cross-sectional study exploring barriers to adverse drug reactions reporting in community pharmacy settings in Dhaka, Bangladesh. BMJ Open. 2016; 6(8): e010912. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSekine S, Pinnow EE, Wu E, et al.: Assessment of the impact of scheduled postmarketing safety summary analyses on regulatory actions. Clin Pharmacol Ther. 2016; 100(1): 102–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMukherjee D: Selective cyclooxygenase-2 (COX-2) inhibitors and potential risk of cardiovascular events. Biochem Pharmacol. 2002; 63(5): 817–21. PubMed Abstract \| Publisher Full Text\n\nSaito M, Hirata-Koizumi M, Miyake S, et al.: Withdrawal of cerivastatin revealed a flaw of post-marketing surveillance system in the United States. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku. 2005; (123): 41–5. PubMed Abstract\n\nEnsuring drug safety: lessons from the thiazolidinediones. Lancet. 2007; 370(9593): 1101. PubMed Abstract \| Publisher Full Text\n\nAbiri OT, Johnson WCN: Pharmacovigilance systems in resource-limited settings: an evaluative case study of Sierra Leone. J Pharm Policy Pract. 2019; 12: 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMammì M, Citraro R, Torcasio G, et al.: Pharmacovigilance in pharmaceutical companies: An overview. J Pharmacol Pharmacother. 2013; 4(Suppl 1): S33–S37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNukala S: Pharmacovigilance: the role of pharmaceutical companies to protect patients from adverse drug reactions. 2017. Reference Source\n\nWysowski DK, Swartz L: Adverse drug event surveillance and drug withdrawals in the United States, 1969-2002: the importance of reporting suspected reactions. Arch Intern Med. 2005; 165(12): 1363–1369. PubMed Abstract \| Publisher Full Text\n\nHoffman KB, Dimbil M, Tatonetti NP, et al.: A pharmacovigilance signaling system based on FDA regulatory action and post-marketing adverse event reports. Drug Saf. 2016; 39(6): 561–575. PubMed Abstract \| Publisher Full Text\n\nOnakpoya IJ, Heneghan CJ, Aronson JK: Post-marketing withdrawal of 462 medicinal products because of adverse drug reactions: a systematic review of the world literature. BMC Med. 2016; 14: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerrewaerts J, Delbecque L, Orban P, et al.: Patient Participation and the Use of Ehealth Tools for Pharmacoviligance. Front Pharmacol. 2016; 7: 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTyler LS, Cole SW, May JR, et al.: ASHP guidelines on the pharmacy and therapeutics committee and the formulary system. Am J Health Syst Pharm. 2008; 65(13): 1272–83. PubMed Abstract \| Publisher Full Text\n\nPappa D, Stergioulas LK: Harnessing social media data for pharmacovigilance: a review of current state of the art, challenges and future directions. Int J Data Sci Anal. 2019; 8(2): 113–135. Publisher Full Text" }	[ { "id": "67201", "date": "31 Jul 2020", "name": "Sue E Jordan", "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 the opportunity to review this interesting short opinion paper.\nThe SmPCs for gabapentin and pregabalin include respiratory problems under ‘undesirable effects’; however, as the authors say, there are no recommendations to apply this to practice. This applies in all countries, particularly post-industrial societies. Our trial and observation study found too many unaddressed respiratory problems 1,2.\nWhilst I concur with the sentiments expressed, I feel these should be placed in the context of full patient monitoring for the full range of possible adverse effects of prescribed medicines. Respiratory depression is extremely important, as indicated, but it is also important to check patients for other possible undesirable effects of medicines and unmedicated problems, including pain, sedation and falls. We have shown the effectiveness of this approach, and would like to see it more widely adopted1,2. Our website gives free access to our strategy. http://www.swansea.ac.uk/adre/.\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": "68652", "date": "17 Aug 2020", "name": "Tetsu Ohnuma", "expertise": [ "Reviewer Expertise Perioperative multimodal analgesics" ], "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 article on a relevant topic. It is well written and important points are clearly presented. Gabapentinoids, gabapentin and pregabalin, are being increasingly dispensed for indications outside US FDA labelling, particularly as part of multimodal analgesia in the perioperative setting. Emerging evidence from pre-clinical, human volunteer, inpatient, and outpatient studies suggests that their use in combination with opioids may increase the risk of respiratory depression. In our recent studies, use of gabapentinoids on the day of surgery was associated with increased risk of postoperative pulmonary complications in patients who underwent colorectal surgery1. We also found that in-hospital use of both gabapentin and pregabalin had dose dependent associations with pulmonary complications in the immediate postoperative period after total hip and knee arthroplasties2. The adverse effect of perioperative use of gabapentinoids can occur in all countries. While differences in risk across different populations are unclear (for example, the prevalence of pulmonary complications after concurrent use of opioids with gabapentinoids may differ among races), we suggest that a caution be warranted when these drugs are used in combination with opioids. Obviously, it is essential to establish effective safety reporting systems to detect signals of adverse events and disseminate the information to all relevant sections.\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? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-32
https://f1000research.com/articles/10-143/v1	24 Feb 21	{ "type": "Software Tool Article", "title": "RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting", "authors": [ "Travis L. Jensen", "William F. Hooper", "Sami R. Cherikh", "Johannes B. Goll", "Travis L. Jensen", "William F. Hooper", "Sami R. Cherikh" ], "abstract": "Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user’s own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).", "keywords": [ "RP-REP", "ribosomal profiling", "RNA-Seq", "transcriptomics", "differential gene translation", "pathway enrichment", "translational efficiency", "reproducible research", "cloud computing", "AMI" ], "content": "Introduction\n\nWhile the principles for ribosomal profiling (RP) were invented decades ago, the application of next-generation sequencing recently set the stage for genome-wide assessments of translation at codon resolution1–3. The technique makes use of the facts that mRNAs that undergo translation in ribosomes can be fixated to each other using certain chemicals and that the joint ribosome/mRNA complexes can be isolated using chromatography after mRNAs not protected by ribosomes have been degraded using ribonucleases. Following isolation, as for RNA-Seq, mRNA fragments are reverse transcribed and sequenced. The resulting reads may not only represent true ribosomal footprints (reads that originated from mRNA bound to a ribosome, typically ranging between 25 to 35 nt in length) but artifacts/contaminants that were not actively translated such as spurious mRNA or rRNA. These artifacts need to be identified and removed before or during the reference genome alignment step. The resulting clean RP data can then be used for multiple purposes including mapping of translation sites such as initiation regions or elongation regions, characterization and timing of protein folding (when combined with ChIP), and quantification of genome-wide translation via counting of ribosomal footprints per gene4. The last can be utilized to assess changes in mRNA translation in individual cells or different groups of cells in response to certain drugs or therapeutics, providing insights into how such treatments work on the gene and pathway level and how these effects differ across patients or patient cohorts. In an ideal scenario, such information could then be utilized to develop predictive biomarkers to personalize treatment.\n\nBefore scientists can readily analyze RP data, key challenges must be overcome5. These include the provisioning of adequate hardware and software resources to meet the data processing and storage requirements for this type of analysis. Depending on the size of the project, both can be substantial. In addition, setting up a suitable RP data processing and analysis workflow requires significant bioinformatics programming resources and careful workflow parameterization. Analysis and visualization of high-dimensional RP data is not trivial requiring a thorough understanding of multivariate data analysis and statistical methods for appropriately modeling the data6. Even if all these challenges are addressed, ensuring fully reproducible results when all steps are being re-executed is very hard to accomplish unless all components are tightly integrated and automated, and software versions, arguments, and reference data are properly controlled.\n\nHere we present RP-REP7, a new software that allows scientists to address these challenges and, at the same time, facilitates full reproducibility starting from the raw data. The software is designed to run on scalable cloud resources via AWS and pre-built AMI is available atami-00b92f52d763145d3. Alternatively, users can install the software on a local Ubuntu machine using our installation script (RPREP/ubuntu/install-software-v2.1.0.sh). The software also allows for joint data processing analysis of both RP and RNA-Seq data leveraging functionality of our previously published RNA-Seq software (RESEQREP)8. We demonstrate the joint capabilities of our RP-REP software for a published dengue virus study that collected cytosol and ER cellular fractions of human Huh7 cells pre-infection and 6 h, 12 h, 24 h, and 40 h post-infection and performed multiple replicate RNA-Seq and RP experiments (GEO:GSE69602)9.\n\n\nMethods\n\nFigure 1 provides an overview of RP-REP software components. The software is organized into four main components: (1) setup (2) pre-processing (3) analysis, and (4) reporting (Figure 1A). The software utilizes a variety of open-source software in combination with custom shell, R, and Perl scripts to process raw sequence data, quantify gene expression, and track storage, CPU, memory, and other runtime metrics. Preprocessing steps are organized into two stages. Stage 1 executes read filtering steps (Figure 1B) while stage 2 executes read mapping and gene level quantification (Figure 1C).\n\n(A) The software is organized into four main components: (1) setup (2) pre-processing (3) analysis, and (4) reporting. Preprocessing steps are organized into two stages. (B) Pre-Processing Stage 1 executes read filtering steps. (C) Pre-Processing Stage 2 executes read mapping and gene level quantification. (B) and (C) depict the respective Snakemake workflow in the form of a directed graph indicating sequential and parallel processing of certain pre-processing components.\n\nStage 1 performs RNA artefact filtering by retaining raw FASTQ reads that fail to map to an alignment index built from known human rRNAs, rRNA pseudogenes, tRNA pseudogenes, mitochondrial rRNAs (mt-rRNAs), mitochondrial tRNAs (mt-tRNAs), and mt-rRNA pseudogenes, as well as other known rRNA sequences from Ensembl and GenBank. Additional read processing such as adapter trimming, quality and read length filtering to retain reads that likely represent true ribosomal footprints (read length 25–35 nt), can be performed (Figure 1B). Stage 2 performs splice-aware human reference genome alignment of reads that have been trimmed and/or filtered during Stage 1 followed by gene expression quantification carried out on the gene level, reference alignment QC including the generation of gene body read coverage plots (Figure 1C). Processing of samples within each stage is parallelized using the Snakemake workflow management system10. Dependencies of steps within each stage are outlined in Figure 1B and 1C and are optimally prioritized based on available computing resources.\n\nThe analysis component is based on R using both custom R programs as well as existing R/Bioconductor packages (Figure 1A). The reporting component is based on R (Version 3.6.0), the knitr R package (Version 1.23), and LaTeX (Version TeX Live 2012/Debian) for reproducible and automatic PDF report and figure/table generation. All components read user-defined arguments from the respective tab in the RPREP/config/config.xlsx spreadsheet.\n\nAll four workflow components can be run in sequence via the RPREP/run-all.sh script7 or can be run individually to update results of the respective component. When running each individual step, the most recent version of the configuration file will be reloaded to ensure that any modifications to the configuration will be reflected. This is particularly useful for optimizing results by removing outliers, adjusting cut offs and for overall report customization such as color-coding.\n\nStep 1. Configuration parsing and setup: The RPREP/run-setup.sh script executes a parsing of the configuration .xlsx file, downloads the genome and gene models, and prepares the preprocessing and analysis/report result directories.\n\nStep 2A. Stage 1 data preprocessing: The RPREP/source/shell/run-pre-processing.sh script initiates the preprocessing workflow, reading in all user-specified arguments provided in the config.xlsx file. Reference data including user-specified versions of the human reference genome sequence and associated gene model information from the Ensembl database are accessed11. Input for pathway enrichment analysis is handled via Gene Matrix Transposed (GMT) files. GMT files, Entrez Gene IDs, Ensembl Gene IDs, and gene symbols are supported and will be automatically mapped to the human Ensembl reference annotations. We recommend that users obtain reference pathway GMT files from the Molecular Signatures Database (MSigDB)12. The MSigDB import is not automated as download requires registration, but the location of downloaded GMT file can be specified in the configuration file. We do provide a script (RPREP/source/shell/download-gene-sets.sh) to automatically download Reactome, Blood Transcriptome Module, and KEGG pathway information and convert this information to GMT files (note, a license may be required prior to downloading the KEGG pathway information). Contaminant sequences of known human rRNAs, rRNA pseudogenes, tRNA pseudogenes, mitochondrial rRNAs (mt-rRNAs), mitochondrial tRNAs (mt-tRNAs), and mt-rRNA pseudogenes, as well as other known rRNA sequences from Ensembl and GenBank are downloaded using biomaRt software (Version 2.40.0) and the Ensembl Perl API (Version 90). Following the reference data download, a Bowtie2 index13 of contaminant masking sequences will be created to optimize reference alignment searches. Based on FASTQ file input specifications in the config.xlsx, workflow execution downloads and decrypts (optional) FASTQ files from AWS S3 cloud storage, a local file location, or directly from SRA29 via file references. Following the download, the script executes sequence data QC (FastQC). Next, 3' and 5' adapter sequences are trimmed from reads using Cutadapt (Version 2.3)14. Reads with Phred quality score of less than 20 for the majority of bases are removed using FASTQ quality filter from the FASTX Toolkit software package (Version 0.0.14)15. During processing of ribosomal profiling data, reads that fall outside the typical length range of ribosomal footprints (25 nt to 35 nt) are removed. Reads are then aligned to the index of contaminant sequences using Bowtie2 (Version 2.3.5) with its local alignment option. Reads that map to contaminant sequences are removed, and those that do not are output to a FASTQ file for alignment to the human reference genome. With the exception of read length filtering, the RNA-Seq data is processed as described above.\n\nStep 2B. Stage 2 data preprocessing: The human reference genome assembly, gene models, and associated gene annotation information in the form of a gene transfer format (GTF) are obtained from the ENSEMBL database. The genomic reference is built by merging all human chromosomes. Sequence reads from the Stage 1 data preprocessing that failed to map to the index of contaminant sequences are re-aligned to the reference genome using the HISAT2 splice-aware read aligner (Version 2.1.0)16 on stranded, unstranded, or paired-end read data as specified in the config.xlsx, as well as reference based compression (samtools17). Ensembl gene models are used to guide the alignment process. For each sample, the quality of reference alignments is evaluated using RSeQC software (Version 3.0.0)18. Gene expression quantification is carried out on the gene level using the featureCounts function as implemented in the Subread software (Version 1.6.4)19. Reads that overlap with multiple genes or map to multiple genomic locations on the reference genome are excluded. This is followed by assessment of gene body coverage to calculate the average read coverage over reference genome gene sequences using the RSeQC software. Additionally, for both Stage 1 and Stage 2, the workflow will track program arguments, program return codes, input and output file names, file size, MDS checksum, wall clock time, CPU time and memory consumption using the built-in Snakemake benchmarking utility.\n\nStep 3. Data Analysis: The RPREP/run-analysis.sh script initializes analysis datasets for the final reporting steps including distance matrix calculations for global multivariate analysis (PCA, MDS, heatmaps), fold change calculations, and differentially expressed gene (edgeR20), co-expressed gene clusters (pvclust21), and enriched pathway (GoSeq22) identification. Interim result files generated as part of these analyses are saved in gzipped .csv format within the analysis directory.\n\nStep 4. Automatics report generation: The PREP/run-report.sh script produces the final results. It runs R analyses on the intermediate analysis files generated in Step 3 and generates a summary PDF report and result tables in gzipped .csv format as well as individual figure files in .pdf and .png format. This script also summarizes key run time statistics that were collected in the Snakemake benchmarking Step 2 in graphical form.\n\nFor local instance storage (storage immediately accessible by the instance’s operating system), a 60 GiB Elastic Block Store (EBS) volume is sufficient for storing the Ubuntu Linux operating system, user accounts, and temporary analysis space for smaller studies like the dengue virus case study. For studies with larger sample sizes and sequence coverage, we recommend adding one or more additional EBS volumes (see information on AWS set-up on GitHub under RPREP/aws/aws_instructions.docx). We found an m5.2xlarge computational Elastic Compute Cloud (EC2) instance type (8 vCPUs, 32 GiB) to be sufficient for processing and analyzing the dengue virus case study data. Our benchmarks showed that the memory-limiting step is the index generation process executed by HISAT2/Bowtie2 during the preprocessing steps. For the dengue virus case study, the maximum memory requirement was 20 GB, and we expect comparable requirements for studies of similar size.\n\nWe provide a pre-configured RP-REP AMI available on AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)) that combines the Ubuntu Linux operating system Version 18.04.2 (long- term support) with all additional software that is required for RP-REP operation (RPREP/software.xlxs). We prepared a manual that provides step-by-step instructions on how to set up the AWS instance including mounting of EBS volumes for local storage and an optional Elastic IP for machine access (RPREP/aws/aws_instructions.docx). Alternatively, we provide installation scripts that can be executed on a local Ubuntu machine (Version 18.04.2) to install necessary dependencies (RPREP/ubuntu/install-software.sh). In both cases, (AWS or local setup) prior to workflow execution, users would need to pull the latest RP-REP source code from GitHub (git clone).\n\nRP-REP configuration is handled via the RPREP/config/config.xlsx file. The first tab allows users to specify sample metadata. Fields include subject ID, sample ID, sampling time point, a flag (is_baseline) that indicate if a sample was collected prior to treatment, the treatment group, specimen type (e.g. B-cells, PBMCs, etc.), FASTQ sequence file location (AWS S3, local, or remote SRA location), and assay type (ribosomal_profling or rna_seq). In addition, color-coding for time points, treatment groups, and specimen types can be defined. The second tab specifies options related to the pre-processing step. This tab uses a two-column key value pair format to define options. For example, to specify the Ensembl version, users can set the value of the ensembl_version key to 95. Other options include the type of data (stranded: yes/no), paths to all software utilities, and options for executing certain workflow processes (read distribution, FastQC). Paired-end experiments can be accommodated for each sample by specifying two input FASTQ files. The third tab allows users to customize analysis and reporting components. Options include specification of cut-offs to define lowly-expressed genes, differentially expressed (DE) genes, and enriched pathways, as well as the distance metric for heatmap and gene clustering analysis. For further information, see descriptions and examples for each of these options in the configuration file (RPREP/config/config.xlsx). We implemented the framework to dynamically adjust the report presentation depending on the number of subjects, time points, specimen types, and treatment group combinations. For example, Venn diagrams are shown for comparisons between up to five sets (e.g. five time points). Larger sets are accommodated via UpSet plots23. The configuration file allows users to subset the data by limiting the metadata file to samples, treatment groups, and time points of interest.\n\nTo demonstrate the functionality of the RP-REP software, we analyzed a public dengue virus (DNV) data set (GEO: GSE69602)9. The study assessed the impact of DNV infection on viral and host transcription (via RNA-Seq) and translation (via RP) in human Huh7 cells after 6 h, 12 h, 24 h, and 40 h post infection. Prior to running RNA-Seq and RP, Huh7 cells were fractionated to extract RNA and ribosome-bound RNA from cytosolic and ER compartments to understand how viral replication impacts each cellular fraction on the transcriptional and translational level. The same was done for mock infected Huh7 cells to determine results for uninfected cells. DNV is a plus-strand virus; as such it depends on the host to replicate and translate itself.\n\nHere, we used RP-REP to assess how the host transcriptional and translational profile changed over time following DNV infection. The mock-infection sample timepoint was labeled with 0h. For RNA-Seq, 2 replicates were run per time point and cellular department for a total of 20 samples. For RP, 4 replicates were run for a total of 40 samples. The results (RP-REP report) and corresponding configuration file with public SRA FASTQ file references can be found as extended data in data files 1 and 2, respectively7. We provide the configuration file to exemplify the use case and to allow users to reproduce the case study analysis on their own RPREP/RSEQREP AWS instance or Ubuntu Linux machine.\n\nThe RP-REP report for this study includes 182 figures and 82 tables (data file 17). Differential gene expression and translation was assessed by comparing pre- vs. post DNV infection read counts using negative binomial models as implemented in the edgeR R package20. Genes with an FDR-adjusted p-value < 0.05 and fold change ≥4 fold were selected as differentially expression (DE) or differentially translated (DT). The high fold change cut off was chosen to accommodate the strong signal post-DNV infection which required more stringent filtering of DE/DT genes. In the following sections we highlight a subset of the key findings (referenced supplemental tables and figures refer to the corresponding results in data file 17).\n\nHost gene transcription following DNV infection. A noticeable increase in differential transcript abundance in the cytosol of infected Huh7 cells was observed at 24 h (213 DE genes) and 40 h (899 DE genes) (Figure 2A). In the ER, DE gene expression increased from 10, to 24, to 82, and 786 DE genes at 6 h, 12 h, 24 h, and 40 h following DNV infection, respectively. While most of the DE genes expressed in the cytosol were up-regulated (98% at 24 h and 85% at 40 h), up-regulation was suppressed in the ER relative to the cytosol, in particular at 40 h (77% at 24h and 54% at 40 h) (Figure 3A). At 24 h and 40 h, 37 (14%) and 285 (20%) DE genes overlapped between the two compartments. All DE gene results are presented in data file 1 Tables 5–127. Pathway enrichment analysis showed that at 40h post DNV infection, transcripts in the Huh7 cytosol were enriched in GO ENDOPLASMIC RETICULUM (178 DE genes), GO IMMUNE SYSTEM PROCESS (146 DE genes), REACTOME IMMUNE SYSTEM (79 DE genes), GO RESPONSE TO ENDOPLASMIC RETICULUM STRESS (59 DE genes), REACTOME INTERFERONG SIGNALING (26 DE genes), and REACTOME UNFOLDED PROTEIN RESPONSE (25 DE genes). While similar immune system pathways were enriched in the ER compartment including GO IMMUNE SYSTEM PROCESS (111 DE genes), REACTOME IMMUNE SYSTEM (58 DE genes), and INTERFERON SIGNALING (25 DE genes), ER-related stress pathways were not enriched. All pathway enrichment results based on DE genes are provided in data file 1 Tables 19–417.\n\nIn red: up-regulated relative to pre-DNV infection. In blue: up-regulated relative to pre-DNV infection. ER: endoplasmic reticulum.\n\nIn red: DE/DT genes. CPM: gene expression measured in counts per million. Blue line: fold change cut off (4-fold on the original scale).\n\nHost gene translation following DNV infection. For the cytosol fraction, 24 h following infection, 10 differentially translated (DT) genes were identified (Figure 2B). This signal increased to 267 DT genes at 40h post-DNV infection. Most of these DT responses were decreased relative to pre-infection. In the ER compartment, 42 and 1047 DT genes were detected at 24 h and 40 h post-DNV infection, respectively. The ratio of genes with increased translation was 100% for cytosol and 100% for the ER at 24h. While the ratio remained similar for cytosol at 40 h (94%), it dropped to 53% in the ER compartment indicating that protein translation in infected Huh7 cells was strongly suppressed in the ER relative to the cytosol compartment between 24 h and 40 h (Figure 3B). The fraction of shared DT responses between compartments was 9/43 (21%) of DT genes at 24h and 192/1122 (17%) at 40 h indicating that in addition to suppression, fewer genes translated in the cytosol were translated in the ER between 24 h and 40 h. All DT gene results are presented in data file 1 Tables 46–497.\n\nPathway enrichment analysis showed that at 40 h post DNV infection, translation in the Huh7 cell cytosol was enriched in GO IMMUNE SYSTEM PROCESS (58 DT genes), GO DEFENSE RESPONSE TO VIRUS (22 DT genes), GO RESPONSE TO TYPE I INTERFERON (12 DT genes), REACTOME INTERFERON SIGNALING (18 DT genes), and REACTOME CYTOKINE SIGNALING IN IMMUNE SYSTEM (22 DT genes). Most DT genes showed increased translation relative to pre-infection indicating that in the cytosol host proteins related to viral defense were actively translated. In contrast, protein translation in the ER 40 h post-DNV infection was characterized by decreased translation of host RNA related to genes involved in ER-related pathways. This included GO LIPID METABOLIC PROCESS (135 DT genes, 117 DT genes decreased), GO ENDOPLASMATIC RETICULUM (159 DT genes, 136 DT genes decreased), go intrinsic component of plasma membrane (124 DT genes, 110 DT genes decreased), GO CELL SURFACE (65 DT genes, 54 DT genes decreased), REACTOME METABOLISM OF LIPIDS AND LIPIDPROTEINS (59 DT genes, 51 DT decreased), and REACTOME POST TRANSLATIONAL PROTEIN MODFICATION (26 DT genes, 25 DT genes decreased). While some immune responses were still active at 40 h post DNV infection in the ER including REACTOME INTEREFERON SIGNALING (24 DT genes, 1 DT decreased), many immune system-related genes were deprioritized (GO IMMUNE SYSTEM PROCESS had 100 DT genes of which 49 were decreased relative to pre-infection). All pathway enrichment results based on DT genes are provided in data file 1 Tables 54–737.\n\nTime trend plots for co-translated DT genes are provided in data file 1 Figures 127–1427. A selection is shown in Figure 4. The first cluster highlights translational activation of a group of known interferon-inducible anti-viral genes (Figure 4A). The trend line indicated that the antiviral response was first triggered between 12 h and 24 h post DNV-infection with an exponential increase in translation between 12 h and 40 h in both the cytosol and the ER. In contrast, translation of several genes encoding for proteins involved in lipid biosynthesis (HACD2), lipid transfer between ER and mitochondria (VPS13A), and transport (ATP13A, SLC35F5) sharply declined between 12 h and 40 h, suggesting increased competition in the ER between viral and host translation (Figure 4B). Translation in the cytosol for this cluster increased over time potentially to account for the loss in the ER. A similar pattern for the ER compartment was seen for a group of genes related to lipid metabolism (Figure 4C).\n\nEach thin line represents the mean log2 cluster response for a certain gene. The thick line indicates the mean across genes. Co-translated gene clustered were identified using a bootstrap-based method as implemented in pvclust21 using the uncentered Pearson correlation distance in combination with complete linkage clustering. ER: endoplasmic reticulum.\n\n\nDiscussion\n\nRNA-Seq and RP are powerful sequencing-based tools to comprehensively assess cellular responses to treatment on the transcriptional and translational level, respectively. To extract meaning from such data is not trivial, requiring both computational resources as well as programming and biostatistical skills. While a multitude of RNA-Seq and RP software tools and R packages are available24–26, software that fully automate all steps starting from the raw sequencing data and ending with publication-ready tables, figures, and report are rare. Here we presented RP-REP, a new cloud-enabled software that enables researchers to analyze and contrast both RP and RNA-Seq data. The benefit of this software is that it facilitates reproducible research by automating key analysis steps including RP-specific data preprocessing including RNA contaminant filtering, reference alignment, expression/translation quantification, data QC, identification of DE/DT genes, co-expressed/translated gene clusters, and enriched pathways, and calculation of per gene translational efficiency. The software can be tailored to project needs and user data via a user-friendly configuration file. The open-source nature of the software allows for further customization.\n\nAnother benefit is that the software was designed to handle large data volumes via utilizing the Snakemake workflow system for parallel data processing. In combination with the available pre-configured AWS virtual machine image (AMI), this allows for vertical scaling of processing to 96 cores (m5.24xlarge instance, largest single instance available at the time of writing). To track computational requirements, RP-REP monitors computational metrics such as CPU and memory utilization. We used this feature to benchmark computational performance of the RP-REP preprocessing step using the dengue virus case study as an example. To evaluate performance we ran the same 60 samples on increasingly powerful but also more expensive AWS EC2 instance types: m5.2xlarge (8 vCPUs; 32 GiB RAM), m5.4xlarge (16 vCPUs; 64 GiB RAM), m5.8xlarge (32 vCPUs; 128 GiB RAM), and m5.16xlarge (64 vCPUs; 256 GiB RAM) (Figure 5). Doubling the computational resources (CPU cores and RAM) reduced the overall runtime by about 50% when running on an m5.4xlarge compared to an m5.2xlarge and an m5.8xlarge compared to an m5.4xlarge. However, we found that the m5.8xlarge (32 vCPUs; 128 GiB RAM) machine marks the ideal convergence of processing time and cost (Figure 5). To generate the summary PDF report for the 60 samples starting from the raw FASTQ files, sample preprocessing took around 9.25 hours on an m5.8xlarge machine (32 vCPUs; 128 GiB RAM), and analysis and reporting steps took around 9.75 hours on an m5.2xlarge machine (8 vCPUs; 32 GiB RAM). Overall, the benchmark showed that software scaled data processing well with available CPU resources.\n\nThe two ribosomal pre-processing stages were run using increasingly larger AWS instances to assess scalability and to estimate runtimes. The following AWS instance type were utilized: 8 Core 32GB Mem: m5.2xlarge AWS instance; 16 Core 64GB Mem: m5.4xlarge AWS instance; 32 Core 128GB Mem: m5.8xlarge AWS instance; 64 Core 258GB Mem: m5.16xlarge AWS instance.\n\nWe demonstrated the utility of RP-REP using published RNA-Seq and RP data by Reid et al.9. Consistent with the authors findings, we found that the largest changes in transcription and translation occurred between 24 h and 40 h post DNV-infection in the cytosol and ER. Reid et al. and others showed that the virus hijacks a cell’s ER to prioritize viral protein synthesis over non-viral membrane proteins9. Consistent with these results, we found that host translation of genes related to the ER, lipid metabolism, and components of the plasma membrane were strongly suppressed in the ER but not in the cytosol compartment at 40 h post-infection relative to pre-infection. To protect the ER from overload and avoid excess numbers of unfolded proteins, cells can activate the unfolded protein response (UPR) regulatory system9. Our pathway enrichment analysis confirmed gene expression activation of the UPR in the cytosol 40 h after DNV infection. In addition, cellular anti-viral defense mechanisms related gene signatures such as those induced following interferon signaling were activated on the transcriptional and translational level in both cellular compartments at 40 h post DNV infection. While interferon signaling related genes showed an exponential increase of translation over time in both the ER and cytosol, translation for around 50 other immune system-related genes was suppressed relative to pre-infection at 40 h post DNV infection.\n\n\nData availability\n\nRNA-Seq and Ribosomal Profiling data for human Hu7 cells before and 6h, 12h, 24h, 40h after dengue virus infection available from NCBI GEO with accession number GSE69602.\n\nZenodo: emmesgit/RPREP: RPREP v1.0.0. http://doi.org/10.5281/zenodo.44288617.\n\nThis project contains the following extended data:\n\n- Data file 1 - case-study: rprep-report-20201230.pdf (RP-REP results for DNV case study)\n\n- Data file 2 - case-study: config-dengue.xlsx (RP-REP configuration file for the DNV case study)\n\n\nSoftware availability\n\nSource code available from: https://github.com/emmesgit/RPREP\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.44288617\n\nThe cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).\n\nLicense: Subject to various licenses, namely, the GNU General Public License version 3 (or later), the GNU Affero General Public License version 3 (or later), and the LaTeX Project Public License v.1.3(c).\n\nA list of the software contained in this program, including the applicable licenses, can be accessed at https://github.com/emmesgit/RPREP/blob/master/SOFTWARE.xlsx", "appendix": "Author contributions\n\n\n\nTJ: Conception and design; Drafting of Manuscript, RPREP Software Development\n\nWH: Conception and design; RPREP Software Development\n\nSC: Drafting of Manuscript, Documentation and testing, RPREP Software Development\n\nJG: Conception and design; Drafting of Manuscript; Statistical considerations;\n\n\nReferences\n\nIngolia NT, Ghaemmaghami S, Newman JRS, et al.: Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science. 2009; 324(5924): 218–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGlincy NJ, Nicholas TI: Transcriptome-wide measurement of translation by ribosome profiling. Methods. 2017; 126: 112–129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIngolia NT, Brar GA, Rouskin S, et al.: The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012; 7(8): 1534–1550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrar GA, Weissman JS: Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol. 2015; 16(11): 651–664. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSboner A, Mu XJ, Greenbaum D, et al.: The real cost of sequencing: higher than you think! Genome Biol. 2011; 12(8): 125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCalviello L, Ohler U: Beyond read-counts: Ribo-seq data analysis to understand the functions of the transcriptome. Trends Genet. 2017; 33(10): 728–744. PubMed Abstract \| Publisher Full Text\n\nGit E: emmesgit/RPREP: RPREP v1.0.0. (Version v1.0.0). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4428861\n\nJensen TL, Frasketi M, Conway K, et al.: RSEQREP: RNA-Seq Reports, an open-source cloud-enabled framework for reproducible RNA-Seq data processing, analysis, and result reporting [version 2; peer review: 2 approved]. F1000Res. 2017; 6: 2162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReid DW, Campos RK, Child JR, et al.: Dengue virus selectively annexes endoplasmic reticulum-associated translation machinery as a strategy for co-opting host cell protein synthesis. J Virol. 2018; 92(7): e01766–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöster J, Sven R: Snakemake--a scalable bioinformatics workflow engine. Bioinformatics. 2012; 28(19): 2520–2522. PubMed Abstract \| Publisher Full Text\n\nHubbard T, Barker D, Birney E, et al.: The Ensembl genome database project. Nucleic Acids Res. 2002; 30(1): 38–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiberzon A, Subramanian A, Pinchback R, et al.: Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011; 27(12): 1739–1740. 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\nMartin M: Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011; 17(1): 10–12. Publisher Full Text\n\nGordon A, Hannon GJ: Fastx-toolkit. FASTQ/A short-reads preprocessing tools. (unpublished) 2010; 5. Reference Source\n\nKim D, Langmead B, Salzberg SL: HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12(4): 357–360. 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): 2078–2079. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang L, Wang S, Li W: RSeQC: quality control of RNA-seq experiments. Bioinformatics. 2012; 28(16): 2184–2185. PubMed Abstract \| Publisher Full Text\n\nLiao Y, Smyth GK, Shi W: The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013; 41(10): e108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuzuki R, Shimodaira H: Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics. 2006; 22(12): 1540–1542. PubMed Abstract \| Publisher Full Text\n\nYoung MD, Wakefield MJ, Smyth GK, et al.: goseq: Gene Ontology testing for RNA-seq datasets. R Bioconductor. 2012; 8: 1–25. Reference Source\n\nLex A, Gehlenborg N, Strobelt H, et al.: UpSet: visualization of intersecting sets. IEEE Trans Vis Comput Graph. 2014; 20(12): 1983–1992. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang H, Wang Y, Xie Z: Computational resources for ribosome profiling: from database to Web server and software. Brief Bioinform. 2019; 20(1): 144–155. PubMed Abstract \| Publisher Full Text\n\nLegendre R, Baudin-Baillieu A, Hatin I, et al.: RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis. Bioinformatics. 2015; 31(15): 2586–2588. PubMed Abstract \| Publisher Full Text\n\nMichel AM, Mullan JPA, Velayudhan V, et al.: RiboGalaxy: a browser based platform for the alignment, analysis and visualization of ribosome profiling data. RNA Biol. 2016; 13(3): 316–319. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "80139", "date": "10 Mar 2021", "name": "Molly Hannigan", "expertise": [ "Reviewer Expertise RNA localization", "translational regulation", "RNA-seq", "Ribo-seq." ], "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 introduces a high utility software/data processing package that is effectively a “soup to nuts” RPF data processing suite. Paraphrasing from the authors presentation, RP-REP works with local FASTQ files, cloud-storage files (AWS S3), and SRA files. The software includes a useful set of customizable steps that are integral to RFP data processing and analysis. It appears to be particularly strong in alignment QC, data visualization and pathway analysis, in addition to parameters such as gene-specific translational efficiency metrics. RP-REP meets a growing need and as an open source tool, this contribution supports community development and standardization of Ribo-seq computational pipelines. The authors do a commendable job of highlighting the challenges of the ribosome profiling data analysis (paragraph 2 of the Introduction) and the utility of RP-REP to the community can be expected to be high.\nMajor Comments: The manuscript would be strengthened by including a short paragraph in the Introduction that informs the reader of currently available resources, e.g. GWIPS-viz, riboSeqR, riboWaltz, RiboCode, and notes that there are limited resources for fully integrated analyses. In the latter category, RiboToolKit, from the Gregory lab, stands out as having similar functionality to RP-REP. In the Discussion, it would be helpful to briefly contrast RIboToolKit and RP-REP, so that the general reader can better appreciate the utility of each. In particular, it would be helpful to note that RP-REP is open source, cloud-enabled, and can run on both virtual machines and locally. RP-REP appears to be better positioned for pathway analysis, biomarker discovery, drug/therapeutic response analyses, etc., whereas RIboToolKit delves more deeply into modes of translational regulation, codon optimality, translational dynamics.\n3) The analysis pipeline does not appear to normalize RPFs (Ribo-seq) to RNA abundance (RNA-seq). Rather, it appears that it processes RNA-seq and ribosome profiling data independently. This should be clarified as such normalization is standard in Ribo-seq analyses. For example, the authors do not describe if they intersect ribosome profiling with parallel RNA-seq (in Figure 1 or within the body of text).\nMinor Comments:\n\nAbstract – clarify the statement “…by sequencing short mRNA fragments undergoing translation in ribosomes.” This should read “…sequencing ribosome-protected mRNA fragments obtained by ribonuclease digestion of polyribosomes. These fragments represent ribosome-occupied regions of the mRNA.”\n\nIntro: the sentence “The technique makes use of the facts that mRNAs that undergo translation in ribosomes can be fixated to each other using certain chemicals and that the joint ribosome/mRNA complexes can be isolated using chromatography after mRNAs not protected by ribosomes have been degraded using ribonucleases” needs rewording. In the technique, polyribosomes are isolated under native conditions and/or after addition of translation elongation inhibitors such as cycloheximide or emetine, which generally stabilize polyribosomes through processing. The mRNAs are not fixed to one another.\n\nUse case. Second paragraph, fifth line – change department to compartment.\n\nData export in .svg formats would also be useful.\n\nDoes the analysis include read length distribution plots? These are useful for distinguishing between true RPFs and RNA binding protein-protected fragments. Similarly, does the analysis include frame periodicity output? Although the utility of this metric is not entirely clear, given that it can be influenced by the nuclease used in the RPF generation stage and whether the periodicity analysis is anchored at the 5’ or 3’ end of the read, it is useful output to include.\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": "150857", "date": "18 Oct 2022", "name": "Jordan A. Berg", "expertise": [ "Reviewer Expertise Metabolism", "ribosome profiling", "software development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor:\nBy using Docker, this is great for reproducibility, but what if improved version of a dependency is released? How easy will it be to update the pipeline?\n\nAre there options to modify the footprint window? For instance, some work has shown that 21 nt RPFs correspond to open A site ribosomes (https://www.cell.com/molecular-cell/fulltext/S1097-2765(18)31063-3).\n\nIt would be helpful to explain more why each dependency was chosen. For instance, by Bowtie2 vs STAR? Why Cutadapt vs something else?\n\nIt would be helpful to discuss existing pipelines for ribosome profiling data and more clearly explain the rationale for creating this pipeline.\n\nMinor:\nSome of the phrasing is unclear in the introduction. For instance, the authors state that “ribosomes can be fixated together” in ribosome profiling. However, I think what is meant that ribosomes can by fixated to transcripts (via cycloheximide, etc).\n\nI think it would be ideal to cite all dependency software within the manuscript/references for the benefit of the authors of those software.\n\nThe output summary PDF is quite large. Is there a way to prioritize output summaries or make them easy to parse? In the case of the case study, 623 pages seems difficult to intuitively parse, especially if I were a user with minimal ribosome profiling experience. For the results, could headers/sections be specified for each analysis type so users could navigate to the results they are interested in easier?\n\nIt would be nice to quantify how large the input FASTQ files were in the benchmarking to help users predict how long processing would take for their project based on the size of their input files. Also, how would a human dataset scale. Presumable, the sequence search space would be different for this organism, and may impact the processing time requirements.\n\nSoftware:\nThe masking sequences in the source code are for humans, but the dataset tested for this work is not. What was the rationale for doing so, and would the user need to modify masking sequences manually for their organism of interest? I may have just not been clear about this in the writing, but I think clarification would be helpful. It seems like other scripts (e.g. download-ensembl-genome.sh) are also hardcoded for humans.\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/10-143
https://f1000research.com/articles/10-142/v1	24 Feb 21	{ "type": "Software Tool Article", "title": "Interactive biomedical segmentation tool powered by deep learning and ImJoy", "authors": [ "Wei Ouyang", "Trang Le", "Hao Xu", "Emma Lundberg", "Trang Le", "Hao Xu" ], "abstract": "Deep learning-based methods play an increasingly important role in bioimage analysis. User-friendly tools are crucial for increasing the adoption of deep learning models and efforts have been made to support them in existing image analysis platforms. Due to hardware and software complexities, many of them have been struggling to support re-training and fine-tuning of models which is essential to avoid overfitting and hallucination issues when working with limited training data. Meanwhile, interactive machine learning provides an efficient way to train models on limited training data. It works by gradually adding new annotations by correcting the model predictions while the model is training in the background. In this work, we developed an ImJoy plugin for interactive training and an annotation tool for image segmentation. With a small example dataset obtained from the Human Protein Atlas, we demonstrate that CellPose-based segmentation models can be trained interactively from scratch within 10-40 minutes, which is at least 6x faster than the conventional annotation workflow and less labor intensive. We envision that the developed tool can make deep learning segmentation methods incrementally adoptable for new users and be used in a wide range of applications for biomedical image segmentation.", "keywords": [ "Deep Learning", "Segmentation", "Image Analysis", "Interactive Machine Learning", "ImJoy" ], "content": "Introduction\n\nDeep learning-based methods have been widely used to analyze biomedical images for common tasks such as segmentation1,2, denoising3,4 and classification5. Despite their potential, building user-friendly deep learning tools and distributing them to non-experts remains challenging. Some attempts to tackle this challenge, like ImageJ plugins3,6,7 or web applications8–10, have been proposed. Among them, due to practical and technical challenges, most tools (e.g. ImageJ plugins) only allow for inference with pre-trained models. While it lowers the barriers for users to test and evaluate advanced tools, pre-trained models can easily fail or generate unexpected artifacts due to mismatch of the data distribution for many applications. Depending on the training data distribution and many other factors, applying pre-trained deep learning models can easily suffer from overfitting or other generalization problems. This makes them vulnerable to subtle changes such as noise patterns generated by different microscopes, or morphological changes from different samples. Therefore, it is important to improve the generalization of the pre-trained models, or to re-train and finetune models with users’ own data. Previous work including nucleAIzer10 and CellPose1 for nuclei and cell segmentation have demonstrated that more generalized models can be trained by using a large amount of labelled data covering many possible variations such as different imaging modalities and object types. The resulting pre-trained models show promising generalization capability and robustness when applied on unseen data without retraining. However, the fact that this requires a much larger and richer training dataset prevents its use in cases where such a dataset is too expensive to obtain. For most cases, it is thus required to re-train or fine-tune models with a user’s own data.\n\nIn a typical workflow for training a deep learning model, the first step is to annotate the objects (e.g. cells) in the image manually and obtain the corresponding masks. Typically, this step is labor intensive and time consuming. The second step is to use the labelled images to train a deep learning model (e.g. a U-Net). After training, one can use the trained model to process new images. Despite some metrics such as the Jaccard similarity coefficient which can be used to monitor the training, a common issue is that it is hard to predict how much annotation is required for training the model. As a result, users often need to iterate several rounds between annotation and training.\n\nMeanwhile, interactive machine learning tools such as ilastik11 can be used to interactively annotate data and train models by combining the two steps in a more efficient way. It works by first annotating a small amount of data to allow the model to start training in the background. While the model is training, users can work on the annotation and add newly annotated data to the training dataset. In a later stage, instead of annotating all the objects in the image, users can use the model to obtain predicted objects and decide to accept or re-annotate them. This greatly improves the efficiency of both annotations and model training, which not only reduces the overall amount of work required but makes the new annotations more targeted to the weaker part of the model. By coupling the processing of active learning12, the total amount of training data can be further reduced. Besides that, the whole process is beneficial for users in helping them to understand the failure modes of the model.\n\nDespite these advantages, many existing interactive machine learning tools are built for classic machine learning methods such as random forest. While ongoing efforts have been made to train deep learning models interactively, building tools that are accessible for non-experts remains challenging due to the increased complexity for annotating data and training deep learning models from an interactive graphical interface. Nevertheless, there have been many existing tools such as Jupyter notebook and many libraries in the Web and Python ecosystem that can be used for building interactive training processes. To combine them and make it even easier to work with, we developed ImJoy8 which is a web application for building interactive and scalable data analysis tools (ImJoy, RRID: SCR_020935).\n\nIn this work, we demonstrate a tool we built with ImJoy for interactive deep learning-based image segmentation. From a web based graphical user interface (GUI), it allows interactive annotations of images and training powerful deep learning models including basic U-Net and CellPose. While the GUI can run in a desktop web browser, a mobile phone, or a tablet with touch screen, the computation server part can run in a local workstation or remote server. In addition, the GUI can be embedded in Jupyter or Google Colab notebooks to allow easy customization and further development for the developers. It also works independently as an ImJoy plugin which can be easily shared with a hyperlink to the end users.\n\n\nMethods\n\nThe tool is implemented as two ImJoy plugins13: a model trainer plugin and an image viewer plugin. The trainer plugin is mainly responsible for performing model training and the viewer plugin is built on top of Kaibu and used for visualizing and annotating the images. There are additional panels in the GUI for controlling and monitoring the trainer. The model trainer plugin requires Python (version 3.7+) along with other python modules, the image viewer plugin runs in a web browser which requires Chrome (version 80+) or Firefox (version 73+). The two plugins can run directly in ImJoy using Jupyter notebooks with ImJoy Jupyter extension installed, or in a Google Colab notebook.\n\nAs shown in Figure 1, the image viewer interface consists of 1) a set of tools for drawing or editing polygons to mark the objects in the image; 2) the image along with the annotation markups shown as different layers; and 3) control panels for listing the samples, controlling the trainer and monitoring the training loss. The viewer can run independently in modern web browsers on a desktop computer or tablets. Meanwhile, the trainer plugin is written in Python and can run through any Jupyter notebook server locally or remotely. Depending on the models, we use either Tensorflow or Pytorch as the base framework. If available, graphical processing units (GPUs) can be used for acceleration. The two plugins are connected via the remote procedure calls (RPC) provided by ImJoy such that the plugins can call each other’s functions and pass data between them in a transparent manner. For example, the viewer plugin can call the predict function in the trainer plugin to infer the labels and the trainer plugin can call a function in the viewer to obtain the user corrected labels. For interactive image annotation, a powerful combination is to use a touchscreen device (e.g. tablet or mobile phone) with a pen to draw the markups, and in the meantime, train the model with a remote Jupyter notebook server with GPU.\n\nIn addition to the ImJoy interface, with our newly developed Jupyter notebook and Google Colab extension for ImJoy, this interactive annotation tool can also be embedded directly in a notebook interface. This allows developers to quickly customize the trainer plugin in a notebook environment, debug and interact with the trainer through Python code. By running the tool on Binder14 or Google Colab with free computing resources (including GPU access), it allows users and developers to share and reproduce interactive workflows without setting up a local computational environment.\n\nIn order to use the tool, the user needs to prepare a set of unlabeled images and organize them into folders as required by the trainer. Then the user should open the plugin with ImJoy (version 0.11.29+) in a web browser such as Chrome (version 80+) and Firefox (version 73+) to see the image annotation interface. To run the trainer plugin, the user needs to connect to a local or remote Jupyter notebook server (Miniconda 3 or Anaconda 3 is recommended for installing the Jupyter notebook server). GPU hardware is recommended but not mandatory. Alternatively, the user can also use the annotation tool in a Google Colab notebook with GPU runtime type selected, and this is recommended for evaluation without any local installation.\n\nTo train a model from scratch, the user can start by loading an unlabeled image into the viewer and start the initial annotation process (Figure 2a). With the markup tool, the user can then draw polygons to outline each object (e.g. cell) in the image. Once done, these polygons will be sent to the trainer plugin and be saved into a text-based format named GeoJSON. Importantly, the image along with the GeoJSON annotation will be moved and added into a training sample pool. After annotating a few images, the user can start training the model. In another thread, a training loop will be started and for each iteration, the trainer will randomly take a batch of samples (e.g. 1–3 images) from the training sample pool, and train the model for one iteration and repeat the process until the user stops the training loop. After being trained for some iterations (e.g. in 2–5 minutes with a GPU from scratch), the user can start to use the model to perform predictions. From then on, instead of annotating the entire image, the user can send the image to the trainer to obtain the predicted labels, fix the wrong labels and keep the correct ones. Since the label correction process is typically much faster than annotating from scratch, the entire workflow is accelerated. The curated labels will be added to the training sample pool and used for training. Video 115 is a screen recording for using our tool to annotate images and train a CellPose segmentation model from scratch in Google Colab.\n\n(a) Interactive annotation and training workflow. Starting with a set of unlabelled images, annotations are gradually made and moved to the training sample pool for training the model. After some iterations, instead of annotating images from scratch, the model can be used to make predictions such that the user can quickly correct the labels. (b) Segmentation results showing the progression of the interactive training. Specifically, a CellPose model being trained from scratch with 4 images at time 0, then we gradually add new samples to the training sample pool (20s/image). We test the model with a fixed image which is not seen by the network.\n\nThe trainer supports different types of segmentation models including U-Nets with various types of encoder variations16 and the CellPose1 model. It is worth noting that when a suitable pretrained model is used as a start, the user can also skip the initial annotation process and start to correct predicted labels.\n\n\nUse cases\n\nThe tool can be generally applied for annotating images and training deep learning models for segmentation. It is easy to customize and extend to support different types of data organization, image formats and model architectures.\n\nFor demonstration purposes, we provide a small example dataset17 from the Human Protein Atlas18 with 163 samples in total. Each sample is a 4-channel (microtubules, endoplasmic reticulum (ER), nuclei and protein of interest) cell image with manually annotated cell mask in GeoJSON format. The samples cover ~20 different cell-line types with variations in morphology.\n\nFor the model training part, we used a slightly modified CellPose model and training process. Specifically, we switched off the style connection in the U-Net used by CellPose and used Adam19 as the optimizer. The same rotation and scaling augmentations were used as in the original CellPose training workflow. Only two channels (ER and Nuclei) were used as input to the model. Starting by using four labelled images in the training sample pool, new images and labels were gradually moved to the training sample pool during training. Figure 2b illustrates the rapid progression of the interactive learning process. Specifically, we started the trainer (at time 0) and gradually added one annotated sample every 20s into the training pool. With the same test image, we checked the predicted labels. As shown in the figure, the result rapidly improved in less than a minute. In order to better understand the learning process, we initialized the model with random weights (i.e. no pretrained model is loaded). Compared to a conventional annotation workflow, the interactive tool can accelerate the annotation by roughly 6 times (it takes ~2 minutes to manually annotate the image from scratch, and ~20s to correct labels with our tool).\n\n\nConclusions\n\nWe demonstrated an interactive annotation and training tool that is capable of accelerating the annotation process for image segmentation. It works by running a training loop in the background while adding new annotations. The feedback loop improves the annotation efficiency and allows more flexibility for the user to control the training and decide on whether new annotation is needed. With the example dataset, we demonstrated improved efficiency for training and annotation. This type of incremental process is more user-friendly for people who want to adopt deep learning-based methods.\n\nWe foresee that the developed model can be further improved to increase its efficiency, reproducibility and areas of applicability. A more advanced version could for example support 3D annotation and segmentation, allow recording of the annotation and training activities, or use more advanced sampling schemes when selecting which images the user should annotate (also known as active learning).\n\n\nData availability\n\nZenodo: HPA Cell Image Segmentation Dataset. https://doi.org/10.5281/zenodo.443089217\n\nThis project contains the following underlying data:\n\n- hpa_cell_segmentation_dataset_v2_512x512_4train_159test.zip\n\n\nSoftware availability\n\nSample dataset, source codes and tutorial are available from: https://github.com/imjoy-team/imjoy-interactive-segmentation\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.446108013\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 Christian Gnann for testing and evaluating the performance of the interactive annotation tool. The example dataset is annotated by Jay Kaimal and Peter Thul.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nStringer C, Wang T, Michaelos M, et al.: Cellpose: a generalist algorithm for cellular segmentation. Nat Methods. 2021; 18(1): 100–106. PubMed Abstract \| Publisher Full Text\n\nFalk T, Mai D, Bensch R, et al.: U-Net: deep learning for cell counting, detection, and morphometry. Nat Methods. 2019; 16(1): 67–70. PubMed Abstract \| Publisher Full Text\n\nWeigert M, Schmidt U, Boothe T, et al.: Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat Methods. 2018; 15(12): 1090–1097. PubMed Abstract \| Publisher Full Text\n\nOuyang W, Aristov A, Lelek M, et al.: Deep learning massively accelerates super-resolution localization microscopy. Nat Biotechnol. 2018; 36(5): 460–468. PubMed Abstract \| Publisher Full Text\n\nOuyang W, Winsnes CF, Hjelmare M, et al.: Analysis of the Human Protein Atlas Image Classification competition. Nat Methods. 2019; 16(12): 1254–1261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGómez-de-Mariscal E, García-López-de-Haro C, Donati L, et al.: DeepImageJ: A user-friendly plugin to run deep learning models in ImageJ. bioRxiv. 2019; 799270. Publisher Full Text\n\nKrull A, Buchholz TO, Jug F: Noise2Void - Learning Denoising from Single Noisy Images. ArXiv181110980 Cs. 2019. Reference Source\n\nOuyang W, Mueller F, Hjelmare M, et al.: ImJoy: an open-source computational platform for the deep learning era. Nat Methods. 2019; 16(12): 1199–1200. PubMed Abstract \| Publisher Full Text\n\nBannon D, Moen E, Schwartz M, et al.: DeepCell Kiosk: scaling deep learning-enabled cellular image analysis with Kubernetes. Nat Methods. 2021; 18(1): 43–45. PubMed Abstract \| Publisher Full Text\n\nHollandi R, Szkalisity A, Toth T, et al.: nucleAIzer: A Parameter-free Deep Learning Framework for Nucleus Segmentation Using Image Style Transfer. Cell Syst. 2020; 10(5): 453–458.e6. Publisher Full Text\n\nBerg S, Kutra D, Kroeger T, et al.: ilastik: interactive machine learning for (bio)image analysis. Nat Methods. 2019; 16(12): 1226–1232. PubMed Abstract \| Publisher Full Text\n\nSener O, Savarese S: Active Learning for Convolutional Neural Networks: A Core-Set Approach. ArXiv170800489 Cs Stat. 2018. Reference Source\n\nOuyang W, Xu H; trangle1302: CellProfiling/imjoy-interactive-segmentation: v0.1.0 (Version v0.1.0). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4461081\n\nJupyter P, Bussonnier M, Forde J, et al.: Binder 2.0 - Reproducible, interactive, sharable environments for science at scale. Proc 17th Python Sci Conf. 2018; 113–120. Publisher Full Text\n\nOuyang W, Le T, Xu H, et al.: Interactive biomedical segmentation tool powered by deep learning and ImJoy. f1000research.com. Media. 2021. https://doi.org/10.6084/m9.figshare.13721410.v1\n\nYakubovskiy P: Segmentation Models. GitHub. 2019. Reference Source\n\nKaimal J, Thul P, Xu H, et al.: HPA Cell Image Segmentation Dataset (Version v2). Zenodo. [Data set], 2021. http://www.doi.org/10.5281/zenodo.4430901\n\nThul PJ, Åkesson L, Wiking M, et al.: A subcellular map of the human proteome. Science. 2017; 356(6340): eaal3321. PubMed Abstract \| Publisher Full Text\n\nKingma DP, Ba J: Adam: A Method for Stochastic Optimization. ArXiv14126980 Cs. 2017. Reference Source" }	[ { "id": "80101", "date": "08 Mar 2021", "name": "Jean-Yves Tinevez", "expertise": [ "Reviewer Expertise image analysis", "microscopy", "imaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMain comments.\nOuyang et al. present in this article a new module of the software ImJoy, focused on offering interactive Deep-Learning training for segmentation of microscopy images. The goal of this work is to offer biologists a way to iteratively train a Deep-Learning model by manually annotating images. Once an object has been annotated, the training phase runs immediately and displays the results to the user, who can then correct them, add new annotations or stop the training phase if the results are satisfactory. As noted by the authors, this takes some inspiration from the workflow of software like Ilastik, which proved successful.\nThe paper is concise and well written. The software works as expected and we only have minor points. We recommend the publication of this article.\nMinor points.\n1. We are both Python users but I seldom use it. I tried to install the software on a Windows machine. Despite getting very clear installation instructions, I failed and the experience was very frustrating. A core library (pytorch) could not be installed. I attach my report of errors at the bottom of this review in case it helps.\n\nI fully understand that the unavailability of a library is not the responsibility of the author, and that a user must be ready to do whatever it takes to get an academic software running. However I am convinced that the impact of the tool would be greater if the installation would become more robust. This could be achieved either by a more thorough testing and installation documentation, or by working around unavailable libraries. This issue is not specific to Imjoy. The frustration against the difficulty of installing Python academic software is shared by many of our colleagues, who are not expert Python developers but still want to benefit from the many software found in this ecosystem for their Research.\n2. On mac OS we could run the tutorial notebook and train. But without GPU it is very slow. Maybe the author could insist on the importance of GPU? And document typical training time for both cases?\n\n3. Page 3, 1st column, 1st paragraph: “Among them, due to practical and technical challenges, ...” Could you give examples of such challenges?\n4. Page 3, 1st column, 1st paragraph: “However, the fact that this requires a much larger and richer training dataset prevents its use in cases where such a dataset is too expensive to obtain.”. Can you detail a bit more this sentence for biological imaging? How many images in common use cases? What “too expensive” means in practical cases?\n5. Page 3, 2nd column, 1st paragraph: “Despite these advantages, many existing interactive machine learning tools are built for classic machine learning methods such as random forest.”. This sentence seems imprecise. Could the authors be more precise in its meaning?\n6. Page 3, 2nd column,last paragraph: “1) a set of tools for etc…”. Perhaps the points 1 to 3 could be shown in Figure 1 for better clarity?\n7. Could the software be extended for more flexibility with:\nAdding custom losses (dice, bce, cbce), Using custom models, Adding a few built-in optimizers, Letting the user select the channel for training?\n\nInstalling the software. On Windows 10, from scratch (no Python install present before review). I failed to install the software and therefore could not evaluate it. Here is the list of steps I took with the error I met, and attempts I made to circumvent them. Following the instructions on https://github.com/imjoy-team/imjoy-interactive-segmentation with Anaconda3.\n1.The command “pip install -r requirements.txt” cannot complete. I get an error message after getting torch:Collecting torch==1.7.0 Downloading torch-1.7.0-cp37-cp37m-win_amd64.whl (184.0 MB) \|████████████████████████████████\| 184.0 MB 1.7 MB/s ERROR: torch has an invalid wheel, .dist-info directory not found I tried to downgrade requirements to 1.6.0 but it did not work either: ERROR: Could not find a version that satisfies the requirement torch===1.6.0 ERROR: No matching distribution found for torch===1.6.0\n2. The command python3 -m ipykernel install --user --name imjoy-interactive-ml --display-name \"ImJoy Interactive ML\" does not work. It should be python instead of python3.\n3. Running this command generate another error:python.exe: No module named ipykernel This is linked to the error #1 above, as ipykernel is listed in the requirements file\n4. The command imjoy --jupyter returns an error 'imjoy' is not recognized as an internal or external command, operable program or batch file. Again linked to the error #1 above.\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": "80102", "date": "20 Apr 2021", "name": "Jan Funke", "expertise": [ "Reviewer Expertise Machine learning", "computer vision", "bioimage analysis", "computational neuroscience" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper introduces software infrastructure for an important up and coming field: the interactive annotation of biomedical images. The authors convincingly introduce the challenges and opportunities of this field, e.g., the difficulty of setting up computational infrastructure for contemporary machine learning and the potential gains of manual interference during the training process. The presented solution consists of a browser-based tool for image annotation, training, prediction, and correction. Crucially, the tool supports several computational backends for the machine learning part, which can easily be selected without requiring deep expertise in distributed computing or machine learning.\nAs it is, the tool should be seen as an important stepping stone towards a truly interactive training paradigm for experimentalists. Further efforts will be needed to lift this contribution from a proof of concept to a tool that can widely be used by experimentalists and easily extended by machine learning researchers.\nThe manuscript itself is well written and provides a high-level introduction of the tool, its underlying infrastructure, and design decisions. Nevertheless, it remains somewhat unclear who the target audience of this article is. For machine learning researchers in the life sciences, the manuscript would be more valuable if it would describe in detail how the infrastructure can be extended and modified to fit different algorithms. For experimentalists, a more detailed explanation of how to apply this tool would be beneficial.\nDepending on who this manuscript is targeting, I would suggest to answer the following questions.\nFor machine learning researchers:\nWhat are the components that make the infrastructure \"easy to customize and extend\"? How do they interact with each other?\n\nAre there any constraints on ML models that can be used?\n\nWhat is the communication protocol between the front- and backend? Can different annotation tools be used if they implement the protocol?\n\nAre prediction (by an ML model) and post-processing (for point and polygon detection) coupled? What is the intended way to implement custom post-processors, e.g., for tracking of cells over video frames?\n\nWhat motivated the choice for GeoJSON for annotation? Does this choice preempt training on 3D data?\n\nWhat are the implicit assumptions about the data? (2D, in-memory storage of training samples).\n\nWhat is the standard that describes trained models for sharing? What needs to be done to convert an existing model to be used by this framework?\nFor experimentalists:\nWhat kind of use cases does the tool support? What are the limitations of the current tool? (E.g., can it be used on 3D data, on movies, on very large datasets, for semantic segmentation, for denoising, etc.).\n\nWhat kind of augmentations are used by the training algorithm? Are they configurable?\n\nHow exactly does one ingest custom datasets and train from scratch? What is the meaning of some of the paramters (\"channels\", \"style_on\", \"default_diameter\") and how should they be picked for a new dataset?\n\nOnce trained, how does one obtain a segmentation for a set of images? \"Save Annotation\" seems to produce a GeoJSON next to the images in the \"data\" directory, but there is no notification in the frontend. How would one turn this into a segmentation for further analysis?\nOther remarks:\nWhat is the difference between \"Save Annotation\" and \"Send for Training\"?\n\nHow to accept a prediction as a true positive to be included in training?\n\n\"Send for Training\": Where does the sample go? It does not seem to appear in the \"train\" folder. Related, how does one remove a sample from the training pool?\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? No\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/10-142
https://f1000research.com/articles/10-141/v1	24 Feb 21	{ "type": "Software Tool Article", "title": "periodicDNA: an R/Bioconductor package to investigate k-mer periodicity in DNA", "authors": [ "Jacques Serizay", "Julie Ahringer" ], "abstract": "Periodic occurrences of oligonucleotide sequences can impact the physical properties of DNA. For example, DNA bendability is modulated by 10-bp periodic occurrences of WW (W = A/T) dinucleotides. We present periodicDNA, an R package to identify k-mer periodicity and generate continuous tracks of k-mer periodicity over genomic loci of interest, such as regulatory elements. periodicDNA will facilitate investigation and improve understanding of how periodic DNA sequence features impact function.", "keywords": [ "DNA periodicity", "gene regulation", "promoter" ], "content": "Introduction\n\nShort DNA sequence motifs provide key information for interpreting the instructions in DNA, for example by providing binding sites for proteins or altering the structure of the double-helix. A less studied but important feature of DNA sequence motifs is their periodicity1–4. Two famous examples are the universal 3bp periodic (RNY)n pattern (R = A or G, Y = C or U, N = any base) in exons5 and 10-bp periodic k-mers in nucleosome positioning (reviewed in 6 and 7). However, despite the wealth of software focusing on motif discovery and analysis, no tool provides an easy way to quantify the periodicity of a given motif, i.e. the extent to which a given motif is repeated at a regular interval in specific sequences. For instance, HeliCis and SpaMo identify conserved distances between two motifs in sequences of interest, but they do not assess larger scale periodic occurrences of motifs8,9.\n\nHere we present periodicDNA, an R package to investigate k-mer periodicity. periodicDNA provides a framework to quantify the periodicity of any k-mer of interest in DNA sequences. It can identify which periods are statistically enriched in a set of sequences by using a randomized shuffling approach to compute an empirical p-value and can also generate continuous linear tracks of k-mer periodicity strength over genomic loci.\n\n\nMethods\n\nThe two main functions of periodicDNA are getPeriodicity() and getPeriodicityTrack(). The getPeriodicity() function interrogates the extent to which a given k-mer periodically occurs, and at which periods, in one sequence or a set of sequences. It uses a randomized shuffling approach to compute an empirical p-value and identify k-mer periods that are statistically enriched in the sequences of interest. getPeriodicityTrack() generates a linear .bigWig track over genomic loci of interest, representing the periodicity strength of a chosen k-mer at a given period.\n\nperiodicDNA is available as an R package on Github. Package dependencies and system requirements are documented here: https://js2264.github.io/periodicDNA/. periodicDNA has been tested using R (version 4.0.2) on Mac OS (versions 10.11 and later) and Ubuntu 18.04.2 machines.\n\nInternally, getPeriodicity() and getPeriodicityTrack() functions both compute the power spectral density (PSD) of an input k-mer in input sequence(s) to estimate its average periodicity. The magnitude of the PSD reflects the strength of the k-mer signal at periodic positions10. To compute the power spectral density (PSD) of a k-mer of interest in one or several sequences, the following steps are executed (Figure 1):\n\n(A) The k-mer of interest is mapped in the input sequence and all pairwise distances are calculated. (B) The distribution of all resulting pairwise distances (also called the \"distogram\") is generated. (C) The distogram is transformed into a frequency histogram and (D) smoothed using a moving window of 3 to mask the universal three-base genomic periodicity11. (E) To normalize the frequency for distance decay, the local average (obtained by averaging the frequency with a moving window of 10) is subtracted from the smoothed frequency. (F) Finally, the power spectral density (PSD) is estimated using a Fast Fourier Transform (F). In this example, TT shows strong 10bp periodicity.\n\n1. The k-mer occurrences are mapped and their pairwise distances are calculated (Figure 1A).\n\n2. The distribution of all the resulting pairwise distances (also called \"distogram\") is generated (Figure 1B).\n\n3. The distogram is transformed into a frequency histogram (Figure 1C) and smoothed using a moving window of 3 to mask the universal three-base genomic periodicity11 (Figure 1D). To normalize the frequency for distance decay, the local average (obtained by averaging the frequency with a moving window of 10) is subtracted from the smoothed frequency (Figure 1E).\n\n4. Finally, the power spectral density (PSD) is estimated using a Fast Fourier Transform (Figure 1F). The magnitude of the PSD values indicates the contribution of a given period to the overall periodicity of the k-mer of interest. In Figure 1, TT dinucleotides are generally 10bp periodic.\n\nThe package relies on BSGenome packages to handle genome assemblies. Genomic loci can be imported from BED files with rtracklayer and are handled in R by the GenomicRanges classes. Biostrings functions are used to map k-mer occurrences in sequences of interest.\n\n\nWorkflow\n\nTo install the current release of periodicDNA from Bioconductor, use:\n\n\n\nAlternatively, the developing branch of periodicDNA can be installed from Github as follows:\n\n\n\nUsing a provided k-mer (e.g. WW, motif argument) and a set of sequences of interest (seqs argument), getPeriodicity() computes several different metrics:\n\n1. The k-mer power spectral density (PSD) values at different periods obtained by Fourier Transform (following the approach described in the Implementation section). For each individual period T, the corresponding PSDT indicates the relative contribution of the period to the overall periodicity of the k-mer of interest10.\n\n2. The log2 fold-change, for each individual period T, between the observed PSDT and the median of the PSDT values measured after shuffling n times the input sequences (l2FC=log2(PSDT,observedmedian(PSDT,shuffled))).\n\n3. Associated empirical p-values and false discovery rates (FDR) indicating, for each individual period T, whether the observed PSDT,observed is significantly greater than PSDT,shuffled values measured after shuffling n times the input sequences (p=∑i=1n(PSDT,shuffled≥PSDT,observed)+1n+1, 12). Note that empirical p-values are only an estimation of the real p-value. Notably, small p-values are systematically over-estimated as their lower bound is 1/(n + 1).\n\nThese metrics are accessible in the periodicityMetrics table obtained when running getPeriodicity(). For each Frequency (or Period) analysed by Fourier Transform, the resulting PSD value, a log2 fold-change, its associated p-value as well as its false-discovery rate (FDR) are returned (see tables in the examples below).\n\nWe ran getPeriodicity() on a set of 6,533 300-bp long sequences centered at all S. cerevisiae TSSs, to investigate WW periodicity, comparing to 500 shufflings as default. Using 12 cores in parallel, this function took approximately 15 minutes to run. The results were then plotted using plotPeriodicityResults() (Figure 2A), demonstrating the known underlying 10-bp WW periodicity present at promoter sequences in the yeast genome13.\n\n\n\nTo identify periodicity of WW dinucleotides, getPeriodicity() was run on (A) a set of 300-bp long sequences centered at 6,533 S. cerevisiae TSSs and (B) a set of 300-bp long sequences centered at 2,295 ubiquitous C. elegans TSSs14. The plotPeriodicityResults() function was run on the getPeriodicity() results to generate three plots as shown. Left, frequency histogram of distribution of pairwise WW distances; middle, normalised frequency histogram of distribution of pairwise WW distances; right, power spectral densities (PSDs) of a set of experimental sequences (red) and 500 iterations of shuffled sequences (grey). Grey ribbon represents the 95% confidence interval of the PSD values obtained after sequence shuffling. Red-filled dots represent PSD values in experimental sequences statistically higher than those from shuffled sequences (FDR < 0.05).\n\nUsing the same approach, we measured the WW periodicity around ubiquitous TSSs in C. elegans, which have been characterized as largely enriched for WW 10-bp periodic sequences14 (Figure 2B). The 10-bp WW periodicity at ubiquitous C. elegans TSSs is stronger than at all S. cerevisiae TSSs.\n\n\n\nThe generatePeriodicityTrack() function calculates the strength of a given k-mer at a particular periodicity across genomic regions of interest, generating a linear genomic track in .bigWig format (Figure 3). The user specifies a genome and a set of genomic loci, a motif and a period of interest, and a sliding window size (window_size, 100 bp by default) and step value (step_size, 2 bp by default). The input genomic loci are split into small sliding windows and for each window, the k-mer periodicity is quantified as described in the Implementation section. The PSD value at the period of interest (e.g. period = 10) is then retrieved and assigned to the center of the corresponding window. Finally, the resulting .bigwig track is smoothed using a rolling window (smooth_track = 20). generatePeriodicityTrac() should be run in parallel across many cores using the BPPARAM argument from BiocParallel. Using 12 cores, this command takes approximately half a day to produce a periodicity track over ~ 15,000 1-kb-long GRanges with default parameters.\n\nATAC-seq signal and 10-bp WW dinucleotide periodicity at three C. elegans promoters. 10bp WW dinucleotide periodicity signal was generated at 1 kb centered at annotated C. elegans promoters14 using the `generatePeriodicityTrack()` function. The data are vizualised using IGV.\n\nAs an example, we generated a WW 10-bp periodicity linear track over annotated promoters in C. elegans genome. In the previous section, we have shown that sequences in the vicinity of ubiquitous TSSs were statistically enriched for WW 10-bp periodicity. Here, the .bigwig track highlights the increased WW 10-bp periodicity in the sequences immediately flanking ubiquitous promoters, where the -1 and +1 nucleosomes are positioned14.\n\n\n\n\nConclusion\n\nperiodicDNA is an R package that provides functions to investigate the periodicity of k-mers of interest in DNA sequences. It is primarily designed to analyse individual or sets of sequences (typically few hundred bases long and up to a kilobase) to identify overall periodicity of a chosen k-mer. It can also generate linear .bigwig tracks of k-mer periodicity at a chosen period (e.g. 10-bp WW periodicity), over genomic loci of interest. periodicDNA is well integrated within the Bioconductor environment and can easily fit in standard genomic analysis workflows.\n\n\nData availability\n\nThis project contains underlying data published in Serizay et al., 202014. All the data are also available from the original reference.\n\n\nSoftware availability\n\nperiodicDNA is released as an R package on Bioconductor: http://www.bioconductor.org/packages/release/bioc/html/periodicDNA.html\n\nSource code available from: https://github.com/js2264/periodicDNA.\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.453370415.\n\nLicense: GPL-3", "appendix": "Acknowledgments\n\nWe would like to thank T. Winder for his help interpreting Fourier Transform analyses.\n\n\nReferences\n\nXiong H, Buckwalter BL, Shieh HM, et al.: Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides. Proc Natl Acad Sci U S A. 1995; 92(14): 6349–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorotkova MA, Korotkov EV, Rudenko VM: Latent Periodicity of Protein Sequences. Molecular Modeling Annual. 1999; 5(6): 103–15. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarsson E, Lindahl P, Mostad P: HeliCis: a DNA motif discovery tool for colocalized motif pairs with periodic spacing. BMC Bioinformatics. 2007; 8(1): 418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitington T, Frith MC, Johnson J, et al.: Inferring transcription factor complexes from ChIP-seq data. Nucleic Acids Res. 2011; 39(15): e98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYin C, Wang J: Periodic power spectrum with applications in detection of latent periodicities in DNA sequences. J Math Biol. 2016; 73(5): 1053–79. PubMed Abstract \| Publisher Full Text\n\nHowe ED, Song JS: Categorical spectral analysis of periodicity in human and viral genomes. Nucleic Acids Res. 2013; 41(3): 1395–405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNorth BV, Curtis D, Sham PC: A note on calculation of empirical P values from Monte Carlo procedure. Am J Hum Genet. 2003; 72(2): 498–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMavrich TN, Ioshikhes IP, Venters BJ, et al.: A barrier nucleosome model for statistical positioning of nucleosomes throughout the yeast genome. Genome Res. 2008; 18(7): 1073–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerizay J, Dong Y, Jänes J, et al.: Distinctive regulatory architectures of germline-active and somatic genes in C. elegans. Genome Res. 2020; 30(12): 1752–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerizay J: periodicDNA (Version 1.0.0). Zenodo. 2021. http://www.doi.org/10.5281/zenodo.4533704" }	[ { "id": "81590", "date": "26 Mar 2021", "name": "Ilya Ioshikhes", "expertise": [ "Reviewer Expertise Bioinformatics", "gene regulation", "nucleosomes", "algorithms" ], "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 software offered is potentially helpful to the researchers in the area. However the paper (and probably the software itself) should be modified to enable its complete evaluation for indexing.\nComparison to other existing state of the art approaches should be added, e.g. to PerPlot and PerScan tools by Mrazek and colleagues1, ID/MPA by Shelenkov et al., 20082. The progress obtained in the present submission should be clarified.\n\nAll the examples shown in the paper and related links are focused on TT dinucleotides. It is not clear whether oligo nucleotide combinations involving W, S, R, Y, N, and others from the IUPAC nucleotide codes are supported by the present software version.\n\nIt is not clear whether there any other restrictions on the software parameters, e.g. oligonucleotide and sequences lengths.\n\nThe software is composed at R language with key functions provided. To make it really practically helpful, the graphic user interface should be provided, to enable entering oligonucleotides of interest and other parameters from a window or dialogue line, without necessitating the changes in the functions text. As a very minimum, the clear instructions for such changes should be provided.\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": "81589", "date": "12 Apr 2021", "name": "Sabarinathan Radhakrishnan", "expertise": [ "Reviewer Expertise Bioinformatics", "Cancer genomics", "mutational processes and gene regulation." ], "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 by Serizay and Ahringer presents an R/Bioconductor package to investigate the k-mer periodicity in DNA. Both the manuscript and the package details are informative and will be useful to the researchers. The following are some minor comments and suggestions:\na) Regarding the shuffling of input sequences, it is not clear if there is an option to preserve the doublet or triplet nucleotide counts. Is the parameter “order” in getPeriodicity() function meant for that (through ushuffle)? If so, please do mention this in the manuscript and its default value.\n\nb) In addition to the shuffling of input sequences, it would also be useful if the function allows for random selection of N number of sequences (with similar GC content) from the genome or an option for the users to define their own set of input random sequences (through DNAstringSet or bed file).\nc) In Figure 1A, under the “Mapping of all dinucleotides” it is not clear why the single occurrence of ‘T’ is also shaded.\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": "81592", "date": "13 Apr 2021", "name": "Eugene Korotkov", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is aimed at solving an important problem - the study of the structure of promoter sequences. The calculations are performed at a good scientific level, the results are beyond doubt. I have only three, as it seems to me, small comments.\n1. It seems to me that it is not entirely correct to represent the data as a file printout. Probably it would be better to present this data in the form of tables.\n2. Figure 3 evokes some skepticism. It seems to me that it is very difficult to draw a conclusion on 3 promoters. Maybe this is a purely coincidence. I believe that it is necessary to study much more promoters in this way in order to draw any conclusions. Probably more promoter sequences need to be studied.\n3. There are a lot of publications on the search for periodicity in promoter sequences. For example, Kravatskaya GI et al. (2011)1 and Nov Klaiman T et al. (2009)2\nIt seems to me that a little comparative analysis could be done in the discussion.\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": "80117", "date": "12 May 2021", "name": "Boris Lenhard", "expertise": [ "Reviewer Expertise Gene regulation." ], "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\nperiodicDNA is a R/Bioconductor package for the detection and visualisation of string-based periodic motifs (k-mers) in DNA sequences. While there are several periodic motifs that are known to occur in genomic sentences, the paper - and, for the most part, the tool itself - focuses on the analysis of by far the most important of them, the 10-bp periodicity of WW dinucleotides that is the hallmark of nucleosome positioning signals, present in the majority of RNA polymerase II promoters, in most eukaryotic genomes analysed so far.\nThe tool implements the calculation of power spectral density over the distances between the instances of k-mers in a set of sequences by normalising the frequencies of pairwise TT distances against \"smoothed\" distances. It has only a couple of main functions, one for performing general power spectrum analyses, with table of power spectral density as a result, and one that produces a genome browser track for a selected set of sequences using a predefined period size. The latter is shown to produce a characteristic double pattern at bidirectional promoters in C. elegans (Figure 3).\nThe tool is simple, clean, and seems to do its job well. I have only a couple of suggestions:\nThe smoothed local average (Figure 1D) uses a 10 bp window. While that is a suitable window size for nucleosome positioning signal, it might not work for exploring other, possibly unknown periodic motifs. It should at least be remarked on, and the authors should consider adding functionality to suggest an optimal or at least minimal window size for averaging.\n\nThe stated running times on a 12-core CPU for the presented examples look surprisingly long to me. Could the authors provide more detail - with or without profiling the code - about which computational step is the reason the whole calculation takes this long?\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": [] } ]	1	https://f1000research.com/articles/10-141
https://f1000research.com/articles/9-1018/v1	21 Aug 20	{ "type": "Study Protocol", "title": "Protocol for a controlled, randomized, blind, clinical trial to assess the effects of transcranial direct current stimulation associated with balance training using games in the postural balance of elderly people", "authors": [ "Andre Issao Kunitake", "João Carlos Ferrari Corrêa", "Klaine Silva Nascimento", "Bianca Barioni Cardoso de Oliveira", "Natalia Maciel Muniz", "Soraia Micaela Silva", "Fernanda Ishida Corrêa", "João Carlos Ferrari Corrêa", "Klaine Silva Nascimento", "Bianca Barioni Cardoso de Oliveira", "Natalia Maciel Muniz", "Soraia Micaela Silva", "Fernanda Ishida Corrêa" ], "abstract": "Aims: This study aims to evaluate the additional effect of transcranial direct current stimulation (tDCS) on training postural balance with the use of video games in aged. Methods: This is a blinded, randomized, controlled clinical trial protocol, with elderly people of both genders. Participants will be randomized into three training groups: Group 1 (videogame balance training), group 2 (videogame balance training associated with active tDCS), group 3 (videogame balance training associated with sham tDCS). The training will be carried out twice a week for four weeks, totaling eight sessions, and will be performed with the Nintendo Wii videogame console, using games that stimulate the postural balance associated with tDCS, with anode applied over the left dorsolateral prefrontal cortex and cathode on the contralateral supraorbital region at 2 mA for 20 minutes. The postural balance will be assessed using the Mini Test of the Balance Assessment System and posturography. Evaluations will be carried out before and after eight training sessions and 30 days after the end of treatment. Discussion: Some studies show favorable results from the use of video games in improving postural balance in the elderly; however, their effect does not remain long-term. TDCS associated with other therapies can potentiate and prolong the effects of these therapies owing to its ability to stimulate neurotrophins important for neurogenesis, facilitating tasks that require attention, and helping to consolidate learning and memory. The effect of the two associated techniques on balance have not yet been tested in this population. Registration: Brazilian Registry of Clinical Trials ID U1111-1213-4266; registered on 15 October, 2018.", "keywords": [ "Elderly", "postural balance", "transcranial direct current stimulation", "video game" ], "content": "Introduction\n\nAging is a physiological occurrence that has several consequences, including an increased risk of falls1,2. The incidence of falls increases proportionally to age, from 28% to 35% in the elderly over 65 years and from 32% to 42% in those over 75 years. Falls can lead to musculoskeletal injuries, and in more severe cases, they can lead to death1,3,4.\n\nPreventive therapies for the risk of falls can be performed with the use of technological resources, such as videogames. Studies using videogames have shown positive effects in improving the postural control, balance, and functional capacity of the elderly when practiced regularly5. They have been widely used by therapists with high acceptance in clinical practice because they are motivating, challenging, and recreational, showing good evidence to improve postural control in the elderly6–9.\n\nAnother technique that has aroused interest in clinical practice is transcranial direct current stimulation (tDCS), which consists of low-intensity current, and can be applied to the scalp by means of rubber electrodes, thus promoting changes in the potential of resting membrane, such as depolarization by the anode electrode or hyperpolarization by the cathode; modulating cortical excitability10,11. Anodic tDCS, when applied over the long term, can stimulate the brain-derived neurotrophic factor (BDNF), which is an important protein for stimulating neuroplasticity, improving attention, consolidating learning, and memory. Thus, its use has aroused interest for the elderly, in order to reduce cognitive and motor declines12–15.\n\nHowever, the protocols for the use of tDCS associated with tasks that involve balance, in order to improve balance are not yet defined, and are being applied in different dosages and locations16–18, therefore, further studies are necessary to verify the benefit of tCDS in association with other therapies. For that reason, the objective of this study is to assess whether tDCS can enhance and prolong the effects of videogame training on improving postural balance in the elderly. This study hypothesizes that the association of tDCS with virtual reality therapy is more effective in improving balance in the elderly than therapy with videogames alone, and that the addition of tDCS will prolong the effects of balance training.\n\n\nMethods\n\nThis is a protocol for a randomized, controlled blind, longitudinal clinical trial, as shown in Figure 1. This project will be carried out at University Nove de Julho in São Paulo, Brazil.\n\ntDCS. transcranial direct current stimulation.\n\nThe inclusion criteria are people between 60 and 80 years old, of both genders, able to stand or walk without the aid of auxiliary devices, with reduced balance (score ≤25 points) evaluated using the Mini-BESTest19. Exclusion criteria are cognitive impairment (scores ≤14 points) corrected for education, measured by the Mini-Mental State Examination (MMSE)20, presence of lower limb fractures or amputations, neurological diseases, cardiovascular diseases that limit the performance of exercises, untreated acute and chronic respiratory diseases and the presence of pain that limits movement; the presence of contraindications to the use of tDCS.\n\nThe informed consent form will be explained to all participants. The volunteers who participate in this study, must sign the form (supplementary file 1), which guarantees the secrecy and confidentiality of data, free access to the final data, explanations of any kind related to the study and possible compensation for those that suffer from participation in the study.\n\nThe protocol was approved in May 2018 by Human Research Ethics Committee of the University Nove de Julho, São Paulo, Brazil (Opinion number 2.962.837), and Brazilian Clinical Trials Registry (ReBec), number: U1111-1213-4266, in accordance with Resolution 466/12 of the National Health Council of Brazil.\n\nParticipants in this study will be community elders elected according to the criteria established for allocation. They will be randomized into 3 groups:\n\nGroup 1: Control Group (Balance training with video game);\n\nGroup 2: Active group (balance training with video games associated with active tDCS);\n\nGroup 3: Sham Group (Balance training with video games associated with sham tDCS).\n\nA researcher not involved in evaluations or training will be responsible for allocating participants via www.randomization.com.\n\nThe sample size was obtained using G*Power 3.0.10 software, based on the outcomes from a pilot study. The calculation was carried out by the two-way repeated measures analysis of variance (ANOVA), considering the mean value (before and after training, and 30-day follow-up) for the control groups (25.46) active (27.46) and sham (24.66) and the pooled standard deviation (SDpooled) (2.89), with α = 0.05, β = 0.2 (80% of power) and the effect size of 0.40. A total of 15 individuals were determined to be required for each group (total sample: 45 individuals). Considering possible dropouts and to ensure a sample size that will demonstrate the effect of the intervention, the sample will be expanded by 25%, resulting in 19 individuals in each group, thus totalling 54 participants.\n\nAll evaluations will be carried out on three occasions: pre-intervention, after eight treatment sessions and 30 days after the end of the training (follow-up). The training will be held twice a week, for 4 weeks, totalling 8 sessions.\n\nPostural balance. The assessment of postural balance will be performed by the Mini BESTest Scale, which consists of 14 functional tasks, such as sitting and getting up from a chair, standing up, balancing on tiptoes, and on one foot, overcoming walking obstacles, and double activities task21.\n\nPerformance can range from 0 to 28 points. Each test can be performed for up to three attempts and the best result will be obtained. If adaptations are necessary to accomplish this, a point will be deducted from the maximum score obtained. This is a very reliable instrument, with an intraclass correlation (ICC) of 0.84)22.\n\nStatic postural balance. Posturography data will be collected using the Wii Balance Board (Nintendo, Kyoto, Japan), which is a validated instrument for posturography evaluation23. The software for postural assessment is available at http://www.rehabtools.org/sway.html.\n\nThe evaluation protocol will be performed in two conditions, standing with eyes open and then with eyes closed. These two postures are reliable for measuring body sway, with eyes open with (ICC: 0.77) and with eyes closed (ICC: 0.89)23.\n\nTo standardize the collections, the initial position of the feet on the evaluation platform will be marked and the same measurement repeated in all evaluations. The tests will last 1 minute, the initial 30 seconds will be to establish the patient's suitability and the final 30 seconds will be to collect the posturography data.\n\nSymptoms of depression. The symptoms of depression will be evaluated and classified according to severity, using the Beck Depression Inventory (BDI), a questionnaire composed of 21 questions, which can be self-applied. Scores from 0 to 10 indicate an absence of depression; 11 to 18 indicate mild depression; 19 to 29 indicate moderate depression and 30 to 63 is considered severe depression24. In studies with the elderly, the BDI score shows a degree of reliability r = 0.7825. Subsequently, it will be assessed whether the emotional state interfered with the results.\n\nQuality of life (QOL). QOL will be measured by the World Health Organization's Quality of Life Instrument, Bref version26, which is composed of 26 questions, scored from 1 to 5, with the worst and the best scores being 26 and 130, respectively. In studies with the elderly, it showed a high degree of reliability to mediate quality of life in the domain of physical capacity (0.89), psychological well-being (0.95), social relationships (0.81), and the environment (0,93).\n\nThe information related to continuous-use medication will be monitored through the application of a questionnaire prepared by the author. Such information will be used to characterize the sample.\n\nBalance training with video games. The balance training sessions will be performed using only the video games in group 1 (control), group 2 will be the balance training with the video games associated with the active tDCS and group 3 will be the balance training with the video games associated with the tDCS sham.\n\nVideo game training will be carried out with the Nintendo Wii and Wii Fit Plus, placed in a slide projector to enlarge the image. The sequence of games will always be the same, and the participant should play an average of 5 to 7 minutes each game, passing the stage according to their learning.\n\nThe sessions will be held twice a week, for four weeks, totalling eight sessions. Each session will last 20 minutes. The selected games are table tilt, penguin slide and ski slalom, which will be performed standing on the Wii Board Balance and require movements that stimulate balance, such as the anterior, posterior and lateral tilt of the body, without moving (Figure 1).\n\ntDCS will be performed with a tDCS device, the NeuroConn DC_STIMULATOR PLUS, from Germany, by means of two non-metallic surface electrodes, cathode 35 cm² (5 × 7 cm) and anode 25 cm² (5 × 5 cm), both wrapped in sponges moistened in a saline solution.\n\nThe intensity will be 2mA with a 20-second ramp up and down for a 20-minute period of stimulation. The montage will be anodal, unbalanced bilateral bipolar, anode positioned over the dorsolateral prefrontal cortex of the dominant hemisphere (F3 or F4) and the cathode electrode on the supraorbital region contralateral to the anode (Figure 2), according to the criteria of the 10–20 electroencephalogram system27.\n\nSource: author’s own, consent received for publication.\n\nAt the end of each session, a questionnaire on the adverse effects of tDCS will be applied to the participant26 (see Extended data, Assessment of ETCC Adverse Events form).\n\nThe NeuroConn DC-STIMULATOR PLUS device has settings that allow the selection of the active stimulation or sham mode, by inserting codes. A researcher not involved in the procedures will program the equipment with the code to which the individual will be allocated. The stimulus mode will not be perceived by the external (supplementary functioning of the device, therefore, neither the researcher who will apply the intervention nor the individual will know what treatment will be applied (double-blind).\n\nParticipants in the group that will only perform training with videogames will not be blind to treatment; however, a researcher will be responsible for the exclusive training of this group and will not be aware of the procedures with the tDCS of the other groups. All assessments will be made by researchers who have not participated in the training of the elderly, and will, therefore, be blind to the intervention. A blinding questionnaire will be applied to guarantee its reliability and satisfaction with the treatment received (supplementary files 3 and 4), without the researcher knowing the training carried out by the participant.\n\nAll statistical tests will be performed using SPSS (V22, IBM Corporation, New York, USA). Initially, we will perform the test of normality of the sample using the Shapiro-Wilk test, considering the significance level defined as a value of α < 0.05. The parametric data will be expressed as a mean ± SD (standard deviation), and the nonparametric data as a median (IQR); the categorical data will be described as absolute values and as a percentage of the total sample.\n\nThe variables of the data of the Mini-BESTest, Posturography, obtained pre-intervention, post-intervention and 30-day follow-up for the three groups (control, active and sham) will be calculated by the two-way repeated measures analysis of variance (ANOVA) for the parametric data and the Friedman’s test for the nonparametric data.\n\nFinally, to verify whether there is a correlation between BESTest values and the symptoms of depression and quality of life, Pearson’s R correlation (parametric) or Spearman correlation (nonparametric) for the three groups in three evaluated times will be performed, verifying the degree of correlation (r) and its significance (p).\n\nThe protocol was approved in May 2018 by the Brazilian Clinical Trials Registry (ReBec) U1111-1213-4266. Submission of the manuscript occurred after this period, with the collection taking place and the completion is expected to happen in December 2020. The outcomes of this study will be subsequently published in a journal of interest.\n\n\nDiscussion\n\nThe incidence of falls has become an aggravating problem with the increase in the number of elderly people and life expectancy. The costs of falling are also high; prevention is an effective and inexpensive alternative when compared to any other procedure28,29.\n\nPreventive therapies can be performed with the use of video games, since they can be used to improve postural control and balance in the elderly, helping to prevent falls. Besides, they are well accepted for being motivating and recreational and can be easily performed at home7–10.\n\nHowever, there are several types of training protocols, which can vary from 4 to 20 weeks22. For some elderly people, this long process of therapy can be tiring; however, for the consolidation of learning and memory to occur, repetitive training is necessary. According to29, the learning and memory process is directly related to the number of repetitions performed by the elderly, with a better effect in the long run. In this sense, the simultaneous application of tDCS can be a resource that can assist in this process of consolidating learning, improving attention for its ability to stimulate neuroplasticity. For the elderly, it can be an important tool in the rehabilitation process, as it is known that, with advancing age, they present cognitive and motor decline11–14.\n\nHowever, one of the limitations of using tDCS for the elderly is that few studies have investigated its effects on this population; some showing positive effects16,18,30,31, while others do not17,32,33. Protocols combining the two resources have already been carried out in children with cerebral palsy34, Parkinson's disease35, spinal cord injury36 and stroke victims37,38. However, studies that associated tDCS and video games in the elderly was not found.\n\n\nConclusion\n\nThis article presents a randomized, controlled and blind protocol developed to show the effect of the combination of transcranial direct current stimulation (tDCS) associated with training using video games in the elderly. The outcomes obtained will be published and their evidences may contribute for new training alternatives in the elderly.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nHarvard Dataverse: A PROTOCOL OF A CONTROLLED, RANDOMIZED, BLIND, CLINICAL TRIAL, TO CHECK THE EFFECTS OF TRANSCRANIAL DIRECT CURRENT STIMULATION (tDCS) ASSOCIATED WITH BALANCE TRAINING USING GAMES IN THE POSTURAL BALANCE OF ELDERLY PEOPLE. https://doi.org/10.7910/DVN/OJBWHJ\n\nFile ‘Suplementary File.docx’ contains the following extended data:\n\nModel informed consent form.\n\nAssessment of ETCC Adverse Events form.\n\nBlinding questionnaire.\n\nSatisfaction questionnaire.\n\nHarvard Dataverse: SPIRIT checklist for ‘Protocol for a controlled, randomized, blind, clinical trial to assess the effects of transcranial direct current stimulation associated with balance training using games in the postural balance of elderly people’. https://doi.org/10.7910/DVN/OJBWHJ\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 of this study thank the University Nove de Julho and its collaborators for supporting the present study.\n\n\nReferences\n\nda Saúde M, Motta LB da: Envelhecimento e Saúde da Pessoa idosa. 1st ed. Brasil: Ministerio da Saúde; BRASIL. 2006. Reference Source\n\nda Saúde M: Atenção à Saúde da Pessoa Idosa e Envelhecimento. Brasil. 2010.\n\nHorak FB, Henry SM, Shumway-Cook A: Postural perturbations: new insights for treatment of balance disorders. Phys Ther. 1997; 77(5): 517–33. PubMed Abstract \| Publisher Full Text\n\nFerreira OGL, Maciel SC, Costa SMG, et al.: Envelhecimento Ativo e Sua Relação Com a Independência Funcional. Texto e Context Enferm. 2012; 21: 513–8. Publisher Full Text\n\nDonath L, Rössler R, Faude O: Effects of Virtual Reality Training (Exergaming) Compared to Alternative Exercise Training and Passive Control on Standing Balance and Functional Mobility in Healthy Community-Dwelling Seniors: A Meta-Analytical Review. Sport Med. 2016; 46(9): 1293–309. PubMed Abstract \| Publisher Full Text\n\nBisson E, Contant B, Sveistrup H, et al.: Functional Balance and Dual-Task Reaction Times in Older Adults Are Improved by Virtual Reality and Biofeedback Training. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYosephi MH, Ehsani F, Zoghi M, et al.: Multi-session anodal tDCS enhances the effects of postural training on balance and postural stability in older adults with high fall risk: Primary motor cortex versus cerebellar stimulation. Brain Stimul. 2018; 11(6): 1239–1250. PubMed Abstract \| Publisher Full Text\n\nMagnani PE, Genovez MB, Porto JM, et al.: Use of the BESTest and the Mini-BESTest for Fall Risk Prediction in Community-Dwelling Older Adults Between 60 and 102 Years of Age. J Geriatr Phys Ther. 2019; 1. PubMed Abstract \| Publisher Full Text\n\nBertolucci PHF, Brucki SMD, Campacci SR, et al.: O Mini-Exame do Estado Mental em uma população geral. Impacto da escolaridade. Arq Neuropsiquiatr. 1994; 52: 1–7. PubMed Abstract \| Publisher Full Text\n\nPotter K, Brandfass K: The Mini-Balance Evaluation Systems Test (Mini-BESTest). J Physiother. 2015; 61(4): 225. 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Brain Stimul. 2016; 9(5): 641–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManor B, Zhou J, Jor’dan A, et al.: Reduction of Dual-task Costs by Noninvasive Modulation of Prefrontal Activity in Healthy Elders. J Cogn Neurosci. 2016; 28(2): 275–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoene D, Lord SR, Delbaere K, et al.: A Randomized Controlled Pilot Study of Home-Based Step Training in Older People Using Videogame Technology. PLoS One. 2013; 8(3): e57734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Ooteghem K, Frank JS, Horak FB: Practice-related improvements in posture control differ between young and older adults exposed to continuous, variable amplitude oscillations of the support surface. Exp Brain Res. 2009; 199(2): 185–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou J, Lo OY, Lipsitz LA, et al.: Transcranial direct current stimulation enhances foot sole somatosensation when standing in older adults. Exp Brain Res. 2018; 236(3): 795–802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhsani F, Samaei A, Zoghi M, et al.: The effects of cerebellar transcranial direct current stimulation on static and dynamic postural stability in older individuals: a randomized double-blind sham-controlled study. Eur J Neurosci. 2017; 46(12): 2875–2884. PubMed Abstract \| Publisher Full Text\n\nDoeltgen SH, Young J, Bradnam LV: Anodal Direct Current Stimulation of the Cerebellum Reduces Cerebellar Brain Inhibition but Does Not Influence Afferent Input from the Hand or Face in Healthy Adults. Cerebellum. 2016; 15(4): 466–74. PubMed Abstract \| Publisher Full Text\n\nNilsson J, Lebedev AV, Lövdén M: No significant effect of prefrontal tDCS on working memory performance in older adults. Front Aging Neurosci. 2015; 7: 230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrecco LAC, Duarte NAC, Zanon N, et al.: Effect of a single session of transcranial direct-current stimulation on balance and spatiotemporal gait variables in children with cerebral palsy: A randomized sham-controlled study. Brazilian J Phys Ther. 2014; 18(5): 419–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchabrun SM, Lamont RM, Brauer SG: Transcranial direct current stimulation to enhance dual-task gait training in Parkinson’s disease: A pilot RCT. PLoS One. 2016; 11(6): e0158497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoler MD, Kumru H, Pelayo R, et al.: Effectiveness of transcranial direct current stimulation and visual illusion on neuropathic pain in spinal cord injury. Brain. 2010; 133(9): 2565–77. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71407", "date": "02 Nov 2020", "name": "Fernando Zanela da Silva Arêas", "expertise": [ "Reviewer Expertise Neurorehabilitation", "neuromodulation", "traumatic brain inury" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well delineated, and well written - it is clear, objective and relevant. The protocols follow all the ethics criteria for reproduction. Also, the literature does not have works that investigate the approach with this methodologic quality.\n\nDoes the project have some finance?\nThis is a good project. I recommend indexing.\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": [] }, { "id": "74514", "date": "01 Dec 2020", "name": "Rodrigo Vitório", "expertise": [ "Reviewer Expertise Movement Science and Neuroscience", "with experience in conducting clinical trials and reporting their findings." ], "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 interesting ongoing randomized controlled trial examines whether anodal tDCS applied over the dorsolateral prefrontal cortex can enhance and/or prolong the effects of videogame training on balance in older adults. The authors report existing approval by the local ethics committee and ongoing training/intervention and data collection. The study is registered at ensaiosclinicos.gov.br. Methods are sound. I have only a few suggestions that I believe will make the manuscript clearer.\n\nPlease mention stimulation type (i.e., \"anodal\" or \"anodic\") and targeted area (i.e., dorsolateral prefrontal cortex) in the title and aims (abstract and main text).\n\nPlease clarify why the study includes a \"control group\" (no electrodes positioning) and a \"sham group\" (placebo tDCS).\n\nPlease provide further details about the sham tDCS (e.g., will any current intensity be applied? For how long? Etc.)\n\nPlease avoid the use of “elderly”. Consider using “older adults/people” throughout the entire manuscript.\n\nThe primary/secondary outcome measures should be explicitly identified.\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": "6220", "date": "23 Dec 2020", "name": "Andre Issao Kunitake", "role": "Author Response", "response": "Dear reviewer. We appreciate your time reading our study and making your considerations. I adapted the article taking into account everything that was requested, to enrich and make this article more quality. Best regards" } ] } ]	1	https://f1000research.com/articles/9-1018
https://f1000research.com/articles/10-135/v1	22 Feb 21	{ "type": "Research Article", "title": "Exploring the physical and psychosocial experience of Immersion Therapy for people living with a disability", "authors": [ "Karlee Naumann", "Jocelyn Kernot", "Gaynor Parfitt", "Kade Davison", "Jocelyn Kernot", "Gaynor Parfitt", "Kade Davison" ], "abstract": "Background: Immersion Therapy (IT), is an underwater experience using self-contained underwater breathing apparatus (SCUBA) equipment for individuals with disabilities. The aim of this study was to produce a descriptive overview of IT and explore measures used to capture physical and psychosocial experiences. Methods: Six participants, two females and four males aged 24-54, with a range of disabilities were recruited. A single session was filmed and analysed, with a selection of outcome measures explored during and post session. Results: A typical session of IT involves both active and inactive time, with a range of observed activities. All participants showed an increase in heart rate, rating of perceived exertion, and affect, however, these results varied. IT is described as ‘fun, challenging and social’, with participants expressing they enjoy the freedom and experience. The overall perception and experience of the activity tends to be positive. Conclusions: More research is required to determine if IT has significant effects on physical and psychosocial outcomes.", "keywords": [ "Disability", "SCUBA", "Therapy", "Physical", "Psychosocial" ], "content": "Introduction\n\nImmersion Therapy (IT), created by the organisation Determined2 in 2015, is a pool based underwater experience using self-contained underwater breathing apparatus (SCUBA) equipment for individuals with injuries or disabilities. Determined2 have established a comprehensive model for medical screening and safe operating procedures for delivering these services, allowing participants to safely experience a weightless underwater environment. Sessions take place in a 2–5m depth swimming pool using standard SCUBA equipment, including a regulator or full-face mask connected to a 10–12L air tank and a buoyancy control device , which may or may not be worn like a backpack by the participants. Additional equipment that may also be used includes flippers, lead weights, foam floats and various other devices to assist with body positioning under water. Staff provide different levels of support in the pool, based on the client’s requirements, with some participants requiring physical assistance to complete all tasks during their sessions, while others participate independently with staff supervision only. Immersion Therapy is advertised to follow the biopsychosocial model, which is outlined as the interactions between biological, psychological, and social factors which determine the cause, manifestation, and outcome of wellness and disease (Engel, 1977).\n\nThe physical properties of water in conjunction with the use of SCUBA equipment result in the greatly reduced effect of gravity on movement and a uniquely altered sensory experience (e.g. altered sound, and an increase in pressure on the body). Anecdotally, positive effects have been reported by most clients of the service, but no formal evaluation of the service has been undertaken (Determined2, 2018). The implementation of the National Disability Insurance Scheme has meant that, for the first time in Australian history, people with a disability have been provided with flexibility in both finances and choice of services (Warr et al., 2017). In the world of therapeutic service delivery, ‘best practice’ interventions are those implementing service with a solid evidence base (Grol & Grimshaw, 2003). Determining if a service is best practice can only occur if the evidence for effectiveness is shown through systematic and rigorous research processes (Grol & Grimshaw, 2003). More importantly, government funding bodies require services to be underpinned by the best available evidence and use this to determine when they will provide funding for interventions. The Australian National Disability Insurance Agency (NDIA) state that they are working hard to ensure that the process of funding disability-related health supports is guided by the best available evidence (NDIA, 2019). Therefore, for services like IT, ongoing evaluation of the service and the incorporation of evidence-based practice is vital for progression and longevity.\n\nThere is limited peer reviewed literature on SCUBA-based interventions for health and wellbeing. A systematic review by Naumann, et al. identified four papers exploring SCUBA interventions (Naumann et al., 2020). The review found that the potential psychosocial benefits of scuba diving for individuals with disabilities include enhanced freedom of movement and social experiences and an increase in physical self-concept (Carin-Levy & Jones, 2007; Williamson et al., 1984). Reported motivators for participation by divers with disabilities include the excitement of the activity and wanting to play and have fun (Yarwasky & Furst, 1996). It has also been found that teaching people with an intellectual disability to dive can increase the participant’s ability to understand instructions and visual attention (Stefania et al., 2019). There were no physical benefits investigated in the included studies. Overall, further research is required to validate the psychosocial and physical benefits of SCUBA interventions. Unlike the above studies, IT does not aim to teach participants to dive recreationally, it instead utilises SCUBA as a form of ‘therapy’, aiming to exert its effects, either through physical movement underwater and/or the psychosocial experience. This study is one of the first to explore SCUBA as a therapeutic experience for participants living with a disability and specifically investigate the physical effects on participants.\n\nIT is a unique new service, and this study aims to provide a snapshot of the type of activities that participants complete as part of a session. Hence the aim of this study was to firstly produce a descriptive overview of a session of IT through objective observation, and secondly to explore the client experience though a series of physical and psychosocial outcome measures.\n\n\nMethods\n\nThis exploratory collective case study provides a descriptive analysis of IT sessions and explores a range of physical and psychosocial outcomes for participants with a range of disabilities. The research is exploratory in nature, intending to describe the service in an objective manner and investigate a range of measures to understand the participant experience, which will provide some guidance for future research in the area (Yin, 2009). People with a wide range of disabilities access the service and sessions are individualised based on the person’s focus and abilities, thus it was not expected that individual results could be grouped. Therefore, the method of exploratory collective case studies was adopted to represent this variety and allow for a more detailed exploration of the service (Crowe et al., 2011). This study design falls under level 5 on the hierarchy of evidence (Haynes et al., 1997).\n\nAll new and existing clients of IT who were over the age of 18 were invited to take part in the research via an information flyer and asked to contact the research team directly if they were interested in participating.\n\nTo meet the inclusion criteria, participants were required to have the ability to provide informed consent and pass the medical screening process set out by Determined2. The medical screening process involves participants attending an IT medical assessment with a qualified South Pacific Underwater Medicine Society doctor. This process was developed by Determined2 management in collaboration with rehabilitation physicians who had expertise in diving, hyperbaric and rehabilitation medicine from the Royal Adelaide Hospital (Wilkinson et al., 2019).\n\nAn initial meeting was conducted to familiarise the participants with the research and gain written informed consent. The accessibility of measurement tools and processes was considered in the research study design. For example, electronic versions of forms were completed on the program JotForm V4.0, and questions were read out by the candidate to participants if required. Answers were only obtained through a proxy if no other form of data collection was feasible.\n\nEthical approval was obtained from the UniSA Human Ethics Research Committee on the 9th of March 2018.\n\nIT sessions were completed at the Adelaide Aquatic Centre. Sessions were allocated a 1 hour timeslot, with the aim of 30–45 minutes being spent in the water. The IT sessions were conducted by trained ‘Immersion Therapy specialists’ (Determined2 staff members) and were independent of the research. IT Specialists undergo in-house training, completing skills from a competency checklist over approximately 400 hours of training, with further on the job mentored experience and training until deemed independently competent. Each session delivered by the IT Specialist is tailored for the person based on their agreed needs and focus, based on an initial interview and ongoing collaboration. Within a session, the participant decides how long they will spend completing each activity and when to move onto the next. For those who require additional support from the IT Specialist, hand signals or eye gaze are used to determine when to move from one activity to the next.\n\nData were collected by the primary investigator (KN). Table 1 details the data collected at each time point.\n\nNotes: HR = heart rate, RPE = ratings of perceived exertion, FS = feeling scale, SEAS = self-reported experiences of activity settings.\n\nOne session of IT was filmed for each participant, with notes taken on the types of activities completed. The focus of filming was to determine the length of time spent being active vs inactive, as well as time spent doing different activities. The filming was completed during session one, no additional data were collected during this session.\n\nThe assessments were intended to ‘observe’ the experiences of IT, so aimed to be minimally invasive. All methods, excluding the survey, which was developed by the research team, were determined to be valid and reliable. All measures and procedures were piloted before commencing the research, ensuring that the process for data collection was acceptable to participants and minimally disruptive to the session. Piloting involved a small group of people with a disability, who had experience with the IT service, providing feedback on the measures over three separate sessions. The underwater environment requires special consideration for data collection, such as the need for a clear method of communication between the researcher and participant (i.e. hand signals), as well as appropriate equipment (i.e. waterproof paper, clip board, pencils) so that data can be obtained and recorded in a timely manner (see Figure 1 for image of underwater data collection).\n\nIn session 1, background information was gathered on the participants through a physical activity and medical form.\n\nHeart Rate (HR), Ratings of Perceived Exertion (RPE) & Feeling Scale (FS) data collection. HR, RPE, and FS were collected simultaneously before the IT session, during the IT session at 5 minute intervals and after the IT session. Participants were required to point to or signal the RPE and FS result whilst under the water and this was recorded on waterproof paper along with the respective HR value.\n\nRPE. RPE values were gathered in order to determine how hard the participant worked during the IT session. As the population group in this study varies in levels of physical and/or intellectual disability, two versions were used: Borg 6–20 scale and the Eston-Parfitt (E-P) 0–10 ratings of perceived exertion scale. The higher the score, the greater the perceived exertion. The Borg scale has been validated for use in participants with a physical disability, while the E-P scale is better suited for participants with intellectual disability (Eston et al., 2009; Goosey-Tolfrey et al., 2010). Participants decided which scale they preferred to use, with the researcher providing advice and guidance.\n\nFS. FS (-5 to +5) was used to identify how the participant was feeling before, during and after a session (Hardy & Rejeski, 1989). Positive affect is determined by a score above 0, while a negative affect is below 0. Positive mood anchors included 1 – fairly good, 3 – good and 5 – very good. Negative mood anchors included -1 – fairly bad, -3 – bad and -5 – very bad.\n\nHR. Participants wore either a Polar RS800x or a Garmin Swim HR monitor for recording HR over the whole session.\n\nSelf-reported experiences of activity settings (SEAS). The SEAS is a 22-item questionnaire exploring the experience of recreational and leisure activities for participants with disabilities (Gibson et al., 2014). The areas explored in the questionnaire include personal growth, psychological engagement, social belonging, meaningful interactions, and choice & control (King et al., 2014). Each item has both a positive or negative worded choice (i.e. excited or bored), allowing the participant to rate which statement they agree with and to what extent. This questionnaire provides valuable information regarding the participant’s experience of the activity and allows the participant to reflect in a systematic way. Participants completed this measure at the end of session 2.\n\nPerceptions of IT survey. This survey instrument, which comprised of 10 items, was developed by the research team to capture the participant’s perceptions of the IT experience. Specifically, the survey gathered information on what activities were completed in the session, their perceptions of the activities and of the service in general. This included some open-ended questions e.g. asking participants to describe the most (least) beneficial part of Immersion Therapy and included items rated on a 10-point Likert scale e.g. how much socialisation were you hoping to get out of involvement in Immersion Therapy? with 0 being ‘not a lot’ and 10 being ‘a lot’. The survey was developed by the research team and from discussions with staff from D2 who live with a disability. Participants completed this measure at the end of session 2 and were able to complete this measure with the help of the researcher or a family member/support worker if required. An online format was used as this was the most inclusive format available.\n\nDescription of IT. Time-motion video analysis was completed through the program SportsCode, version 11 (11.3.0). SportsCode is a video analysis software, focused on the capturing, coding and analysis of performance sports (typically at an elite level). The program aims to determine speed, distance, and duration of various locomotive activities as well as provide information on work to rest ratios, energy use and movement patterns (Dobson & Keogh, 2007). While SportsCode is not typically used in a therapeutic or recreational activity setting, its ability to identify and track movements is useful in any setting where there is a change from one task to another. An open source alternative to SportsCode, PhysMo, can be found here. For the research, video files were uploaded and then specific ‘events’ (different activities completed in the session) were coded into the categories of active and non-active time. Active time was divided into the following subcategories displayed in Table 2.\n\nTable 2 does not provide a definite list of all the activities that can be completed in IT sessions but does include all the activities completed by participants involved in this research. The variable level of independence in performing each of the above activities is dependent on the level of ability of each individual.\n\nInactive time was subcategorised into different types of rest:\n\nResting on the surface (such as talking to the specialist or adjusting gear set up)\n\nResting underwater (relaxing, stillness)\n\nStatistical analysis. Results for this study are presented using descriptive statistics and non-parametric statistical comparison where possible. Specifically, descriptive analysis was used for SportsCode data, demographic information, and for the reporting of physical experience of the sessions (HR, RPE and FS). Non-parametric statistical analysis included reporting of the SEAS, and some aspects of the survey of perceptions. The survey of perceptions (Naumann et al., 2021) gave an opportunity for the participant to provided extended responses if they desired. As a result, a small portion of the research data is mixed methods, therefore, verbatim qualitative findings are presented in Table 5.\n\n\nResults\n\nThe results are reported first by summarising the characteristics of the participants and then providing an overview of the sessions across cases for comparison. Following this, the data displaying the case by case physical and psychosocial experience is presented. Note, HR, RPE, and FS were not collected for case study 3, as this participant was unable to provide responses underwater due to vision impairment and HR monitor dysfunction.\n\nThe participants included two females aged in their 30s and four males between the ages of 24 to 54. The participants had accessed IT services between 18 months and three years of the time of the research, attending up to two sessions a week. All participants reported that they were not experiencing short term illness at the time of completing the research. Table 3 provides further detail on the participant characteristics and includes the participant’s general ‘focus’ for the session (as determined in consultation with their IT specialist). Participants were asked to keep their session the same as usual and not alter behaviour due to the research involvement.\n\nNote: descriptive factors are reported in a manner that protects the identity of the participants.\n\nFigure 2 provides a visual representation of the time spent in each activity for the individual sessions to be compared to one another. The results are displayed as a timeline representation of the full session. Please note, case study 3 required physical assistance from an Immersion Therapy specialist for all activities in her session. While it may seem that she has completed a great variety of activities, she did not physically move herself but experienced these different types of activities nonetheless. For example, ‘swimming’ involved the IT specialist pulling her through the water in a straight line, while ‘acrobatics’ involved the IT specialists spinning and turning her in the water. For simplicity, inactive time is reported overall in Figure 2, but discussed in detail separately in Table 4.\n\nNote: CS = Case study.\n\nParticipants were active between 57–97 per cent of the time (median 78.2 per cent) and inactive 3–43 per cent of the time (median 21.8 per cent). Swimming without fins was the most completed activity (completed by 4 out of 6 cases), followed by swimming with fins (2 out of 6), the scooter (2 out of 6), and water acrobatics (2 out of 6).\n\nFigure 3 to Figure 7 display the individual SportsCode analysis, HR, RPE and FS results for case studies 1, 2, 4, 5 and 6. It should be noted that the session analysis (filming) was completed prior to the session collecting RPE, FS and HR data. While the sessions were aimed to be kept the same the timescale varied.\n\n3a (session), 3b (HR), 3c (RPE) and 3d (FS): Case study 1.\n\n4a (session), 4b (HR), 4c (RPE) and 4d (FS): Case study 2.\n\n5a (session), 5b (HR), 5c (RPE) and 5d (FS): Case study 4.\n\n6a (session), 6b (HR), 6c (RPE) and 6d (FS): Case study 5.\n\n7a (session), 7b (HR), 7c (RPE) and 7d (FS): Case study 6.\n\nFigure 8 displays the responses to the 22 questions of the SEAS questionnaire. Responses have been displayed as the positively worded statements, with negatively worded answers being reversed in order to reflect the participant's response.\n\nNote: Each of the above statements were rated by the six participants. Red colour is ‘strongly disagree’ anchored from the left, neutral response is in light grey, while dark blue colour is ‘strongly agree’ anchored from the right.\n\nBoth positive and negative responses to questions were reported. The top 5 statements with the most positive responses were ’I felt valued‘, ’I belonged‘, ’got along with others‘, ’I was supported and encouraged‘ and ’good mood‘. The three least positive statements were ’learned new skills‘, ’tried new things‘ and ’challenged’.\n\nIn the survey of perceptions, participants were asked to report ‘what were you hoping to get out of being involved in Immersion Therapy’, with five statements being rated out of 10. The responses from all 6 of the participants were group together into the box and whisker plots below (Figure 9).\n\nParticipants were asked to rate how beneficial they found the activities completed in their session out of 10. While some participants completed numerous activities, the most prominent activities from their sessions are presented in Table 5. Additionally, participants were asked to comment on the most and least beneficial aspects of Immersion Therapy, with this response also displayed in Table 5.\n\n\nDiscussion\n\nImmersion Therapy is a novel SCUBA-based intervention. This research is the first to explore this service and one of the first to explore the use of SCUBA as a therapeutic intervention for people with a disability. The aim of this study was to produce a descriptive overview of what is involved in a session of IT and to explore a series of physical and psychosocial outcomes used to capture the experience of participation.\n\nIT can be described as a unique underwater experience provided to participants with a range of different conditions or disabilities, utilising SCUBA equipment in a controlled pool setting. Tailored to the individual, the service can vary in many factors, including the session length and activities completed. Each session is delivered based on the participant’s personal focuses and preferences and involves both ‘active’ and ‘inactive’ aspects. IT appears to be more than just a physical experience, with participants reporting a range of different physical and psychosocial experiences from participation.\n\nOverall, the average IT session length was 45 minutes, with in-water time ranging from 22 minutes to 43 minutes. Active time typically ranged between 57–97 per cent (median 78.2 per cent) and inactive time 3–43 per cent (median 21.8 per cent) of the session. Common factors in service delivery depended on whether the individual’s focus was on physical fitness outcomes or social outcomes, this focus was decided by the participant in consultation with the IT specialist. Case studies 1, 2, and 5, who were aiming for an improvement in physical capacity, spent their session completing activities such as swimming, walking or exercises. Case studies 3, 4 and 6, who were looking for social activity, to have fun or relax, spent their session completing activities such as the bubble gun, using the scooter or throwing darts. It would be expected that the common patterns of behaviour displayed by those with a physical focus in their session would include exercising at higher intensities and taking fewer rest breaks. However, CS1 and CS2 both swam laps for the duration of their sessions, but the patterning of time swimming and in rest differed. CS1 had 11 moments of rest underwater (total of 3 mins 58 secs), compared to CS2 who only had 3 (over 6 mins 15 secs). This suggests that the intensity of the session differed, with CS2 reaching an intensity (RPE 15) that was less sustainable, resulting in the need for a longer rest period. As for the sessions with focuses on social aspects or having fun, the expected patterns of behaviours would include completing a larger range of activities, as well as working at a lower intensity in the session. This can be seen in CS6, who completed 6 different activities (and a total of 32 events) and whose RPE values stayed consistently low throughout the session. As for rest patterns, CS3-6 all spent very minimal time resting underwater (ranging from 12 secs to 46 secs total). As for rest above water, CS4-6 all spent a considerable amount of time completing this (ranging from 5 mins to almost 12 mins, and across 6–7 moments each). Rest breaks above water were often used to adjust gear and to discuss skills or ideas for the session with the Immersion Therapy specialist. As CS3’s session was completed with an instructor physically moving them around, rest was only taken when the instructor changes position for holding onto the client.\n\nAt the time of this study, the SportsCode program, used to analyse a session of IT for each case, was typically used to analyse sports during training or games. While time-motion analysis was not typically used in therapeutic or recreational activities that were not sport-related, there was no reason to suggest that it cannot be used to help define novel activities such as this. Timing movements and coding into specific categories means that any activity can be defined and quantified to understand an individual’s participation in specific activities and to compare between individuals or groups. This study has shown that the program can be applied in the context of therapeutic or recreational activity and should be considered for other similar research purposes, particularly for understanding how clients use their time between different activities. A study by English et al. (2014), used video analysis to explore tasks completed in physical rehabilitation for stroke patients. In the study, 79 sessions from both individual or circuit-based classes were recorded and analysed, with valuable information being extracted on the type of activities completed and the amount of time being spent on active tasks, ultimately advising practitioners on the most efficient form of rehabilitation (English et al., 2014). While this study did not use a specific video analysis program, it demonstrates that the exploration of time spent active and inactive during rehabilitation or recreational settings has great benefit for health professionals when prescribing exercise and creating recommendations for the wider community.\n\nThis research has demonstrated six different ways an IT session may be spent. The amount of inactive time varies depending on the participant, along with the time spent doing an activity and the number of activities completed. This self-selected variation in the sessions reinforces the client-centredness of the Immersion Therapy service. While the six case studies provide a great deal of insight into the nature of IT, the results of this study are not a full representation of the activities that a participant may complete in a session.\n\nThrough the six participants’ experiences of IT, a selection of physical and psychosocial outcomes were explored. Specifically, this study explored HR, RPE, FS, participation, and perceptions following a single session of IT. Varied responses were seen for all outcome measures, confirming the client-centred nature of the service and reflective of the different conditions experienced by participants.\n\nThe overall trend of HR compared to RPE was similar to that seen in other forms of exercise, there appeared to be a linear increase in HR and RPE (Chen et al., 2002; Eston et al., 1987). However, the participants appeared to have lower HR to begin with in relation to RPE during IT sessions. This was possibly due to a combination of the temperature of the water and the depth of the pool. Research suggests that a water temperature of 30° C can decrease HR by 8 bpm, while 25° C can decrease it by 15 bpm (Lees, 2007). The temperature of the IT pool is kept at 28° C year-round, suggesting that HR may be affected somewhere between 8–15 bpm. Depth is also suggested to decrease HR by 10–15 bpm, due to an increase in stroke volume from an increase in hydrostatic pressure (Lees, 2007). As participants were able to go as deep as 2 m in their sessions, the hydrostatic pressure and temperature have likely also decreased HR response. A study by Matthews & Airey (2001), explored athlete’s RPE and HR responses in deep water running compared to land treadmill running. It was discovered that at the same HR output, RPE ratings were significantly higher. The authors attributed the increase to ‘localized fatigue associated with the unfamiliarity of the task’ and suggested that HR based prescription in a water environment should be reduced by 12–17 bpm (Matthews & Airey, 2001). While these findings do not confirm that the temperature and depth of the pool were the reason to alter the HR prescription, it is clear that the water environment does affect HR. Additionally, as the participants involved in this study had varied disabilities and health conditions, this may have also impacted HR response, specifically if the participant had impaired neurological function affecting their ability to exercise at higher intensities. Research on participants with neurological conditions such as multiple sclerosis and acquired brain injury has shown that they have decreased exercise tolerance (Collett et al., 2011; Dawes et al., 2006).\n\nIt is also important to note that there has been no research found that validated RPE in a completely underwater environment. As for environments with head above water, the Borg 6–20 scale is suggested to be useful in controlling the intensity of aquatic activity (Graef & Kruel, 2006). While it is unclear whether the relationship between HR and RPE is the same as that during land-based activity, the results of this study did show good face validity, with the potential for the use of these measures in setting and monitoring or self-regulation of exercise in this underwater environment.\n\nWhen looking at the individual figures (Figure 3a–d to Figure 7a–d), both CS1 and CS2 had RPE, FS and HR increases as expected for someone working at a moderate to high intensity. Both participants reported high RPEs during their session, with their HR increasing and FS decreasing at this workload. As both participants were aiming to improve their fitness, it appears that the focus of their sessions were being achieved. For CS2, his FS rating was relatively high even during a higher intensity activity, with a drop in his FS response being seen shortly after stopping this activity. As CS2 was diagnosed with a psychosocial disability, it is speculated that this affected his ability to work at the same intensity week to week. The water environment likely acts as a space to escape from thoughts/negative emotions, and to exercise. A study by Haghayeghi et al. (2016), suggests that participation in hydrotherapy can have a positive effect on mental health and quality of life. While it is unclear whether this can be transferred to IT, it was suggested that the improvement was due to increased socialisation, the properties of water and learning of new skills, all of which are integral to an IT session (Haghayeghi et al., 2016). For CS4, the reported HR, RPE and FS reflect that periods of both higher intensity exercise and rest were completed. CS4 discussed that her IT sessions provided both physical and psychosocial ‘relaxation’ effects.\n\n‘I always feel ‘stretched out’ at the end of my session and like my muscles don’t feel as bunched up and tense. I have always loved the water but physically have difficulty getting in and out, so it was too risky to go swimming without considerable help. Immersion Therapy allows me the chance to do that and to focus on myself with no stress or distractions’\n\nAs for CS5, the session began with an elevated HR, with a possible explanation being a higher body temperature as he wore a thermal top before getting into the pool and the outside air temperature was above 28 degrees. Once in the water and his temperature decreased, so did his HR. During the session, CS5 worked at a consistent rate and this is reflected in a steady HR and RPE. Finally, CS6 had a relatively low and consistent HR, RPE and FS, suggesting his session was low intensity. This shows that even though CS6 was ‘active’ for a large period of the session and completed a high number of activities, RPE and HR cannot necessarily be correlated with a specific activity.\n\nDue to the linear relationship between HR and RPE (Chen et al., 2002), it would be expected that RPE and HR response related to how physically hard the participant worked in the session. While the HR response in water appears to be dampened compared to land, it is unclear whether the water environment affects a person’s perception of an activity. The FS results appear to show that in most cases, participants working at a high RPE will also have a decrease in FS. A review by Ekkekakis et al. (2011) demonstrates that working at a high intensity (at ’supra-threshold‘) results in a negative affective response. It is likely that participants experience positive changes at low-moderate (‘sub-threshold‘) intensities, and a large variability in affective responses at moderate-high intensity (close to ventilatory or lactate threshold) (Ekkekakis et al., 2011). While the individual results on HR, RPE and FS from this study are unable to be grouped to look for an overall effect of the service, these results are valuable as they have provided evidence of how varied a session can be and that each activity is experienced differently person to person.\n\nRegarding participation and perception, the six case studies had mostly positive responses. The reported positive aspects of the service included being valued, belonging, getting along with others, improved mood and feeling supported and encouraged during IT sessions. IT is described as exhausting, fulfilling, challenging, fun, enjoyable, freeing, calming, interesting, social, and hard work. Additionally, some of the positive responses go beyond the water-based service itself. Feeling supported, encouraged, getting along with others and being valued suggests that external factors such as the staff, organisation and other participants/attendees are playing their part to provide a positive environment.\n\nIt is important to note that the individualistic nature of IT is likely to result in different responses to the SEAS questionnaire. Different activities and the intensity in which they are completed would affect the responses. Additionally, the responses would also vary depending on whether participants were new to the service or they had been participating for a prolonged period. All participants in this research had been participating for longer than 18 months which, along with the subjective data, concurs that they were finding the activity enjoyable and beneficial. If this was not the case, it seems highly unlikely that they would have participated in the service for such an extended period. The participants reported that IT helped them improve their physical skills/mobility, giving them a sense of freedom and that the service was fun/enjoyable. As for reported negative aspects, the responses suggested that participants did not share their own ideas, learn new skills or try new things. As the case studies have been attending IT sessions for a prolonged period, they were likely very familiar with the service and not necessarily trying new activities each week.\n\nThe results of this research show that IT is a physical, psychological and social experience, providing evidence that the service fits the biopsychosocial model (Engel, 1977). In the case of IT, the ‘bio’ (biological) aspect includes the physical aspect of the session. Participants are experiencing varying sessions that are physically affecting their mood, RPE and HR in different ways. For some participants, they are experiencing what would be considered a high level of physical activity, and for others, they are completing lower intensity activity. It appears that this physical aspect may alter the participant’s overall health and wellness, but more research is required to determine how. The ‘psychosocial’ (psychological and social) aspects are evident in the responses from the survey and the SEAS questionnaire. Responses relating to ‘freedom’, ‘enjoyment’ and other positive factors when completing an IT session, show that participants are experiencing changes to their psychological health. Research has suggested that SCUBA interventions may help to enhance participant’s physical self-concept (Carin-Levy & Jones, 2007; Williamson et al., 1984). While self-concept was not directly measured in this study, the results from the SEAS and survey (particularly the social aspects) support the likelihood of improvements to this for IT participants, which may result in increases to overall health and wellbeing (Tam, 1998). Additionally, participants reported social connections with staff, other participants and the wider community involved in IT sessions. These appear to be valuable and cherished social interactions, with participants indicating feeling valued and supported in this environment.\n\nOverall, this research is valuable as it provides a systematic and objective description of a SCUBA intervention using a novel approach. To the best of our knowledge, no other research has provided this level of detail into the exact breakdown of a SCUBA therapy for people living with a disability. Additionally, no other research has explored the physical experience of SCUBA interventions for participants living with a disability. This research has provided valuable insight into how to conduct physical outcomes in an underwater setting and provides a framework for future research. In an area where there has been a lack of research, both the systematic description and the exploration of the physical and psychosocial effects of IT are important. Future recommendations include exploring the effects of this service over an extended period, particularly in people who are naive to the service beforehand.\n\nThe participants volunteered to be part of the research which may introduce a bias. Determined2 advertises perceived benefits and client testimonies through their website and social media. This bias could result in participants having an expectation for a positive result and therefore affecting responses to the self-reported measures. This is a bias that the research team could not control.\n\nMany valuable lessons were learnt from completing data collection in an underwater setting. Not only is this a novel experience, made more difficult by the water environment, but it can result in methodological limitations. Collection of RPE, FS and HR were limited to 5-minute intervals in order to minimise the burden on the participant, prevent a disruption to the flow of the session and allow time for the researcher to collect the desired information. Collection in 5-minute blocks may have resulted in ‘missed data’, as not all activities completed were able to have a corresponding RPE, FS and HR. If the collection of HR, RPE and FS was conducted in shorter time intervals (i.e. at the end of every minute), the disruptive effect on the session would be greater than the potential for missed data.\n\nAnother lesson learnt is that HR is difficult to measure in an underwater setting. There were a number of technical issues that occurred when using the HR monitors and, while both the Polar RS800x and Garmin Swim HR monitors are able to be used in water (with the Garmin HR monitor specifically designed for swimming), these monitors were not necessarily designed for full underwater immersion. This study identified techniques that helped minimise HR connection issues, including using a strap with a silicon-based electrode and making sure the strap is firm on the skin (to ensure it is not dislodged underwater). For CS3, no matter how much troubleshooting was completed, the HR monitor did not work for them once submerged. Ways to combat the HR limitations are limited by the technology available.\n\nGiven the limited evidence on SCUBA-based interventions for people with disabilities and the heterogenous nature of the sample population, an exploratory collective case study (hierarchy 5 on level of evidence) was chosen for this study. Caution should be given to generalising these results. Due to the results being gathered at a single time point (one session) and across varying participants completing different activities, they were unable to be grouped together to see if there is an overall effect or change. Triangulation was performed where possible in order to provide support between quantitative and qualitative results, however limited triangulation of results could occur due to the nature of the data gathered. The research team would like to acknowledge that a limitation of this paper is the study design and that it is important to be mindful of this limitation when interpreting the results. Future research focused on completing qualitative interviews would provide an opportunity for participants to express their thoughts, feelings and experiences in more depth. Additionally, future research should consider involving a larger number of participants with data collection over numerous timepoints to determine the effect of the intervention.\n\n\nConclusion\n\nImmersion Therapy is an individualised service with a range of different activities offered depending on the person’s preferences and focuses. The service can be summarised as a positive, physically rewarding and socially supportive experience. Six participants involved in the research experienced varying physical and psychosocial effects from a single session of IT. This study provides valuable lessons learnt for collecting data in an underwater setting but as this research is exploratory in nature, results need to be interpreted with caution.\n\n\nData availability\n\nOpen Science Framework: Exploring the physical and psychosocial experience of Immersion Therapy for people living with a disability\n\nhttps://doi.org/10.17605/OSF.IO/FHRMW (Naumann et al., 2021)\n\nThis project contains the following underlying data:\n\nRaw Data.xlsx (Raw underlying data of paper)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nOpen Science Framework: Exploring the physical and psychosocial experience of immersion therapy for people living with a disability\n\nhttps://doi.org/10.17605/OSF.IO/FHRMW (Naumann et al., 2021)\n\nThis project contains the following extended data:\n\nIT Survey of Perceptions.pdf (Full copy of survey of perception)\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\nThis study was conducted during the degree of Masters by Research (Author KN). The authors would like to acknowledge the Australian Government Research Training Program Scholarship, the Lifetime Support Authority, the Future Industry Accelerator Program and the University of South Australia. The research team would also like to acknowledge the participants and thank them for their involvement.\n\n\nReferences\n\nCarin-Levy G, Jones D: Psychosocial aspects of scuba diving for people with physical disabilities: an occupational science perspective. Can J Occup Ther. 2007; 74(1): 6–14. PubMed Abstract \| Publisher Full Text\n\nChen MJ, Fan X, Moe ST: Criterion-related validity of the Borg ratings of perceived exertion scale in healthy individuals: a meta-analysis. J Sports Sci. 2002; 20(11): 873–99. PubMed Abstract \| Publisher Full Text\n\nCollett J, Dawes H, Meaney A, et al.: Exercise for multiple sclerosis: a single-blind randomized trial comparing three exercise intensities. Mult Scler. 2011; 17(5): 594–603. PubMed Abstract \| Publisher Full Text\n\nCrowe S, Cresswell K, Robertson A, et al.: The case study approach. BMC Med Res Methodol. 2011; 11: 100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDawes H, Scott OM, Roach NK, et al.: Exertional symptoms and exercise capacity in individuals with brain injury. Disabil Rehabil. 2006; 28(20): 1243–50. PubMed Abstract \| Publisher Full Text\n\nDetermined2: Immersion Therapy Service. Determined2, Adelaide, South Australia, viewed 4th of April, 2018. Reference Source\n\nDobson BP, Keogh JWL: Methodological Issues for the Application of Time-Motion Analysis Research. Strength Cond J. 2007; 29(2): 48–55. Reference Source\n\nEkkekakis P, Parfitt G, Petruzzello SJ: The pleasure and displeasure people feel when they exercise at different intensities: decennial update and progress towards a tripartite rationale for exercise intensity prescription. Sports Med. 2011; 41(8): 641–71. PubMed Abstract \| Publisher Full Text\n\nEngel GL: The need for a new medical model: a challenge for biomedicine. Science. 1977; 196(4286): 129–36. PubMed Abstract \| Publisher Full Text\n\nEnglish C, Hillier S, Kaur G, et al.: People with stroke spend more time in active task practice, but similar time in walking practice, when physiotherapy rehabilitation is provided in circuit classes compared to individual therapy sessions: an observational study. J Physiother. 2014; 60(1): 50–54. PubMed Abstract \| Publisher Full Text\n\nEston RG, Davies BL, Williams JG: Use of perceived effort ratings to control exercise intensity in young healthy adults. Eur J Appl Physiol Occup Physiol. 1987; 56(2): 222–24. PubMed Abstract \| Publisher Full Text\n\nEston RG, Lambrick DM, Rowlands AV: The perceptual response to exercise of progressively increasing intensity in children aged 7-8 years: Validation of a pictorial curvilinear ratings of perceived exertion scale. Psychophysiology. 2009; 46(4): 843–51. PubMed Abstract \| Publisher Full Text\n\nGibson BE, King G, Kushki A, et al.: A multi-method approach to studying activity setting participation: integrating standardized questionnaires, qualitative methods and physiological measures. Disabil Rehabil. 2014; 36(19): 1652–60. PubMed Abstract \| Publisher Full Text\n\nGoosey-Tolfrey V, Lenton J, Goddard J, et al.: Regulating intensity using perceived exertion in spinal cord-injured participants. Med Sci Sports Exerc. 2010; 42(3): 608–13. PubMed Abstract \| Publisher Full Text\n\nGraef FI, Kruel LFM: Heart rate and perceived exertion at aquatic environment: differences in relation to land environment and applications for exercise prescription - a review. Rev Bras Med Esporte. 2006; 12(4): 221–28. Publisher Full Text\n\nGrol R, Grimshaw J: From best evidence to best practice: effective implementation of change in patients' care. Lancet. 2003; 362(9391): 1225–30. PubMed Abstract \| Publisher Full Text\n\nHaghayeghi M, Kalani N, Nikseresht A: The effect of eight weeks of hydrotherapy on life quality and depression in elderly women over 50 years of jahrom city in Iran. IIOAB J. 2016; 7: 588–92. Reference Source\n\nHardy CJ, Rejeski WJ: Not what, but how one feels: the measurement of affect during exercise. J Sport Exerc Psychol. 1989; 11(3): 304–17. Publisher Full Text\n\nHaynes RB, Sackett DL, Richardson WS, et al.: Evidence-based medicine: How to practice & teach EBM. Canadian Medical Association Journal. 1997; 157(6): 788. Reference Source\n\nKing G, Batorowicz B, Rigby P, et al.: Development of a measure to assess youth self-reported experiences of activity settings (SEAS). Intl J Disabil Dev Educ. 2014; 61(1): 44–66. Publisher Full Text\n\nLees TA: Heart-rate response to exercise in the water: Implications for practitioners. International Journal of Aquatic Research and Education. 2007; 1(3): 11. Publisher Full Text\n\nMatthews M, Airey M: A comparison of ratings of perceived exertion during deep water running and treadmill running: considerations in the prescription of exercise intensity. Sports Medicine, Training and Rehabilitation. 2001; 10(4): 247–56. Publisher Full Text\n\nNational Disability Insurance Agency (NDIA): Disability related health supports. 2019. Reference Source\n\nNaumann K, Kernot J, Parfitt G, et al.: What are the effects of SCUBA-based interventions for clients with neurological disability, autism or intellectual disability? A systematic review. University of South Australia, Diving and Hyperbaric Medicine Journal, (Submitted for review). 2020.\n\nNaumann K, Davison K, Parfitt G, et al.: Exploring the physical and psychosocial experience of immersion therapy for people living with a disability. 2021. http://www.doi.org/10.17605/OSF.IO/FHRMW\n\nStefania M, Marsico E, Cassese FP: Assessment of the impact of an inclusive diving program, on subjects with cognitive disability: analysis of the enhancement of cognitive processes. Journal of Physical Education and Sport. 2019; 19(5): 1937–42. Publisher Full Text\n\nTam SF: Comparing the self-concepts of persons with and without physical disabilities. J Psychol. 1998; 132(1): 78–86. PubMed Abstract \| Publisher Full Text\n\nWarr D, Dickinson H, Olney S, et al.: Choice, control and the NDIS: Service users' perspectives on having choice and control in the new National Disability Insurance Scheme. Melbourne: Melbourne Equity Insitute. 2017. 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Reference Source" }	[ { "id": "118780", "date": "10 Jan 2022", "name": "Kerri Morgan", "expertise": [ "Reviewer Expertise Rehabilitation", "Disability", "and Physical Activity." ], "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 overview: This manuscript focuses on a unique therapeutic intervention for individuals with disabilities. The authors present a thorough overview of what activities were involved in the IT sessions as well as exercise and psychosocial related outcomes following participant engagement in one session. Important implications and lessons learned are presented at the end. Below are a few areas that could benefit from clarification or further explanation.\n\nTitle: Consider adding the study deign (case studies) to the title.\n\nAbstract: Under the methods consider describing the types or areas of outcomes measured, such as exercise/activity and psychosocial related outcomes.\n\nIntroduction: The introduction does a nice job of introducing IT and setting up the rest of the manuscript. However, the paper does not mention how immersion therapy may be the same and/or different as another therapeutic intervention in the water (aquatic therapy) and how the benefits and risks may the same and/or different as aquatic therapy.\n\nMethods: The methods section provides a thorough outline of the IT sessions, data collection, and data analysis. A few more details would be helpful …\n\n“All methods, excluding the survey, which was developed by the research team, were determined to be valid and reliable.” This statement or more descriptive statements later on for each measure should be accompanied by actual data or statistics supporting validity and reliability.\n\n- “The survey… gave an opportunity for the participant to provided extended responses if they desired.” Fix typo.\n\nThe authors mention “non-parametric statistical comparisons where possible” but this is not discussed in the manuscript. This statement on statistical comparisons should be deleted if they are not reported on later in the results section.\n\nResults: The results are presented in a clear and comprehensive manner.\n\nDiscussion: The authors should more clearly describe the limitation that participants’ goals/desired focus of the IT session likely dictate how much of each session would be spent engaging in intense exercise. For example, the participant’s desire to focus on “exploring movements in water, going fast and having fun” will likely affect the HR and RPE outcomes. Just as the authors pointed out, participants who do lower-intensity activities for the entire session will have lower HR and RPE outcomes, but the participants’ goals were what ultimately shaped the activities which then shaped HR, RPE, etc. Also, there has been some research on aquatic therapy, how does the results of this study relate to studies of engaging persons in water therapy differ from fully immersing them or not.\n“It was discovered that at the same HR output, RPE ratings were significantly higher.” Need further explanation, deep water running or land treadmill running group had higher RPE? Otherwise, this sentence is confusing.\n\n“…particularly in people who are naïve to the service beforehand.” Consider changing the wording to “new” or “unfamiliar” rather than “naïve.”\n\n“…a limitation of this paper is the study design…” Please be more specific and explain what aspects of the design are deemed by the authors to be a limitation.\n\nConclusion: In bringing the manuscript to a close, it may be worth restating in some way that Immersion Therapy involves water rather than solely focusing on the activities and client-centeredness.\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/10-135
https://f1000research.com/articles/7-1483/v1	18 Sep 18	{ "type": "Case Report", "title": "Case Report: Two cases of rare head injuries from Nepal", "authors": [ "Joe M. Das", "Apar Pokharel", "Rashmi Sapkota", "Manish Mishra", "Ashish Babu Aryal", "Apar Pokharel", "Rashmi Sapkota", "Manish Mishra", "Ashish Babu Aryal" ], "abstract": "Background: There are a number of ways in which one can sustain a head injury. Even if you are doing simple household activities or going out for a morning walk, you cannot be sure of what type of injury awaits you. The source of injury may be a pressure cooker whistle acting as a projectile or a hailstone falling from the sky. Such injuries are common in Nepal, considering the socio-demographic and geographic conditions. In this article, we present two such very rare cases of head injury. Case Reports: The first case is a middle-aged woman who sustained an accidental injury to the face associated with fracture of frontal sinus and frontal contusion, following the impact from a high momentum projectile in the form of the pressure regulator of a pressure cooker. She underwent craniotomy and removal of the foreign body. In the second case, an elderly man sustained minor injury to the head following the fall of hail. The abrasions and contusions produced by the hail were managed conservatively. Since he did not have any clinical evidence of head injury, other than multiple abrasions with contusions in the scalp, he did not undergo any imaging studies. He did not have any neurological deficits. The postoperative period was uneventful for the first patient and she was followed up for one month. The second patient was lost to follow-up. Conclusion: Successful management of two very rare cases of head injuries from Nepal are reported. Proper care and maintenance of the house-hold utensils that are constantly used may protect people from head injuries. Though natural calamities cannot always be avoided, simple measures like using an umbrella while going outdoors may protect individuals from head injuries due to hailstones.", "keywords": [ "Craniocerebral trauma", "Craniotomy", "Skull base", "Frontal sinus", "Cerebrospinal fluid", "Contusions" ], "content": "Introduction\n\nThe pressure cooker (PC) is an essential utensil for cooking in Asian kitchens, especially in Nepal and India, and are mostly handled by women. Accidental injury from such a commonly used utensil can sometimes be grievous. Most of the reports of injuries from PCs concern burns due to sudden opening of the lid and releasing of steam under very high pressure1. There is one previous report in which a mandible fracture occurred along with burns2 and one in which brain damage occurred due to the cooker blast3. However, there are only five reports of accidental head injury due to a pressure regulator projectile to date4–8. Ours is the sixth case, and the second producing a craniofacial injury, published in the literature, which describes successful management.\n\nHail is a variant of weather which can be occasionally harmful as well as dangerous. It is a form of precipitation and consists of balls or irregular lumps of ice known as hailstones. Hailstones are composed mostly of ice and measure 5–50 mm in diameter. Hail is produced by cumulonimbus clouds otherwise known as thunderstorm clouds, which are transparent ice or made up of alternating layers of transparent and translucent ice, at least 1 mm thick9. Though hailstones are of small size usually, sometimes they are large enough to kill a person if it falls on the head. A previous newspaper report of a “hailstone massacre”, which occurred centuries ago details this scenario10. The second case reported here is a case of mild head injury produced by falling hailstones. Surprisingly, head injury produced by hailstone has never been reported previously in the literature, to the best of our knowledge.\n\n\nCase 1\n\nA 55-year-old woman, with no known comorbidities, was cooking dal using a pressure cooker at night. The patient doubted why there was no whistling after the expected time, and so tried to gently lift the pressure regulator, which suddenly gave way and was thrust into her face, like a projectile, near the right eye. There was no history of loss of consciousness, nasal bleed or seizures, though she had one episode of vomiting. She was a chronic smoker and occasional drinker of alcohol.\n\nWhen she reached our emergency room in June 2018, her vital signs were stable. She had a wound of size 4×2 cm in between the right eye and root of nose, which was deep and still had the cooker whistle lodged. There was cerebrospinal fluid (CSF) mixed with blood in the periphery of the wound and there was mechanical ptosis of right eye. The patient was conscious and oriented. Emergency X-ray of skull (Figure 1) and computed tomography (CT) of the head (Figure 2) were done, which showed the foreign body just lateral to the root of nose on the right side with the right lateral wall of the nostril fractured and pushed inside. There was fracture involving the right frontal sinus with pneumocephalus and 1×1 cm sized left frontal contusion (Figure 3).\n\nThe patient underwent emergency bifrontal craniotomy. The right frontal sinus fracture with dural tear of size 0.5×1 cm was noted and the foreign body (Figure 4) was retrieved through the fracture from the cranial aspect (Figure 5). The frontal sinus was exteriorized and packed with muscle and bone wax applied across the defect. Anterior cranial fossa was carpeted with pedicled pericranial flap and the wound was closed after replacing the bone flap. The wound at the site of foreign body was seen by our otorhinolaryngologist and was closed in layers. The nasal mucosa was intact and there was no injury to nasolacrimal duct. The fractured nasal bone was not disturbed as it could cause stenosis of the right nostril.\n\nThe patient was started on Meropenem 2 g thrice a day, Vancomycin 1 g twice a day and Metronidazole 500 mg thrice a day as antibiotics, and levetiracetam 500 mg twice a day as anticonvulstant, which were continued for one week. The patient’s vision was fully preserved. The post-operative period was uneventful. Post-operative CT scan of brain showed resolving frontal contusion and she was discharged on post-operative day 8. When she came for follow-up after one month, she was asymptomatic, the wound had healed fully and there was no CSF leak.\n\n\nCase 2\n\nAn 85-year-old man presented to our neurosurgery out-patient department in May 2018 with complaint of headache following falling of hailstones on his head three days previously. He was a chronic smoker and alcoholic and did not have any comorbidities. He had no history of previous hospital admissions.\n\nFollowing the incident, the patient did not lose consciousness, vomit, or have a seizure. He also did not have any nasal or ear bleed. On clinical examination, he was neurologically intact and there was no papilledema on examination of the fundus. The patient had three healing abrasions with contusions, each the size of around 1 × 1 cm with local tenderness. Two abrasions were located in the left frontal region, behind hairline and one in the right parietal region, behind hairline (Figure 6). There was no clinical evidence of infection or skull fracture. Radiological imaging was not warranted and he was managed with 37.5 mg of tramadol hydrochloride and 325 mg of acetaminophen twice a day for five days. Though he was instructed to come for follow-up after one week, he did not turn up.\n\n\nDiscussion\n\nThe pressure cooker (PC), invented by the French-born British physicist Denis Papin in 1679, is a hermetically sealed pot that produces steam heat to cook food quickly. The PC heats water to produce very hot steam and as a result, the temperature inside it will increase to around 130° C, which is much higher than the maximum heat produced by ordinary cookware. The main advantage of this much high temperature is that it penetrates food quickly so that cooking time is reduced without diminishing vitamin and mineral content11. The problem faced high altitude areas, like Nepal, is that boiling happens at low temperature due to reduced atmospheric pressure. PCs will increase the pressure so that cooking occurs at the appropriate temperature.\n\nPC pressure regulator projectile injury to the face was first reported by Chattopadhyay et al in 20104. Altogether, there are five reports of this type of case (Table 1), four of them with significant ocular trauma. The case reported by Gupta et al. was similar to our case and had significant head injury5. In that case, the foreign body lodged transorbitally and was operated on promptly, even though the patient’s vision was lost by the injury and led to the development of a brain abscess subsequently. Our patient was lucky enough for her eye to escape from the direct impact of foreign body and from further complications of surgery.\n\nCT – Computed tomogram, y – years, M – Male, F – Female, PL – Perception of light, GCS – Glasgow Coma Scale Score, LMN – Lower motor neuron\n\nPCs can maintain high temperature (121°C) and proper pressure (1 kg/cm2) inside for cooking. The pressure is controlled inside by the vent weight (pressure regulator) and its spring action - excess steam goes out through the vent tube. Sometimes it can get blocked due to imperfect cleaning, excessive volume of water or overfilling with green leaves12. Then excess steam will accumulate inside, which can push the PC’s lid or pressure regulator out with huge force. Such accidents can be reduced by proper maintenance of the cooker, cleaning the lid and vent valve and filling the objects inside the cooker up to the appropriate level.\n\nThe projectile of a pressure regulator will almost always be directed towards the face (especially orbit) and skull. As has been reported previously, it is always safe to perform a craniotomy followed by removal of the foreign body for all foreign bodies, which have breached the dura, to prevent inadvertent damage to vital structures13. The patient had pneumocephalus with frontal sinus fracture, which was suggestive of a breach in the dura. If the foreign body is directly taken out blindly without exposing it through craniotomy, there is a chance that injury can occur to the brain as well as to the bridging vein at anterior skull base, if any14. This was the rationale for approaching the foreign body via cranium.\n\nHail is a form of frozen precipitation (hydrometeor) which originates in a thunderstorm cloud, scientifically known as cumulonimbus (thundercloud), which is composed of water droplets and ice crystals. There are upward forces in such clouds known as updrafts, and they carry raindrops upward into very cold areas of the atmosphere. In such areas, water droplets become super-cooled and freeze when coming into contact with condensation nuclei (small aerosols), thus forming small hailstones. The updraft then dissipates and these hailstones fall down. But these will be brought back into another updraft, and will be lifted up again. A layer of ice will get added to the hailstone and it grows in size with each ascent. Once a hailstone becomes too heavy to be supported by the updraft, it falls down from the cloud15. The main factors present in thunderstoms that are favorable to hail formation are strong updrafts, large liquid water contents, large cloud-drop sizes, and great vertical height16. Hail usually falls during severe thunderstorms in the warm season, when the temperature on the surface of the earth rises above 20 °C9.\n\nHailstone is an individual unit of hail. By convention, any frozen precipitation having a diameter of 5 mm or more is classified as hailstone, whereas smaller particles of similar origin are known as either ice pellets or snow pellets17. In the Cambridge dictionary, hailstone is defined as “a small, hard ball of ice that falls from the sky like rain”18. Most of the hail storms are made up of hailstones of different sizes. Usually only the large ones pose serious risk to people caught in the open. According to the Guinness book of world records, the heaviest hailstones ever recorded weighed approximately 1 kilogram and are reported to have killed 92 people in the Gopalganj area of Bangladesh on 14 April 198619. The largest hailstone recently recovered in the USA fell in Vivian, South Dakota on June 23, 2010 with a diameter of 8 inches and a circumference of 18.62 inches. It also weighed almost 1 kilogram20. One of the most lethal hailstorms in history, leading to the death of hundreds of nomads, occurred around AD 850 close to the glacial Roopkund Lake in the remote Himalayan Gahrwal region10.\n\nEven though there is a high frequency of occurrence of thunderstoms in the tropics, hail is actually less common in these regions, compared to the mid-latitudes, as the atmosphere over the tropics is warmer over a much greater height. Hail is common in mountain ranges because mountains force horizontal winds to move suddenly upwards (orographic lifting). This intensifies the updrafts within thunderstorms which makes hail more likely21. Hence hail is relatively common in Nepal.\n\nThere has been no report of hail falling on the head and producing injury, though hail is known to cause widespread damage to farms, houses, animals and humans. The present case did not have any criteria for radiological imaging as per the Canadian CT rule22. Moreover, the patient presented three days after the incident. In Nepal, patients may not be presenting immediately after the injury, either because they have only mild symptoms or they have to travel a long distance to reach a tertiary care hospital. The main limitation of this case is that there is no proper follow-up.\n\n\nConclusion\n\nHead injury can occur wherever you are – it does not matter whether you are indoors or outdoors. Here this fact is stressed with the help of two different and extremely rare types of head injuries. Some simple manoeuvres, such as proper maintenance of equipment and utensils you are working with or taking an umbrella while going for a morning walk, may protect individuals from such calamities.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from both 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.", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nPerera VA, Karunadasa K, Perera C: A case series of domestic pressure cooker burns. Ceylon Med J. 2012; 57(1): 49. PubMed Abstract \| Publisher Full Text\n\nGundeslioglu AO, Yenidunya MO: Burn and mandible fracture due to pressure cooker explosion. J Craniofac Surg. 2010; 21(5): 1631–3. PubMed Abstract \| Publisher Full Text\n\nCalderon-Miranda WG, Escobar-Hernandez N, Moscote-Salazar LR, et al.: Traumatic brain injury due to pressure cooker explosion in a child: case report. Romanian Neurosurgery. 2016; 30(2): 300–302. Publisher Full Text\n\nChattopadhyay SS, Mukhopadhyay U, Saurabh K: An unusual case of penetrating ocular trauma with a pressure cooker. Oman J Ophthalmol. 2010; 3(2): 89–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta OP, Roy K, Ghosh S, et al.: An unusual penetrating transorbital craniocerebral injury. The Indian Journal of Neurotrauma. 2014; 11(1): 53–6. Publisher Full Text\n\nDobariya V, Sheikh KM, Shastri M, et al.: An unusual case of penetrating ocular trauma with a pressure cooker whistle. Delhi J Ophthalmol. 2014; 24: 207–208. Publisher Full Text\n\nAtreya A, Kanchan T, Nepal S: Pressure Cooker-A Potential Hazard in Domestic Setting. Kathmandu Univ Med J (KUMJ). 2016; 14(54): 181–3. PubMed Abstract\n\nSingh AK: An Unusual Intraorbital Foreign Body. Delhi J Ophthalmol. 2016; 27(3): 213–4. Reference Source\n\nAkhmedovic IS: About the Mechanism of the Hail Formation. Science Discovery. 2014; 2(2): 27–33. Publisher Full Text\n\nOrr D: Giant Hail Killed More than 200 in Himalayas. The Telegraph. 2004; accessed June 22, 2018. Reference Source\n\nThe Editors of Encyclopaedia Britannica: Pressure cooker. Encyclopædia Britannica. accessed June 22, 2018. Reference Source\n\nSandhir RK, Sandhir M: Accidental pressure cooker lid blow-out. Burns. 1992; 18(5): 438. PubMed Abstract \| Publisher Full Text\n\nDas JM, Chandra S, Prabhakar RB: Penetrating brain injury with a bike key: a case report. Ulus Travma Acil Cerrahi Derg. 2015; 21(6): 524–6. PubMed Abstract \| Publisher Full Text\n\nZhang D, Chen J, Han K, et al.: Management of Penetrating Skull Base Injury: A Single Institutional Experience and Review of the Literature. Biomed Res Int. 2017; 2017: 2838167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe National Severe Storms Laboratory. accessed June 22, 2018. Reference Source\n\nConsidine DM, Considine GD: Precipitation and hydrometeors. In: Considine DM, Considine GD, eds. Van Nostrand’s Scientific Encyclopedia. 8th ed. New York: Springer Science+Business Media; 1995; 2538. Publisher Full Text\n\nGlossary of meteorology. accessed on June 22, 2018. Reference Source\n\nCambridge dictionary. accessed on June 22, 2018. Reference Source\n\nGuinness Book of World Records. accessed on June 22, 2018. Reference Source\n\nArndt D: Official Documentation of the Verification of the Vivian, SD record setting hailstone of 2010. 2010; accessed on June 22, 2018. Reference Source\n\nHail. National Environment Agency. accessed on 22 June, 2018. Reference Source\n\nDavey K, Saul T, Russel G, et al.: Application of the Canadian Computed Tomography Head Rule to Patients With Minimal Head Injury. Ann Emerg Med. 2018; pii: S0196-0644(18)30301-9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "40762", "date": "03 Dec 2018", "name": "Amit Agrawal", "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\nIn this article the authors report two interesting cases of head injuries. One is due to accidental explosion of a man-made device and another one is due to a natural calamity.\nIn the second case, it is interesting to note that hailstorm is a natural phenomenon, and there are number of incidences reported in news ranging from minor injuries to fatal incidences both to humans as well as to livestock.\nAs the authors have suggested, the systematic information for such injuries is lacking. This single case can be taken as message that we need to further expand the scope of the incidence and impact of injuries due to natural calamities on human life.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6115", "date": "22 Feb 2021", "name": "Joe M Das", "role": "Author Response", "response": "In this article the authors report two interesting cases of head injuries. One is due to accidental explosion of a man-made device and another one is due to a natural calamity. In the second case, it is interesting to note that hailstorm is a natural phenomenon, and there are number of incidences reported in news ranging from minor injuries to fatal incidences both to humans as well as to livestock. As the authors have suggested, the systematic information for such injuries is lacking. This single case can be taken as message that we need to further expand the scope of the incidence and impact of injuries due to natural calamities on human life. Thank you for understanding the major reason for including the second report in our article." } ] }, { "id": "67439", "date": "12 Aug 2020", "name": "Amos O Adeleye", "expertise": [ "Reviewer Expertise Clinical neurological surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is generally an interesting clinical vignette by these authors, especially the first case. We are of the opinion actually that the authors have enough literature base as well as the clinical materials of the first case to do this write-up simply as a case-based literature review of head injury/traumatic brain injury from projectiles from house-hold pressure cookers.\nThe second case does not warrant any scientific report in our opinion, not by the near normal clinical acute-event / immediate post-event period status of the patient, as well as the very minimal body harm to the patient.\n\nHaving said that, we like to make some of the observations below concerning the authors’ otherwise well-written paper.\n\nThe figure 2, the plain 3-D reconstruction of the cranial CT bone windows, contains more information that just ‘the foreign body and its relation to the orbit’. There is the impression of a right naso-orbital fracture extending from the inferomedial orbital rim across the maxilla (just medial to the zygomasticofaical foramen) to the right nasal wall. One would expect all this to reflect in the authors’ case description (and the figure legend) by way of emphasis on the apparent destructive impact of the house-hold utensil turned projectile.\n\nCase 1 “A 55-year-old woman, with no known comorbidities, was cooking dal using a pressure….” What’s ‘dal’ please, for us international readers?\n\n“There was fracture involving the right frontal sinus with pneumocephalus and 1×1 cm sized left frontal contusion (Figure 3).” The figure 3 referred to here is a brain, not a bone, window of the cranial CT. The frontal sinus fracture being described could therefore hardly be appreciated. We had actually wondered why the authors did not render some of the images at this frontobasal level in the bone windows. That’s what would have been more instructive for this part of the work\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": [ { "c_id": "6076", "date": "22 Feb 2021", "name": "Joe M Das", "role": "Author Response", "response": "Thank you for looking into our article in detail and providing constructive feedback. The article has been amended as suggested. The term \"dal\" has been explained. A detailed description of the facial fracture is included. A new figure showing the fractured frontal sinus has been provided." }, { "c_id": "6114", "date": "22 Feb 2021", "name": "Joe M Das", "role": "Author Response", "response": "This is generally an interesting clinical vignette by these authors, especially the first case. We are of the opinion actually that the authors have enough literature base as well as the clinical materials of the first case to do this write-up simply as a case-based literature review of head injury/traumatic brain injury from projectiles from house-hold pressure cookers. - Thank you. The second case does not warrant any scientific report in our opinion, not by the near normal clinical acute-event / immediate post-event period status of the patient, as well as the very minimal body harm to the patient. - We still would like it to be published as there are not many reports on the harms this natural calamity can do to humans. Having said that, we like to make some of the observations below concerning the authors’ otherwise well-written paper. The figure 2, the plain 3-D reconstruction of the cranial CT bone windows, contains more information that just ‘the foreign body and its relation to the orbit’. There is the impression of a right naso-orbital fracture extending from the inferomedial orbital rim across the maxilla (just medial to the zygomasticofaical foramen) to the right nasal wall. One would expect all this to reflect in the authors’ case description (and the figure legend) by way of emphasis on the apparent destructive impact of the house-hold utensil turned projectile. - We have added the details of the fracture in the case description and figure legend, as suggested. Case 1 “A 55-year-old woman, with no known comorbidities, was cooking dal using a pressure….” What’s ‘dal’ please, for us international readers? We have added a simple definition of dal. “There was fracture involving the right frontal sinus with pneumocephalus and 1×1 cm sized left frontal contusion (Figure 3).” The figure 3 referred to here is a brain, not a bone, window of the cranial CT. The frontal sinus fracture being described could therefore hardly be appreciated. We had actually wondered why the authors did not render some of the images at this frontobasal level in the bone windows. That’s what would have been more instructive for this part of the work. We have added a new image of the bone window at the frontobasal level." } ] } ]	1	https://f1000research.com/articles/7-1483
https://f1000research.com/articles/9-1502/v1	23 Dec 20	{ "type": "Case Report", "title": "Case Report: Bilateral mandibular buccal bifurcation cysts", "authors": [ "Ashwag Aloyouny", "Hamad Albagieh", "Soad Mansour", "Fahmy Mobarak", "Hamad Albagieh", "Soad Mansour", "Fahmy Mobarak" ], "abstract": "Buccal bifurcation cyst (BBC) is a rare inflammatory odontogenic cyst, which commonly affects children in the first decade of life. We report a case of a seven-year-old healthy boy with bilateral BBC, which involved unerupted incomplete permanent mandibular first molars. A review of the literature in English language revealed few similar cases. We reviewed 16 manuscripts of bilateral mandibular BBC, reporting a total of 20 cases since 1970. The clinical features of bilateral mandibular BBC summarized here could assist specialists with an accurate diagnosis and provide patients with optimal management.", "keywords": [ "Buccal Bifurcation Cysts", "Bilateral mandibular cysts", "Children", "Oral Cyst" ], "content": "Introduction\n\nBuccal bifurcation cyst (BBC) is a rare inflammatory odontogenic cyst. The first case of BBC was reported in 1983 by Stoneman and Worth1. Children between the age of 4 and 14 years are most commonly affected. In addition, BBC occurs more in the mandible and most likely involves the permanent mandibular first molar. Typically, BBC causes many undesirable oral manifestations such as buccal swelling at the affected area, delayed tooth eruption or partially tooth eruption associated with deep periodontal pockets. In some cases, pain and infection associated with pus could be present2.\n\nRadiographically, BBC is illustrated as a well-defined radiolucent lesion surrounded by sclerotic rim. The lesion either involves the roots of a partially erupted tooth or surrounds an unerupted tooth; usually the permanent mandibular first molar3. Histopathologically, the BBC cyst wall is lined by a non-keratinized stratified squamous epithelium with inflammatory lymphocytes infiltrate. Surgical excisional procedure is performed for complete removal of the cysts with the involved teeth to reduce the risk of recurrence of the epithelial cysts4.\n\nHere, we report a case of a seven-year-old healthy boy with a chief complaint of painful, slowly growing lower jaw swellings on both right and left sides consistent with bilateral BBC, which involved unerupted incomplete permanent mandibular first molars. A review of the literature revealed few similar cases. We report this case to add to an additional case of bilateral mandibular BBC to the literature.\n\n\nCase presentation\n\nA seven-year-old healthy boy presented to the Oral and Maxillofacial Surgery Clinic with a chief complaint of painful, slowly growing lower jaw swellings on both right and left sides. The patient had no past medical, surgical, or dental history. His guardian reported no known drug and food allergies. The guardian also denied having family history of genetic related diseases or syndromes. The swellings caused esthetic disfiguring of the patient’s face, which led to social exclusion.\n\nOn examination, extraoral, bilateral asymmetric swellings of the lower part of the face with no lymph node involvement were noted. Intraorally, painful bilateral, hard, bony mandibular swellings covered with normal color mucosa, extending from the mesial aspect of the mandibular second primary molars on both sides including the retromolar areas, was observed. On palpation, the affected site revealed expansion of the buccal cortical plates on both sides. Both mandibular permanent first molars were not clinically erupted.\n\nDiagnostic radiographs were taken. A panoramic radiograph illustrated bilateral, well-defined radiolucencies surrounded by sclerotic margins and including the unerupted incomplete permanent mandibular first molars (36 and 46, according to the FDI World Dental Federation Notation). The panoramic image showing right radiolucency measured around 1 cm in its greatest dimension, and involved the unerupted permanent mandibular first molar (46, according to the FDI World Dental Federation Notation), and not involving the inferior mandibular cortical bone and the adjacent areas. On the other hand, the left radiolucency measured around 3cm in its greatest dimension, and involved the unerupted permanent mandibular first molar (36, according to the FDI World Dental Federation Notation), the inferior mandibular cortical bone and the adjacent areas. Moreover, the left radiolucent cyst distally displaced the permanent mandibular second molar tooth bud (37, according to the FDI World Dental Federation Notation) (Figure 1). Provisional diagnosis of the bilateral lesions suggested bilateral BBC, dentigerous cyst, and paradental cyst.\n\nThe guardian was informed about the patient’s condition, possible management and complications. Then, a consent form was signed before starting the complete surgical removal of the lesions under general anesthesia. An incision was made, then a buccal mucoperiosteal flap was raised in the mandible at the mandibular permanent first molar area on both right and left sides to expose the bone. The buccal plate was expanded and thinned; especially in the left side. An access to the cyst area was made by performing an ostectomy on both sides. The whole cyst lining was removed with the attached mandibular first molars. Then, the cavity was enucleated and curetted thoroughly (Figure 2), to reduce the risk of recurrence.\n\n(a and b) Clinical intraoral photographs show bilateral surgical removal of the bilateral BBC with the involved permanent teeth.\n\nThe specimens were sent to the histopathology laboratory in two containers. The histopathologic analysis of the multiple serial sections of the two specimens revealed multiple pieces of cyst wall lined by small strip of non-keratinized stratified squamous epithelium with mixed inflammatory reaction and surrounding edematous granulation tissue formed of numerous proliferating capillary type vascular spaces. Also, the specimens under the microscope showed bland looking spindle-shaped fibroblasts and degenerated bony spicules, which was consistent with cystic lesions (Figure 3). Based on the clinical examination, radiographic interpretation, and histopathologic analysis, the final diagnosis was bilateral BBC.\n\nSix months later, the clinical and radiographic examination of both surgery sites showed signs of gradual healing and resolution and increased bone density in the areas. One year later, no clinical evidence of lesion recurrence, and radiographic imaging illustrated that the affected areas were completely filled up with bone.\n\n\nDiscussion\n\nBilateral BBC is an uncommon inflammatory odontogenic cyst. It usually arises from the buccal side of the mandibular permanent first molar in children. The pathophysiology of BBC is still undetermined. It is claimed that the inflammation is caused by the deep periodontal pocket caused by tilted tooth. Other authors claim that the reason behind the cyst formation could be because of a defect in the tooth eruption, which may lead to inflammation, proliferation of the epithelial cells and cyst formation1–5. It is also postulated that enamel projections at the area covered by reduced enamel epithelium between the cementoenamel junction and the furcation of the tooth could lead to cyst formation6.\n\nClinically, bilateral BBC has few challenging similarities with dentigerous cyst, and paradental cyst. For example, all previously mentioned cysts are commonly associated with a tooth. However, paradental cysts and dentigerous cysts usually occur in adults in the mandibular third molar region7–8. Dentigerous cyst is classified as a developmental cyst that may also occur in the mandibular first molar areas and sometimes interfere with the process of normal tooth eruption in children. On the other hand, paradental cyst is considered as an inflammatory cyst, which commonly occurs in the lateral surface of a vital tooth usually the first and second mandibular molars of primary teeth.\n\nRadiographic interpretation is essential to assist recognize and differentiate between different cystic lesions. Normally cystic lesions have a well-defined, unilocular radiolucency with a sclerotic margin; however, cysts sometimes present with different features. For instance, paradental cysts present on the lateral crown of an incompletely erupted tooth, while dentigerous cysts usually surround the crown of an unerupted tooth. Every rule has an exception, it is challenging to differentiate between the circumferential dentigerous cyst and the BBC radiographically as both illustrated as radiolucent cysts involving a completely unerupted tooth. Histopathologically, the cyst wall under the microscope shows chronic inflammatory lymphocytes and two to four cell layers thick of proliferating lining of nonkeratinized stratified squamous epithelium.\n\nThe clinical examination, radiographic interpretation and histopathologic analysis of both lesions displaying cystic origin with bilateral buccal expansion involving both mandibular first molars in a seven-year-old child associated with bilateral BBC was presented here. This reported case had similar clinical, radiographical and histopathological features of bilateral mandibular BBC. It is worth mentioning that the case presented here is very rare because it describes bilateral mandibular BBC. To the best of our knowledge, the bilateral characteristic of BBC is very rare; there are only few cases have been reported in the English-language literature. In this article, we reviewed 16 manuscripts of bilateral mandibular BBC, reporting a total of 20 cases in the period between 1970 to 2019. Furthermore, these cases occurred in different ages starting from 4 years to 13 years with predilection to male patients (Table 1).\n\nM: Male; F: female\n\nThrough the past years different approaches have been performed to manage the BBC. In some cases, the way of management was only by following up the lesion with no interventions; some lesions showed different degrees of regression and others needed intervention5–9. On the other hand, other cases underwent surgical enucleation of the cysts either with the involved tooth or maintaining the involved tooth. In the current case, we chose to perform a surgical procedure under general anesthesia to enucleate both bilateral mandibular BBCs with the involved teeth to reduce the risk of recurrence and to fulfill the patient’s parent’s desire.\n\n\nConclusion\n\nBBC typically affects children in the first decade of life. BBC occurs in the buccal area of the mandibular first molar. Only a few bilateral mandibular BBC cases were reported in the literature. Although bilateral mandibular BBC is uncommon, the diagnosis would be less challenging if it is established by the correlation of the clinical examination, radiographic interpretation and histopathological analysis. Moreover, the clinical features of bilateral mandibular BBC summarized in this review could assist specialists to an accurate diagnosis and provide patients with optimal management.\n\n\nPatient perspective\n\nThe parents mentioned that the surgery has an excellent impact on their son’s life. Also, the outcomes met their expectations as the facial swellings disappeared.\n\n\nConsent\n\nWritten informed consent was obtained from the patient’s father for publication of this case report and accompanying images.\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\nThis research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program.\n\n\nReferences\n\nStoneman DW, Worth HM: The mandibular infected buccal cyst--molar area. Dent Radiogr Photogr. 1983; 56(1): 1–14. PubMed Abstract\n\nPompura JR, Sándor GK, Stoneman DW: The buccal bifurcation cyst: a prospective study of treatment outcomes in 44 sites. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1997; 83(2): 215–221. PubMed Abstract \| Publisher Full Text\n\nWhite S, Pharoah M: Oral Radiology Principles and Interpretation. 7th ed. 2013; 696. Reference Source\n\nNeville BW, Damm DD, Allen CW, et al.: Oral and Maxillofacial Pathology. 3rd Edition. 2008; 984. Reference Source\n\nDavid LA, Sándor GK, Stoneman DW: The buccal bifurcation cyst: in non-surgical treatment an option? J Can Dent Assoc. 1998; 64(10): 712–717. PubMed Abstract\n\nCraig GT: The paradental cyst. A specific inflammatory odontogenic cyst. Br Dent J. 1976; 141(1): 9–14. PubMed Abstract \| Publisher Full Text\n\nPhilipsen HP, Reichart PA, Ogawa I, et al.: The inflammatory paradental cyst: a critical review of 342 cases from a literature survey, including 17 new cases from the author’s files. J oral Pathol Med. 2004; 33(3): 147–155. PubMed Abstract \| Publisher Full Text\n\nJones AV, Craig GT, Franklin CD: Range and demographics of odontogenic cysts diagnosed in a UK population over a 30-year period. J oral Pathol Med. 2006; 35(8): 500–507. PubMed Abstract \| Publisher Full Text\n\nCorona-Rodriguez J, Torres-Labardini R, Velasco-Tizcareño M, et al.: Bilateral buccal bifurcation cyst: case report and literature review. J oral Maxillofac Surg. 2011; 69(6): 1694–1696. PubMed Abstract \| Publisher Full Text\n\nStanback JS: The management of bilateral cysts of the mandible. Oral Surg Oral Med Oral Pathol. 1970; 30(5): 587–91. PubMed Abstract \| Publisher Full Text\n\nSwerdloff M, Alexander SA, Ceen RF, et al.: Bilateral mandibular dentigerous cysts in a seven-year-old child. J Pedod. 1980; 5(1): 77–84. PubMed Abstract\n\nVedtofte P, Praetorius F: The inflammatory paradental cyst. Oral Surg Oral Med Oral Pathol. 1989; 68(2): 182–188. PubMed Abstract \| Publisher Full Text\n\nPackota GV, Hall JM, Lanigan DT, et al.: Paradental cysts on mandibular first molars in children: report of five cases. Dentomaxillofac Radiol. 1990; 19(3): 126–132. PubMed Abstract \| Publisher Full Text\n\nBohay RN, Weinberg S, Thorner PS: The paradental cyst of the mandibular permanent first molar: report of a bilateral case. ASDC J Dent Child. 1992; 59(5): 361–365. PubMed Abstract\n\nMartinez-Conde R, Aguirre JM, Pindborg JJ: Paradental cyst of the second molar: Report of a bilateral case. J Oral Maxillofac Surg. 1995; 53(10): 1212–1214. PubMed Abstract \| Publisher Full Text\n\nShohat I, Buchner A, Taicher S: Mandibular buccal bifurcation cyst: enucleation without extraction. Int J Oral Maxillofac Surg. 2003; 32(6): 610–613. PubMed Abstract \| Publisher Full Text\n\nGallego L, Baladrón J, Junquera L: Bilateral mandibular infected buccal cyst: a new image. J Periodontol. 2007; 78(8): 1650–1654. PubMed Abstract \| Publisher Full Text\n\nRamos LMA, Vargas PA, Coletta RD, et al.: Bilateral Buccal Bifurcation Cyst: Case Report and Literature Review. Head Neck Pathol. 2012; 6(4): 455–459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorgonovo AE, Reo P, Grossi GB, et al.: Paradental cyst of the first molar: Report of a rare case with bilateral presentation and review of the literature. J Indian Soc Pedod Prev Dent. 2012; 30(4): 343–348. PubMed Abstract \| Publisher Full Text\n\nBoffano P, Gallesio C, Roccia F, et al.: Bilateral buccal bifurcation cyst. J Craniofac Surg. 2012; 23(6): e643-e645. PubMed Abstract \| Publisher Full Text\n\nIssler A, Bornert F, Clauss F, et al.: Mandibular buccal bifurcation cyst treatment: Report of two cases and literature review. Med Buccale Chir Buccale. 2013; 19(2): 77–84. Publisher Full Text\n\nBautista CRG, Milhan NVM, Ankha MDVEA, et al.: Bilateral mandibular buccal bifurcation cyst: a case report emphasizing the role of imaging examination in the diagnosis. Autops Case Rep. 2019; 9(2): e2018073. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77339", "date": "18 Jan 2021", "name": "Denise Tostes Oliveira", "expertise": [ "Reviewer Expertise Oral pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and well-documented case report of bilateral buccal bifurcation cysts (BBC). It is important to include in the manuscript that the etiopathogenesis of BBC is the same of paradental cyst, according to World Health Organization Classification of Head and Neck Tumours (2017). In addition, the diagnosis should be established based in the clinical/radiographic and microscopic characteristics.\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/9-1502
https://f1000research.com/articles/7-1984/v1	28 Dec 18	{ "type": "Method Article", "title": "Assembling cheap, high-performance microphones for recording terrestrial wildlife: the Sonitor system", "authors": [ "Kevin F.A. Darras", "Bjørn Kolbrek", "Andreas Knorr", "Volker Meyer", "Bjørn Kolbrek", "Andreas Knorr", "Volker Meyer" ], "abstract": "Passive acoustic monitoring of wildlife requires microphones. Several cheap, high-performance open-source solutions currently exist for recording sounds, but all of them are still reliant on commercial microphones. Commercial microphones are relatively expensive, specialized on particular taxa, and often have opaque technical specifications. We designed Sonitor, an open-source microphone system to address all needs of ecologists that sample terrestrial wildlife acoustically. We evaluated the cost of our system and measured trade-offs that are seldom acknowledged but which universally limit microphones' functions: weatherproofing versus sound attenuation, windproofing versus transmission loss after rain, signal loss in long cables, and analog sound amplification and directivity with acoustic horns. We propose three microphone configurations suiting different budgets, sound qualities, and flexibility requirements, which all cover the entire sound frequency spectrum of sonant terrestrial wildlife at a fraction of the cost of commercial microphones.", "keywords": [ "autonomous sound recorders", "passive acoustic monitoring", "signal-to-noise ratio", "self-noise", "acoustic horn", "Song Meter", "Swift recorder", "Bioacoustic recorder" ], "content": "Introduction\n\nPassive acoustic monitoring of terrestrial wildlife is nowadays a firmly established field of study. It has many advantages over classical human observation methods1 and bears considerable potential for further development2. Birds, bats, amphibians, insects, and primates are often surveyed using autonomous sound recorders. A wide range of open-source devices and commercial products exists for recording sound in terrestrial habitats (Table S1)3. Established manufacturers offer products to cover all needs, and non-profit organisations also build and sell autonomous sound recorders. Raspberry-Pi based solutions, as well as dedicated autonomous sound recorders offer cheap alternatives to commercial products4.\n\nAs transducers of mechanical energy into electrical signals, microphones are the most important components of a sound recorder. They are the first step in the sound recording process, and through their frequency response, they determine which animals can be recorded. A recent meta-analysis demonstrated the crucial importance of microphone specifications and underlined how microphone signal-to-noise ratio, a measure of its inherent noise level, affected the sound detection space5, which is also determined by external factors6.\n\nDespite the many different sound recorders that are available, their owners are usually restricted to the microphones of the manufacturers or the recommendations of recorder builders due to compatibility or warranty issues. Outdoor microphones rapidly degrade as they are exposed to rain ingress, animal damage, ultraviolet radiation, and wide temperature ranges7. Users usually only have the choice of expensive microphone replacements as repair instructions are not available, components are unknown, and the design is not disclosed. Microphone specifications are rarely complete, and sensitivity is stated more often than the more important signal-to-noise ratio. In many cases, the microphone element that is used is unknown. In some cases, microphone signals are filtered at the source only for commercial reasons, to enable either bird or bat recordings and sell multiple specialised products. Currently, no microphone is available to record both bats and birds, although the recorders that can record ultrasound theoretically could sample the entire frequency range of interest.\n\nTo provide alternatives to the sound-recording community of ecologists, we designed a cheap, open source, high-performance, and modular microphone system called Sonitor. The system can be used to record all terrestrial wildlife. We first present the basics of microphone parts, then present the Sonitor system, and assess its performance. We show the trade-offs between weatherproofing and transmission loss, between wind-proofing and drying time, between cable length and signal loss, between directivity and analog amplification, and evaluate the temporal and financial cost of assembly. We built microphones for the most common audio connector system used in current recorders of established manufacturers: Wildlife acoustics (Song Meter), FrontierLabs (Bioacoustic recorder), and Cornell University (Swift). We present three different microphone configurations for different needs and budgets.\n\n\nMethods\n\nSound consists of pressure waves travelling through a medium, in our case air. Audible sound makes the air vibrate at frequencies between 20 Hz and 20 kHz. Ultrasound, which is not audible for humans, extends beyond 20 kHz. Insects and bats can emit and perceive ultrasound up to 200 kHz8. Microphones are transducers of mechanical energy (pressure waves) into electrical energy (a voltage). A variable voltage is created as sound waves move mechanical parts of microphones, which can be a polarized membrane (electret condenser), or a piezoelectric element. The role of the recorder is mainly to increase the minimal voltage differences with amplifiers, digitize them with analog-to-digital converters, and record them to a digital storage medium (mostly solid-state memory secure digital cards).\n\nOutdoor microphones are electrical devices which need to be protected against water ingress, and climatic and mechanical shocks. Protection comes from solid housings, often metal tubes in which the microphone element is inserted. The microphone element (often ambiguously called simply \"microphone\") is the centerpiece of the microphone and consists only of the acoustic sensor which transduces sound to a variable voltage, and it is not usable as is. Microphone housings need to be open to allow sound to reach the microphone element through their acoustic port. Since an opening would allow water to penetrate the microphone, corrode its components, and block the sound path, protection is needed. Acoustic vents are used: they are transmissive for sound while being impermeable to water or hydrophobic, and thus fulfil a crucial function for outdoor microphones. Then, microphones need to transmit their output voltage to a recorder via electrical wires. When microphones are interchangeable, they use an audio connector as interface, which needs to be weatherproof too. A minimal microphone assembly only requires soldering of microphone elements and cables, as well as sealing of the other microphone parts using glue if used outdoors.\n\nBasic microphone properties can be augmented with attachments. Windscreens, usually made of synthetic foam or fur, reduce unwanted wind noise which comes from friction of air against the microphone. They also reduce potentially damaging water pressure from rain drops. Furthermore, parabolic reflectors or horns can be used to gather sound over a larger area before concentrating it to the microphone element, but the gained amplification is traded off against higher directivity: the sound pickup pattern becomes narrower.\n\nMicrophone element. We chose to use microelectromechanical (hereafter MEMS) microphones due to their high performance at small sizes, the potential of that newer technology to mature and offer higher performance than conventional microphone capsules, and their lower part-to-part variation and sensitivity to temperature variations (Lewis et al. 2013). Different elements exist that can fulfil different requirements by prioritizing low-noise recording, a wide frequency response, or weatherproofing. We are using microphone elements from different manufacturers. We used a tried-and-tested element from Knowles (SPU0410LR5H-QB), which was used by the company Biotope.fr inside the now discontinued BIO-SMX-US microphone as a substitute for SMX-US microphones by Wildlife acoustics. We also used it inside our own housings since 2017 for recording birds and bats. We tested Invensense's ICS-40720 element, which features low-noise recording (specified signal-to-noise ratio of 70 dB) and also Vesper's VM1000, which is a piezo-electric element that is waterproof and resistant to various environmental stresses.\n\nPrinted circuit board (PCB). Microphone elements can be directly soldered to cables, but this requires great care and dexterity for a precise soldering result that does not exceed the temperature tolerance of the element. Moreover, a precise alignment of the microphone within the housing and with the acoustic vent is needed for compatibility with external attachments and for enabling consistent part-to-part quality. It is thus preferable to reflow-solder MEMS elements to printed circuit boards, which can be made in electronic laboratories or workshops equipped with reflow ovens. This is readily available as a paid service and is a burgeoning business satisfying the needs of electronic equipment manufacturers and electronics hobbyists in need of prototypes. Cables can then be more easily soldered to PCBs without damaging the microphone element. The microphone and conductive tracks can be attached on the bottom side of the PCB, which guarantees a result that is flush with the housing. PCBs can be ordered in any size and shape with a variety of support materials.\n\nHousing. We chose to integrate the microphone elements into simple metal tubes, which can be made out of stainless steel or lighter aluminium. These metals offer high resistance to weather and mechanical shocks, are cheap and readily available, and easy to glue. They can be painted to reduce their visibility in natural environments. Due to their hardness, metals can also be lathed with high precision to ensure stable results within tight tolerances so that any attachment can easily fit the housing.\n\nWires and connector. We chose standard 30 AWG stranded wires for more flexibility compared to solid wires. On one end, the cables are connected to the PCB, which is connected to the microphone element. On the other end, the wires are connected to Mini-Con-X series waterproof connectors without the grommet, which is needed to release the tension when the connector is attached to flexible cables. This connection form is commonly used in most autonomous sound recorders. Mini-Con-X connectors can withstand some abuse and are ingress-protection rated at IP67 (dust tight and protected against water up to 1 m deep).\n\nAcoustic vent. We use Gore acoustic vents to protect the element against solid and liquid ingress. Different products in varying sizes and protection levels against water are available. GAW112 vents can be used, they appear identical to the ones used in SMX-US, SMX-U1, and SMX-II microphones from Wildlife acoustics. They need to be coupled with windscreens, as GAW112 vents let water pass after immersion or drop projection. We also tested GAW325 vents, which are IP67 rated. Freshwater ingress per se only temporarily blocks microphone elements that are not waterproof from vibrating, but will not short-circuit the microphones due to the low conductivity of water. However, water leads to corrosion, which will destroy microphones and conductive tracks, given enough time. The GAW3XX series also have a support material, which can be made of woven or non-woven PET material. The PET (woven) support elements are better suited as they absorb water less.\n\nAll assessments of the microphones’ technical qualities were performed with SM2Bat+ recorders (Wildlife acoustics), which allow to record two channels up to a maximum sampling frequency of 192 kHz. We used a battery-powered one-driver Anker SoundCore loudspeaker for emitting audible pure test tones at 1 and 10 kHz (generated using Audacity 2.2.2) and an ultrasonic calibrator (Wildlife Acoustics) that emits chirps at 40 kHz. Test sounds were emitted to the front of the microphones and when needed also to the side at a 90° angle. We measured the amplitude of test tones in recordings with a sampling frequency of 96 kHz in Audacity by exporting the frequency spectra with a Hanning window size of 1024 and choosing the frequency window that included our tone's base frequency.\n\nWeatherproofing vs. sound attenuation. The only point that is permeable to sound is the acoustic vent, and its permeability to water ingress is given by its specifications. The sound attenuation at 1 kHz is usually also indicated in the product specifications given by the manufacturer in decibels (dB), as this is the frequency most relevant for recording human speech. However, terrestrial wildlife sounds span frequencies from 20 Hz to 200 kHz, so we measured the transmission at three representative frequencies: 1 kHz (birds and amphibians), 10 kHz (insects), and 40 kHz (bats) to quantify the acoustic vents’ trade-off between sound transmission and ingress protection.\n\nWe compared sound attenuation of 2 GAW113 and 2 GAW325 vents with an open setting without vent, outdoors (Figure 1). We recorded the US calibrator and loudspeaker tones at 3 m from the microphones, to the front and to the side at a 90° angle to the side. Four Knowles microphones were used, first open, then with the vent holders, and then two of them were covered with the GAW112 vent and the other two with the GAW325 vent.\n\nFoam strips reduced ultrasound echoes and the microphones were approximately 1 m above the ground and parallel to each other.\n\nWindproofing vs. drying after rain. We used Knowles elements; one was protected by a GAW112 vent and a windscreen (Wildlife Acoustics), one had a 6 mm long horn attached, and one had a GAW325 vent outdoors. All three configurations represented similar levels of water ingress protection, but we used the Knowles microphone with the 6 mm horn instead of the Vesper microphone (for which it was designed) to equalize the microphone model. We emitted test sounds with the loudspeaker and the calibrator at approximately 4 m. We placed a 62 W fan at approximately 30 cm from the microphones, to the front and to the side (90 degrees) to simulate wind. We recorded the test sounds to check how prone to noise the vent-only and horn-only microphones are in comparison to the microphone with the windscreen. Then, we drenched all microphones in distilled water to simulate heavy rain. We continued recording test sounds immediately after, as well as 1, 3, 18, and 66 hours after the simulated rain to check how long sound transmission was attenuated by the different wet attachments. We measured the sound level of the 1, 10, and 40 kHz tones recorded by each microphone relative to the sound level recorded after 66 hours of drying.\n\nCable length vs. signal loss. The latest microphones of Wildlife Acoustics usually advertise built-in amplifiers to strengthen the relatively low voltage signals of the microphones so that they do not degrade over long cable distances. High frequencies are more prone to signal degradation because the capacitance of the cable causes more attenuation at high frequencies. We tested whether the output signals of the Knowles microphones were affected by long cables, which are sometimes needed for installing microphones far apart or in different locations than the recorders themselves. We attached two Knowles microphones to the recorder, one via a 5 m cable and the other one via a 52.5 m long cable. They were close to each other and pointing in the same direction. We recorded test sounds emitted with the loudspeaker and the ultrasound calibrator at 6 m from the recorder. We recorded the same test sounds after switching the cables to check whether the results were driven by the microphone itself. We measured 20 ultrasound chirps for each microphone with each configuration.\n\nDirectivity vs. amplification. We built different horns for amplifying the acoustic input signal before it is transduced by the microphone (Figure 2). Doing this results in an increased signal-to-noise ratio and ultimately greater detection ranges. However, acoustic horns are generally directive: At high frequencies, horns will mainly respond to sounds within their opening angle, where direct sound can reach the throat of the horn. Outside the opening angle, low-frequency sounds reach the throat of the horn by diffraction.\n\n1 EUR coin for scale.\n\nThe reasoning behind using horns is that in stereo deployments, there is a redundancy of recorded data: omnidirectional microphones pointing in opposite directions are recording much of the same data twice. To make better use of them, one can use acoustic horns that amplify the sound from the front and decrease sound from the back or the sides. Ultrasound, which propagates less far, benefits especially from horns, because even very small horns can achieve considerable amplification. For ultrasound, horn dimensions can also be held as small as the existing microphone housings. Also, microphones usually suffer from a drop in the frequency response and/or signal-to-noise ratio in the ultrasound range, thus horns help to attain a desirable, more linear frequency response.\n\nWe chose horn designs with steadily increasing amplification with frequency starting approximately from 10 kHz and minimal directivity. Conical horns are generally more suitable than exponential horns, which do not amplify sound much above a certain threshold. Horn dimensions were chosen by calculating and simulating the theoretical analogue amplification in-axis and off-axis using numerical methods to choose the most favourable designs. The gain of the horns was calculated using one-dimensional equations for conical horns9. Since the one-dimensional calculations could not predict directivity, Boundary Element Method models10 were set up to model the directivity of the horns. The ultimate gain depended mainly on the ratio of the areas between the mouth and throat of the horn, while the frequency range depended on the length of the horn. A long and narrow horn will also be resonant, which will increase the gain but reduce the fidelity of the recorded sounds.\n\nWe investigated whether ultrasonic horns could amplify the signal enough to compensate for the transmission loss due to the acoustic vents. We also tested how much amplification could be gained with different horns placed in front of the Vesper microphones, which do not require vents.\n\nThe Knowles and Invensense microphones require the use of the GAW112 or GAW325 vents for ingress protection. The diameter of the vents’ active surface (through which sound travels) dictates the maximum mouth diameter and theoretical amplification of the horn. The resulting horns were named after the vent they were designed to hold (GAW112 and GAW325 horns). We compared sound attenuation of three GAW112 and three GAW325 horns with and without vent to the open microphones. We tested three horns of each type on three different Knowles microphones, by first recording with open microphones, then with the horns attached, and finally with the vents pasted onto them. We recorded the US calibrator and loudspeaker tones at 3 m from the microphones.\n\nFor the waterproof Vesper microphone, we were free to test three different horns whose mouth diameter was only limited by the diameter of the housing but tested varying lengths. We also tested 3 other ultrasonic horn types designed for the Vesper element (thus not holding vents) on three different Knowles microphone elements (for consistency with our measurements of the vent-holding horns). We had 3, 6, and 12 mm long horns, with a throat diameter of 0.75 mm and a mouth diameter of 12 mm. We first recorded open microphones, and then successively attached horns of increasing length to each microphone. We recorded the US calibrator and loudspeaker tones at 6 m from the microphones due to the greater amplification of these longer horns.\n\nCost. We assessed the cost in working time and money at each step of the creation process for 100 microphones. We contrasted the cost for 3 microphone designs presented later. We considered the ordering of individual parts, components assembly, and microphone testing. We estimated labour and prices from our own purchases and working time. For the costs of building the PCBs and metal housings and horns, we asked three different suppliers for quotes and chose the best offer.\n\n\nResults\n\nThe GAW112 vent reduces ultrasound transmission from the front only slightly, by almost 2 dB, while sounds from the side are reduced by more than 7 dB, which is partly due to the vent holder itself (Figure S1)3. The GAW325 vent reduces ultrasound transmission by almost 15 dB but relatively less for sounds coming from the side (almost 11 dB).\n\nThe windscreen significantly reduced wind friction noise (Figure 3). The vent-only and 6mm horn configurations were affected by wind friction noise at up to 3 kHz, greatly masking the 1 kHz test tones, although they were still audible and visible in spectrograms. Data for windproofing and weatherproofing are available on OSF3.\n\nWithout windscreen, 1 kHz test sounds are masked by wind noise.\n\nThe GAW112 vent with windscreen combination needed much longer to dry than the 6 mm horn (Figure 4). When wet, from one to three hours after drenching, high audible frequencies (10 kHz) were attenuated around 20 dB and ultrasound around 30 dB more than the 6 mm horn. After at most 18 hours, the droplet that could have blocked sound from reaching the microphone acoustic port had evaporated and the microphone recorded sound levels as high as when entirely dry. Low audible frequencies (1 kHz) were not impeded even by water-logged windscreens. The waterproof, hydrophobic GAW325 vent ensured that no water blocked the sound path: sound of all frequencies were recorded at approximately the same level, irrespective of the time after drenching.\n\nWe found that the 52.5 m cables decreased the sound level of our 40 kHz test chirps by 1.2 to 1.3 dB compared to 5 m cables. Data for signal loss with increasing cable length are available on OSF3.\n\nThe GAW112 and GAW325 horns were capable of mitigating but not completely offsetting the ultrasound transmission loss caused by the acoustic vents (Figure S1)3. The longer the ultrasound horns for the Vesper microphone, the higher the achieved transmission, but the losses for sounds coming from the side also increased, as the horns were more directional (Figure 5). Data for directivty/amplification assessment are available on OSF3.\n\nIn accordance with the theoretical predictions, we found no measurable positive or negative impact of the ultrasound horns on audible frequencies. Our open microphones were also directive, with ultrasound levels around 5 dB lower to the side compared to the front.\n\nWe calculated the costs for each of our three recommended microphone designs (Table 1), which are presented in the discussion. The costs ranged from 12 to 33 EUR per unit, with a bulk assembly of 100 units. Required labour was slightly lower for our budget \"Bufo\" design. Data for material and labor costs are available on OSF1.\n\nComplete data are provided in the supplementary information raw data table.\n\n\nDiscussion\n\nThe best microphone configuration will depend on the organisms of interest, the presence of wind and rain, and the need for directional recording. Many different combinations are possible, all of which have not been tested or built here. We compiled a list of microphone configurations that would be optimal for recording different taxa and named them after representative genera (Figure 6).\n\nFor recording birds, manufacturers like Wildlife Acoustics couple GAW112 vents with windscreens to achieve high protection levels against water ingress and wind noise. However, in habitats or regions with little wind, it becomes worthwhile to use only high-performance vents like the GAW325, thus avoiding sound transmission losses when windscreens are drenched with water after rain.\n\nFor recording bats, high degrees of protection come at the expense of ultrasound transmission. The high-performance waterproof Gore vents muffle ultrasound too much, and the GAW325 horn cannot offset that loss. The classical approach with unprotected microphone elements would be to use GAW112 vents with windscreens: ultrasound is only slightly attenuated with the GAW112 vent. Notably, GAW112 horns only offer minimal amplification, so that manufacturing costs are not justified. However, windscreens are not needed for bats because wind noise only reaches frequencies around 3 kHz, which explains why Wildlife Acoustics forewent the decision to include those on their latest SMM-U2 microphone for bats. Moreover, drenched wind screens block ultrasounds much more than audible frequencies. Thus, a sensible approach would be to use waterproof elements like the VM1000, coupled only with a GAW112 vent that prevents droplets to block the acoustic port. Interestingly, since all microphones are able to record sounds underwater and record normally thereafter (see supplementary materials), the Vesper microphone seems to attain waterproofing only because of the tight solder pattern around the acoustic port, which prevents water to get inside the housing.\n\nWe recommend using microphones with high signal-to-noise ratios whenever possible5. To date, the Invensense element has the highest specified signal-to-noise ratio (70 dB) among our microphones. At a price point of 2.58 EUR, it is roughly four times more expensive than the Knowles element (0.62 EUR), and the waterproof Vesper element (1.58 EUR) is almost three times more expensive. However, all units are so cheap that replacing broken ones would not be an economic consideration, and they represent only a fraction of the price of commercial microphones (at most 1%). According to a preliminary assessment, the Invensense and Vesper elements perform as well as the Knowles element in the audible range, while the Vesper element trails behind for recording ultrasound. However, the Vesper element has the advantage that it does not require a high-performance vent or a windscreen when recording bats, and it can be easily combined with horns.\n\nWe would like to stress the benefit of using acoustic horns to amplify sound \"for free\". The horns we tested considerably improved signal-to-noise ratios, essentially transforming average elements into high-quality microphones. The advantage of such horns has seldom been exploited (but see ultrasonic horn of Wildlife acoustics and Petterson M500 microphone), although the only downside seems to be the loss in directivity.\n\nSurprisingly, we did not find a large signal loss when using long cables. Including pre-amplifiers in microphones (like some manufacturers do) seems unnecessary, which simplifies microphone design.\n\nThe minimalist: Bufo. This microphone is the cheapest, simplest, and, like its namesake, ugliest design. It is easy to assemble, as it only consists of an audio connector, wires, the Vesper microphone on its PCB with a GAW112 vent glued onto it, and epoxy glue. The glue is required to make the microphone waterproof and hold the module in place. Only the Vesper microphone is suitable for this design as it can withstand higher environmental stress due to its piezoelectric design. The downsides of that design are that it is not repairable (due to the epoxy glue, only discardable), and not modular (horns and vent holders cannot be attached). The Bufo is equally suitable for birds and bats.\n\nThe silent one: Otus. Like its namesake, this is the most silent microphone with the lowest specified self-noise, enabling recordings of maximum signal-to-noise ratio in the audible range. It consists of an audio connector, a simple metal tube enabling only vents to be attached, and the Invensense element. The recommended configuration for birds would be with a GAW325 vent. In regions and habitats where winds are prominent, a windscreen can optionally be fastened to it with a cable tie.\n\nNote that when using a GAW112 vent with the necessary windscreen, you would essentially get a microphone similar to Wildlife Acoustic's SMM-U1. However, the Otus can also record audible sound and could have higher-quality recordings: The SMM-U1 probably uses the same Knowles FG element as the SMX-U1 that we tested and found to have shorter detection ranges. We only recommend this configuration when single omnidirectional microphones are required and rain is not too frequent as to avoid ultrasound transmission losses due to water-logged windscreens. We next present a microphone that does not require a wind screen, which is more modular than the Otus and better suited for bats.\n\nThe allrounder: Myotis. This microphone would be intended mainly for bats. Even though it records the entire sound spectrum, the audible sound interval is recorded slightly less cleanly than with the Knowles or Invensense elements due to the lower specified signal-to-noise ratio. The microphone consists of an audio connector, a metal tube designed for attachments, and a waterproof Vesper microphone with a GAW112 vent glued onto it. The microphone can be used without or with horns to narrow and amplify the pickup area to the desired degree, which is often desirable for bat surveys to focus on flyways. This combination is particularly useful when doing stereo recordings, where the redundancy of recording with two omnidirectional microphones can be reduced while also increasing the detection ranges. This design without a windscreen enables microphones to dry quickly to record sounds soon after rain. Wind friction is restricted to low frequencies and thus not problematic when recording bats, but it is still possible to attach windscreens in areas prone to wind when low-frequency sound recordings are desired.\n\nF1000Research allows for article versioning. We welcome prospective co-authors to continue develop our open-source microphone system. Further technological improvements will lead to new products, and there are many development opportunities.\n\nWe found significant variations in the amplification attained by different microphone-horn combinations, which are probably caused by variable micro-alignment of the horn with the microphone's acoustic port. Our PCBs were slightly too small for the space they had in the housing but this has been corrected in the PCB design files provided in the supplementary materials.\n\nWe need to design a screwable attachment system that allows horns to be easily attached and removed. It should feature rubber rings for waterproofing. We need lighter, attachable audible horns of similar dimensions as the ones used here, which would be usable in the field. We are designing larger ultrasonic horns that are less directive while still offering similar amplification levels.\n\nMore acoustic vents should also be tested to find high-performance acoustic vents that do not reduce ultrasound transmission too much. However, they are difficult to source as they can only be purchased in batches of 1000 from the manufacturer Gore, and ultrasound transmission is also not tested by the manufacturer. This also underlines the fact that we could only test ultrasound transmission at 40 kHz, although several bat species vocalise well above 100 kHz. However, no affordable, commercial ultrasound emitters are available to our knowledge.\n\nTo allow our microphones to be used on a broader range of recorders, we should also design housings for other acoustic connectors. The signal loss in even longer cables should be tested, and if substantial, small amplifiers should be designed to compensate that loss. Finally, testing the microphones in freshwater systems could reveal new opportunities in that field.\n\n\nData availability\n\nRaw data for microphone assessment are available on OSF in folder: Microphone assessment. Data for different cable lengths, cable drying, cost and labor, and transmission are available in the indicated csv files.\n\nDOI: https://doi.org/10.17605/OSF.IO/HEZKW3.\n\nExpanded microphone building instructions are available on OSF in folder: Building instructions.\n\nTable S1. Available open-source devices and commercial products for recording sound in terrestrial habitats. Available in folder: Microphone assessment.\n\nFigure S1. Absolute amplitude of all the different microphone attachments measured at 3 and 6 m from the microphone. Available in folder: Microphone assessment, File: Extended data – Microphone assessment.\n\nDOI: https://doi.org/10.17605/OSF.IO/HEZKW3.\n\nAll data 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\nKD was supported by the DFG (CRC990 EFForTS) [SFB990/2].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Ilham for carrying out repeated cable tests and Deice Wayong for her support in field recordings. We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the Göttingen University.\n\n\nReferences\n\nShonfield J, Bayne EM: Autonomous recording units in avian ecological research: current use and future applications. Avian Conserv Ecol. 2017; 12(1): 14. Publisher Full Text\n\nGibb R, Browning E, Glover‐Kapfer P, et al.: Emerging opportunities and challenges for passive acoustics in ecological assessment and monitoring. Methods Ecol Evol. 2018. Publisher Full Text\n\nDarras KFA: Sonitor: Open-Source Microphone System for Terrestrial Ecology. OSF. 2018. http://www.doi.org/10.17605/OSF.IO/HEZKW\n\nSethi SS, Ewers RM, Jones NS, et al.: Robust, real-time and autonomous monitoring of ecosystems with an open, low-cost, networked device. Methods Ecol Evol. 2018; 9(12): 2383–2387. Publisher Full Text\n\nDarras K, Batáry P, Furnas B, et al.: Comparing the sampling performance of sound recorders versus point counts in bird surveys: A meta-analysis. J Appl Ecol. 2018; 55(6): 2575–86. Publisher Full Text\n\nDarras K, Pütz P, Fahrurrozi, et al.: Measuring sound detection spaces for acoustic animal sampling and monitoring. Biol Conserv. 2016; 201: 29–37. Publisher Full Text\n\nTurgeon P, Van Wilgenburg S, Drake K: Microphone variability and degradation: implications for monitoring programs employing autonomous recording units. Avian Conserv Ecol. 2017; 12(1): 9. Publisher Full Text\n\nAdams RA, Pedersen SC, editors: Ontogeny, Functional Ecology, and Evolution of Bats. 1 edition. Cambridge; New York: Cambridge University Press; 2000; 398. Publisher Full Text\n\nStewart GW, Lindsay RB: Acoustics: A text on theory and applications. New York, NY: D. Van Nostrand Company; 1930. Reference Source\n\nKirkup S: The Boundary Element Method in Acoustics. 2nd ed. Integrated Sound Software; 2007; 8. Reference Source" }	[ { "id": "42343", "date": "15 Jan 2019", "name": "Sarab S. Sethi", "expertise": [ "Reviewer Expertise Autonomous ecosystem monitoring", "bioacoustics", "time series 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\nI enjoyed reading this paper, in particular the thorough nature of the methodology to test various configurations and their effect on amplification, directionality and quality of the audio signal recorded by the microphones. Furthermore, the three clear recommended designs will be particularly useful for ecologists to immediately start incorporating this research into their projects. Commercially available microphones rarely appreciate their weaknesses with such honesty, and as such it’s difficult to find the correct solution for each situation without specialist knowledge.\nI only have a small number of comments as outlined below, but generally I believe this paper is welcome, and should add to the growing appetite for high quality engineering in the field.\nGeneral\nThe structure of the paper didn’t quite flow from the Results to Discussion sections. On page 7, in the text under ‘Cost’ and in Table 1 mention is made to Bufo, Otis and Myotis whilst full descriptions of each of these configurations is only given a lot later. I’d recommend moving the recommended designs to the end of the Results section of this paper rather than Discussion The lowest frequency tested in all your examples is 1kHz, however you also mention that audible range goes as low as 20Hz. Many terrestrial mammals vocalise with fundamental frequencies under 1kHz (e.g. gibbons, elephants). Ideally we could see results starting at 100Hz or so, or if not this limitation should be made clear in the text\nIntroduction\n“microphone signals are filtered at the source only for commercial reasons, to enable either bird or bat recordings and sell multiple specialised products” – I can believe this, but would like to see a citation\nMethods\nYou only compare MEMS microphones for well justified reasons. However, I would still like to see this mentioned more clearly in the introduction or even the abstract. Some mention of Electret Condenser Microphone (ECM) drawbacks would be appreciated “This connection form is commonly used in most autonomous sound recorders” – which ones?\nResults\n“The vent-only and 6mm horn configurations were affected by wind friction noise at up to 3 kHz, greatly masking the 1 kHz test tones” – I expect this will be a lot worse for lower frequencies I suggested testing above?\nDiscussion\nIt is possible to keep windscreens mostly dry – if they are mounted under a sheltered place (e.g. under a solar panel in Sethi et. al.). They will still get wet, but nowhere near the submerged drenching described here. If this is possible, would this change recommendations? Table 1: give per unit costs too please “We would like to stress the benefit of using acoustic horns to amplify sound “for free”.” – but later you appreciate the added directionality. This is very important in mono setups and definitely not a free amplification\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": "4954", "date": "05 Nov 2019", "name": "Kevin Darras", "role": "Author Response", "response": "Thank you for your appreciation of our work. We respond to your review comments below but please note that there are extensive additional improvements throughout, summarised in the “Changes from the previous version” text. We improved the flow of the paper: we moved the costs section into the discussion, as it did not fit well within the assessment of the acoustic properties of the microphones, and thus avoided to show results about designs that were not presented yet. We integrated the Sonitor designs better into the discussion to show that they are derived from our results. We also added a summarising paragraph to the start of the discussion to announce our structure better. We clarified why we did not assess acoustic performance below 1 kHz: “Since we did not have access to dedicated anechoic rooms, all tests were conducted outdoors, and we also refrained from using test tone frequencies below 1 kHz, as lower-frequency anthropogenic noise was constantly present.” We were unsure whether we should specifically mention that Wildlife Acoustics ultrasonic microphones filter audible signals using PCBs integrated into their housings. Their component microphone elements are actually well-suited by design for the audible range as well and we cannot think of any technical reason why this is done; the commercial reason might be to create multiple product niches. We now write in a less derogatory but more specific way: “In some cases, audible sound frequencies are filtered at the source to enable only bat recordings although the underlying microphone elements record the entire acoustic spectrum from amphibians to bats (Wildlife Acoustics, e.g. SMX-U1, SMM-U2 microphones).” We now propose a new design using an ECM (re-named Bufo as it focuses on audible sound) and integrated the ECM concept into the article. We specify which recorders use the Mini-Con-X connectors: “This connection form is commonly used in most autonomous sound recorders (Wildlife Acoustics and Frontier Labs recorders, Swift, Arbimon)” Indeed wind noise will affect interest sounds below 1 kHz even stronger, we mention this now: “However, detectability of target sounds below 1 kHz should be even more negatively affected.” Now we also recommend a conventional microphone design with a windscreen - which is actually very similar to the one we use in the field – and acknowledge and cite that solar panels can also protect microphones from rain: “It is also possible to reduce the rain ingress by blocking it with shelters or solar panels (6), although this can block sounds of interest coming from above the microphone to some extent.“ We choose not to include the per unit cost in Table 1, as this is an unrealistic number for ecological studies, and also because of the following reason: “Prices (as of December 2018) do not increase proportionally with the number of units due to economies of scale.” Thus, prices are higher for lower amounts and lower for higher amounts, and we mention this in the discussion too now. We clarify that the amplification of horns comes “for free” now with this amendment: “[…] when using stereo deployments“. Indeed, we stated before: “[microphones] can be used without or with horns to narrow and amplify the pickup area […] This combination is particularly useful when doing stereo recordings, where the redundancy of recording with two omnidirectional microphones can be reduced while also increasing the detection ranges.”" } ] }, { "id": "42344", "date": "16 Jan 2019", "name": "Holger Klinck", "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 very interesting paper which provides very useful information for researchers in the field of terrestrial bioacoustics, especially those involved in remote passive acoustic monitoring efforts.\n\nGeneral comment: The English in the present manuscript requires improvements. Please carefully proof read and spell check the manuscript to eliminate existing grammatical errors. For example, “As transducers of mechanical energy into electrical signals” is not proper English. Another example is “Commercial microphones are relatively expensive, specialized on particular taxa, and often have opaque technical specifications.” Specialized on should be replaced with specialized for. Also, technical specifications cannot be opaque. This sentence needs to rephrased. For example: Technical specifications on the microphones are often not publicized. Language issues like these exist throughout the manuscript and need to be addressed.\n\nA few detailed comments: Which MEMS type is being used in the recommended designs? Sounds like the Bufo is based on the Vesper 1000 MEMS but it is not mentioned which MEMS was used for the Otus and Myotis.\n\nWhat really should be included in the manuscript are frequency response curves for the various designs indicating the sensitivity across the entire frequency range of interest. For example, the gain of the horns will be frequency dependent and alter the frequency response of the actual MEMS. A single frequency test is informative but doesn’t provide enough information. This is especially true for frequencies in the 50-100 kHz range.\n\nThe authors emphasize the importance of the microphone’s SNR. The MEMS mics used in the designs feature SNRs between 60 and 70 dB. However, most of the recording system listed in Table 1 feature mics with a SNR of 80 dB. The authors should include talk about these differences in the discussion section.\n\nIn addition, microphone sensitivity is also an important parameter. How do the selected MEMS mics differ in sensitivity (and compare to the mics listed in Table 1)? Again, a comparative frequency response curve would answer many of these questions.\n\nMost MEMS these days can be wired differentially or single-ended. Differential outputs are typically lower noise and in case of the MEMS and increase the sensitivity. Is this something which could be accommodated in your design? Should this be considered?\n\nMany autonomous systems aim for low power consumption. How do MEMS compare to traditional mic designs in that regard?\n\nBTW, TDK recently released the ICS-40730 MEMS with a SNR of 74dB. To my knowledge this is currently the MEMS with the best SNR.\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": "4955", "date": "05 Nov 2019", "name": "Kevin Darras", "role": "Author Response", "response": "Thank you for critically reviewing our article. We respond to your review comments below but please note that there are extensive additional improvements throughout, outlined in the “Changes from the previous version” text. We corrected all English mistakes that we could spot and also those that you pointed out, thank you. We did not fully write out the microphone model names throughout the manuscript, we do that now. We do not have the necessary hardware for carrying out frequency sweeps in the ultrasound range, so we focused on the three representative frequencies of 1, 10, and 40 kHz. We discuss the fact that our MEMS have lower specified signal-to-noise ratios than some of the other recorders’ microphones now: “It appears that in theory, our microphones do not reach the specified signal-to-noise ratios of most commercial audible range recorder alternatives (4). However, there is much variation between manufacturers’ specified signal-to-noise ratios due to loosely standardised measurement protocols, and the Knowles SPU0410LR5H-QB was measured to be on par with ECMs that had specified signal-to-noise ratio values of 80 dB (PUI Audio AOM-5024L-HD-R), with the Vesper VM1000 and the Invensense ICS-40720 following closely behind (unpubl. data). Also, microphone signal-to-noise ratio is also almost never measured in the ultrasound range. According to our measurements, the Vesper VM1000’s signal-to-noise ratio element trails behind its Knowles and Invensense counterparts for recording ultrasound (unpubl. data).” This is based on our study which is in review elsewhere (for 7 months), but has been provided as additional material for the reviewers to consider. We also detail microphone sensitivity now: “All three MEMS elements have a typical sensitivity of -38 dB and thus require relatively strong amplification from the recorder for soundscape recordings.” We also mention differential output microphones in the discussion. We mention the ICS-40730 microphone (which has a bigger form factor) with a 74 dB signal-to-noise ratio now and encourage peers to design a PCB for it." } ] }, { "id": "42345", "date": "17 Jan 2019", "name": "Catharina Karlsson", "expertise": [ "Reviewer Expertise Acoustic Ecology" ], "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 outlines three open-source hardware solutions to microphones for bioacoustics monitoring. The authors justly acknowledge the lack of transparency from current manufacturers of the technical details of the components, especially the microphone element. Another important note is the decrease in cost in comparison to off-the-shelf products which as the authors note, are not only expensive but difficult and impractical to get repaired. Considering the increasing use of open-source hardware in the bioacoustic field this article is a very nice addition to allow practitioners an easier time of assembling their own equipment.\n\nEven though this is a nice article, it is a bit difficult to follow at times and it doesn’t flow that well. The introduction is good, but I recommend the authors to look through the structuring of the methods and the results section.\n\nThe justification for only testing MEMS elements is not thorough enough as it is not compared to the other types of elements that are on the market. What are the benefits of using a condenser element for example?\n\nFor the sentence below (methods section) I think it is better to just state the representative frequencies rather than giving readers (especially inexperienced ones) the impression that these are representative frequencies for those taxonomic groups (amphibians and birds go above 10kHz depending on species for example). Especially as it is stated in another section that insects can also hear sound up to ultrasonic frequencies. \"1 kHz (birds and amphibians), 10 kHz (insects), and 40 kHz (bats)\"\n\nThe English need to be proof read up throughout, three examples follow (especially in the methods section, I think it makes it a bit stodgy and difficult to follow).\n\n“This microphone would be intended mainly for bats.” reads better as “This microphone is intended for bat recordings”.\n\n“This microphone is the cheapest, simplest, and, like its namesake, ugliest design.” Sentences like this one can be cleaned up a bit, Bufonides are not ugly (you just have not looked closely enough), and there are better words to use than ugly (i.e. basic design, rough etc).\n\n“It is thus preferable to reflow-solder MEMS elements to printed circuit boards, which can be made in electronic laboratories or workshops equipped with reflow ovens.” Sentences like this one does not make a lot of sense (after the comma), I had to re-read several times before I understood what you meant as the way the sentence is structured it could refer to the MEMs element or the circuit board (but you mean neither). I would use a full stop instead of using a comma then re-write the second part to something along the lines of “Reflow-soldering can be performed in reflow equipped electronic laboratories or workshops”.\n\nThe section I was really interested in was to see how the weatherproofing affected the performance of the microphones – however, there is only mention of how it affects the ultrasonic frequencies? It would be nice to see a graph of how the frequencies are affected by different levels of waterproofing (it would even be interesting to see how complete waterproofing, such as a plastic bag compares to vents and no proofing as I’ve worryingly seen that used at times). In general, the result section feels a bit rushed and not developed enough. It was especially difficult to follow, both in the methods and results section, which element and which vent was used where. Occasionally only the vent is mentioned and no element (remind the reader again).\n\nI think you missed a bit in the discussion, it is worth mentioning that the first time all these things must be sorted out they will take considerable time and I think your estimate of labour is on the low side. It often takes considerable effort to figure out where to source everything the first time – in addition it needs to be highlighted that these cost estimates are for Europe (Germany to be precise, it is in the supplementary information but I do not think it is mentioned in the text), there can be considerable variation depending on where you are based (both lower and higher). The sourcing time is also a labour and it can take considerable time to find suppliers, sort out shipments etc so it should at least get a mention.\n\nI am pleased that someone has managed to find waterproof vents that come in smaller batches than 10,000 pieces. I also acknowledge the quite comprehensive supplementary material that is attached with more in-depth details of assembly – the article itself is really the tip of the iceberg of the amount of work that has gone into this study. I do believe that comprehensive manuals and instructions like these are a necessity to ensure other people use it. All in all, this is a very nice and timely article and I hope we start seeing more of this kind of work that is written for field scientists coming out.\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": "4956", "date": "05 Nov 2019", "name": "Kevin Darras", "role": "Author Response", "response": "Thank you for taking your time to critically evaluate our article. We respond to your review comments below but please note that there are extensive additional improvements throughout, outlined in the “Changes from the previous version” text. You will see that the methods and results have been re-structured and re-written in parts. Hopefully this meets your expectations. In this new version, we acknowledge the benefits of using ECMs and propose a new design based on ECMs (re-named Bufo). We also discuss their characteristics versus MEMS elements. We acknowledge that the frequencies we tested are only approximately representative but would like to continue pointing out that they stand for different taxa: “However, terrestrial wildlife sounds span frequencies from 20 Hz to 200 kHz, so we measured the transmission across three frequencies that can roughly be assigned to different taxa: 1 kHz (birds and amphibians), 10 kHz (insects), and 40 kHz (bats)“. These point frequencies should be representative of nearby frequencies as abrupt changes of acoustic properties along the frequency range are physically very unlikely. Thank you for pointing out English language mistakes, we corrected them accordingly. Your question as to how the lack of waterproofing affects microphone performance was addressed in our new long-term test: microphones break, even the water-proof ones. We do not recommend using plastic bags but since this is what many users have with the Audiomoth, we added this: “Since the Audiomoth was introduced, hundreds of these recorders are protected simply by placing the recorder inside a ziplock bag, which can protect the equipment some time, but severely impacts ultrasound transmission, while frequencies in the audible range are better preserved (7).” We reference a forum post by myself where we field-tested different protection levels. But to keep up with the latest improvements, we added: “Fortunately, a new weatherproof case that uses acoustic vents has recently be developed (8).“ We underlined that our labor estimates are conservative: “Our labour estimates are conservative as it can take considerable and variable time for finding suppliers, choosing the design, and setting up of the microphone building.” and acknowledge the local context: “ […] these prices are representative for Germany and countries with similar supply chains”" } ] } ]	1	https://f1000research.com/articles/7-1984
https://f1000research.com/articles/9-632/v1	22 Jun 20	{ "type": "Brief Report", "title": "biogitflow: development workflow protocols for bioinformatics pipelines with git and GitLab", "authors": [ "Choumouss Kamoun", "Julien Roméjon", "Henri de Soyres", "Apolline Gallois", "Elodie Girard", "Philippe Hupé", "Julien Roméjon", "Henri de Soyres", "Apolline Gallois", "Elodie Girard" ], "abstract": "The use of a bioinformatics pipeline as a tool to support diagnostic and theranostic decisions in the healthcare process requires the definition of detailed development workflow guidelines. Therefore, we implemented protocols that describe step-by-step all the command lines and actions that the developers have to follow. Our protocols capitalized on two powerful and widely used tools: git and GitLab. They address two use cases: a nominal mode to develop a new feature in the bioinformatics pipeline and a hotfix mode to correct a bug that occurred in the production environment. The protocols are available as a comprehensive documentation at https://biogitflow.readthedocs.io and the main concepts, steps and principles are presented in this report.", "keywords": [ "development workflow", "bioinformatics pipeline", "quality management", "healthcare" ], "content": "Introduction\n\nThe importance of best practices for bioinformatics analysis and software have been highlighted by many authors who proposed very valuable guidelines for better reproducibility and traceability (Georgeson et al., 2019; Hamburg & Mogyorodi, 2019; Noble, 2009; Sandve et al., 2013). However, reproducing an analysis is often a challenge (Kim et al., 2018). Therefore, guidelines have to be promoted by the computational labs and bioinformatics core facilities to federate the bioinformaticians across common practices for software development. This is especially essential when the bioinformatics pipeline is used in precision medicine to support the diagnostic and theranostic activities. Indeed, many hospitals worldwide use High-Troughput Sequencing in routine clinical practice to guide the therapeutic decision. This new era of genomic medicine is even promoted in healthcare systems at a large scale within several national initiatives, such as France, USA, UK or Australia (Stark et al., 2019).\n\nThis evolution has brought Bioinformatics at the forefront of the healthcare process with the bioinformatics pipeline being a fully integrated component of the clinical decision. Compliance with the healthcare laboratory accreditation standards and regulations is thus required for the development and exploitation of the bioinformatics pipeline. In this context, several authors (Hume et al., 2019; INCa, 2018; Matthijs et al., 2016; Roy et al., 2018) recommended in their guidelines that an appropriate code repository tool should be used to enforce version control. It ensures to track the different releases of the bioinformatics pipeline, their validation and the developers involved in their implementation. This must be integrated in a quality management process with standardized protocols approved for a diagnostic use.\n\nThere is a wide ecosystem of tools (see Perez-Riverol et al., 2016; Riesch et al., 2020; Zolkifli et al., 2018) that can support the implementation of such protocols. Among them, we capitalized on git for the version-control system as it became a very popular tool in the software engineering community and GitLab for the repository manager as it can be self-hosted. Both tools offer a large set of very powerful functionalities that can be used and combined in multiple ways thus increasing their usage complexity. It is thus mandatory to formalize through detailed guidelines how to use them on a daily basis for the development and deployment of the bioinformatics pipeline. Therefore, we implemented the set of protocols biogitflow (Kamoun et al., 2020) that describes step-by-step all the command lines and actions to be performed by the developers. A comprehensive documentation available at https://biogitflow.readthedocs.io provides all the technical details. We introduce here the main concepts, steps and principles.\n\n\nMethods\n\nThe development workflow consists of four main steps. The first step is software development, which includes code writing and testing. The second step is acceptance testing by the end-users who validate that the expected functionalities have been correctly implemented. The third step is check the installation process and new testing to ensure that the bioinformatics pipeline can be installed in a similar environment than the one used in production. During this step, a new testing is performed such that bugs can be corrected before installing the bioinformatics pipeline in production. Finally, the fourth step is production deployment. During this last step, the new release of the bioinformatics pipeline with the new functionalities is installed in the production environment.\n\nThe four different steps have to be performed in separated environments in order to i) ensure that a stable version can be used in production, ii) allow the end-users to validate a new release without any impact on both the version used in production or the version under development and iii) allow the software developers to add new functionalities and modify the code without any impact on the end-users who are validating a new version and/or using the current version in production. Therefore, three deployment environments are used: a development (dev), a validation for pre-production (valid) and a production environment (prod). Besides these environments, each developer can deploy the bioinformatics pipeline in a local workspace to test the new functionalities that have been developed.\n\nOur biogitflow protocols capitalized on the gitflow model proposed by Driessen (2010). The management of the different bioinformatics pipeline versions is based on four different git branches. Depending on the context and the step of the development workflow the following branches on the remote repository are used:\n\ndevel contains the code of the current version under development.\n\nrelease contains the code with both candidate and official releases. The release branch comes from the devel branch.\n\nhotfix is a mirror of the release branch and is used to patch the code that is in production. If a critical bug occurs in production, this branch is used to fix the issue.\n\nmaster is only used to archive the code from the release and hotfix branches. This branch is not used for development.\n\nAmong these four branches, the release, hotfix and master are protected branches such that only the developers with the Maintainer role in the GitLab repository can directly push code on these branches (the other users have to use the GitLab merge request functionality to push on protected branches).\n\nIn addition to these four branches, the developer can create local branches to i) implement a new feature (the branch is named with a prefix feature plus any meaningful suffix) and ii) fix a bug in production or resolve a problem during the third step (these branches are either named with the prefix release or hotfix depending on the use case plus some relevant contextual information).\n\nTwo levels of roles and permissions are considered. Firstly, in the GitLab remote repository a developer is either assigned to the role Developer (D) to push the developments only on the non-protected branches or Maintainer (M) to push the developments on any branches. Secondly, in the deployment environments a user is either granted with the permission UD to deploy in the dev environment only or UVP to deploy in the valid and prod environments (any user with the UVP permissions is also granted with the UD permissions).\n\nTesting the bioinformatics pipeline occurs during all the steps of the development workflow. It involves all stakeholders including the developers, the users in charge of the deployments and the end-users. This should be done as often as possible to identify and resolve any unexpected behavior early in the development process. The International Software Testing Qualifications Board provides comprehensive guidelines for software testing (Hamburg & Mogyorodi, 2019). Among them, we strongly recommend to include the following testing. The unit testing confirms that a piece of code provides the expected output according to the input parameters. The integration testing checks that the interfaces of the different bioinformatics pipeline components are consistent with each other and that the result of their integration allows the expected functionalities to be performed. The system (or functional) testing validates that the full bioinformatics pipeline works and fits well the end-user’s needs. The regression testing checks that the correction of bugs or the development of new functionalities did not introduce defects in unchanged areas of the bioinformatics pipeline. In addition, we highlight the importance of the operational testing to check that the bioinformatics pipeline provides the expected results on a reference dataset (golden dataset) in the production environment. This testing is systematically launched prior to any new analysis by the bioinformatics pipeline in the production environment. This ensures that the results are reproducible as long as the exact same version of the bioinformatics pipeline is used.\n\n\nResults\n\nTwo use cases have been addressed with dedicated protocols that detail the different actions step-by-step. The first one is the nominal mode (Figure 1) in which a new feature is implemented to improve the bioinformatics pipeline based on requirements and expectations from the end-users who operate it for their daily clinical practice. The second one is the hotfix mode (Figure 2) in which there is a bug in the bioinformatics pipeline in the production environment that hampers the delivery of the results for the patients. The biogitflow documentation provides all the technical details, on how to configure the remote repository in GitLab to develop a new bioinformatics pipeline, how to use git and GitLab depending on the roles and permissions during the time frame of the development workflow for both use cases.\n\nThis graphical synopsis provides an overview of the different steps of the development workflow when a new feature is implemented in the bioinformatics pipeline according to the role and permissions of the developer. It describes the different git actions that are performed on the different branches and the deployments in the different environments.\n\nThis graphical synopsis provides an overview of the different steps of the development workflow when a bug occurred in the production environment in the bioinformatics pipeline according to the role and permissions of the developer. It describes the different git actions that are performed on the different branches and the deployments in the different environments.\n\nWhatever the use case, the bioinformatics pipeline is deployed in production and operated by the end-users once it has successfully passed all the testing. Whenever new patient data has to be analyzed to deliver results for diagnostic or theranostic purposes, the bioinformatics pipeline can be launched by the end-users only if the operational testing reproduces the results from the golden dataset, otherwise it is blocked by internal control mechanism. In this case, the developers investigate the reason of the failure and have to fix it using the hotfix mode.\n\nAs a real example, we provided a biogitflow template of the repository that is available for any registered user at GitLab.com. The user can fork the repository in a personal workspace and apply the nominal mode (Figure 1) and hotfix mode (Figure 2) following the proposed protocoles.\n\n\nDiscussion\n\nWriting these protocols required some feedback from the developers in order to decide what was the best way to capitalize on git and GitLab taking into account our internal constraints and organization. The need of formalization required by the laboratory accreditation agency such that a bioinformatics pipeline can be used in healthcare was a great catalyst to implement these protocols. It was a major step to improve our daily practice of software engineering towards better quality management and was a source of motivation to change our work habits.\n\nAs a corollary of these protocols was the necessity to train the users involved in the development workflow to demonstrate to the laboratory accreditation agency that the technical protocols are known, understood and mastered by the developers. Therefore, an internal accreditation process of our developers was implemented. The training consists of a series of exercises that cover all the different use cases, roles and permissions of the protocols. The exercises are first realized in pair with the tutor and the trainee, then the trainee performs the exercises alone twice, and finally the trainee performs the exercises alone but in the presence of the tutor who ask additional questions to ensure that the tricky parts of the protocols are understood. To be endorsed, the trainee has to perform the exercises fluently in full autonomy. All the actions of the training process are tracked in a dedicated GitLab remote repository. Endorsement is valid for one year that can be extended if the developer still masters the protocols. This is assessed during a dedicated yearly interview with the tutor.\n\n\nConclusions\n\nWe described two protocols that are used on a daily practice to develop bioinformatics pipelines compliant with the accreditation standards for healthcare. While some choices were made to match our internal constraints and organization, the protocols can be easily transposed in other institutes as the main concepts, steps and principles hold in most of the contexts. According to the principle of the Deming wheel of continuous improvement in quality management, the protocols we described are intended to evolve in order to address future requirements, handle new risk management, integrate new tools or technical frameworks offering better efficiency of the overall process. We strongly encourage the promotion of such protocols not only for the healthcare activities but also for research activities.\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\nbiogitflow documentation is available at: https://biogitflow.readthedocs.io/.\n\nArchived source of the biogitflow documentation at time of publication: https://doi.org/10.5281/zenodo.3885463\n\nLicense: CeCILL Version 2.1.\n\n\nAuthor contributions\n\nCK, HDS, JR and PH conceived the protocols. PH wrote the protocols and the manuscript that were reviewed by AG, CK, EG, HDS and JR. PH supervised the study.", "appendix": "Acknowledgments\n\nWe thank the French Bioinformatic Network for NGS Cancer Diagnosis and the Institut National du Cancer (INCA) for the fruitful discussions.\n\n\nReferences\n\nDriessen V: A successful git branching model. 2010. Reference Source\n\nGeorgeson P, Syme A, Sloggett C, et al.: Bionitio: demonstrating and facilitating best practices for bioinformatics command-line software. GigaScience. 2019; 8(9): giz109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamburg M, Mogyorodi G: Standard glossary of terms used in software testing. Technical report, International Software Testing Qualifications Board. 2019. Reference Source\n\nHume S, Nelson TN, Speevak M, et al.: CCMG practice guideline: laboratory guidelines for next-generation sequencing. J Med Genet. 2019; 56(12): 792–800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nINCa: Conception de logiciels pour le diagnostic clinique par séquençage haut-débit. Technical report, collection Outils pour la pratique. 2018. Reference Source\n\nKamoun C, Roméjon J, De Soyres H, et al.: bioinfo-pf-curie/biogitflow: version-1.0.1. Zenodo. 2020. Publisher Full Text\n\nKim YM, Poline JB, Dumas G: Experimenting with reproducibility: a case study of robustness in bioinformatics. GigaScience. 2018; 7(7): giy077. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatthijs G, Souche E, Alders M, et al.: Guidelines for diagnostic next-generation sequencing. Eur J Hum Genet. 2016; 24(10): 1515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoble WS: A quick guide to organizing computational biology projects. PLoS Comput Biol. 2009; 5(7): e1000424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerez-Riverol Y, Gatto L, Wang R, et al.: Ten simple rules for taking advantage of git and github. PLoS Comput Biol. 2016; 12(7): e1004947. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiesch M, Nguyen TD, Jirauschek C: bertha: Project skeleton for scientific software. PLoS One. 2020; 15(3): e0230557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoy S, Coldren C, Karunamurthy A, et al.: Standards and guidelines for validating next-generation sequencing bioinformatics pipelines: A joint recommendation of the association for molecular pathology and the college of american pathologists. J Mol Diagn. 2018; 20(1): 4–27. PubMed Abstract \| Publisher Full Text\n\nSandve GK, Nekrutenko A, Taylor J, et al.: Ten simple rules for reproducible computational research. PLoS Comput Biol. 2013; 9(10): e1003285. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStark Z, Dolman L, Manolio TA, et al.: Integrating genomics into healthcare: A global responsibility. Am J Hum Genet. 2019; 104(1): 13–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZolkifli NN, Ngah A, Deraman A: Version control system: A review. Procedia Comput Sci. 2018; 135: 408–415. Publisher Full Text" }	[ { "id": "72478", "date": "22 Oct 2020", "name": "Bernard Pope", "expertise": [ "Reviewer Expertise Bioinformatics. Genomics. Cancer. Software Development. Pipelines." ], "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\nMotivated by laboratory accreditation requirements for the development of bioinformatics workflows related to high throughput sequencing in a clinical setting, the authors have developed a software development process formalisation based on gitflow, a popular model in mainstream software engineering practice.\nThe formalisation prescribes a branching, testing, merging and release process, with set roles and permissions for developers, and methods for managing the development of new features (nominal mode) and bug fixing (hotfix mode).\nIn addition to describing the biogitflow workflow, the paper also contains a short section on testing bioinformatics software pipelines, however, this is largely a recapitulation of standard testing terminology, and does not appear to be particularly bioinformatics-specific.\nThe paper has a brief discussion about the motivation for adopting the workflow and some unsubstantiated claims about improving their software engineering practices. It also has a brief discussion about the need to train developers in the workflow and formal accreditation.\nTwo large figures are included in the paper that attempt to illustrate the proposed workflow in nominal and hotfix modes.\nWhile the topic of this paper is interesting and highly relevant to bioinformatics software developers working in areas of clinical application, where process accreditation is a key concern, there are a number of areas where I think the paper could be improved:\nThe current version of the paper is lacking in critical analysis of the proposed workflow. One does not have to look far to find criticism (positive and negative) of gitflow. However, this paper does not provide any significant discussion of the pros and cons of the approach. One of the main criticisms of gitflow is that it is overly complex (for some software development projects) and thus prone to error and misapplication. This complexity is illustrated in Figures 1 and 2 and the discussion in the paper about the need to train developers in its use. Given the apparent regimented nature of the process, it seems plausible that some of the complexity could be avoided by automation. Are there any useful tools that can help developers apply the model successfully and avoid the common criticisms of gitflow? If developers are finding it difficult to follow, especially the \"tricky\" parts then that may be a sign of limitations in the process or limitations in the tools supporting its application.\n\nIt is not clear how significantly biogitflow differs from gitflow. In what sense does the proposed workflow differ from established software engineering practices?\n\nWhat alternative competing development models exist?\n\nHow can the authors be sure that the workflow is in fact making a positive difference to their software development?\n\nIt would be useful to clarify the intended audience of the paper. How can they apply and benefit from this work?\n\nThe discussion of the provided biogitflow templates sounds useful, but the discussion in the paper is unclear. Perhaps this could be expanded upon?\n\nThe paper ends with \"We strongly encourage the promotion of such protocols not only for the healthcare activities but also for research activities.\" However, due to the aforementioned lack of critical analysis, it is not clear from the paper why this is true.\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? Partly", "responses": [ { "c_id": "6150", "date": "04 Dec 2020", "name": "Philippe Hupé", "role": "Author Response", "response": "First, we would like to thank the reviewer. We are very grateful for his time, contribution and very valuable comments that significantly helped to improve the article. You will find below a detailed answer to the different issues that we have addressed in the revised manuscript. Best regards, Choumouss Kamoun, Julien Roméjon and Philippe Hupé Detailed response to the reviewer1's comments > Motivated by laboratory accreditation requirements for the development of bioinformatics workflows related to high throughput sequencing in a clinical setting, the authors have developed a software development process formalisation based on gitflow, a popular model in mainstream software engineering practice. > The formalisation prescribes a branching, testing, merging and release process, with set roles and permissions for developers, and methods for managing the development of new features (nominal mode) and bug fixing (hotfix mode). > In addition to describing the biogitflow workflow, the paper also contains a short section on testing bioinformatics software pipelines, however, this is largely a recapitulation of standard testing terminology, and does not appear to be particularly bioinformatics-specific. > The paper has a brief discussion about the motivation for adopting the workflow and some unsubstantiated claims about improving their software engineering practices. It also has a brief discussion about the need to train developers in the workflow and formal accreditation. > Two large figures are included in the paper that attempt to illustrate the proposed workflow in nominal and hotfix modes. > While the topic of this paper is interesting and highly relevant to bioinformatics software developers working in areas of clinical application, where process accreditation is a key concern, there are a number of areas where I think the paper could be improved: > 1. The current version of the paper is lacking in critical analysis of the proposed workflow. One does not have to look far to find criticism (positive and negative) of gitflow. However, this paper does not provide any significant discussion of the pros and cons of the approach. One of the main criticisms of gitflow is that it is overly complex (for some software development projects) and thus prone to error and misapplication. This complexity is illustrated in Figures 1 and 2 and the discussion in the paper about the need to train developers in its use. Given the apparent regimented nature of the process, it seems plausible that some of the complexity could be avoided by automation. Are there any useful tools that can help developers apply the model successfully and avoid the common criticisms of gitflow? If developers are finding it difficult to follow, especially the \"tricky\" parts then that may be a sign of limitations in the process or limitations in the tools supporting its application. We fully agree that the protocols are complex and it is not straightforward to master them. Of note, we pointed out in our reply to you comment number 5 that the protocols are mainly relevant in the context of production to deliver to process data on the fly which is obviously a challenge especially when the bioinformatics pipelines have to deal with a very high-throughput of data. The complexity of the workflow just reflects the reality of the development and deployment cycle in the context of production as we intend to promote industrialized processes for the software implementation in order to offer better service, better reactivity, better traceability to end-users. Formalism was thus required and this is the reason why we wrote the biogitflow documentation. You are also fully right when you say that it is \"prone to error and misapplication\" and that some \"complexity could be avoided by automation\". This is clearly an improvement according the Deming wheel as the next step would be to capitalize on additional functionalities of git/gitlab using git hooks or gitlab-CI/CD (continuous integration, continuous deployment) and gitlab Operations features for example. Anyway, as you have mentioned, this critical analysis and limitations were absolutely not highlighted. We, therefore, added a new paragraph at the end of the discussion to explain limitations and foreseen improvements with automation. > 2. It is not clear how significantly biogitflow differs from gitflow. In what sense does the proposed workflow differ from established software engineering practices? This comparison was clearly missing. We added a new paragraph at the beginning of the discussion that explains the main differences. > 3. What alternative competing development models exist? Other alternatives exist such as GitHub flow and GitLab flow. We added them in the section \"Version control and branching model\". > 4. How can the authors be sure that the workflow is in fact making a positive difference to their software development? This is a good question that is difficult to answer. This would require something like an experimental design comparing two or more workflows based on the assessment of objective performance metrics, and then compare the metrics to figure out if they are statistically different. We did not perform such an approach. However, we can say that the benefit of the biogitflow protocols was two-fold: 1/ \"harmonization of the development practices\" and 2/ the \"constructive and positive pressure\" for \"better quality\".These points have been added at the end of the second paragraph of the discussion. > 5. It would be useful to clarify the intended audience of the paper. How can they apply and benefit from this work? Indeed, the target audience was not clearly mentioned. Therefore, we added in the introduction that the protocols are appropriate for software developers in the \"context of production\" to \"support core facilities\" that generate \"high-throughput of data\". The protocols aim at promoting better \"industrialized processes\". The \"context of production\" has been reminded in the conclusion as well. Obviously, any bioinformatician involves in a custom development to analyze data in a research project would not straightforwardly benefit from our work : for this use case, it might just be sufficient to track the developments with git using a single branch. However, we encourage anyone to read the proposed protocols for general knowledge as it can also give ideas for more elaborated needs. > 6. The discussion of the provided biogitflow templates sounds useful, but the discussion in the paper is unclear. Perhaps this could be expanded upon? We realized that both the link to the template and its content were indeed missing. We, therefore, added the url to the template and a more detailed description that also refers to the dedicated section in the biogitflow documentation website. > 7. The paper ends with \"We strongly encourage the promotion of such protocols not only for the healthcare activities but also for research activities.\" However, due to the aforementioned lack of critical analysis, it is not clear from the paper why this is true. As mentioned in point number 5, we have detailed in the introduction who was the targeted audience and highlighted that these protocols are useful in the \"context of production\". We have modified the quoted sentence above highlighting again in the conclusion the usefulness of the protocols in the \"context of production\"." } ] } ]	1	https://f1000research.com/articles/9-632
https://f1000research.com/articles/10-131/v1	19 Feb 21	{ "type": "Data Note", "title": "Development of the Wits Face Database: an African database of high-resolution facial photographs and multimodal closed-circuit television (CCTV) recordings", "authors": [ "Nicholas Bacci", "Joshua Davimes", "Maryna Steyn", "Nanette Briers", "Joshua Davimes", "Maryna Steyn", "Nanette Briers" ], "abstract": "Forensic facial comparison is a commonly used, yet under-evaluated method employed in medicolegal contexts across the world. Testing the accuracy and reliability of facial comparisons requires large scale controlled and matching facial image databases. Databases that contain images of individuals on closed-circuit television (CCTV), with matching formal and informal photographs are needed for this type of research. Although many databases are available, the majority if not all are developed in order to improve facial recognition and face detection algorithms through machine learning, with very limited if any measure of standardisation. This paper aims to review the available databases and describe the development of a high resolution, standardised facial photograph and CCTV recording database of male Africans. The database is composed of a total of 6220 standardised and uncontrolled suboptimal facial photographs of 622 matching individuals in five different views, as well as corresponding CCTV footage of 334 individuals recorded under different realistic conditions. A detailed description of the composition and acquisition process of the database as well as its subdivisions and possible uses are provided. The challenges and limitations of developing this database are also highlighted, particularly with regard to obtaining CCTV video recordings and ethics for a database of faces. The application process to access the database is also briefly described.", "keywords": [ "face database", "CCTV", "facial photographs", "facial identification", "facial comparison", "morphological analysis", "facial recognition" ], "content": "Introduction\n\nFacial comparison is utilised by law enforcement to associate two sets of images, captured on video or photographically, to one another. Although different approaches exist, facial comparison by morphological analysis is currently considered the most reliable method1. In this method, a target image, such as a snapshot from closed circuit television (CCTV) recordings obtained during criminal activity, and a standardised optimal image, such as a police mugshot, are compared to ascertain whether the two individuals are the same person. Facial comparison by morphological analysis has no directed standardised stepwise procedure as a validated methodology2. Although facial comparison still does employ the analysis, comparison, evaluation and verification (ACE-V) tenets of forensic examinations1,3, its accuracy and reliability has not been tested extensively. As such, assessing its reliability should be considered a priority4. The lack of validation can potentially be attributed to the logistic complexity required for rigorous scientific testing, of which a considerable limitation is the lack of standardised and actualistic databases to use5. While several facial image databases exist (e.g. 5,6), the incongruity of their composition is a major limiting factor.\n\nThe composition of many face image databases tends to be specific to the original intended use with a focus on various controlled conditions which makes them difficult to use for general purposes6. For example, there is a variety of pre-landmarked databases available for use in the field of facial recognition (e.g. 7–11) with many variations and controlled conditions. Some of the most commonly controlled-for conditions are orientation of the head/pose, illumination/lighting conditions, facial expressions, and age-related variations (e.g. 6,7,12). Despite the large number of databases available (Table 1), there is a tendency towards either highly controlled data sets captured under very specific conditions with limited actualistic applications (e.g. 13,14) or highly randomised images collected under inconsistent conditions (e.g. 7–9,15). In addition, most of these databases include a limited number of subjects with many replications under niche conditions and no standard baseline control images. Some of the datasets with a limited number of unique individuals include under 100 subjects (e.g. 13,16,17), with a handful including only 10 to 15 subjects (e.g. 14,18–20). Lastly, many of these databases are, by today’s standards, of subpar resolution with only two databases including images of resolutions greater than 640 x 480 pixels21,22. Between the highly specialised and varied conditions of capture, the lack of controlled images with matching realistic informal photographs of the same subjects, the low resolution, lack of methodological standardisation in image capture, and limited subject numbers, these facial image datasets provide very limited use in a forensic facial comparison context or more generalised facial studies.\n\nRecently, databases consisting of video recordings of faces have become more common (e.g. 21,23–26) due to the increase in CCTV surveillance cameras. Generally, these databases have either been collated via recording of known participants in controlled environments (e.g. 21,23,26), or were based on pre-recorded videos obtained from various media on the internet or movies24,25. To the best of the authors’ knowledge, only a single other database (SCface – Surveillance Cameras Face Database) published includes both facial photographs of high resolution and corresponding still images extracted from CCTV recordings of varying resolutions21. These databases are primarily used in testing and developing head/face detection and tracking or automated face annotation systems under a variety of video recording conditions.\n\nThe majority of facial image databases are primarily or exclusively inclusive of males of either European or East Asian descent (e.g. 17,27,28). Only a few databases contain individuals from other ancestry groups, particularly African individuals (e.g. 6,7,13), and they have a limited number of individuals. This is evident when considering there is only a single other South African face database29. Although a great initiative for a large, landmarked face database specifically developed to increase the variety of lighting, ethnicity and age available, it consists primarily of low-resolution webcam-based images with often very distorted lighting conditions. In addition, a total of 276 subjects were used for this database with no control images or demographic specifications provided.\n\nThe majority of existing facial databases have been developed for the purposes of facial recognition and machine learning training and do not contain target and control images of the same individual. These databases tend to be sourced from public, internet images containing faces in a variety of inconsistent conditions (e.g. 9,11,12). To the best of the authors’ knowledge, no databases exist that are intended specifically for use with facial comparison by morphological analysis. As such, we aimed at creating a system for developing a consistent face database with corresponding individuals across various photographic and video recording conditions, resulting in the Wits Face Database (WFD). This database is intended to be a functional, actualistic African database of facial images that can be utilised for facial comparison analyses and research in craniofacial identification. This database is intended to be a free resource strictly for non-commercial scientific research, provided access has been cleared by an ethics committee overseeing its use.\n\n\nMaterials and methods\n\nThe database was established by collecting photographs of willing participants on the University of the Witwatersrand campus, Johannesburg, South Africa, including corresponding CCTV recordings. The database is comprised of CCTV recordings and photographs gathered on university premises, between July 2018 and October 2019, via the pre-existing CCTV systems used by campus security. Facial photographs were standardised and in five different views. The CCTV recordings were captured in a variety of conditions, such as from different quality cameras, in different formats and heights of recording, and with disguises (sunglasses and caps).\n\nEthical approval was obtained from the Human Research Ethics Committee (Medical) of the University of the Witwatersrand (clearance certificate No.: M171026). Permits from the campus head of security and deputy registrar were obtained prior to data capture, in their capacity as site managers. Facial photographs were captured by an experienced photographer following recruitment of participants. CCTV recordings of matched participants were collected through the university’s security systems. The original target of participants intended for the database were 600. However, due to data loss as a result of power failures and data transfer corruption a greater number of participants had to be recruited for redundancy purposes. Potential participants were identified and recruited near the data collection sites based on facial anthropological features resembling males of South African descent and being older than 18 years of age. Approached participants were then informed of the greater project orally and with an information sheet40 and asked for voluntary participation in the study and database. Once agreed, they signed an informed consent form40 prior to being photographed or recorded. If participants requested to be removed from the database, all their photographs and recordings were erased and their recruitment information shredded. None of the personal details and images given by the participants are to be freely distributed. As agreed with the ethics committee, and according to the consent forms signed by the participants, no identifiable images of any individuals are to be published without following up with participants for additional consent. As a result, for publication purposes to indicate examples of the face database, one of the author’s images have been utilised.\n\nFacial photographs and recordings were collected at three designated access-controlled locations with a large influx of potential participants on the Braamfontein Campus of the University of the Witwatersrand, Johannesburg, South Africa. The following three collection sites were set-up and utilised:\n\nSite A: outdoors CCTV camera (installation height: 3100 mm) with a view of a student card terminal near the food concourse and Student Union Building, the Matrix.\n\nSite B: outdoors CCTV camera positioned at eye-level height (1700 mm) in one of the more frequented pedestrian entrances in proximity to the Oppenheimer Life Sciences building.\n\nSite C: indoors analogue CCTV camera (installation height: 2500 mm) in the administration building concourse in view of the cashier’s offices, Solomon Mahlangu House.\n\nIn the vicinity of the installed CCTV cameras at each site, a photography station was set up in a standard manner as demonstrated in Figure 1 and Figure 2. Within this set-up, facial photographs were captured in five different views using two different cameras with slightly different parameters and conditions. All cameras were arranged in a marked fixed location near the CCTV field of view on tripods at a fixed height of 1600 mm. This height was maintained to attempt centring the field of view of the photographs on the face, as the mean height for black South African males41 is 1710 mm. Specifically, the eye level of each participant was composed on the top horizontal rule of thirds line for all photographs taken. All photographs were taken in portrait orientation.\n\nST = standardized photograph; WT = wildtype photograph.\n\nArrangement of cameras and backdrop for standardised and wildtype photographs at site A (a), site B (b) and site C (c).\n\nAn example of the arrangements for database image capture and recording on pre-existing CCTV systems is shown in Figure 2. Distances were controlled for participants to be photographed and recorded at each site. Standardised (ST) photographs, with a solid black backdrop and participant clothing covered, were captured at a distance of 1500 mm. Wildtype (WT) photographs were captured at a 5000 mm distance. These photographs included a mixed background that was intentionally meant to simulate real-life photographic conditions. WT photographs were taken in a simulated scenario of suboptimal facial images with a comparable quality camera and facial poses. The background was purposefully not controlled for with a mixed environment visible and varied based on the location site of data collection. This background was intentionally out of focus to simulate suboptimal photographic conditions. Despite a minor level of variation, consistency was maintained across all photographs with regard to distance to subject, aperture, and composition.\n\nThe first set of photographs were captured under standardised conditions using a Canon 1300D 18MP DSLR camera (18–55 mm DC Canon lens) with the following settings: image sensor sensitivity (ISO) of 800, aperture F/9, shutter speed between 1/125 and 1/40, focal length of 55 mm and daylight white balance. For these standard photographs, the objective to face distance was fixed at 1500 mm. A set of standardised photographs were taken, with a black backdrop and with the participants’ clothing covered by a black velvet cloth – similar to the backdrop – in order to prevent matching participants based on clothing appearance. These standard photographs were captured in the following five views (Figure 3):\n\na. Anterior frontal view\n\nb. Right 45-degree view\n\nc. Right lateral view\n\nd. Left 45-degree view\n\ne. Left lateral view\n\nThe five views of facial photographs are demonstrated in this image, showing anterior (a), right 45-degree (b), right lateral (c), left 45-degree (d) and left lateral (e) views.\n\nThe second set of WT photographs captured for each participant, corresponded to the same views as described above, using a Sony SLT A57 (18 – 250 mm Sony Zoom Lens) with the following settings: image sensor sensitivity (ISO) of 200, aperture F/9, shutter speed between 1/125 and 1/40, focal length of 250 mm and daylight white balance. Indoor photographs at site C were captured at a range of ISOs between 400 and 1600 depending on the varying light conditions.\n\nAll photographs were captured as both native .jpeg and RAW format. Images were then downloaded from the SD cards of each camera, stored, and sorted on a desktop computer. All images were then batch cropped at a 4x5 (8x10) aspect ratio, using Adobe Photoshop CS6, to only include the participants as centred in the frame of the photograph. The resulting image resolution for the ST photographs was 3456 x 4320 pixels at 300 dpi and sRGB colour representation. The WT photographs’ resolution was 3264 x 4080 at 350 dpi and with sRGB colour representation. Following batch cropping, the standardised images were imported into Adobe Lightroom (v. 5.3) for basic editing. The only adjustments made included exposure level, highlights, and shadow correction. Additionally, removal/spot healing of any exposed clothing or background features were done if the cloth or backdrop did not fully obscure the participant and surroundings. The wildtype images were left unaltered post cropping. The above discussed image processing (batch cropping) and adjustments (exposure level, highlights, shadows correction and spot healing) can alternatively be performed using open source software such as GNU Image Manipulation Program (GIMP), Photivo, and darktable.\n\nCCTV recordings from internet protocol (IP) cameras were transferred live to a HikVision (model: DS-9664NI-I16) server for storage. Videos from the analogue camera were stored on a DS-ENC-V120B20121026-7054D2ABA6FB digital video recorder (DVR) device. All recordings were then extracted in .mp4 format from the University of the Witwatersrand’s CCTV systems through the university’s protection services software (iVMS 4200 v. 2.71.9). Only footage recorded during data collection times was extracted from the CCTV systems. During recording, participants were asked to stand at a marked location and rotate through the same five views that the photographs were captured in for approximately 5 seconds. Following this, the participant acted out the process of utilising a student card terminal in view of the camera (sites A and C) or attempting to exit the campus (site B).\n\nThe outdoor CCTV camera at site A was an IP camera (HikVision, model: DS-2CD2142FWD-I, 4-megapixel, 4 mm fixed lens, aperture F/2). This camera was installed at a height of 3100 mm and a floor distance of 2690 mm from the marked location where participants stood in the process of video capturing. The resulting distance between the participants’ face and the IP camera objective was 3030 mm (Figure 4). This distance was calculated based on the mean height of the average black South African male of 1710 mm41. The angle of incidence from the camera objective to the face was approximately 27°.\n\nVertical distance indicates the mean height of a South African male (1710 mm) and the oblique distance indicates the calculated approxiamte camera to face distance at Site A (3030 mm).\n\nThe outdoor “eye-level” IP CCTV camera at site B had the same model and specifications of the camera at site A and was installed at a height of 1700 mm from the ground, marked location to floor distance of 800 mm. The resulting distance from target face to camera was 800 mm and the angle of incidence was virtually zero.\n\nThe indoor analogue CCTV camera at site C (Securi-Prod 1/3” Sony Effio E 700TVL indoor dome, model: CC217, 2.8 – 12 mm vari-focal lens) was installed at a height of 2500 mm and at a floor distance from the marked area of 2810 mm. The resulting distance between camera and face was 2920 mm and the angle of incidence was approximately 22°.\n\nA total of approximately 30 seconds of footage was recorded for each participant and out of the 30 seconds five still images were captured from the footage at each of the five views previously described (Figure 5,Figure 6,Figure 7 and Figure 8). The photographs and recordings of each individual were coded with a participant number to maintain anonymity of the participants and a separate record of the identity of the participants was retained. Complete anonymity of the participants included in the facial image database was maintained. Images and videos included in the database were stored on three separate password protected desktop computers and encrypted on a constantly monitored external hard disk drive. In addition, the dataset was also transferred to the University of the Witwatersrand’s Library repository under restricted access to data management services. This repository retains the database in triplicate for cataloguing and in order to allocate searchable metadata to it to facilitate use.\n\nThe five views of facial photographs are demonstrated in this image, showing anterior (a), right 45-degree (b), right lateral (c), left 45-degree (d) and left lateral (e) views.\n\nThe five views of facial photographs are demonstrated in this image, showcasing anterior (a), right 45-degree (b), right lateral (c), left 45-degree (d) and left lateral (e) views.\n\nThe five views of facial photographs are demonstrated in this image, showing anterior (a), right 45-degree (b), right lateral (c), left 45-degree (d) and left lateral (e) views.\n\nThe five views of facial photographs are demonstrated in this image, showing anterior (a), right 45-degree (b), right lateral (c), left 45-degree (d) and left lateral (e) views.\n\n\nDatabase composition\n\nThe database was composed strictly of male South Africans of African descent. Participants were over the age of 18, for consent purposes, and in the young adult age-ranges, between 18 and 35 years of age. The participants were subdivided into a series of cohorts depending on the type of matched analysis possible, as outlined in Table 2. All participants were photographed in both the standardised and wildtype setting at the various sites. The first two groups were only photographed under natural outdoor lighting at site A (n=120) as well as with artificial indoor fluorescent lighting at site C (n=99). For each participant 10 photographs were captured, totalling 1200 photographs under outdoor conditions with natural lighting conditions and 990 photographs under indoor fluorescent lighting conditions. A second group, in addition to being photographed as above, was recorded on an outdoors setting CCTV camera at site A (n=98, n=86 with corresponding footage). A third group was recorded at site B with the eye-level camera (n=108, n=76 with corresponding footage). A fourth cohort was recorded at site C with the analogue CCTV camera and lastly a final group of participants were recorded with obstructive accessories, namely caps (n=45, n=34 with corresponding footage) or sunglasses (n=41, n=31 with corresponding footage), using the same IP camera at site A. Due to data corruption or data loss in the CCTV recording process, not all photographed individuals have corresponding footage associated to them. Overall, the database is inclusive of over 6200 facial photographs and 334 corresponding video recordings from various types of CCTV cameras within an African sample (Table 2).\n\nCCTV= closed-circuit television.\n\n\nDatabase utility and applications\n\nA database of this scale can be utilised in a variety of training and research applications, particularly when considering this as the first database of facial images with such a large complement of facial photographs of African individuals. Among a variety of possible applications, the primary intended use is testing various methodologies and conditions for forensic facial comparison. Furthermore, this database can be utilised as a prime tool for training facial comparison experts to develop a high rate of competency and proficiency. Having an appropriate level of training is a crucial aspect of the judicial process1,4. In addition, the faces in this database could be used to generate stimuli for studies in psychology and marketing sciences relating to facial recognition. Likewise, the images in this database could be modified as required on an ad hoc basis and implemented to train and develop future machine learning and artificial intelligence systems for the purpose of facial recognition in a forensic context. Although a potentially laborious process, standardised landmarks could be added to allow for an estimated dimension calculation of the various facial proportions and features as required by facial recognition systems. However, the database is only available for bona fide researchers affiliated with academic research institutions and not for commercial use. An example use the dataset was designed for was validating the use of morphological analysis in forensic facial comparison across a photographic and CCTV sample27. This was achieved by sub-setting selected photographs and CCTV recording stills from the database into facial image pools that were independently analysed27 following the Facial Identification Scientific Working Group morphological analysis feature list42.\n\n\nChallenges and limitations\n\nDeveloping a database of facial photographs and surveillance footage is a logistically complex and tedious process. It can be difficult to recruit volunteers even in highly trafficked areas in a major university (the University of the Witwatersrand has over 39,500 students across five campuses), although people on a campus tend to feel more secure and therefore more willing to participate in this type of study. The recruitment and photography process require a large amount of manpower with a minimum of three to four volunteers needed per day as assistants for an efficient image acquisition process. The location management and site selection are also a highly demanding task as one needs to limit variations between recorded images as much as possible. This entire process needs to be carried out while still collecting images that are representative of a somewhat realistic scenario. The variations in conditions of recordings vary based on camera type, quality, and installation. The majority of CCTV recordings, for example, were collected in an outdoors area with sunny daylight conditions which is by design an actualistic sample, although inconsistent due to weather and lighting conditions changing throughout a given day.\n\nSimilarly, the photography collection was affected by uncontrollable varying lighting conditions for both the indoor and outdoor settings. These include dim lighting indoors leading to lower quality, higher noise images, and dappled light and sun position outdoors leading to highlights, shadow, and contrast artefacts. The objective to face distances were controlled as much as possible, without compromising the actualistic nature of the data, in order to minimise perspective distortion due to the nature of relaying a three-dimensional scene/object into a two-dimensional medium of photography43. Despite the intent, at greater distances the focal plane of a camera lens requires adjusting and can result in lower clarity images43, as is evident in the varied focal lengths of the CCTV cameras.\n\nFurthermore, the nature of CCTV cameras depends on the entirety of the surveillance system installation. A variety of complications, including inconsistent IP camera network connectivity and power outages caused occasional complete data loss or footage corruption in the recording process, resulting in the overall reduced numbers of corresponding recordings. This data loss was particularly evident in the IP cameras, as in the particular set-up used by the university, they do not locally store footage but transfer it immediately to a central server. In the process, any interruption or fluctuation in local area network speeds or connectivity would result in data loss or corruption. Even though analogue cameras by default record at lower resolutions, the immediate local storage on a DVR device resulted in reduced data loss and no data corruption. In fact, a total of 710 participants were originally recruited and photographed and/or recorded. Following data loss, participants that requested to be excluded from the database and data corrupted during transfer from the CCTV cameras to the servers, only 622 of the 710 could be included in the database.\n\nMale individuals were selected specifically as males are more commonly involved in criminal activity, both as victims and perpetrators, in both developed and developing nations44,45. However, other demographic factors such as age and ancestry were not strictly controlled, and sample composition resulted varied across all cohorts. This lack of strict control was primarily due to the sensitivity of labelling groups of individuals as belonging to specific descent groups, making requesting this information from participants one of the ethical limitations considered originally.\n\nThe intention of the authors is for this database to be further expanded to include female individuals, additional disguises in the form of make-up and face masks, as well as more variations of CCTV camera recordings, such as infrared night vision CCTV, to provide a better and more varied selection for its applications.\n\n\nEthics of face databases\n\nOverall, face databases are quite common with 27 photographic image databases available for use in the fields of head and face detection, tracking and recognition. These databases are usually created with specific criteria, such as particular facial expressions and lighting condition variations for testing a specific aspect of facial recognition. Amongst the 27 databases outlined in Table 1, seven have been collected from various media of personal photographs available on the internet7,9,11,12,15,24,37, which is a legal yet perhaps ethically questionable practice, as the individuals included in these face databases have not provided consent for that inclusion. This is particularly common in the most recently developed databases (e.g. 7,8,15), most likely due to the broad availability of facial images and the highly efficient and accurate search engines. This practice can result in privacy-intrusive practices of freely available and commercialised application of facial recognition28, which is an ever-increasing concern for the public with specific regard to privacy of data and images46. The database compiled here is an example of a complex yet more ethical approach that limits commercialised application and potential research.\n\n\nData availability\n\nThe WFD is stored on the Wits Institutional Repository environment on DSpace (WIReDSpace) and published under the following unique identifier: http://doi.org/10.17605/OSF.IO/WMA4C (this registration also contains the PhD study protocol that led to the development of the WFD and an addendum to the protocol registration highlighting the major changes to the methodological approach of the original protocol). A sample of the dataset is freely accessible at https://hdl.handle.net/10539/29924.\n\nThe database is an open access resource for use in strictly non-commercial research. In order to access the WFD, prospective users will have to apply for access to the Institutional Review Board overseeing ethical and scientific use of the database in order to safeguard the privacy and decency of the database’s participants. Once approved a researcher may use the database free of charge. Database access is restricted and limited to following the above-mentioned procedure, due to the nature of the data including potentially identifying information (facial physiognomy) of participants. In addition, strict limitations were imposed by the Human Research Ethics Committee (Medical) as well as the consent permissions agreed upon with the participants, which assign responsibility to the School of Anatomical Sciences to review access applications in ethical and scientific merit in order to exclusively conduct research. The access procedures and limitations are governed by a legally binding Conditions of Use document available on https://hdl.handle.net/10539/2992440 in conjunction to the freely accessible sample. Data will be made available to successful applicants under a temporary restricted licence guided by the aforementioned conditions of use document.\n\nOpen Science Framework: Wits Face Database: Description. https://doi.org/10.17605/OSF.IO/Q8V2R40\n\nThis project contains the following extended data:\n\n- Participant information sheet\n\n- Participant consent form\n\n- Conditions of use\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Authors' contributions\n\nConceptualization: Nicholas Bacci, Maryna Steyn, Nanette Briers; Data curation: Nicholas Bacci, Joshua Davimes; Methodology: Nicholas Bacci, Joshua Davimes, Maryna Steyn, Nanette Briers; Investigation: Nicholas Bacci, Joshua Davimes; Project Administration: Nicholas Bacci, Joshua Davimes; Resources: Nicholas Bacci, Joshua Davimes; Visualisation: Nicholas Bacci, Joshua Davimes; Writing - original draft preparation: Nicholas Bacci; Writing - review and editing: Nicholas Bacci, Joshua Davimes, Maryna Steyn, Nanette Briers; Funding acquisition: Nicholas Bacci, Maryna Steyn, Nanette Briers; Supervision: Maryna Steyn and Nanette Briers.\n\n\nAcknowledgements\n\nThanks are due to all the participants who agreed to be photographed for the development of this database. Particular recognition is due to all the volunteers who assisted in participant recruitment for this study: Jesse Fredericks, Kiveshen Pillay, Rethabile Masiu, Sameerah Sallie, Daniel Munesamy, Laurette Joubert, Jordan Swiegers, Betty Mkabela, Johannes P. Meyer, Amy Spies, Natasha Loubser, Nicole Virgili, Dan-Joel Lukumbi, Tamara Lottering, Mathabatha Ntjie, Claudia Landsman, Raheema Dalika, Merete Goosen, Stephanie Souris, Rabelani Negota, Mahlatse Mahasha, Jessica Manavhela. Special thanks are due to Gideon LeRoux for his assistance in the capturing and extracting of the CCTV recordings from the University security systems. A great deal of thanks are also due to Nina Lewin who aided in policy development and establishing the database repository. All named persons above have granted permission to be included within the acknowledgements.\n\n\nReferences\n\nFacial Identification Scientific Working Group: Facial Comparison Overview and Methodology Guidelines. 2019. 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Anthropol Rev. 2018; 81(2): 202–23. Publisher Full Text\n\nLe V, Brandt J, Lin Z, et al.: Interactive Facial Feature Localization. In: European Conference on Computer Vision. 2012; 679–92. Publisher Full Text\n\nJesorsky O, Kirchberg KJ, Frischholz RW: Robust face detection using the Hausdorff distance. In: International Conference on Audio- and Video-based Biometric Person Authentication. 2001; 90–5. Publisher Full Text\n\nMarszalec E, Martinkauppi B, Soriano M, et al.: Physics-based face database for color research. J Electron Imaging. 2000; 9(1): 32–28. Publisher Full Text\n\nLewin NS, Bacci N, Davimes J, et al.: Wits Face Database: Description. 2021. http://www.doi.org/10.17605/OSF.IO/Q8V2R\n\nSteyn M, Smith JR: Interpretation of ante-mortem stature estimates in South Africans. Forensic Sci Int. 2007; 171(2–3): 97–102. PubMed Abstract \| Publisher Full Text\n\nFacial Identification Scientific Working Group: Facial Image Comparison Feature List for Morphological Analysis. 2018. 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Publisher Full Text" }	[ { "id": "79950", "date": "05 Mar 2021", "name": "Ching-Yiu Jessica Liu", "expertise": [ "Reviewer Expertise Facial Anthropology" ], "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 described the development of the 'Wits Face Database'. The authors collected CCTV footage from three different environments, along with standardised and uncontrolled facial photographs detailed in Table 2. A total of 622 adult male individuals with facial anthropological features resembling South African descent were recruited.\nIt will be beneficial to document the framerates of the CCTV recordings.\n\nThe mean height for black South African males was used for the photography station for ST images. Did you have to make any adjustments for different heights or were all participants within the capture range?\n\"individuals with facial anthropological features resembling South African descent were recruited.\" I am not sure I fully understand what this includes and excludes. You mentioned that age and ancestry were not strictly controlled along with the sensitivity of labelling groups. Does this mean anyone who was an adult male was recruited?\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": [ { "c_id": "6460", "date": "18 Mar 2021", "name": "Nicholas Bacci", "role": "Author Response", "response": "Dear Dr Liu, Thank you for your prompt and positive review. We appreciate your insights for the missing information and points requiring clarification. We will be including the following information in a new version of the manuscript to provide the information to all readers: Comment: It will be beneficial to document the framerates of the CCTV recordings. Response: The framerates and codec information for the IP camera CCTV recordings will be included. However, the analogue camera did not store framerate or codec information in its metadata or the manufacturer website. Amendment: The following statements will be included in the revised manuscript version for clarity: “The recordings from these two cameras were encoded in MPEG-H Part2/HEVC (H.265) codec and had a frame rate of 20 frames per second.”. “No codec and framerate information was available for this camera both on the manufacturer website and the recording metadata.” Comment: The mean height for black South African males was used for the photography station for ST images. Did you have to make any adjustments for different heights or were all participants within the capture range? Response: The height of the tripod for the ST and WT photographs was not adjusted, it was maintained fixed at the average height of black South African males throughout the data collection process. Only a handful of participants were significantly above or below the mean height in order to affect the composition of the photographs. In those cases the angle of the camera was adjusted slightly in the vertical axis to retain the same composition. Amendment: The following clarification will be included in the manuscript: “In all cases, for both ST and WT photographs, the tripod height was not adjusted and left at the fixed height of 1600mm. The majority of participants were within the capture range; however, for a few of the individuals who were either vastly below or above average South African male height, the camera was tilted in the vertical axis of the tripod in order to align their eyes along the uppermost 1/3rd of the portrait composition. No other changes were made in terms of focal length or post crop editing.” Comment: \"individuals with facial anthropological features resembling South African descent were recruited.\" I am not sure I fully understand what this includes and excludes. You mentioned that age and ancestry were not strictly controlled along with the sensitivity of labelling groups. Does this mean anyone who was an adult male was recruited? Response: Clarification on the participants included in the study will be included. Male South Africans of facial appearance resembling African descent were specifically recruited for the database. Amendment: The statement speaking to participant descent will be amended for clarity as follows: “Potential participants were identified and recruited near the data collection sites based on facial anthropological features resembling South African males of African descent and being older than 18 years of age.”. Best Regards, Nicholas Bacci" } ] }, { "id": "80712", "date": "22 Mar 2021", "name": "Won-Joon Lee", "expertise": [ "Reviewer Expertise Craniofacial Identification" ], "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 stated the procedure and results of developing a facial image database from South African males. Also, the authors discussed the applicability and limitations of their research.\nThis study is regarded to produce the best results from given conditions which they stated objectively in the section of challenges and limitations. It is considered that the database will benefit the research and researchers into facial recognition using CCTV images, especially South African males which available database has not been enough in this field.\n\nConsequently, I would like to recommend this manuscript to be indexed.\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/10-131
https://f1000research.com/articles/9-1443/v1	10 Dec 20	{ "type": "Brief Report", "title": "A theoretical model for the prevention of Banana Moko (Musa AAB Simmonds)", "authors": [ "Marly Grajales-Amorocho", "Anibal Muñoz Loaiza", "Anibal Muñoz Loaiza" ], "abstract": "A population simulation model with non-linear ordinary differential equations is presented, which interprets the dynamics of the banana Moko, with prevention of the disease and population of susceptible and infected plants over time. A crop with a variable population of plants and a logistic growth of replanting is assumed, taking into account the maximum capacity of plants in the delimited study area.", "keywords": [ "Mathematical model", "Moko", "Banana", "Ralstonia solanacearum", "Logistic growth" ], "content": "Introduction\n\nThe banana is a fruit of great economic importance and food sovereignty, because it is found in the shopping basket of people across different social strata and because of its nutritional content. However, its production is threatened by re-emerging diseases such as Moko, caused by the bacterium Ralstonia solanacearum race 2 philotype II (Fegan & Prior, 2006), which causes wilting and deterioration of the plant. Symptoms are usually visible when there is a great spread of bacteria and adjacent plants may have already been infected (Viljoen et al., 2016). Moko is a peculiar manifestation of bacterial wilt; it is a quarantine pest that, once inside the host, moves through the vascular bundles. Being a vascular disease, this bacterium not only affects the vegetative part but also the daughter hill, promoting the spread of the disease, which is accelerated by the high optimal minimum, optimal and maximum temperatures of 10°C, 35°C and 41°C, respectively, infecting triploid plantains, heliconia (Heliconia spp.) and other ornamental Musacea plants (Jiang et al., 2017).\n\nThe development of mathematical models has contributed through the use of a wide range of techniques to the study of epidemics and diseases, helping to answer biological questions and raising new questions related to the epidemiology and ecology of pathogens and the diseases they cause; in most cases, mathematical models lead directly to applications in the control of the disease (Jeger et al., 2018). Some prediction models that calculate the propagation threshold R0 have evaluated the control of some diseases in plantain and bananas, such as banana wilt by Xanthomonas (BXW) using mathematical models, describing a deterministic SI-type epidemic model for control of BXW focusing on the integrated management of the disease through cultural control as in Nannyonga et al. (2015), who considered the optimal control strategies associated with the prevention of transmission by the use of contaminated tools. The researchers assumed a model with three modes of transmission: vertical (from the mother plant to its child), horizontal (indirect) from the vector to plant, and through contaminated agricultural tools (Nannyonga et al., 2015).\n\nLikewise, Nakakawa et al. (2016) presented a mathematical model for BXW propagated by an insect vector. The mathematical model they formulated takes into account inflorescence infection and vertical transmission from the mother corm to the daughter hills, but not tool-based transmission by humans (Nakakawa et al., 2016). In this context, a dynamic system is formulated based on ordinary two-dimensional differential equations that interprets the dynamics of incidence of banana Moko disease, including prevention and treatment.\n\n\nThe model\n\nA population model with nonlinear ordinary differential equations is presented, which interprets the dynamics of the banana Moko, including a constant rate of disease prevention in the population of susceptible plants over time. A variable population of plants and a logistic growth of replanting are assumed, taking into account the maximum capacity of plants in the study region. The variables and parameters of the model are: x(t), the average number of susceptible banana plants; y(t), the average number of diseased banana plants; and P(t) = x(t) + y(t), total number of banana plants at one time t, shown in Figure 1.\n\nThe model parameters are: γ, constant overseeding rate; k, load capacity (maximum capacity) of banana plants in the study region; and β, probability of transmission of infection. Preventive controls are: g, fraction of infected banana plants removed; and f, fraction of susceptible banana plants that receive prevention of contagion of the bacteria. The dynamic system that interprets the infectious process including prevention and elimination, is formed by the following two nonlinear differential equations:\n\n\n\n\n\nWith initial conditions x(0) = x0, y(0) = y0, P(0) = x(0) + y(0), γ, k > 0, 0 < f, g, β < 1, P ≤ k, x(t) ≡ x, and y(t) ≡ y.\n\nThe region of eco-epidemiological sense is defined where the trajectories of the plant infection dynamics make sense,\n\n\n\n\nStability and sensitivity analysis\n\nWe start by finding the equilibrium populations, the constant solutions of the system, where the population variation of susceptible plants and variation of infected plants become zero, that is, dxdt=0;dydt=0\n\n\n\n\n\nWe solve this non-linear algebraic system for x and y, determining the equilibrium point with agronomic sense, free of infected plants E1 = (k, 0). A breakeven point with disease invasion without susceptible plants E2 = (0, k), and a balance point with susceptible plants and infected plants E3=(x^,y^), with\n\n\n\nConsidering,\n\n\n\nWe write x and y like this\n\n\n\nIn coexistence of populations x^>0 and y^>0, which is true when ξ0 > 1 and ρ < 1.\n\nSince P = x + y, the total plant population in equilibrium is, P^=x^+y^. That is,\n\n\n\nTherefore, P^=k(ρ−1).\n\nξ0, indicates the average number of infected plants that an infected plant produces during the infectious period (before being killed) in the population of susceptible plants and is considered the threshold of infected plants. We can consider this threshold as a function that depends on f and g,\n\n\n\nTo determine the stability of each equilibrium point (E), we apply the Hartman-Grobman theorem (Perko, 2011), linearizing the system of non-linear Equation (1) – Equation (2), obtaining the linearization matrix (Jacobian matrix) of the form:\n\n\n\nWith the following partial derivative elements,\n\n\n\n\n\n\n\n\n\nThese elements of the matrix J (E) are the coefficients of the linear system\n\n\n\nWhere, U = (u, v)t (transposed vector).\n\nWe analyze the balance points with an agronomic sense E1 = (k, 0) y E3 = (x^, y^). For E1, we obtain the Jacobian matrix,\n\nBecause it is a triangular matrix, the eigenvalues (λi, i = 1,2)\n\n\n\nwhere λ2 < 0 since the threshold ξ0 < 1.\n\nWe conclude that the free equilibrium point of Moko disease is locally and asymptomatically stable.\n\nFor case E3=(x^,y^), in matrix (10) we obtain the trace and the determinant of J(E3), respectively,\n\n\n\nWe conclude that the equilibrium point with susceptible plants and infected plants is locally and asymptomatically stable if the threshold inequalities (7) and the inequalities are met,\n\n\n\n\n\nThese analytical results are shown in the phase planes of Figure 2, made with Maple 18 software (free trial available; SageMath is an openly available alternative), for different scenarios varying initial conditions.\n\n\nResults and conclusions\n\nLocal sensitivity is a measure of the relative change in a variable when its parameters change (Chitnis et al., 2008; Hamby, 1994; Rodrigues et al., 2013). That is,\n\n\n\nWhere,\n\n\n\ny p: β, g, f, are previously defined parameters.\n\nThe indices of local sensitivity of the epidemic threshold with respect to each parameter are Iξ0β = 1, Iξ0f = -0,43 y Iξ0g = -1. These values indicate that the parameter that most influences the threshold value is β proportionally and g inversely proportional.\n\nIt is concluded that mathematical simulation models are a useful tool for research in banana Moko disease. With them it was determined that the elimination of banana plants infected with the disease plays an essential role in the good agronomic management of the crop.\n\nAs a research perspective, consider the problem of a simulation model including a piecewise function for the rate of elimination of infected plants, in the form\n\n\n\nWhere T is the time it takes for agricultural institutions to confirm the presence of plantain Moko and suggest the elimination of infected plants.\n\nThe analysis of the following optimal control problem is also proposed as a research perspective, applying the principle of the Pontryaguin maximum (Kopp, 1962).\n\nThe functional objective of direct and indirect costs is proposed:\n\n\n\nLinked to the system of differential equations:\n\n\n\n\n\nWith initial conditions ps(0) = and ps0, pi(0) = pi0, P(0) = ps0 + pi0, γ, k > 0, 0 ≤ u1, u2, β ≤ 1, P ≤ k, ni > 0, i =1,2,3, Ps≡x and Pi≡y.\n\nIt is about finding optimal control (u¯1(t),u¯2(t)) such that:\n\n\n\nWhere,\n\n\n\nis the space of admissible controls and L2 is the space of integrable functions.\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\nChitnis N, Hyman JM, Cushing JM: Determining important parameters in the spread of malaria through the sensitivity analysis of a mathematical model. Bull Math Biol. 2008; 70(5): 1272–1296. PubMed Abstract \| Publisher Full Text\n\nFegan M, Prior P: Diverse members of the Ralstonia solanacearum species complex cause bacterial wilts of banana. Australas Plant Path. 2006; 35: 93–101. Publisher Full Text\n\nHamby DM: A review of techniques for parameter sensitivity analysis of environmental models. Environ Monit Assess. 1994; 32(2): 135–154. PubMed Abstract \| Publisher Full Text\n\nJeger MJ, Madden LV, Van Den Bosch F: Plant virus epidemiology: Applications and prospects for mathematical modeling and analysis to improve understanding and disease control. Plant Dis. 2018; 102(5): 837–854. PubMed Abstract \| Publisher Full Text\n\nJiang G, Wei Z, Xu J, et al.: Bacterial wilt in China: History, current status, and future perspectives. Front Plant Sci. 2017; 8: 1549. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKopp RE: Pontryagin Maximum Principle. Mathematics in Science and Engineering. 1962; 5: 255–279. Publisher Full Text\n\nPerko L: Differential Equations and Dynamical Systems. Third edition. Springer Science and Business Media LLC. 2011. Reference Source\n\nNakakawa J, Mugisha JYT, Shaw MW, et al.: A mathematical model for the dynamics of banana Xanthomonas wilt with vertical transmission and inflorescence infection. J Biol Syst. 2016; 24(1): 147–165. Publisher Full Text\n\nNannyonga B, Luboobi LS, Tushemerirwe P, et al.: Using contaminated tools fuels outbreaks of Banana Xanthomonas wilt: An optimal control study within plantations using Runge-Kutta fourth-order algorithms. Int J Biomath. 2015; 8(5). Publisher Full Text\n\nRodrigues HS, Monteiro MTT, Torres DFM: Sensitivity Analysis in a Dengue Epidemiological Model. Hindawi Publ Corp. Conference Papers in Science. 2013. Reference Source\n\nViljoen A, Mahuku G, Massawe C, et al.: Banana Pests and Diseases Field Guide for Disease Diagnostics and Data Collection: Improvement of banana for smallholder farmers in the Great Lakes Region of Africa. 2016. Reference Source" }	[ { "id": "76029", "date": "23 Dec 2020", "name": "Dalia M. Muñoz", "expertise": [ "Reviewer Expertise Public health", "environment", "sustainability", "environmental management", "econometrics" ], "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 simulation model to treat banana Moko dynamics, which constitutes an essential topic in food safety. The model seems to be correctly implemented. However, the manuscript lacks some clarity, and more discussion on the results is strongly encouraged. The manuscript needs corrections before any decision. A point by point list with the concerns raised after my revision is listed in the next lines.\nThe abstract just mentioned how they performed the model and some other conditions like replanting. The abstract needs to be strengthened to discuss the main results and the implications of treating banana plants with some prevention measures.\n\nThe introduction needs to be strengthened to depict the state of the art on the banana Moko study. There exist other simulation models based on differential equations to treat the banana Moko? Why did you choose such a model?\nAddressing these aspects contributes to the authors pointing out the scarce literature in this regard and their study's relevance.\n\nThe following paragraph lacks clarity. Please rewrite it.\nSome prediction models that calculate the propagation threshold R0 have evaluated the control of some diseases in plantain and bananas, such as banana wilt by Xanthomonas (BXW) using mathematical models, describing a deterministic SI-type epidemic model for control of BXW focusing on the integrated management of the disease through cultural control as in Nannyonga et al. (2015), who considered the optimal control strategies associated with the prevention of transmission by the use of contaminated tools. The researchers assumed a model with three modes of transmission: vertical (from the mother plant to its child), horizontal (indirect) from the vector to plant, and through contaminated agricultural tools (Nannyonga et al., 2015).\n\nThe following sentences should be included in the section Model\nLocal sensitivity is a measure of the relative change in a variable when its parameters change (Chitnis et al., 2008; Hamby, 1994; Rodrigues et al., 2013). That is….\n\nWhat do authors mean when they talk about agronomic sense?\n\nThe results need to be expanded, including a better description. In the case of figure two, the authors do not present any description.\n\nConclusions generally present the main findings. Please rearrange it to meet the high-quality standards of the journal.\n\nThe presentation of the research perspective seems messy and needs to be improved. The authors present a research perspective, but it is not considered in the paper's main body. The author should consider it as part of the analysis. The optimal control problem should be in the Section Model, describe the development in the results and the implications or expected results with this analysis in conclusion.\n\nThe list of references requires an update to include recent studies.\n\nAuthors are encouraged to look for other models about diseases in banana crops and discuss their results to see advantages and differences.\n\nOverall, the paper looks interesting, but it needs a major revision before any consideration.\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? 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? Partly", "responses": [] }, { "id": "76032", "date": "04 Jan 2021", "name": "Ana Maria Pulecio Montoya", "expertise": [ "Reviewer Expertise Mathematical biology", "applied Mathematics" ], "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 deals with analytical and numerical study of a model of Ordinary Differential Equations to treat banana Moko dynamics. I submit the following suggestions in order to improve this article:\nThe abstract needs to explain the main results of the article.\n\nThe authors should explain better the description in the differential equations of the model.\n\nThe authors should explain better how they obtained the equilibrium points and why the equilibrium point E2 is not of interest in the analysis presented.\n\nSensitivity analysis should be written as a section and the authors should explain better the results.\n\nThe conclusions should highlight the main results of the model analysis without giving new results or repeating what has already been said.\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? 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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1443
https://f1000research.com/articles/9-1373/v1	26 Nov 20	{ "type": "Software Tool Article", "title": "Effective image visualization for publications – a workflow using open access tools and concepts", "authors": [ "Christopher Schmied", "Helena Klara Jambor" ], "abstract": "Today, 25% of figures in biomedical publications contain images of various types, e.g. photos, light or electron microscopy images, x-rays, or even sketches or drawings. Despite being widely used, published images may be ineffective or illegible since details are not visible, information is missing or they have been inappropriately processed. The vast majority of such imperfect images can be attributed to the lack of experience of the authors as undergraduate and graduate curricula lack courses on image acquisition, ethical processing, and visualization. Here we present a step-by-step image processing workflow for effective and ethical image presentation. The workflow is aimed to allow novice users with little or no prior experience in image processing to implement the essential steps towards publishing images. The workflow is based on the open source software Fiji, but its principles can be applied with other software packages. All image processing steps discussed here, and complementary suggestions for image presentation, are shown in an accessible “cheat sheet”-style format, enabling wide distribution, use, and adoption to more specific needs.", "keywords": [ "Image Publication", "FIJI", "Good principles of figure design", "Beginner's workflow", "Image processing", "open source", "Visualization", "Image analysis" ], "content": "Introduction\n\nEvery day, around 2000 biomedical articles appear, 500 of which contain images. These published images provide new insights, but each day also the number of problematic images increases. While intentionally manipulated images are rare1,2, erroneous handling of images is more common. Problematic is also that methods often insufficiently report on image acquisition and processing3. Last, images frequently have low legibility, as only 10–20% of published images provide all key information (annotation of color/inset/scale/specimen)4. In the long run, problematic images may undermine the trust in scientific data and, when published in emerging image archives, reduce the value of such repositories5,6.\n\nToday’s scientists face rapidly evolving technologies and employ many methodologies, with microscopy and image analysis7 just one among many. Problematic images thus partially arise from: 1) lack in training, as ethical and legible processing of microscopy data is not systematically taught3, 2) lack in local expertise, as image facilities are restricted to a few research hubs, and 3) while publishers established guidelines for handling image forgeries8–10, actionable and clear instructions for legible image publishing are lacking.\n\nHere, we introduce an image processing workflow to effectively and ethically present images. The step-by-step workflow enables novice users, with no image processing experience and occasional microscopists, with no intention towards specializing in image processing to take the first steps towards publishing truthful and legible images.\n\n\nMethods\n\nObtaining high quality bioimages starts with optimal microscope settings and must be adapted to subsequent quantitative or qualitative analyses11–16. After acquisition, bioimages can be processed and prepared for publication using the workflow below (Figure 1), which is visually summarized in cheat-sheet style (Figure 3 and Figure 4). Both are based on Fiji17, an open source, free image analysis program for bioimages.\n\nLoad images into Fiji and make sure metadata (see Table 1: Glossary), such as the scale, are correct. Save images with a new name to keep raw images untouched. After processing, save images in TIFF format, which preserves the entire information and enables measurements. For presentation, save images in PNG format, which irreversibly merges the image with annotations and saves multichannels as 24-bit RGB. Beware of incorrect or unintentional bit-depth conversions18.\n\nAfter opening, images with a large gray value range may appear black11. To properly display such data19, adjust the brightness contrast by pressing the auto button or using the sliders. When performing comparisons between images, we recommend using the same fixed values using the set button. This linear intensity adjustment is acceptable if key features are not obscured. Pressing apply/saving images as PNG changes the intensity range irreversibly and makes images unsuitable for intensity measurements. Non-linear adjustments i.e. histogram equalizations or gamma correction need to be explained20,21.\n\nOften further processing is necessary. Be familiar with these methods to decide if subsequent image intensity quantification is still truthful. A maximum intensity projection is acceptable for visualization of a 3D stack, but intensity measurements should use ‘sum’ or ‘average’ projections. Similarly, noise is problematic for visualizations and is reduced with linear filters such as a Gaussian blur. Clearly state the image processing methods12,20.\n\nImage rotation sometimes helps for better comparisons, to reduce unnecessary information, or for aligning specimens. Rotation, but also decreasing or increasing the size of images in pixels, may degrade the image quality by interpolation. Such loss of information may be acceptable for visualization, but quantification and measurements must be done beforehand20,21.\n\nOften larger fields-of-view are captured than are required. Cropping is then not only permissible, it is necessary to focus the reader on the relevant result. In contrast, it is not ethical to crop out data that would change the interpretation of the experiment, or to “cherry-pick” data20,21. When a larger field of view and a magnification of detail (‘inset’) need to be shown side-by-side, indicate inset position in the original image.\n\nScientific cameras capture each wavelength (channel) with grayscale images. If one channel is shown, grayscale, which has the best contrast with black background, is favorable. To visualize several channels of a specimen (e.g. colocalization studies), encode channels with different colors. A look-up table (LUT) determines how gray values are translated into a color value. For color selection, always consider visibility for color-blind, and traditions of scientific fields. Apply color-schemes consistently.\n\nImages represent physical dimensions and can depict different scales ranging from nanometer to millimeter, which is often not obvious22 thus providing scale information is essential. Further, annotate what each color and symbol represents in an image.\n\nWe tested the workflow on fluorescently-stained microscope images of Drosophila egg chambers (RRID:BDSC_5905;23) and the HeLa (RRID:CVCL_0030) ImageJ sample image24. For generating a “poor” image example, we processed the raw microscope images minimally, only converting the bit depth from 16-bit to 8-bit and retained default color schemes. We did not add annotations, performed no image cropping, rotation, or specific brightness contrast adjustments as these often lack in poorly visualized images4. We thus simulated images as they are typically “processed” in the majority of current publications4. To perform a qualitative assessment, we tested image visibility to color blind (deuteranopia) audiences using the color blindness simulator (RRID: SCR_018400;25).\n\n\nResults\n\nUsing our example microscope images, we qualitatively compared the readability of images processed with or without the workflow described. Images for which the steps of the workflow were implemented contained the key information, were cropped to maximize focus, and sufficiently annotated (color channels, scale, organism), while images processed minimally without following the workflow have a “poor” readability (Figure 2A, B). Furthermore, we demonstrated that images processed according to our workflow (‘color’) are still accessible to color blind readers (Figure 2C).\n\nA. Schematic of typical errors in published bioimages and improved version of exemplary image without compression artifacts, and with accessible color-code, annotation, and scale. B. Example images poorly visualized and after processing with the workflow presented here. C. Color blind (deuteranopia) rendering of the images shown in B. Poorly visualized images are inaccessible to color blind readers.C. Color blind (deuteranopia) rendering of the images shown in B. Poorly visualized images are inaccessible to color blind readers.\n\nOur workflow is based on the open source software Fiji, but its principles are applicable to other software. The workflow steps and accompanying suggestions for image presentation are available as accessible “cheat sheets” (Figure 3 and Figure 4) for wide distribution and adoption to more specific needs.\n\nAfter completing the workflow, images may be assembled for publication and legends added26. Layouting images on a page can be done with design software or in Fiji plugins27,28. Consider the final dimensions and orientation (landscape/portrait) and save figures for print with 300 dots per inch (DPI). This workflow is iterative and feedback from colleagues helps to identify possible hurdles.\n\n\nConclusion\n\nIf followed, the workflow helps avoiding common problems of published 2D images, but principles are also applicable to 3D stacks and movies. Indeed, lack of truthful scientific communication and reproducibility are among the biggest problems faced by science today29 and considering that an estimated 500 publications with images are published daily, improving image quality could have a profound impact in tackling this issue.\n\n\nData availability\n\nHeLa cell test images are available at: https://imagej.nih.gov/ij/images/hela-cells.zip. D.melanogaster egg chamber cells images are available on Open Science Framework.\n\nOpen Science Framework: Effective image visualization for publications – a workflow using open access tools and concepts. https://doi.org/10.17605/OSF.IO/SDPZK30.\n\nOpen Science Framework: Effective image visualization for publications – a workflow using open access tools and concepts. https://doi.org/10.17605/OSF.IO/SDPZK30.\n\nThis project contains the following extended data:\n\n- Processing_images_cheatsheet_SchmiedJambor.png (printable image of cheat sheet 1)\n\n- SchmiedJambor_Figures3_Cheatsheet1.eps (modifiable version of cheat sheet 1)\n\n- Publishing_ images_cheatsheet_SchmiedJambor.png (printable image of cheat sheet 2)\n\n- SchmiedJambor_Figures4_Cheatsheet2.eps (modifiable version of cheat sheet 2)\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 thank Robert Haase, Hella Hartmann, Florian Jug, Anna Klemm, and Pavel Tomancak for constructive comments on manuscript and cheat sheets. Font awesome icons were used in preparing the figures.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nBik EM, Casadevall A, Fang FC: The Prevalence of Inappropriate Image Duplication in Biomedical Research Publications. mBio. 2016; 7(3); e00809–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossner M, Yamada KM: What's in a picture? The temptation of image manipulation. J Cell Biol. 2004; 166(1): 11–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarques G, Pengo T, Sanders MA: Imaging methods are vastly underreported in biomedical research. eLife. 2020; 9: e55133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJambor H, Antonietti A, Alicea B, et al.: Creating Clear and Informative Image-based Figures for Scientific Publications. bioRxiv. 2020. Publisher Full Text\n\nEllenberg J, Swedlow JR, Barlow M, et al.: A call for public archives for biological image data. Nat Methods. 2018; 15(11): 849–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams E, Moore J, Li SW, et al.: The Image Data Resource: A Bioimage Data Integration and Publication Platform. Nat Methods. 2017; 14(8): 775–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEliceiri KW, Berthold MR, Goldberg IG, et al.: Biological imaging software tools. Nat Methods. 2012; 9(7): 697–710. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnhancing the quality and transparency of reporting. Nat Cell Biol. 2017; 19(7): 741. PubMed Abstract \| Publisher Full Text\n\nCSE’s White Paper on Promoting Integrity in Scientific Journal Publications [Internet]. Wheat Ridge, CO; 2012 [cited 2020-10-23]. Reference Source\n\nTeare MD: Transparent reporting of research results in eLife. eLife. 2016; 5: e21070. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown CM: Fluorescence microscopy--avoiding the pitfalls. J Cell Sci. 2007; 120(Pt 10): 1703–5. PubMed Abstract \| Publisher Full Text\n\nJonkman J, Brown CM, Wright GD, et al.: Tutorial: guidance for quantitative confocal microscopy. Nat Protoc. 2020; 15(5): 1585–611. PubMed Abstract \| Publisher Full Text\n\nNorth AJ: Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. J Cell Biol. 2006; 172(1): 9–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPawley J: The 39 steps: a cautionary tale of quantitative 3-D fluorescence microscopy. Biotechniques. 2000; 28(5): 884–6, 8. PubMed Abstract \| Publisher Full Text\n\nPawley J: Points, Pixels, and Gray Levels: Digitizing Image Data. Pawley J, editor. Boston, MA: Springer; 2006; 59–79. Publisher Full Text\n\nWaters JC: Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol. 2009; 185(7): 1135–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBankhead P: Analyzing fluorescence microscopy images with ImageJ: Types & bit-depths. 2016 [2020-09-30]. Reference Source\n\nRuss J: Seeing the Scientific Image. John C. Russ. Proc Roy Microsc Soc. 2004; 39/2:97-114; 39/3:179-193; 39/4: 267-281. Reference Source\n\nCromey DW: Avoiding twisted pixels: ethical guidelines for the appropriate use and manipulation of scientific digital images. Sci Eng Ethics. 2010; 16(4): 639–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCromey DW: Digital images are data: and should be treated as such. Methods Mol Biol. 2013; 931: 1–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZoppe M: Towards a perceptive understanding of size in cellular biology. Nat Methods. 2017; 14(7): 662–5. PubMed Abstract \| Publisher Full Text\n\nJambor H, Surendranath V, Kalinka AT, et al.: Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. eLife. 2015; 4: e05003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernie J: NVK. Color Oracle Version 1.3. 2018 [2020-10-20]. Reference Source\n\nYu H, Agarwal S, Johnston M, et al.: Are figure legends sufficient? Evaluating the contribution of associated text to biomedical figure comprehension. J Biomed Discov Collab. 2009; 4: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAigouy B, Mirouse V: ScientiFig: a tool to build publication-ready scientific figures. Nat Methods. 2013; 10(11): 1048. PubMed Abstract \| Publisher Full Text\n\nMutterer J, Zinck E: Quick-and-clean article figures with FigureJ. J Microsc. 2013; 252(1): 89–91. PubMed Abstract \| Publisher Full Text\n\nBelluz JBP, Resnick B: The 7 biggest problems facing science, according to 270 scientists: Vox Media in 2014. 2017 [2020-09-30]. Reference Source\n\nJambor H: Effective image visualization for publications – a workflow using open access tools and concepts. 2020. http://www.doi.org/10.17605/OSF.IO/SDPZK" }	[ { "id": "75494", "date": "09 Dec 2020", "name": "Guillaume Jacquemet", "expertise": [ "Reviewer Expertise Cell biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Christopher Schmied and Helena Klara Jambor provide an excellent overview of the steps and concept that should be taken in consideration when preparing figures containing microscopy images. The article is of very high quality and will be very useful for students and seasoned microscopists alike. I will certainly make it a mandatory read for my lab members.\nBelow are several comments that came to mind while reading the manuscript that the authors may want (or not) to consider. Several of them are perhaps out of the scope of this work but I will write them down in any case for the authors' consideration.\nWhile the manuscript is intended to educate and improve image display in figures, it may be useful to add a disclaimer that images are illustrations and should be associated with a quantification of the phenomenon displayed.\n\nIn the manuscript, the authors discuss changing the brightness contrast to improve the images visually. But other manipulations such as changing the gamma or the colour balance should probably also be mentioned. While the appropriateness of these manipulations is often debated for scientific images, it would be useful to educate the readers about their existence and their pitfall. Especially since the correct gamma to use depends on the display used to visualise the images.\n\nOn a similar note, it may be useful to inform that performing \"background clipping\" may also not be an appropriate image manipulation.\n\nOn a similar note, it may be useful to add a warning message in the LUT section indicating that not all LUT display the image intensities linearly.\n\nIt may be useful to explain the benefit of using PNG over TIFF images when preparing figures.\n\nI would personally not recommend annotating and cropping images directly in ImageJ but rather do these operations in the software used to build the figures themselves. This ensure that the annotations are easily changed (font, size) and are scalable (when exported to PDF). But that is likely a personal preference.\n\nIt would be useful to list a few software solution that can be used to create the figures. For instance if done directly in ImageJ, FigureJ is a popular option. I use Inkscape to build figure panels. In this context, it would be useful to indicate that software may modify images when importing them and that this should be avoided (image artefacts due to compression or upscaling).\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": "6335", "date": "18 Feb 2021", "name": "Helena Jambor", "role": "Author Response", "response": "We thank Guillaume Jacquemet for these constructive suggestions to improve our paper. The reviwers comments are shown in italic text. Our detailed responses and an indication of the implemented modifications to the manuscripts are below. 1) While the manuscript is intended to educate and improve image display in figures, it may be useful to add a disclaimer that images are illustrations and should be associated with a quantification of the phenomenon displayed. RESPONSE: We agree that qualitative images and quantifiable data from images are distinct and have now included a sentence in the methods section, which also refers to a workflow for reproducible image quantification: “Images are quantitative data. While image visualizations allow qualitative assessments, it is important to accompany them with quantitative measurements and appropriate statistical analysis. This workflow strictly addresses the image processing necessary for presentations and figures. Images prepared for presentation (e.g. 8-bit, RGB) are unsuitable for subsequent quantification such as intensity measurements. We therefore recommend separating image quantification and visualization workflows. Finally, documentation of any imaging and image processing workflows is key for reproducibility [21].” 2) In the manuscript, the authors discuss changing the brightness contrast to improve the images visually. But other manipulations such as changing the gamma or the colour balance should probably also be mentioned. While the appropriateness of these manipulations is often debated for scientific images, it would be useful to educate the readers about their existence and their pitfall. Especially since the correct gamma to use depends on the display used to visualise the images. and 3) On a similar note, it may be useful to inform that performing \"background clipping\" may also not be an appropriate image manipulation. RESPONSE: Excellent point. We already mention in step 2 that ‘non-linear adjustments, .. e.g. gamma correction need to be explained’ and have now expanded the section to indicate this more clearly. We also agree with the reviewer that background clipping, especially when done excessively, may be misleading audiences. As both points are prominently highlighted in the CSE’s White Paper [Ref 9] and in the milestone blog by the Journal of Cell Biology, we have clearly referenced these resources for further guide audiences. The section now reads as follows (also integrating comments by reviewers 2): “Images with a large gray value range may appear black when opening them in FIJI [12]. To properly display such data for the purpose of presentation/communication [24], adjust the brightness and contrast. For comparisons of intensities across images, it is recommended to use the same fixed intensity values (‘set’). For adjustments, avoid auto-buttons as, depending on the software packages the underlying code may differ, resulting in display differences. Linear intensity adjustment is acceptable, as long as key features are not obscured and minimal background signal is still visible to provide audiences with a sense for signal specificity. Entirely eliminating the background signal, or completely ‘clipping’ high intensities, is misleading (see also [10, 19, 25]). Some saturated pixels in the image are acceptable, if this helps the visualization. To identify problems with intensity sampling, or seeing if the image has been processed, the image histogram can be used to show its gray value distribution (Fig. 3). Briefly, good unprocessed images should have some offset in the low intensity range (Fig. 3: Histogram A). The distribution should not be cut off in the high range (Fig. 3: Histogram B) and the range should be continuous (Fig. 3: Histogram C) “ 4) On a similar note, it may be useful to add a warning message in the LUT section indicating that not all LUT display the image intensities linearly. RESPONSE: We thank the reviewer for bringing up this interesting point. After educating ourselves on this topic, we have included a note in the paper and cheat sheet and referenced a useful resource. We now include the sentence below in Step 6 and also expanded the glossary. “Additionally, we often sometimes see the use of false color LUTs for visualizing image data; when used improperly, false color LUTs can be highly misleading [26] and therefore should be explicitly mentioned in methods and figure legends.” 5) It may be useful to explain the benefit of using PNG over TIFF images when preparing figures. RESPONSE: We agree with the reviewers to include a statement of the benefit of PNG file format for publications and, on that note, also contrast it with the JPEG format. We now provide more details in the relevant sentence of ‘Step 1’ and also included a section of file formats in the glossary. “When processing is complete, several options exist (see glossary): saving images in TIFF format preserves the entire information. TIFF files however can rarely be properly used in programs for figure assembly (e.g. Inkscape, PowerPoint). For image presentation (figures, slides, online), save images in PNG format, which irreversibly merges the image with annotations, permanently applies brightness/contrast settings, and saves multiple channels as 24-bit RGB image. Another common image presentation file format is JPEG, which should be rarely used due to its lossy compression [19]. Beware of incorrect or unintentional bit -depth conversions [23].” 6) I would personally not recommend annotating and cropping images directly in ImageJ but rather do these operations in the software used to build the figures themselves. This ensure that the annotations are easily changed (font, size) and are scalable (when exported to PDF). But that is likely a personal preference. RESPONSE: We personally agree with the reviewer and indeed the authors themselves produce legible scale bars with the described procedure (see our post on theNode). We also already mention this as a TIP in the figure 4/Cheat sheet for publishing images. We are however also aware opponents of this method, who fear that it may result in improper scales. We have now amended ‘step 7’ in the paper and added a cautionary note in the FIJI cheat sheet (Fig. 3). Step 7 now says: “Images represent physical dimensions and can depict different scales ranging from nanometer to millimeter, which is often not obvious [36]. Adding scale information, ideally a scale bar with dimension, onto or next to the image, therefore is essential for self-explanatory figures. Also annotate what each color and symbol represents in an image, again best in the image itself or next to it. The aim is to provide sufficient information to the reader to understand the presented result at a glance. Ensure that scale bar, dimensions and annotations are legible in the final figure to be published; it may be more time efficient to adjust scale bar and add dimensions/annotations in the figure preparation software (e.g. as described here [37]).” 7) It would be useful to list a few software solution that can be used to create the figures. For instance if done directly in ImageJ, FigureJ is a popular option. I use Inkscape to build figure panels. In this context, it would be useful to indicate that software may modify images when importing them and that this should be avoided (image artefacts due to compression or upscaling). RESPONSE: We agree with the reviewer that audiences appreciate suggestions for open source software and have expanded the results section of the manuscript, where we already referenced, without explicitly naming, FigureJ. The second paragraph of the results section now states: “After completing the workflow, images may be assembled for publication and legends added [41]. Layouting images on a page can be done with design software such as the free and open source Inkscape (https://inkscape.org) or the proprietary Adobe Illustrator. Several options also exist to prepare publication-ready figures directly in ImageJ/FIJI, for example ScientiFig and FigureJ [42, 43].”" } ] }, { "id": "75495", "date": "14 Dec 2020", "name": "Georgina Fletcher", "expertise": [ "Reviewer Expertise Bioimage analysis (David Barry) and cell and developmental biology (Georgina Fletcher)" ], "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:\nThe authors present a suggested workflow to assist researchers in the life sciences in preparing images for presentation and/or publication. How images should be edited and/or adjusted in a suitable manner prior to submission to journals is certainly a source of confusion for many biologists, as they typically lack training in fields such as microscopy and image analysis, as noted by the authors. Therefore, a simple guide on what constitutes appropriate image adjustment would certainly be a valuable resource for the community. However, there are a number of technical points that require revision if the intended target audience (i.e those lacking a good understanding of image processing are to carry out the workflow correctly). There are also a number of revisions we would suggest to improve clarity and readability.\nSpecific Points:\nWith the greatest of respect to the authors, the manuscript would benefit from proof-reading by a native English speaker.\n\n\"Every day, around 2000 biomedical articles appear, 500 of which contain images.\" - a reference is needed to support this statement.\n\nIt would be good to have more emphasis in the introduction on quality image acquisition being crucial to obtaining good images and that often processing afterwards will not make up for this. In particular, in terms of zoom, brightness/contrast, image alignment and treating all samples in the same way.\nStep 1: Start with safely saving and storing raw images, and making a duplicate before opening it in FIJI. It would be helpful to explain image formats more fully (i.e why TIFF and not JPEG is preferred). It may be useful to add Image Compression to the Glossary terms. Probably worth mentioning Bio-Formats here to ensure correct reading of metadata. In addition, should \"open\" and \"save\" not be two different steps? It may also be worth mentioning that saving with Bio-Formats allows full metadata preservation (and permits lossless TIFF compression).\nStep 2: There's an ambiguity here between preparing an image for publication/presentation and preparing an image for quantification - these are two different things and should be treated as distinct. In terms of contrast adjustment, it should perhaps be mentioned that a little saturation here is ok if it aids visualisation. Probably state that it’s good practice to avoid any auto buttons to ensure that all images are treated the same way as software packages can differ in what auto actually means. Explain either in the main text or the Glossary what a histogram and linear/non-linear adjustments are, and note what is acceptable for presentation but not quantification. Add to the last sentence “in the figure legend and the methods”.\nStep 3: \"Often further processing is necessary\" - no processing has been performed as yet? This whole section is quite confusing and again, there's too much reference to quantification here - the purpose of the pipeline should be solely on adjustments necessary for publication. I would argue that image processing such as maximum intensity projection or Gaussian filtering should only be performed if the intention is to illustrate an analysis pipeline.\nStep 4: Further reference to quantification. Image rotation does not reduce unnecessary information. Do you mean image resizing? Aligning specimens again would be best done whilst imaging. Explain why steps of 90 degrees rotation are acceptable, but any other angles bring up issues.\nStep 5: Probably makes sense to crop before resizing?\nStep 6: It’s also better for black and white printing to have grayscale images. Each channel separated and grayscale, with a composite (it’s in the cheat sheet, but it should be here too). Here it’s written that a black background is preferred, but in the cheat sheet it states that an inverted image gives the best contrast, please resolve.\nStep 7: Mention that good annotations allow the reader to understand the results without reading any text? Results: Reference to DPI is confusing. It should be explained that pixels-per-inch in the context of printing is distinct from microns-per-pixel in the context of image acquisition.\nFigure 2: I'm not sure there's a need for Figure 2C? Why not just show the colour-blind friendly colour scheme in Fig 2B?\nFigure 3:\nI'm not sure there's a need to reference update sites here?\n\n”Tip” is misspelled throughout.\n\nUnder \"Open & Save\", I would suggest always using Bio-Formats to open images.\n\nUnder \"Brightness & Contrast\", I would remove all references to acquisition settings, instead referring to other publications on the matter1,2.\n\nAlso, I don't understand what is meant by \"background not cropped\"? I would expect all microscopy images to have a non-zero offset?\n\nI think the “Typical problems” needs further explanation in the text as a novice user may not understand the concepts from the diagrams alone. Please explain what is being looked at in the histograms.\n\nRemove references to image processing - why would the images be processed if the intention is purely for illustration?\n\nUnder \"Rotating & Resizing\", it should be mentioned that rotating by anything other than a multiple of 90 degrees should be discouraged as this will lead to interpolation.\n\nUnder \"Color\", the simultaneous reference to viewing images as composites and splitting channels is confusing. I think there's probably a little too much information under this heading and certain things could be dispensed with, such as inverting for better visibility (or perhaps just mention it in the text).\n\nUnder \"Annotate\", there should no need for Analyze > Set Scale if the metadata has already been verified.\nFigure 4:\nExamples images used under \"Magnification\" and \"Zoom, Insets\" are not great. I feel like Figures 3 and 4 could be combined into one single cheat sheet?\nReference 30 cites this paper but without its first author – is this correct?\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": "6336", "date": "18 Feb 2021", "name": "Helena Jambor", "role": "Author Response", "response": "We thank the reviewers for their constructive suggestions, which have substantially improved the manuscript and cheat sheets. Below we respond to the reviewers comments (in italics) in detail and describe the modifications we implemented in text and figures/cheat sheets. Specific Points: With the greatest of respect to the authors, the manuscript would benefit from proof-reading by a native English speaker. RESPONSE: We indeed aren’t native speakers and have now convinced an American partner to once more proof-read, with greater care, the manuscript. \"Every day, around 2000 biomedical articles appear, 500 of which contain images.\" - a reference is needed to support this statement. RESPONSE: We agree with the reviewers that this sentence warrants a citation indicating the source of these numbers. We have now amended the sentence to state: “Every year, about 800,000 articles are indexed at Pubmed (https://www.nlm.nih.gov/bsd/index_stats_comp.html) of which 25% contain images (Lee et al. 2018); this amounts to about 500 new articles with images every single day.” It would be good to have more emphasis in the introduction on quality image acquisition being crucial to obtaining good images and that often processing afterwards will not make up for this. In particular, in terms of zoom, brightness/contrast, image alignment and treating all samples in the same way. RESPONSE: We agree that image acquisition is always the critical step in producing good images. The microscopy community has extensively covered many aspects of going from specimen to image; while the message might not have reached everyone, resources and guidelines on image acquisition are plentiful. On the other hand, we see a lack in resources to making such images legible for audiences, and this is what our main focus is; We have now expanded the first sentence of the ‘Methods’ to emphasize further the importance of good image quality before setting out to create publishable image figures. We now say: “Obtaining high quality bioimages starts with specimen preparation such as fixation, labelling and clearing. To acquire and resolve the biological structure of interest, choose a microscopy system with an objective lens that allows suitable resolution, optical sectioning and spatial sampling. It is vital to sample intensity information properly by choosing a sufficient bit depth and avoiding saturation of high intensities. If the microscope-system allows changing the detector offset, low intensities should not be cut off. Rather than down sampling and cropping the image data, choose an appropriate magnification. When possible, align or rotate the sample to avoid image rotations. For comparison of image data, sample preparation and image acquisition settings need to be the same [12-19]. “ Step 1: Start with safely saving and storing raw images, and making a duplicate before opening it in FIJI. It would be helpful to explain image formats more fully (i.e why TIFF and not JPEG is preferred). It may be useful to add Image Compression to the Glossary terms. Probably worth mentioning Bio-Formats here to ensure correct reading of metadata. In addition, should \"open\" and \"save\" not be two different steps? It may also be worth mentioning that saving with Bio-Formats allows full metadata preservation (and permits lossless TIFF compression). RESPONSE: We thank the reviewers for these suggestions. - Indeed, in the cheat sheet the first suggested step is making a duplicate of the raw image and we now include this in the manuscript too. - We indeed now expanded the glossary to explain image file-formats including TIFF, JPEG, PNG, Bio-Formats and Image compression, a point also raised by reviewer 1. - the section is named ‘open & save’ to highlight the first point of the reviewer, that the first step after opening an image should be to make a copy, and also highlight upfront the consequence of certain file formats. Step 1 now says: “Duplicate the raw image to retain the original, untouched image as raw data and only process the duplicate. Load image-duplicate into Fiji and make sure metadata (see Table 1: Glossary), such as the scale, are correct. When possible use the Bio-Formats plugin for import, as this reads key image metadata (e.g. scale) automatically along with the image [22]. When processing is complete, several options exist (see glossary): saving images in TIFF format preserves the entire information. TIFF files however can rarely be properly used in programs for figure assembly (e.g. Inkscape, PowerPoint). For image presentation (figures, slides, online), save images in PNG format, which irreversibly merges the image with annotations, permanently applies brightness/contrast settings, and saves multiple channels as 24-bit RGB image. Another common image presentation file format is JPEG, which should be rarely used due to its lossy compression [19]. Beware of incorrect or unintentional bit depth conversions [23].” Step 2: There's an ambiguity here between preparing an image for publication/presentation and preparing an image for quantification - these are two different things and should be treated as distinct. In terms of contrast adjustment, it should perhaps be mentioned that a little saturation here is ok if it aids visualisation. Probably state that it’s good practice to avoid any auto buttons to ensure that all images are treated the same way as software packages can differ in what auto actually means. Explain either in the main text or the Glossary what a histogram and linear/non-linear adjustments are, and note what is acceptable for presentation but not quantification. Add to the last sentence “in the figure legend and the methods”. RESPONSE: We thank the reviewers for these points and have incorporated them into a new Step 2 description, which also incorporates a suggestion by reviewer 1. Intensity adjustment (i.e. brightness and contrast) and 2 examples of nonlinear adjustments are now also incorporated here and the glossary. We added image histograms as a topic to the glossary and mention that one can analyze image intensity sampling problems using the image histogram. The new step 2 now says: “Images with a large gray value range may appear black when opening them in FIJI [12]. To properly display such data for the purpose of presentation/communication [24], adjust the brightness and contrast. For comparisons of intensities across images, it is recommended to use the same fixed intensity values (‘set’). For adjustments, avoid auto-buttons as, depending on the software packages the underlying code may differ, resulting in display differences. Linear intensity adjustment is acceptable, as long as key features are not obscured and minimal background signal is still visible to provide audiences with a sense for signal specificity. Entirely eliminating the background signal, or completely ‘clipping’ high intensities, is misleading (see also [10, 19, 25]). Some saturated pixels in the image are acceptable, if this helps the visualization. To identify problems with intensity sampling, or seeing if the image has been processed, the image histogram can be used to show its gray value distribution (Fig. 3). Briefly, good unprocessed images should have some offset in the low intensity range (Fig. 3: Histogram A). The distribution should not be cut off in the high range (Fig. 3: Histogram B) and the range should be continuous (Fig. 3: Histogram C) Non-linear adjustments of brightness and contrast, for example histogram equalizations or gamma correction [19, 26] must be explained in both figure legend and method section [19, 26, 27]. Miura and Nørrelykke nicely describe why intensity adjustments (linear and non-linear) must be applied with special caution when images have already been pre-processed, e.g. cropped [21]. Once images have been adjusted, ‘apply’ and ‘save as PNG’ irreversibly change the intensity range, which makes images unsuitable for intensity measurements.” Step 3: \"Often further processing is necessary\" - no processing has been performed as yet? This whole section is quite confusing and again, there's too much reference to quantification here - the purpose of the pipeline should be solely on adjustments necessary for publication. I would argue that image processing such as maximum intensity projection or Gaussian filtering should only be performed if the intention is to illustrate an analysis pipeline. RESPONSE: We thank the reviewers to help us clarify this section. Our intention was to leave a buffer zone in the work process where readers can then perform their specific processing - as these vary dramatically, we cannot possibly start commenting. We are also of the opinion that carefully applied image processing in specific and informed instances is unavoidable for proper image visualization. For instance in advanced systems, reconstruction has to be performed to visualize the biological structure of interest. Also the important information is the actual signal of the biological specimen, which can be degraded, even with highly optimized acquisition, by imaging artefacts such as noise and blur. Here appropriate image processing, made transparent in the publication and guaranteeing the access to the raw image data can promote visualization (Cromey 2010, 2013 Jonkman et al. 2020). It is really crucial that researchers are aware that appropriately used image processing can be fine in specific well defined circumstances, under the condition that necessary information is provided for reproduction AND are not used as a replacement for optimized imaging. We made this section clearer by properly specifying and citing examples for standard or advanced image processing with the aim of allowing or promoting visualization. Also we strongly emphasizes the key points that methods need to be used only in specific conditions, are not replacement for good imaging and need to be made transparent. “Depending on your specific scientific question and goal, further image processing may be necessary for image visualization. For instance, advanced systems such as lightsheet microscopy require extensive image processing workflows to obtain a reconstructed volume of the biological specimen for visualization [28-32]. Large 3D volumes of data are also hard to visualize in two dimensional figures and require the use of projection or rendering [33]. Finally, microscopy systems also add artefacts (noise, blur), which image processing using linear filters [13] and deconvolution [34, 35] can help to reduce. Any processing for representing the image data needs to be carefully applied where necessary and is no replacement for an optimized imaging setup [12-18]. The processing needs to be clearly stated in the methods section, advanced or non-linear adjustments also in the figure legends [13, 19].” Step 4: Further reference to quantification. Image rotation does not reduce unnecessary information. Do you mean image resizing? Aligning specimens again would be best done whilst imaging. Explain why steps of 90 degrees rotation are acceptable, but any other angles bring up issues. Step 5: Probably makes sense to crop before resizing? RESPONSE: We thank the reviewers for requiring us to disentangle image rotation and resizing, we now have included Step 4: Image rotation and Step 5: Cropping and resizing. We have also updated Figure 1 accordingly. In the new section 4 we also explain why rotation is necessary and the specific case of 90-deg rotation (which depends on the software used if it indeed is lossless rotation or not). The new Steps 4 and 5 say: “Step 4: Image rotation Image rotations are sometimes necessary to compare image content properly. For instance, when comparing specimens, it helps to align them in the same anatomical orientation. Image rotations however result in a redistribution of the intensity values within the fixed image pixel grid: for rotations by less than 90 degrees, new intensity values are computed by interpolation, and thus information is lost (Fig. 3). For rotations in multiples of 90-degree steps, pixels can be reordered rather than interpolated, however this depends on the specific implementation of the rotation algorithm (Fig. 3). Loss of information by image rotation may be acceptable for image visualizations, however all image quantifications should be done beforehand [19, 26]. Step 5: Cropping & resizing Often larger fields-of-view are captured on the microscope than are required in the figure. Cropping is then not only permissible, it is necessary to focus the reader on the relevant result. In contrast, it is not ethical to crop out data that would change the interpretation of the experiment, or to “cherry-pick” data [10, 19, 26]. We discourage adjusting the intensity of individual crops especially for comparisons [21]. When a larger field of view and a magnification of detail (‘inset’) need to be shown side-by-side, indicate inset position in the original image. Adjust the size of the image in the figure preparation software, not during image processing: Image size adjustments by upsampling or downsampling an image, requires interpolation and thus may degrade image quality.” Step 6: It’s also better for black and white printing to have grayscale images. Each channel separated and grayscale, with a composite (it’s in the cheat sheet, but it should be here too). Here it’s written that a black background is preferred, but in the cheat sheet it states that an inverted image gives the best contrast, please resolve. RESPONSE: We thank the reviewers for catching our inconsistency and have adapted to the text to fit the cheat sheet. The first section of step 6 now says: “Step 6: Color In fluorescence microscopy, cameras usually capture each wavelength (channel) with a separate grayscale image. Here, no signal is shown as black, and intensities of the fluorescent signal are displayed in steps of grey values with saturated pixels shown in white. When only one fluorophore/wavelength/channel is shown in a figure, grayscale, which has the best contrast, is favorable. Consider also inverting the grayscale images as human brightness perception is logarithmic and can best differentiate bright areas [27]. Inverted grayscale images are also printer-friendly and have better visibility on a white page/slide. To visualize several channels of a specimen (e.g. colocalization studies), encode channels with different colors. A look-up table (LUT) determines how gray values are translated into a color value. Additionally, we often sometimes see the use of false color LUTs for visualizing image data; when used improperly, false color LUTs can be highly misleading [27] and therefore should be explicitly mentioned in methods and figure legends.” Step 7: Mention that good annotations allow the reader to understand the results without reading any text? RESPONSE: We thank reviewers for this suggestion and have added it to the adapted Step 7, which now reads: “Images represent physical dimensions and can depict different scales ranging from nanometer to millimeter, which is often not obvious [36]. Adding scale information, ideally a scale bar with dimension, onto or next to the image, therefore is essential for self-explanatory figures. Also annotate what each color and symbol represents in an image, again best in the image itself or next to it. The aim is to provide sufficient information to the reader to understand the presented result at a glance. Ensure that scale bar, dimensions and annotations are legible in the final figure to be published; it may be more time efficient to adjust scale bar and add dimensions/annotations in the figure preparation software (e.g. as described here [37]).” Results: Reference to DPI is confusing. It should be explained that pixels-per-inch in the context of printing is distinct from microns-per-pixel in the context of image acquisition. RESPONSE: We thank reviewers for noting our imprecise description - and indeed, as we write in the section, feedback from colleagues helps! We stayed with using dots per inch as a term instead of pixel-per-inch (screen res) since for figure preparation the output of many programs (Illustrator, Photoshop) as well as author guidelines usually specify this term. The section in the results now says: “Figure resolution is usually referred to as dots per inch (DPI). For an ‘unpixelated’ display of microscopy images in an electronic publication, publishers require 300 DPI images in RGB color mode. (Note that the dots-per-inch do not correspond to the physical dimension of the microscopy object and scale bar but solely refer to image size in print or on the screen). This workflow is iterative and feedback from colleagues helps to identify possible hurdles.” Figure 2: I'm not sure there's a need for Figure 2C? Why not just show the colour-blind friendly colour scheme in Fig 2B? RESPONSE: We thank reviewers for this suggestion and based on their suggestion have simplified Figure 2. Figure 3: I'm not sure there's a need to reference update sites here? RESPONSE: We agree and have now removed the comment from the cheat sheet. ”Tip” is misspelled throughout. RESPONSE: We thank the reviewers (and several readers on twitter) for catching this mistake and have updated it in Figure 3 and 4. Under \"Open & Save\", I would suggest always using Bio-Formats to open images. RESPONSE: We now specify Bio-Formats as the recommended opening and specify in the text that the Bio-Formats plugin is metadata friendly. Under \"Brightness & Contrast\", I would remove all references to acquisition settings, instead referring to other publications on the matter 1,2. RESPONSE: We agree and remove this section of the TIP instead refer to the publications for further information about image acquisition. Also, I don't understand what is meant by \"background not cropped\"? I would expect all microscopy images to have a non-zero offset? RESPONSE: This is exactly what we wanted to express, since in our practice it can happen that people incorrectly set a detector offset cutting of low values. We agree that this was not written clearly. We adjusted the text next to the Figure to indicate how an acceptable histogram of a raw image could like and that the other histograms represent that the image was altered or is problematic. I think the “Typical problems” needs further explanation in the text as a novice user may not understand the concepts from the diagrams alone. Please explain what is being looked at in the histograms. RESPONSE: We agree and have added an explanation in the text. “Briefly, good unprocessed images should have some offset in the low intensity range (Fig. 3: Histogram A). The distribution should not be cut off in the high range (Fig. 3: Histogram B) and the range should be continuous (Fig. 3: Histogram C).” Remove references to image processing - why would the images be processed if the intention is purely for illustration? RESPONSE: In principle we agree that as little processing as necessary should be applied to images for visualizations. However, as we tried to lay out in the rebuttal (Step 3 - Image processing) in some instances image processing cannot be avoided and for some instances of microscopy artefact it might also be beneficial for visualization (Cromey 2010, 2013, Jonkman et al. 2020). Thus, image processing done correctly and specified transparently in the methods as well as figure legends can be in our opinion permissible. Under \"Rotating & Resizing\", it should be mentioned that rotating by anything other than a multiple of 90 degrees should be discouraged as this will lead to interpolation. RESPONSE: We agree and added an explanatory sentence. Under \"Color\", the simultaneous reference to viewing images as composites and splitting channels is confusing. I think there's probably a little too much information under this heading and certain things could be dispensed with, such as inverting for better visibility (or perhaps just mention it in the text). RESPONSE: We changed the place for showing the tip as indeed this is not relevant for the very first command. Under \"Annotate\", there should no need for Analyze > Set Scale if the metadata has already been verified. RESPONSE: We agree that if the cheat sheet is linearly read, this information is not strictly required at that specific stage. We however expect that users will also read the cheat sheet to look up a specific action or possibly use drag-and-drop for file import. We therefore feel a slight redundancy does not do any harm and has the benefit that users can double check the information. Figure 4: Examples images used under \"Magnification\" and \"Zoom, Insets\" are not great. RESPONSE: We thank reviewers for this comment and have updated the images. I feel like Figures 3 and 4 could be combined into one single cheat sheet? RESPONSE: We’d rather prefer to have two individual Figures in the manuscript that can be individually referenced. The first cheat sheet serves as a general template independent of a specific implementation and is thus crucial for people that use any of the many other open source or proprietary software solutions. The second cheat sheet implements these general principles with FIJI. Thus, both cheat sheets are crucial and can work stand-alone. Also, we hope that readers will print each figure as an A4/Letter cheat sheet. Combining the figures would inevitably result in too small printed versions. We changed the text in the Results section to explain our intentions better: “The workflow steps and accompanying suggestions for image presentation are available as accessible “cheat sheets” (Fig. 3, 4) for wide distribution and adoption to more specific needs. Our workflow is based on the open source software Fiji (Fig. 3), but its principles are applicable to other software (Fig. 4).” Reference 30 cites this paper but without its first author – is this correct? RESPONSE: Yes, as only one author is registered with OSF. In the new version the second author also signed up for OSF and is now listed as co-author." } ] } ]	1	https://f1000research.com/articles/9-1373
https://f1000research.com/articles/10-126/v1	18 Feb 21	{ "type": "Research Article", "title": "Treatment of child wasting: results of a child health and nutrition research initiative (CHNRI) prioritisation exercise", "authors": [ "Chloe Angood", "Marko Kerac", "Robert Black", "André Briend", "Kerstin Hanson", "Stephen Jarrett", "Mark Manary", "Marie McGrath", "Noël Zagre", "Natasha Lelijveld", "Amy Mayberry", "CHNRI collaborators", "Council of Research & Technical Advice on Acute Malnutrition (CORTASAM)", "Marko Kerac", "Robert Black", "André Briend", "Kerstin Hanson", "Stephen Jarrett", "Mark Manary", "Marie McGrath", "Noël Zagre", "Natasha Lelijveld", "Amy Mayberry" ], "abstract": "Background: Child wasting is highly prevalent, with around 49.5 million children under five years affected globally. More evidence is needed to inform the scale up of effective treatment of wasted children worldwide. The aim of this study was to identify and prioritise the main outstanding research questions relating to the treatment of wasting to inform future research agendas. Methods: A research prioritisation exercise was undertaken using the Child Health and Nutrition Research Initiative method. Research gaps were identified from multiple sources, grouped in themes and condensed into a list of 53 research areas by a group of experts. An online survey was developed and circulated globally to individuals working in the global nutrition sector. Participants evaluated each research area according to four agreed criteria. Research areas were then ranked according to an overall research priority score. Results: A total of 394 individuals from 63 countries participated in the survey. Research areas prioritised by the group focused on the effective detection and diagnosis of ‘high risk’ wasted children in the community; provision of a continuum of care; and early life course interventions. The group also prioritised evidence to inform guidance on the impatient management of wasted children with diarrhoea; prevention of post-treatment relapse and mortality; and the optimisation of ready-to-use therapeutic foods in treatment programmes. Conclusions: Critical gaps in our understanding of the treatment of wasting must be filled to inform guidance, policy and programming to ensure that all wasted children receive the treatment services that they need. A coordinated research agenda across treatment and prevention is urgently needed to maximise the impact of funding investments towards the meeting of global targets to reduce child wasting.", "keywords": [ "acute malnutrition", "severe acute malnutrition", "moderate acute malnutrition", "undernutrition", "nutrition", "research priorities", "treatment", "wasting" ], "content": "Introduction\n\nChild wasting is highly prevalent, with approximately 49.5 million children under five years affected globally (Joint malnutrition estimates, 2019). Wasting is associated with a high risk of death, with severely wasted children 9 to 12 times more likely to die compared to their well-nourished counterparts1. Those who survive one or more episodes of wasting have increased risk of childhood infection and detrimental consequences to their healthy growth, development and cognition2. The long term consequences of this can be severe and include: reduced economic productivity; negative health outcomes into adulthood; for women, risk of giving birth to infants who are born wasted and/or stunted (low birth weight (LBW)) who then themselves have heightened risk of illness and death3–5. In this way, the consequences of childhood wasting can pass from one generation to the next.\n\nThe global prevalence of wasting has decreased very little over the past 20 years, particularly when compared to reductions of other manifestations of malnutrition, such as stunting6,7. Currently, only 37 out of 194 countries are on track to reach the World Health Assembly (WHA) 2025 target to reduce and maintain prevalence of wasting below 5% (Global Nutrition Report, 2018) which aims to contribute to the sustainable development goal (SDG) of ending preventable child deaths and ensuring health, progress and opportunities for all children by 2030.\n\nEfforts to prevent wasting must be urgently accelerated to reduce the global burden8. There is also an ethical and moral imperative to ensure universal access to life-saving treatment to wasted children who need it. Significant advances have been made in the treatment of severe wasting in children over six months of age over the last three decades, particularly through the development of ready-to-use therapeutic foods (RUTF). This has substantially changed the treatment of severe wasting and is an approach that is supported by substantial programmatic evidence9. Nevertheless, it is estimated that currently less than 20% of children with wasting receive the treatment that they need (No Wasted Lives, 2018). The management of wasted infants under six months of age has lagged even further behind this10. There has also been a lack of attention afforded to those on the moderate end of the wasting spectrum, underweight children (defined by low weight-for-age), and children with multiple anthropometric deficits who are all also at increased risk of death11,12.\n\nIn response to the pressing need to rapidly scale up and improve wasting treatment, a research prioritisation exercise was carried out with the support of No Wasted Lives (www.nowastedlives.org), a multi-agency coalition that aims to combat acute malnutrition. The objective was to establish a clear set of research priorities that, in the context of limited time and resources, would guide financial investments in research with high potential to translate into meaningful programme and policy action towards the meeting of the SDGs.\n\n\nMethods\n\nThe Child Health and Nutrition Research Initiative (CHNRI) method, described in detail elsewhere13,14, was used as a framework for this research prioritisation exercise. In brief, this involves the identification and listing of many possible research options within a defined context, which are systematically and transparently scored by experts against agreed criteria. The result is a list of priorities that can be used to inform decisions about research investments by governments, international agencies and donors.\n\nThe context and scope of the exercise was first outlined by the Council of Research and Technical Advice on Acute Malnutrition (CORTASAM), a panel of technical experts and advisory group for No Wasted Lives. A decision was made to focus predominantly on the treatment of wasting in children under five years of age, including aspects of prevention only where explicitly linked to treatment. A timeline was set to identify research priorities that would be actionable by 2020 with the support of CORTASAM, No Wasted Lives and partners before the end of the current No Wasted Lives project cycle (2015–2020).\n\nA non-systematic review was conducted to identify previously published priority research questions within the specified context. Several hundred priorities were identified across multiple sources, including the World Health Organization (WHO)’s 2013 guideline updates for the management of SAM15, subsequent focused publications16,17 and other related CHNRI exercises18–21. A consultation was held with members of the No Wasted Lives Coalition, CORTASAM, and other regional and country-level stakeholders to condense this list. The decision was made to focus on areas of research that could each encompass several research studies, rather than specific research questions, in order to include as wide a scope of research needs as possible within an actionable survey. This approach also allowed for high-level strategic prioritisation that could feed into research strategies and incentivise funding for portfolios of research, and flexibility for investors and researchers to respond to emerging evidence and changing needs. The result was a list of 53 research areas, categorised into 11 broader research themes and further into two sections: ‘technical and operational’ (n=30) and ‘epidemiological’ (n=23) (extended data file 3). Criteria against which the research areas would be judged (Table 1) were then selected from the long list of possible criteria described in CHNRI methodology.\n\nThe table presents the agreed criteria by which each research area was judged by survey participants.\n\nThe research questions framed for this exercise use the term ‘acute malnutrition’ to describe wasting, defined as weight-for-height z-score (WHO) <-2, and bilateral pitting oedema and/or mid-upper arm circumference (MUAC) <125 mm. To reflect direction of travel in accepted nomenclature, this paper uses the term ‘wasting’ in place of ‘acute malnutrition’; for the purposes of this paper, when the term ‘wasting’ is used, this also implicitly refers to oedematous malnutrition.\n\nThe research areas and criteria were used to create a survey in English and French using the online tool ‘Survey Monkey’ (www.surveymonkey.co.uk) (extended data file 222). The order that research areas were presented to respondents was randomised to avoid question bias caused by respondent fatigue. An invitation to participate in the survey was circulated in English and French to CORTASAM members; global, regional and country staff within the No Wasted Lives Coalition; and other researchers, implementers, academics and donors through relevant listservs, social media and relevant websites (extended data file 122). Non-probability (convenience) sampling was used with no minimum quota of participants and no specific eligibility criteria was applied; participants took part based on their interest in the study given that all viewpoints were counted as valid and useful. The survey was open between 3rd April and 5th May 2017.\n\nBasic information on the country and region and type of work was collected from each individual. Respondents were then asked to judge how each research area might meet each of the four judging criteria by indicating “Yes” (which was allocated one point); “No” (0 points); “Undecided” (0.5 points); or “Insufficiently informed” (missing input). While we aimed to provide clear and concise guidance and criteria, as with other CHNRI exercises, it was ultimately up to the respondent to interpret the question and respond based on how they envisioned it. Results of the survey were downloaded into Excel and a research priority score (RPS) computed for each criterion for every research area ranging from 0–100%. From this, an overall RPS was computed for each research area (mean of the RPS of all four criteria) globally and for three regions most represented in the data and relevant in terms of burden of wasting, grouped as follows: West and Central Africa; East Africa; and South Asia. The level of agreement or controversy between participants’ answers for each research area was assessed by calculating the average expert agreement (AEA). The AEA is a proportion of scorers who gave the most common score (mode) for a question, divided by the total number of scorers who scored that question. This is computed as follows:\n\n\n\nwhere q is a question that experts are being asked to evaluate each research area against.\n\nThe AEA is unaffected by ‘undecided’ responses and variances in the number of scorers for each survey question. In AEA computation all four possible responses are treated as valid, including ‘insufficiently informed’ to reflect all responses in the level of overall agreement.\n\nThis CHNRI exercise did not involve any personal or otherwise sensitive data and deals with professional individuals solicited through established professional networks. Participants who completed the questionnaire were asked if they would like to be named as part of the group author list, and only those answering “yes” are listed in this paper. All findings were anonymised and individual answers to the questions are not presented.\n\nAs is standard for CHNRI exercises13,21, this research does not require formal ethical committee review. The intent to make results of the survey publicly available and publish the analysis was clearly laid out in the participant instructions (extended data file 122); voluntary participation in the survey was taken as consent. Participants were given the option to be listed as group authors of this paper (“CHNRI collaborators”); those who gave their consent are named in the acknowledgements section.\n\n\nResults\n\nA total of 394 survey responses were received. A total of 81 responses were removed as they were either repeat submissions or no survey questions were answered. Of the final 313 respondents, 143 (46%) answered the full survey and the rest answered only some of the questions. Responses represented 63 different countries (53 of which were low-middle income). There was broad geographical representation in the responses; according to UNICEF regional classifications, 72 (23%) of respondents worked at a global level, (71) 22.7% East Africa, (58) 18.5% West Africa and (46) 14.7% South Asia (Figure 1). Respondents represented a total of 167 different organisations. In total, 149 (47.6%) respondents described themselves as working in operational/programmatic areas; 49 (15.7%) academic; 36 (11.5%) government; 6 (2.2%) policy and 72 (23%) ‘other’ (Figure 2).\n\nThe figure represents the proportion of survey participants by geographical region.\n\nThe figure represents the proportion of survey participants by type of work that they are engaged in.\n\nGlobal analysis. The distribution of RPS was relatively narrow. The average score was 83 (on a scale of 0 to 100) and 95% of scores fell between 73 and 94. The top ten priority research areas according to the overall RPS are presented in Table 2 (the full list is available in Supplementary Material 2). The AEA in this top ten was high (80.3 to 91.6), indicating a high-level of agreement between experts on the top priorities.\n\nThe table lists the top 10 research areas prioritised by survey participants and includes priority scores according to each of the agreed judging criteria and overall scores: O = operational and technical; E = epidemiological; I = impact; E = effectiveness; A = answerability; S = sustainability; RPS = research priority score; AEA = average expert agreement.\n\nThe top ten priorities have a strong focus on programming - seven out of the top ten are technical and operational in nature and only three are epidemiological. The top ranked research area (and with the highest AEA) relates to the effective detection of acute malnutrition in community settings, also echoed in the area ranked eighth in the list around the need for more evidence to inform tools used for community-based detection. The second priority relates to the inpatient management of children with severe wasting with diarrhoea and the need for evidence to inform effective treatment protocols for these children. The third priority identified is to identify strategies and protocols to support scale-up of treatment of acute malnutrition in infants under six months of age. The next two research areas (ranked fourth and fifth) are epidemiological questions on the same theme of understanding the natural course of malnutrition, specifically relating to the process of relapse after treatment and the relationship between maternal and child nutrition. The question ranked sixth is around entry and discharge criteria of treatment programmes to ensure optimum outcomes, which is also relevant to the issue of relapse. The area ranked seventh relates to effective approaches to support infant and young child feeding (IYCF) practices and the need for evidence of the impact of these approaches on CMAM programming and vice versa. The area ranked ninth speaks to the need for more evidence around the safety and cost-effectiveness of locally produced RUTF to support scale up of therapeutic treatment. This relates to the area ranked twelfth around the need for evidence of optimum doses of RUTF. Question ten is the final of the three epidemiological research areas included in the top ten list and relates to the need for evidence to inform strategies to reduce risk of post treatment mortality.\n\nOther high ranked questions that did not make the top ten were around vitamin A supplementation for uncomplicated cases of acute malnutrition (eleventh) and the integration of wasting treatment services into routine health systems (thirteenth). Research areas relating to long-term outcomes, burden estimation and risk factors (all epidemiological in nature) appear much lower in the prioritised list. The lowest priority research areas (ranked 51, 52 and 53) all relate to body composition (either as a predictor or outcome of acute malnutrition), were highly controversial (low AEA levels) and ranked particularly low in terms of their likelihood to result in sustainable interventions.\n\nRegional analyses. Sub-analyses by region (East Africa, West and Central Africa and South Asia) reveal some differences in priorities between regions, although these results must be interpreted with caution given that the survey was not designed to be regionally representative. In the East Africa responses (n=71), questions related to detection were ranked lower than in global and other regional results. Those related to early intervention (in the under six month age group, IYCF and maternal nutrition) and achieving a continuum of care (understanding the process of deterioration from moderate to severe wasting and management of patients between inpatient and outpatient services) were ranked higher. Novel research areas that appeared in the East Africa top ten were on the cognitive effects of acute malnutrition and vitamin A supplementation for uncomplicated cases of acute malnutrition. West and Central African responses (n=76) largely reflected the global results, except for the addition of evidence around the strengthening of surge capacity for wasting treatment. South Asia responses (n=53) demonstrated the need for evidence around maternal nutrition, wasting in infants under six months and detection, diagnosis and estimation of the burden which all scored higher in this region than elsewhere. Three priorities were included in the top ten in South Asia alone that related to community perceptions of treatment, generation of demand for treatment and improving health-seeking behaviours.\n\n\nDiscussion\n\nThis exercise brought together a wide group of participants to prioritise areas of technical, operational and epidemiological research most likely to contribute to the delivery of effective treatment of wasting at scale. Findings reflect that, despite considerable progress in the treatment of child wasting, our knowledge of what to do and how to do it to effect greater change is incomplete. Top priority research areas identified by this group of experts relate to the effective detection and diagnosis of ‘high risk’ wasted children in the community; inpatient management of wasted children with diarrhoea; the scaling up of effective management of infants under six months of age; prevention of post-treatment relapse and mortality; support for maternal nutrition and IYCF as means of improving the nutrition status of wasted infants; and the optimisation of RUTF for wasting treatment. Differences in priorities between regions demonstrate the need for research to inform context-specific approaches.\n\nAn important theme brought out in the results is the need to understand the characteristics of ‘high risk’ children in the community in different contexts to aid early detection and treatment. Results also support the recent rise in research studies to test simplified and/or combined protocols for wasting treatment to help streamline services, improve access to treatment for high risk moderate cases, and support continuity of care to discharge23–25. The prioritisation of areas related to relapse and post-treatment mortality demonstrates recognition among this group of experts that anthropometric recovery is important to achieve and contributes to recovery but is not in itself a cure. This is consistent with a recent review of the literature on relapse that indicated poor post-discharge outcomes after initial recovery (mortality and morbidity) and the need for appropriate, scalable solutions to tackle this problem26.\n\nResults of the present exercise accord with those of other recent CHNRI exercises that place a high priority on research to inform the prevention and management of wasting in infants under six months of age8,18,19. It is now recognised that there are high burdens of wasting in this age group10, that these infants have higher risk of death compared to wasted older children27, and that interventions are urgently needed to avoid their further deterioration to maximise survival and reduce later burden of immediate and longer term care18,19. A previous CHNRI exercise on the management of ‘at risk’ mothers and infants under six months highlights in more detail priority research questions in this area that need to be addressed19. The priority placed on research around wasted infants under six months in this exercise, as well as research around the impact of maternal nutrition and IYCF practices on child wasting, highlights the felt need to tackle malnutrition with an early life course approach. This kind of approach aims to address nutritional needs at key stages within first 1,000 days window to prevent LBW and the deterioration of wasted infants as a means of preventing wasting into childhood and associated long term deleterious effects28.\n\nAnother research area identified as a key gap by this group of experts is the treatment of severely wasted children with diarrhoea in inpatient settings. This reflects continuing uncertainty around the evidence base underlying the current WHO treatment guidelines in this area. A recent review of related studies concluded that the use of ReSoMal for the oral rehydration of severely wasted children with diarrhoea as opposed to ORS may be potentially harmful29. Another review on intravenous rehydration of children with severe wasting and diarrhoea found no support of the current recommendation to only use IV rehydration in case of shock and found that withholding IV fluids in case of heart failure in these children may be more harmful29. More operational research on the management of rehydration in severely wasted children in different settings is needed to inform an update of WHO guidelines.\n\nA final theme in the global results is the need for research to enable the optimisation of RUTF for the management of wasting. Experts prioritised the need for further investigation into the safety and effectiveness of formulations that rely on local ingredients to improve the cost-effectiveness of treatment programmes to support greater coverage. Although there has been an increase in trials of alternative RUTF formulations since this exercise was carried out30, important questions remain, including around the wider economic implications of using local products and producing locally, and on the efficacy of alternative formulations in settings outside of sub-Saharan Africa. Experts in this study also prioritised the need for research to investigate the safety, effectiveness and cost-effectiveness of reduced dosages of RUTF in treatment programmes. Recent trials have been undertaken to this end, some of the results of which have now been published24,31,32. Questions remain, however, on the longer-term impact of reduced dosage and risk of relapse, answers for which are urgently needed to inform national and global guidance.\n\nRegional analyses demonstrate that experts in South Asia place more importance than those from other regions on research into the generation of demand for wasting treatment, community perceptions of acute malnutrition and improving health-seeking behaviours. This may reflect the slower progress that has been made in South Asia compared to Africa to date in the scale up of wasting treatment services, as well as the need for evidence to inform scalable treatment options tailored specifically to the South Asia context (UNICEF, 2018). This is particularly important given that half the global burden of wasting is from South Asia (Joint malnutrition estimates, 2019). Regional analyses also reveal the importance of evidence to inform the development of models of integration of treatment services into routine health systems: experts from South Asia and East Africa prioritised research on the leveraging of existing opportunities within routine health systems to facilitate scale up and experts from West and Central African prioritised research on operational models to strengthen surge capacity of the health system to treat wasting. This suggests the need for models of integration that can be tailor-made to specific health systems and capacities and that reflect context-specific patterns of wasting.\n\nFollowing this exercise, preliminary findings were immediately applied to the development of a research agenda to guide the efforts of CORTASAM, the No Wasted Lives Coalition, and partners (No Wasted Lives, 2018). A portfolio of operational research has since been funded and developed to build evidence around ways to improve the quality, coverage and cost-effectiveness of wasting treatment programmes. Multi-country studies are now in various stages of completion on simplified approaches for the treatment of wasting; community-based models for detection, diagnosis and treatment in the community and understanding treatment success and non-respondence. Although progress has been made, a recent landscape review of progress against the research agenda found many of the gaps identified in this original prioritisation exercise remain outstanding and require further investment (No Wasted Lives, 2020). Now that the United Nations Global Action Plan on Wasting has been released (UN GAP on child wasting) there is now more than ever a need to step up the generation of evidence to inform the development of guidance to successfully operationalise this plan. The World Health Organization (WHO) has a crucial role to play in coordinating a research agenda to fill critical evidence gaps across treatment and prevention identified by this and other recent CHNRI exercises8,18,19. A coordinated approach will maximise returns on research investments and ensure that findings contribute to programme and policy action towards reductions in child wasting.\n\n\nStrengths and limitations of the study\n\nThis study used a validated method designed to maximise the predictive value of a group of experts33. Research ideas were systematically listed and independently and transparently scored by a large group of experts, most of whom have first-hand experience working on acute malnutrition treatment programmes. Participants in the survey were spread across several geographic regions, with good representation from areas of the world with high burdens of wasting (East Africa, West and Central Africa and South Asia). The CHNRI method allowed for the measurement of collective priorities, agreement and controversy of this large, diverse group. The rating of research areas rather than specific questions enables results of this study to feed into research strategies and inform high level decisions about investments into portfolios of research. Results are therefore less sensitive to emerging evidence and changing needs and so remain relevant for a longer time period.\n\nThe primary limitation of this study is that the source of the data is the opinions of individuals working in the field of childhood malnutrition. These opinions are often formed by the experiences of these individuals, and ideas outside of the imagination of those surveyed are not represented. The quality of the resulting recommendations is therefore only as good as the creativity and insight of the individuals surveyed. Respondents working in programming were much more heavily represented among respondents than those working in academia, policy or government; this may be reflected in the bias of results towards operational research rather than epidemiological. The broader political economy of scale-up, and what factors may influence uptake and application of evidence generated across these research priorities, is also not fully reflected. Although we have included a large number of participants (larger than most published CHNRI surveys), this is not representative of the nutrition community as a whole and therefore we cannot assume generalisability of results.\n\nAs this was an online survey with invitations via email, websites and social media, only respondents using these media could be included. This may have limited inclusion of potential respondents working at sub-national levels. Some respondents did not complete the survey; however, question bias was mitigated by the randomisation of the order that questions appeared to each respondent and by discounting non-responses from the computation of the RPS. Regional sub-analyses may be affected by sampling bias and vulnerable to confounding factors, given that regional sampling calculations were not designed into the survey. Furthermore, the proportion of respondents from each region do not accurately represent the regional spread of the burden of wasting. For example, while the current burden of wasting is highest in South Asia, this region was represented least in the results of all three regions included in the sub-analyses. The regional analyses are therefore purely descriptive and must be interpreted with caution.\n\nAnother limitation of the study is possible bias in the selection of research areas. As this is a large topic area spanning many geographical contexts, the list could not be exhaustive and some important research areas had to be removed to make the survey an actionable size. For example, research areas cover operational, technical and epidemiological questions, but not those relating to the wider political economy, which is an important contextual dimension that can hinder or facilitate scale up in any given context. The envisaged results of prioritised research are therefore unlikely alone to inform the scale up of effective wasting treatment programmes and need to be considered within the broader contexts that they are applied.\n\nLastly, given the time that has passed since this exercise was carried out and the rapidly evolving nature of research in this area, terminologies to describe this form of malnutrition have changed, and new evidence has emerged. This must be taken into consideration when decisions are made about which research areas to invest in and the way in which research questions are framed. However, the results remain important and directly relevant to many organisations to inform discussions on funding investments.\n\n\nConclusion\n\nChild wasting is a global problem that requires urgent public health attention. The results of this research prioritisation exercise demonstrate the most critical gaps in our understanding that must be addressed to inform guidance, policy and programming to enable the meeting of global wasting targets. More evidence is urgently needed to inform the effective detection and diagnosis of ‘high risk’ wasted children in the community and to inform a continuum of care for their effective management, as well as to inform early life course interventions, particularly those targeted to mothers and infants under six months of age to prevent deterioration into child wasting. Evidence is also needed to inform guidance on the impatient management of wasted children with diarrhoea, prevention of post-treatment relapse and mortality and to enable the optimisation of RUTF in treatment programmes. Variance in the priorities identified by experts from different regions demonstrate the need for research to inform context-specific approaches that can be applied to specific health systems and patterns of wasting. The results of this exercise have since been used to inform a global research agenda on the treatment of wasting. However, many critical gaps in knowledge remain. A coordinated research agenda across treatment and prevention is urgently needed to maximise the impact of funding investments towards the meeting of global targets to reduce child wasting.\n\n\nData availability\n\nLSHTM Data Compass: Treatment of child wasting: Child Health Research Initiative (CHNRI) prioritisation exercise dataset, https://doi.org/10.17037/DATA.0000188222.\n\nThis project contains the following underlying data:\n\n- Underlying data file 1: dataset (NWL-CHNRI-dataset) (restricted access)\n\n- Underlying data file 2: dataset description (NWL-CHNRI-dataset-codebook) (unrestricted access)\n\nDue to the fact that open posting of data on a repository was not included in the study information sheet at the time the survey was done, data access will be granted once users have consented to the data sharing agreement and provided written plans and justification for what is proposed with the data. Data access may be obtained by submitting a request to the No Wated Lives, Action Against Hunger authors via the LSHTM Data Compass repository. Requests will be reviewed by Action Against Hunger/ No Wasted Lives (they lead agency for this study) and key collaborators as named on the repository.\n\nLSHTM Data Compass: Treatment of child wasting: Child Health Research Initiative (CHNRI) prioritisation exercise dataset, https://doi.org/10.17037/DATA.0000188222.\n\nThis project contains the following extended data:\n\n- Extended data file 1: Participant introduction sheet (CHNRI-Introduction-2017)\n\n- Extended data file 2: Blank copy of survey (Surveymonkey-CHNRI-final- English)\n\n- Extended data file 3: Full list of research questions (CHNRI-wasting-treatment-research-questions)\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\nThe well recognised Child Health and Nutrition Research Initiative (CHNRI) method was followed in this exercise.", "appendix": "Acknowledgements\n\nThe CHNRI exercise documented in this article was led by the Council of Research & Technical Advice on Acute Malnutrition (CORTASAM). Full membership of the CORTASAM is as follows: André Briend, Elhadj Issakha Diop, Ferew Lemma, Marie McGrath, Mark Manary, Marko Kerac, Kerstin Hanson, Noël Marie Zagre, Purnima Menon, Robert Black, Steve Jarrett, Susan Shepherd, Tahmeed Ahmed and Zita Weise Prinzo.\n\nCHNRI collaborators participated in the survey that created the underlying data for this work. Those collaborators that agreed to be named in this paper are:\n\nMd Akhter, Lulseged Tolla, Marie McGrath, Amy Mayberry, Mahamane Laouali Manzo, Abdirahman Saeed, Fahdullah Shakir, Claude Chigangu, Elh Hallarou Mahaman, Concetta Brugaletta, Carlos Grijalva-Eternod, Facely Camara, Philip James, Marko Kerac, Jillian Emerson, Colleen Emary, Emily Keane, Thembekile Dhlamini, Amador Gomez, Indi Trehan, Mohamed Al-Ghaberi, Ahmed Salim, Beatrice Amadi, Handrea Njovo, Marina Kalisky, Eric Muthoka, Emily DeLacey, Abdullah Channa, Alison Talbert, Sophie Woodhead, Walton Beckley, Fekri Dureab, Kathryn Beck, Sarah Abdul Karim, Asif Soomro, Moikabi Matsoai, Alfred Zerfas, Abdul Rahman Kargbo, Souley Abdou, Richmond Aryeetey, Seydou Sangare, Victoria Sauveplane, Oluwaseun Ariyo, Kerri Wazny, Rogers Wanyama, Sheila Reed, Jessica Bliss, Mayom Atem, Regina Keith, Paul Kahitari, Jay Berkley, Ifeanyi Maduanusi, Abdulrashid Mohamed Omar, Kioko Kiamba, Sayed Muhib Shah Sadat, Boureima Kelly, Rhyan Buettner, Claire Orengo, George Waliomuzibu, Jerry Ogak, Sher Singh Dahit, Shemsa Msellem, Aboubacar Mahamadou, Arvind Singh, Solomon Abrha, Norbert Peshu, Ahmed Mohamed Abdullahi, Ali Hassan Maalim, Patricia Hoorelbeke, Marieme Diaw, Muhammad Ali Raza, Debashis Sinha, Babikene Rasi, Rabiu Umar, Susan Mhango, Anirudra Sharma, Moh Moh Hlaing, Eucabeth Onyango, Rodrigue Hervé Nguetsa Nguelemo, Benedikte Grenov, Sharon Cox, Pritish Kumar Nayak, Idda Katigula, André Briend, Samuel Murage, Ammar Altagani, Saeed Rahman, Lisa OBrien, Farah Vaughn, Abdinoor Hassan, Ben Allen, Neha Gehi Arora, Khushbu Gandhi, Fridah Mutea, Haile Girmay Samisie, Patrick Webb, Marylyne Malomba, Balkrishna Chaudhary, Amjad Ali Bacha, Ramesh Shrestha, Bridget Chiwaya, Maryan Dualle, Sanne Sigh, Ashiqullah Hassanzad, Mark Manary, Hussaini Usman, Endris Muhye, Michael Adedotun, Kemal J. Tune, Ashenafi Mamo, Alison Fleet, Andrew Masele, Thithidi Diaho, Abdillahi Ilmi Gueddi, Abdullahi Nor Mohamed, Collins John, Fitsum Minamo, Tanimoune Mahamadou, Dingase Mlewah, Hashim Jelle, Douglas Taren, Catherine Igoh, Shahzad Ajmal, Said Yassin, Mickey McRoberts, Abdirehman Yusuf Ali, Faruk Md. Omar, Layal Mohamad, Gihan Ahmad, John Phuka, Haoua Aouami, Zakayo Kimuge, Sunil Chohan, Nadeem Shahid, Natasha Lelijveld, Jaqueline Frize, Aminata Shamit Koroma, Cecile Basquin, Newman Dieyi, Mathias Altmann, Reuel Mungai Kirathi, Shraddha Manandhar, Anjili Midala, Tekar Jallah-Bundor, Md Bayazid Khan, Namukolo Covic, Murjanatu Kabir, Tatiana Gil, Elhadji Issakha Diop, Barada Mahapatra, Neil Stephen, Sharon Cox, Takele Teshome Demissie, Sinead O'Mahony, Kanda Traore, Urmila Deshmukh, Asnake Ararsa Irenso, Moses Cowan, Stefania Moramarco, Jogie Agbogan, Edwin Kangethe, Nathalie Avril, Alem Abay, Tsedey Beyene, Muhammad Mazhar Alam, Desta Zeweldi, Sunil Parajuli, Simon Karanja, Victoria Kaburu, Moe Zaw, Alfred Yambasu, Nagiba Al Mahdi, Jonathan Nsamba, Suruchi Gupta, Phuong Huynh, Eljaili Eltahir, Khalid Mohammed, Kennady Pulipati, Aida Hassan, Philomena Irene, Aliyu Hassan, Carolyne Kipkoech, Timon Choro, Ali Abdulkarim Ezedeen, Abdikani Mohamed, Kabir Mansur Fagge, Osman Abdi Mohamed, Abdi Moge Mohamed, Ajay Ciciliya, Arjan de Wagt, Wundow Oldman, Mara Nyawo, James Majaha, Robert Kawonga, Judith Kimambo, Ayobami Oyedeji, Ismail Younus Mohammed Ali, Durria Osman, Nader Makki, Moriah McArthur, Nancy Mock, Saadia Khan, Peter Hynes, Ritu Rana, Gloria Adobea Odei, Abigail Perry, Bernice Amadotor, Nicky Dent, Gloria Adobea Odei, Ellyn Yakowenko, Kemal Jemal Tunne, Mark Myatt, Samson Njagi, Hedwig Deconinck, Kajali Paintal, David Doledec, Alebel Yaregal Desale, Kebede Shitaye, Jasinta Hyachits Achen, Fesseha Demessie, Loraine Perraudin, Jean Claude Mahoro, Hamidine Hassane, Cheikh Mohamed El Hafed Dehah, Coffi René Mewatin, Hamidine Hassane, Macky Kyusa, Dominique Diarra, Rodrigue Magloire Eyma, Paluku Bahwere, Tamba Flavien Mamadouno, Chantal Autotte Bouchard, Mohamed Ali Darsa, Marion Junca, Abdillahi Ilmi Gueddi, Justin Mafuko, El Hadji Momar Thiam, Dieynaba Sophie N'Diaye, Issa Malam Kanta, Gaspard Kara, Oumarou Maidadji, Jean Claude Mahoro, Augustin Ndongmo Nanfack, François Stephan Tcheutchoua Noule, Cheikh Mohamed El Hafed Dehah, André Izacar Gael Bita, Ousmane Mouhamadou, Felicien Niada, Ousmane Ouedraogo, Florence M. Turyashemererwa, Richa Bisht, Silvia Barbazza and Barbara Bobba.\n\n\nReferences\n\nOlofin I, McDonald CM, Ezzati M, et al.: Associations of suboptimal growth with all-cause and cause-specific mortality in children under five years: a pooled analysis of ten prospective studies. PLoS One. 2013; 8(5): e64636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack RE, Victora CG, Walker SP, et al.: Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013; 382(9890): 427–51. PubMed Abstract \| Publisher Full Text\n\nGrantham-McGregor S, Baker-Henningham H: Review of the evidence linking protein and energy to mental development. Public Health Nutr. 2005; 8(7A): 1191–201. PubMed Abstract \| Publisher Full Text\n\nWells JCK: The capacity-load model of non-communicable disease risk: understanding the effects of child malnutrition, ethnicity and the social determinants of health. Eur J Clin Nutr. 2018; 72(5): 688–97. PubMed Abstract \| Publisher Full Text\n\nMcDonald CM, Olofin I, Flaxman S, et al.: The effect of multiple anthropometric deficits on child mortality: meta-analysis of individual data in 10 prospective studies from developing countries. Am J Clin Nutr. 2013; 97(4): 896–901. PubMed Abstract \| Publisher Full Text\n\nAnnan R, Webb P, Brown R: Management of moderate acute malnutrition (MAM): Current knowledge and practice. CMAM Forum Technical Brief. 2014. [cited 2020 13 Jun]. Reference Source\n\nDevelopment Initiatives: Global Nutrition Report 2018: Shining a light to spur action on nutrition. Bristol, UK. 2018. [cited 2020 13 Jun]. Reference Source\n\nFrison S, Angood C, Khara T, et al.: Prevention of child wasting: Results of a Child Health & Nutrition Research Initiative (CHNRI) prioritisation exercise. PLoS One. 2020; 15(2): e0228151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhutta ZA, Das JK, Rizvi A, et al.: Evidence-based interventions for improvement of maternal and child nutrition: what can be done and at what cost? Lancet. 2013; 382(9890): 452–477. PubMed Abstract \| Publisher Full Text\n\nKerac M, Mwangome M, McGrath M, et al.: Management of acute malnutrition in infants aged under 6 months (MAMI): Current issues and future directions in policy and research. Food Nutr Bull. 2015; 36(1 Suppl): S30–S34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGrath M, Shoham J: Editorial perspective on the continuum of care for children with acute malnutrition. Field Exchange. 2019; (60): 2. Reference Source\n\nKhara T, Mwangome M, Ngari M, et al.: Children concurrently wasted and stunted: A meta-analysis of prevalence data of children 6-59 months from 84 countries. Matern Child Nutr. 2018; 14(2): e12516. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRudan I: Setting health research priorities using the CHNRI method: IV. Key conceptual advances. J Glob Health. 2016; 6(1): 010501. PubMed Abstract \| Free Full Text\n\nRudan I, Gibson JL, Ameratunga S, et al.: Setting priorities in global child health research investments: guidelines for implementation of CHNRI method. Croat Med J. 2008; 49(6): 720–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: WHO Guideline: Updates on the management of severe acute malnutrition in infants and children. Geneva, World Health Organization. 2013. [cited 2020 13 Jun]. PubMed Abstract\n\nAlvarez JL, Dent N, Browne L, et al.: Putting Child Kwashiorkor on the map. CMAM Forum Technical brief. 2016. [cited 2020 13 June]. Reference Source\n\nBlanchet K, Sistenich V, Ramesh A, et al.: An evidence review of research on health interventions in humanitarian crises. 2015. [cited 2020 13 Jun]. Reference Source\n\nAngood C, Khara T, Dolan C, et al.: Research Priorities on the Relationship between Wasting and Stunting. PLoS One. 2016; 11(5): e0153221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngood C, McGrath M, Mehta S, et al.: Research Priorities to Improve the Management of Acute Malnutrition in Infants Aged Less Than Six Months (MAMI). PLoS Med. 2015; 12(4): e1001812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrudhon C, Maclaine A, Hall A, et al.: Research priorities for improving infant and young child feeding in humanitarian emergencies. BMC Nutr. 2016; 2: 27. Publisher Full Text\n\nWazny K, Sadruddin S, Zipursky A, et al.: Setting global research priorities for integrated community case management (iCCM): Results from a CHNRI (Child Health and Nutrition Research Initiative) exercise. J Glob Health. 2014; 4(2): 020413. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerac M, Angood C, Mayberry A, et al.: Treatment of child wasting: Child Health Research Initiative (CHNRI) prioritisation exercise dataset. London School of Hygiene & Tropical Medicine, London, United Kingdom. 2020. http://www.doi.org/10.17037/DATA.00001882\n\nMarron B, Onyo P, Musyoki EN, et al.: ComPAS trial in South Sudan and Kenya: Headline findings and experiences. Field Exchange. 2019; (60): 19. Reference Source\n\nDaures M, Phelan K, Issoufou M, et al.: New approach to simplifying and optimising acute malnutrition treatment in children aged 6-59 months: The OptiMA single-arm proof-of-concept trial in Burkina Faso. Br J Nutr. 2020; 123(7): 756–767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLelijveld N, Hendrixson DT, Godbout C, et al.: Defining and treating “high-risk” moderate acute malnutrition using expanded admission criteria (Hi-MAM Study): A cluster-randomised controlled trial protocol. Field Exchange. 2019; (60): 64. Reference Source\n\nStobaugh HC, Mayberry A, McGrath M, et al.: Relapse after severe acute malnutrition: A systematic literature review and secondary data analysis. Matern Child Nutr. 2019; 15(2): e12702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrijalva‐Eternod CS, Kerac M, McGrath M, et al.: Admission profile and discharge outcomes for infants aged less than 6 months admitted to inpatient therapeutic care in 10 countries. A secondary data analysis. Matern Child Nutr. 2017; 13(3): e12345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Essential nutrition actions: mainstreaming nutrition through the life-course. Geneva: World Health Organization. 2019. Reference Source\n\nHouston KA, Gibb JG, Maitland K: Intravenous rehydration of malnourished children with acute gastroenteritis and severe dehydration: A systematic review [version 1; peer review: 3 approved]. Wellcome Open Res. 2017; 2: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoonees A, Lombard MJ, Musekiwa A, et al.: Ready-to-use therapeutic food (RUTF) for home-based nutritional rehabilitation of severe acute malnutrition in children from six months to five years of age. Cochrane Database Syst Rev. 2019; 5(5): Cd009000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKangas ST, Salpéteur C, Nikièma V, et al.: Impact of reduced dose of ready-to-use therapeutic foods in children with uncomplicated severe acute malnutrition: A randomised non-inferiority trial in Burkina Faso. PLoS Med. 2019; 16(8): e1002887. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKangas ST, Kaestel P, Salpéteur C, et al.: Body composition during outpatient treatment of severe acute malnutrition: Results from a randomised trial testing different doses of ready-to-use therapeutic foods. Clin Nutr. 2020; 39(11): 3426–3433. PubMed Abstract \| Publisher Full Text\n\nYoshida S, Rudan I, Cousens S: Setting health research priorities using the CHNRI method: VI. Quantitative properties of human collective opinion. J Glob Health. 2016; 6(1): 010503. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "82098", "date": "13 Apr 2021", "name": "Carl Lachat", "expertise": [ "Reviewer Expertise nutrition" ], "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 provides the findings on research priorities for the treatment of child wasting. Although the manuscript is well written and informative, there are in my opinion, some additional elements that require consideration.\nThe focus of the exercise is quite narrow, restricted to the treatment options. Although it is acknowledged, it is important to provide perspective and highlight this bias early on. It would be informative for the reader to know how this exercise fits into a larger research agenda of optimal child nutrition, including options of prevention.\nThe largest area of concern for this manuscript is how the selection research options and criteria used to score them were determined. Although large efforts were made to ensure a wide representation to stakeholders in the appraisal of the research priorities against the criteria, it is not clear to the reader how the pre-selection of the research options and criteria was determined. How was a consensus at this initiatively stage achieved, how were dissenting views considered and how were participants selected for this purpose (with consideration of potential conflict of interest).\nFor instance, why was a decision made “to focus on areas of research that could each encompass several research studies, rather than specific research questions, in order to include as wide a scope of research needs as possible within an actionable survey.” Why was it decided to narrow down the list of options using this criterion before sending it out to the wider group? Why for instance not use this as a criterion for the wider group to score the research options? Some of these decisions obviously may have biased overall findings (e.g. a higher preference for operational aspects compared to for instance research on body composition).\nIn addition, the decision on the approach used (e.g. weighting of the criteria to compute the overall score) entails a decision on values and what is considered important for research. Did the smaller group that made the pre-selection consider such values e.g. by accommodating stakeholders (e.g. funders, decision-makers)? Researchers might not be well placed to decide on these values.\nFinally, a limitation of the approach used is the quantitative approach to scoring and listing of priorities. The difference between the options is essentially due to small differences in (sub)scores. Items are ranked based on numerical scores, which essentially reflect a qualitative appraisal. Using this approach, each criterion is given an equal weight to arrive at the final score. How was this decided? and was this clear to the participants who scored the options? How different criteria are weighed and combined is essentially a value-driven decision that might not have been captured well by the consultation process. Although stakeholders (e.g. funding agencies) that need to act on the research priorities provided may have a different value framework to decide on what to select as a priority for action.\nTo illustrate: can a research option (number 4) with an overall score of 89.7 be really considered a higher priority compared to an option (number 5) that was scored 89.5 even if that option had a higher score for sustainability and effectiveness? The absolute ranking might be misleading to the reader in this sense. A more qualitative presentation of the overall leading list of research priorities (without exact ranking) might have been more appropriate.\nThe practical aspects of the CHNRI approach are clear but a critical discussion on the limits of this approach and the potential bias for the overall findings would be informative to the reader.\nLastly, the paper aims to inform funding on research. Before funding new research, however, it would be necessary to review the literature on the topic. Several of the proposed research questions have been addressed in studies. It might be that participants considered the research options as priorities but, before recommending funding, a careful account of existing evidence and knowledge gaps is necessary. It would be good to clarify that funding might also be necessary to take stock of existing evidence on the topic.\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": "97960", "date": "14 Dec 2021", "name": "Noemí López-Ejeda", "expertise": [ "Reviewer Expertise Nutritional 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\nI believe that the paper is of great interest to implementers, policy-makers, and academics involved in the finding of better ways to manage acute malnutrition. Many of my comments or concerns have already been brought out by the other reviewer of the paper, and I will add only a few lighter aspects that have not been mentioned.\nI consider that the objective of the article is well justified and relevant. The methodology is adequate and well described. The results are described in sufficient detail although there is some redundancy between the info in Figure 2 and the text. I recommend the authors redo this sentence so that it is a more general summary of the information (e.g., Most of the respondents belonged to XXX and with a low percentage of XXX).\n\nIn the methodology section, 2nd paragraph, the authors write 'A decision was made to focus predominantly on the treatment of wasting in children under five years of age, including aspects of prevention only where explicitly linked to treatment'. Could the authors clarify what is meant by \"aspects of prevention only where explicitly linked to treatment\" (perhaps by explicitly adding some examples of such prevention activities that were included as options in the survey).\nThe authors use different labels as \"extended data file\" or \"supplementary material\". It would be convenient to unify the naming of all those supplementary files and also reorder them according to the order of appearance in the main text (the first one mentioned is extended data file 3, this should be 1).\nIn general, the discussion is pertinent, with adequate and recent bibliography. However, given that the data collection was in 2017 and the paradigm shift after the COVID-19 crisis, I think it would be helpful if the authors to reflect further on the applicability of these results to the current context (where the pandemic has accelerated the implementation of simplified protocols and there are documents from international agencies that already review the implementation of these activities that have been identified as priorities).\n\nOtherwise, I believe that the paper is of great interest to the journal's readers and after those clarifications (and that from the other reviewer) the article will meet the required scientific and writing quality to be approved.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-126
https://f1000research.com/articles/10-124/v1	17 Feb 21	{ "type": "Case Report", "title": "Case Report: Ziprasidone induced neuroleptic malignant syndrome", "authors": [ "Yub Raj Sedhai", "Alok Atreya", "Prabin Phuyal", "Soney Basnyat", "Sagar Pokhrel", "Yub Raj Sedhai", "Prabin Phuyal", "Soney Basnyat", "Sagar Pokhrel" ], "abstract": "Neuroleptic malignant syndrome (NMS) is a well-recognized neurologic emergency. It presents with classic features including hyperthermia, autonomic instability, muscle hypertonia, and mental status changes. The syndrome is potentially fatal and is associated with significant morbidity due to complications such as rhabdomyolysis, acute kidney injury, and ventricular arrhythmias due to the trans-cellular electrolyte shift. NMS is conventionally associated with the first-generation antipsychotic agents, however, has been described with the use of atypical and novel antipsychotics including Ziprasidone. A case of NMS with Ziprasidone use at the therapeutic dose is reported here.", "keywords": [ "Neuroleptic Malignant Syndrome (NMS)", "Neurologic emergency", "Parkinson’s disease", "Ziprasidone" ], "content": "Introduction\n\nNeuroleptic malignant syndrome (NMS) is a well-recognized neurologic emergency associated with antipsychotic (neuroleptic) drugs. NMS is characterized by hyperthermia, autonomic instability, severe muscle hypertonia, and mental status changes1. NMS has been conventionally associated with typical antipsychotics like Haloperidol and Fluphenazine1. However, recent studies have shown that every class of antipsychotic can cause NMS including low potency agents like Chlorpromazine, and second-generation antipsychotics like Clozapine, Risperidone, and Olanzapine, and antiemetics like Metoclopramide and Promethazine2,3. NMS results in muscle breakdown, leading to potential sequelae of rhabdomyolysis and acute kidney injury4. Muscle breakdown results in a large transcellular shift which can result in ventricular arrhythmias and can be potentially fatal with a reported mortality of 10–20%4. NMS can result from the use of high-potency or depot formulations of antipsychotics, however, clinicians should be well aware that the use of parenteral antipsychotics for sedation and control of agitation is an important cause of NMS in the inpatient setting5. Intravascular volume depletion and dyselectrolytemia are important risk factors of NMS5. Herewith we present a case of an elderly male, with a history of parkinsonism, who was admitted with toxic metabolic encephalopathy related to urinary tract infection (UTI). He developed NMS after administration of Ziprasidone for control agitation and combative behavior.\n\n\nCase presentation\n\nA 78-year-old male was admitted for urinary tract infection complicated with toxic metabolic encephalopathy. He had past medical history of parkinsonism along with three vessel coronary artery bypass graft (CABG). He was on prescription medications including levodopa/carbidopa. On the night of hospitalization, he developed agitation and combative behavior which was treated with several doses of intramuscular Ziprasidone. He received a total of 30 mg intramuscular Ziprasidone over 12 hours. The following morning the on-duty nurse noted the patient to have generalized muscular rigidity. Assessment of vital signs revealed the temperature of 107° F, heart rate of 120 beats per minute, blood pressure 160/90 mmHg, with oxygen saturation of 94% on room air. A rapid response alert was called.\n\nOn further evaluation, he had generalized muscular rigidity, involving both flexor and extensor compartments in the bilateral upper and lower extremity, muscles of the abdominal wall, back, neck, and jaw suggestive of generalized hypertonia. He had diaphoresis with hyperthermia. Neurologic examination, revealed unresponsiveness, with no eye-opening in response to pain, no verbal response, and no motor response accounting for a Glasgow coma scale of three. Examination of the cardiovascular system revealed sinus tachycardia and was otherwise unremarkable. Chest auscultation revealed minimal mid-inspiratory crackles at the base. Systemic examination was otherwise unremarkable.\n\nEmergent bedside electrocardiography (ECG) was performed, which revealed a QTc of 458 milliseconds (Figure 1). ECG also revealed symmetrical T-wave inversions in leads V1 through V6, which was chronic changes related to his underlying coronary disease. Laboratory evaluation revealed, elevated CPK measuring 18000 units/L, Troponin I measuring 7.12 ng/ml. Serum calcium was 13 mg/dL, serum creatinine of 1.6 mg/dL, bicarbonate of 17 millimole/L with the lactate of 4 millimole/L. Metabolic parameters including serum sodium, potassium, magnesium, phosphate, and liver function panels were within normal limits.\n\nImminent airway compromise with worsening hypertonia was prevented with rapid sequence endotracheal intubation. Considering NMS as the most pertinent differential diagnosis, the patient was treated with intravenous dantrolene sodium at a dose of 1 milligram per kg body weight. Other supportive measures included volume resuscitation with intravenous fluid boluses along with acetaminophen and external cooling blankets for temperature control and intravenous lorazepam for agitation.\n\nAfter initial stabilization alternative causes of acute change in mental status with generalized hypertonia and hyperthermia were considered. Emergent computed tomography of the brain was negative for acute intracranial bleed, there were no signs of elevated intracranial pressure, midline shift, or hydrocephalus. CNS infection including meningoencephalitis was evaluated with lumbar puncture and cerebrospinal fluid (CSF) analysis. CSF analysis revealed normal protein, glucose, RBCs, and WBCs. CSF was negative for cryptococcal antigen, herpes simplex virus DNA, and west-Nile virus. Brain imaging with MRI could not be performed due to metallic artifacts from a prior bullet injury. The patient did not have a preceding history of seizure before hospitalization and did not have a seizure-like activity after hospitalization. Electroencephalography (EEG) with 24-hour video EEG monitoring was negative for epileptiform activity. As the patient had elevated troponin I, with previous history of CABG, transthoracic echocardiography was performed, which was negative for new regional wall motion abnormality. Thus, elevated troponin was attributed to skeletal muscle sources.\n\nDuring hospitalization, generalized muscle rigidity and other signs of NMS continued to improve. Anti-dopaminergic agents were avoided and levodopa/carbidopa for parkinsonism was resumed. There was an elevation in serum creatinine consistent with acute kidney injury stage II which was likely secondary to rhabdomyolysis. It was treated conservatively with volume expansion. He did not require renal replacement therapy, and renal function continued to resolve back to baseline. He was extubated on the fourth day of ICU admission. Urinary tract infection, which was his initial admitting diagnosis, was treated with a five-day course of intravenous ceftriaxone based on the pan-sensitive Escherichia coli on urine culture. He had an otherwise uneventful clinical recovery except for generalized weakness from deconditioning likely related to skeletal muscle injury from NMS related rhabdomyolysis. He was discharged to a subacute rehabilitation facility for post ICU rehabilitation.\n\nThe patient was followed up at the primary care internal medicine clinic and neurology clinic two weeks after hospital discharge. He was noted to have a significant improvement in generalized weakness/deconditioning. Renal function recovered back to baseline. He did not have any worsening of his parkinsonian symptoms including tremor, rigidity, and bradykinesia.\n\n\nDiscussion\n\nZiprasidone is a novel atypical antipsychotic drug that acts as an antagonist at human dopamine D2 receptors and serotonin, 5-HT2A, receptors and an agonist at human serotonin, 5-HT1A, receptor, and also selectively inhibits the reuptake of norepinephrine and serotonin in the presynaptic cell6. Clinical studies have shown Ziprasidone as a well-tolerated drug with high efficacy in the treatment of schizophrenia and schizoaffective disorder6,7. Additionally, the intramuscular use of Ziprasidone has shown good efficacy in the treatment of acute agitation6. It commonly causes adverse effects such as headache, nausea, or drowsiness and rarely been reported to cause extrapyramidal symptoms and QTc prolongation7.\n\nThe systematic review of antipsychotics-induced NMS showed that Ziprasidone-induced NMS occurs abruptly displaying typical symptoms of NMS such as mental status changes, hyperpyrexia, dysautonomia features such as diaphoresis, tachycardia, and fluctuating blood pressure, leukocytosis, and tremor4. The diagnostic criteria set by the DSM-IV includes the presence of hyperpyrexia and severe muscle hypertonia following exposure to neuroleptics and the presence of two or more of the following features: tremor, dysphagia, diaphoresis, altered mental status, incontinence, tachycardia, mutism, labile blood pressure, elevated creatine kinase, and leukocytosis8. Following exposure to repetitive doses of intramuscular Ziprasidone, our patient developed hyperthermia, severe muscle hypertonia, and had tachycardia, tachypnea, diaphoresis, altered mental status, elevated creatine kinase, and elevated troponin, meeting criteria for the diagnosis of the neuroleptic malignant syndrome.\n\nNMS is well recognized with typical antipsychotics; Ziprasidone induced NMS is uncommon and evidence is limited to case-reports9–12. The classical symptoms such as hyperthermia, muscle rigidity, altered mental status, diaphoresis, tremor, stupor, tachycardia, tachypnea, and diaphoresis, were common in all these cases. The onset of NMS varied among these cases, ranging from abrupt onset to onset after eight weeks of Ziprasidone use. The occurrence of NMS due to antipsychotic drug use is idiosyncratic. The symptoms may appear anytime following exposure to antipsychotic drugs, occurring after exposure to single-dose to occurring after exposure to the same dose of an antipsychotic drug for years13. Despite the diagnostic criteria, Ziprasidone-induced NMS can be diagnostically challenging as it oftentimes presents with atypical clinical features.\n\nThe exact pathophysiologic mechanism behind the development of NMS is unclear, but the blockade in dopamine receptors induced by antipsychotics use is believed to play a crucial role in precipitating the syndrome1,4. This theory is supported by numerous shreds of evidence, such as NMS has been reported with the use of drugs with dopamine blockade properties or with the withdrawal of dopamine receptor agonists. The hypothalamic dopamine blockade results in hyperthermia and dysautonomia while the nigrostriatal pathway blockade results in extrapyramidal symptoms such as tremors and muscle hypertonia. It is also believed that the muscle hypertonia and the rhabdomyolysis occur from a direct alteration in the skeletal muscle’s mitochondrial function from the use of antipsychotics14. Sympathoadrenal dysfunction is also believed to have contributed to the development of the clinical features of NMS, as an increased adrenergic and serotonergic activity is associated with the NMS and elevated catecholamine levels have been found in NMS cases15. Our patient was showing combative behavior, agitated, and had altered mental status. The possible development of dehydration and physical exhaustion from his combative behavior, which are the risk factors for the development of NMS5, probably had played a role in the precipitation of the NMS following administration of Ziprasidone. Although there isn’t a particular diagnostic test for NMS, and laboratory tests can help confirm the diagnosis, rule out other differentials, and monitor patients for potential complications from NMS. Although NMS is a nearly fatal condition, no fatal or lethal outcome has yet been reported with Ziprasidone-induced NMS4.\n\nIt is important to recognize NMS as it is associated with significant morbidity and can occur abruptly after the start of antipsychotic agents such as Ziprasidone. Increasing pieces of evidence advocate the use of antipsychotics such as Ziprasidone for delirium although clinical trials have shown questionable benefits16. So, clinicians should be aware of the potentially life-threatening adverse reaction such as NMS and should remain vigilant to identify NMS anytime, if occurs, throughout the use of conventional antipsychotic drugs and novel agents such as Ziprasidone.\n\n\nConclusion\n\nNeuroleptic malignant syndrome (NMS) is a potentially fatal reaction that can result from the use of atypical antipsychotic agents such as Ziprasidone. NMS is associated with significant morbidity and mortality from rhabdomyolysis, acute renal failure, and sepsis. Increasing body of evidence advocate for the use of Ziprasidone in psychiatric conditions like schizophrenia, bipolar disorder, and delirium, so, clinicians should be aware of the NMS associated with use of Ziprasidone.\n\n\nConsent\n\nWritten informed consent was obtained from the patient’s son for publication of this case report and accompanying images because the patient was unable to provide consent and the son was next of kin.\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\nStrawn JR, Keck PE Jr, Caroff SN: Neuroleptic malignant syndrome. Am J Psychiatry. 2007; 164(6): 870–6. PubMed Abstract \| Publisher Full Text\n\nCaroff SN, Mann SC: Neuroleptic malignant syndrome. Med Clin North Am. 1993; 77(1): 185–202. PubMed Abstract \| Publisher Full Text\n\nKogoj A, Velikonja I: Olanzapine induced neuroleptic malignant syndrome--a case review. Hum Psychopharmacol. 2003; 18(4): 301–9. PubMed Abstract \| Publisher Full Text\n\nMurri MB, Guaglianone A, Bugliani M, et al.: Second-Generation Antipsychotics and Neuroleptic Malignant Syndrome: Systematic Review and Case Report Analysis. Drugs R D. 2015; 15(1): 45–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerardi D, Amore M, Keck PE Jr, et al.: Clinical and pharmacologic risk factors for neuroleptic malignant syndrome: A case-control study. Biol Psychiatry. 1998; 44(8): 748–54. PubMed Abstract \| Publisher Full Text\n\nGunasekara NS, Spencer CM, Keating GM: Ziprasidone: A review of its use in schizophrenia and schizoaffective disorder. Drugs. 2002; 62(8): 1217–51. PubMed Abstract \| Publisher Full Text\n\nCaley CF, Cooper CK: Ziprasidone: The fifth atypical antipsychotic. Ann Pharmacother. 2002; 36(5): 839–51. PubMed Abstract \| Publisher Full Text\n\nBerman BD: Neuroleptic Malignant Syndrome: A Review for Neurohospitalists. Neurohospitalist. 2011; 1(1): 41–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGray NS: Ziprasidone-related neuroleptic malignant syndrome in a patient with Parkinson’s disease: A diagnostic challenge. Hum Psychopharmacol. 2004; 19(3): 205–7. PubMed Abstract \| Publisher Full Text\n\nLewis AL, Pelic C, Kahn DA: Malignant catatonia in a patient with bipolar disorder, B12 deficiency, and neuroleptic malignant syndrome: One cause or three? J Psychiatr Pract. 2009; 15(5): 415–22. PubMed Abstract \| Publisher Full Text\n\nOzen ME, Yumru M, Savas HA, et al.: Neuroleptic malignant syndrome induced by ziprasidone on the second day of treatment. World J Biol Psychiatry. 2007; 8(1): 42–4. PubMed Abstract \| Publisher Full Text\n\nStrawn JR, Keck PE Jr: Early bicarbonate loading and dantroline for ziprasidone/haloperidol-induced neuroleptic malignant syndrome. J Clin Psychiatry. 2006; 67(4): 677. PubMed Abstract \| Publisher Full Text\n\nPope HG Jr, Aizley HG, Keck PE Jr, et al.: Neuroleptic malignant syndrome: long-term follow-up of 20 cases. J Clin Psychiatry. 1991; 52(5): 208–12. PubMed Abstract\n\nAdnet P, Lestavel P, Krivosic-Horber R: Neuroleptic malignant syndrome. Br J Anaesth. 2000; 85(1): 129–35. PubMed Abstract \| Publisher Full Text\n\nSpivak B, Maline DI, Vered Y, et al.: Prospective evaluation of circulatory levels of catecholamines and serotonin in neuroleptic malignant syndrome. Acta Psychiatr Scand. 2000; 102(3): 226–30. PubMed Abstract \| Publisher Full Text\n\nGirard TD, Exline MC, Carson SS, et al.: Haloperidol and Ziprasidone for Treatment of Delirium in Critical Illness. N Engl J Med. 2018; 379(26): 2506–2516. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "79895", "date": "05 Mar 2021", "name": "Prajwal Ghimire", "expertise": [ "Reviewer Expertise Neurological disease", "Neurosurgery" ], "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 presented a rare case report of a well recognised drug induced neurologic emergency of Neuroleptic malignant syndrome due to Ziprasidone. Sedhai et al. have highlighted major challenges and salient points during management of these conditions including the current knowledge regarding its pathophysiology. The case report raises the awareness regarding this potentially life-threatening condition during use of an emerging drug which is now more commonly used for neuro-psychiatric conditions of schizophrenia and bipolar disorders.\nThe case report is well written and highlights the current knowledge and brief literature review in the discussion section with relevant references. It certainly adds a vital information regarding the drug to the current available knowledge in the literature.\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": "6438", "date": "11 Mar 2021", "name": "Alok Atreya", "role": "Author Response", "response": "Dear Dr. Ghimire, We appreciate your time you took to go through our article and write such a detailed review. We would further thank you for your positive and encouraging comments on our article." } ] }, { "id": "79894", "date": "09 Mar 2021", "name": "Ashish Saraf", "expertise": [ "Reviewer Expertise Forensic medicine", "Clinical Forensic Medicine", "Forensic Science." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is well written. The authors have given detailed description of the case mentioning the clinical features, the diagnostic workup and treatment given. The other causes of the rigidity have been ruled out during the diagnostic workup. The discussion is also well written and highlighted the importance of this case report.\n\nThis case report will definitely make the clinicians aware of the fact of NMS in newer drugs and hence making them vigilant.\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": "6439", "date": "11 Mar 2021", "name": "Alok Atreya", "role": "Author Response", "response": "Dear Dr. Saraf, We thank you for your time to go through our article. We further thank your for highlighting the important findings and positive comments. We are really encouraged." } ] } ]	1	https://f1000research.com/articles/10-124
https://f1000research.com/articles/10-122/v1	17 Feb 21	{ "type": "Research Article", "title": "Magnitude of parasitic infections and associated factors among pregnant women at health facilities in Hawassa, Southern Ethiopia", "authors": [ "Demelash Wachamo", "Fisseha Bonja", "Bamlaku Tadege", "Siraj Hussen", "Fisseha Bonja", "Bamlaku Tadege", "Siraj Hussen" ], "abstract": "Background: Intestinal parasitic infections (IPIs) are common problems during pregnancy, with adverse outcomes including low birth weight and prenatal mortality. The burden of parasitic infections and its impacts are high among pregnant women in developing countries like Ethiopia. Therefore, this study aimed to assess the burden and associated factors of parasitic infections. Methods: A facility-based cross-sectional study was conducted among 365 randomly selected women attending antenatal clinic at five selected health facilities. Data was collected by a pre-tested questionnaire and stool specimens were collected in clean plastic containers. A combination of direct microscopy and the formol-ether concentration technique was used as soon as the specimen collected. Data entry and analysed for descriptive and logistic regression models by SPSS v.23. The result declared as statistically significant at p < 0.05.\n\nResults: The overall prevalence of IPI was 161 (45.9%). The most frequently identified parasites were Ascaris lumbricoides (27.9%), Schistosoma species (13.7%), Trichuris trichiura (5.1%), Hookworm (4.8%), and Taenia species. (1.4%). The IPIs were associated with women having no formal education [AOR=2.19, 95% CI: 1.05-4.57] or elementary school education [AOR=1.90, 95% CI: 1.11-3.27], as compared with high school educated and above. Monthly income of less than 1920 Ethiopian birr [AOR=2.06, 95% CI: 1.28-3.31], sharing a latrine with neighbours [AOR=1.83, 95% CI: 1.14-2.93], using lake water for washing clothes [AOR=2.24, 95% CI: 1.34-3.74], habit of eating raw vegetables [AOR=2.26, 95% CI: 1.30-3.92] were associated with IPI as compared to their counterparts. Conclusions: Nearly half of the pregnant women were infected with IPs. The health facilities and clinicians need to focus on prevention of IPIs by early diagnosis, treating lake water before use, promote proper latrine utilization and provision of pertinent health education as part of ante-natal care service. It is important to minimize the impact of IPIs on pregnant women and their child.", "keywords": [ "Intestinal Parasitic Infection", "Pregnant Women", "Hawassa", "Southern Ethiopia" ], "content": "Introduction\n\nIntestinal parasitic infections, mainly soil-transmitted helminths, and water-related parasitic diseases are is the common problem during pregnancy in low- and middle-income countries (LMICs)1. More than 50% of pregnant women in LMICs suffer from anaemia, and low birth weight and prenatal mortality2. Ascaris lumbricoides, Trichuris trichiura, and hookworms are the most common causes of intestinal parasitic infections (IPIs), which affect more than 2 billion people worldwide3. The prevalence and impacts of these parasites are high among pregnant women in developing countries like Ethiopia4,5. IPIs were the second most predominant cause of outpatient morbidity in the LMICs, particularly in sub-Saharan Africa including Ethiopia6,7.\n\nThose in developing countries are more likely to suffer from IPIs due to a lack of resources, poverty, low literacy rates, lack of safe drinking water, poor hygiene, malnutrition and hot and humid tropical climate8. Many pregnant women in developing countries are more vulnerable to IPIs due to poverty. In addition to this, they can’t afford a safe water supply, shoes, nutrition or sanitation practices7,9.\n\nHowever, the Transformation Plan, the Health Sector Transformation Plan, the Neglected Tropical Diseases (NTDs) Master Plan, the National Hygiene and Environmental Health Strategy, and the targets of the One WASH National Programme working to reduce NTDs or parasitic disease10. Even though nationally representative and comprehensive data regarding the magnitude of prevalence of IPIs among pregnant women lack in Ethiopia, There is high burden of parasitic infection in Ethiopia; some regional studies have shown the prevalence ranging from 27% to 70.6% in Ethiopia4,11. The main identified IPIs are Ascaris lumbricoides, Hookworms, Trichuris trichiura and Schistosoma mansoni12. Therefore, it is crucial to identify the magnitude of IPIs and their determinant factors to reduce the burden and impacts of these parasites on pregnant women and their children. This is important for researchers, clinicians, and health planners.\n\nThis study sought to assess the magnitude of intestinal parasitosis in the study area, which of the identified species was the most dominant, and which of the risk factors are pregnant women mainly exposed to for intestinal parasitosis in the study area.\n\n\nMethods\n\nA facility-based cross-sectional study was conducted among pregnant women attending antenatal care (ANC) clinics at five selected health centres in Hawassa, in Southern Ethiopia from September 01, 2019, up to Jan 30, 2020, which is one of the most densely populated areas in Ethiopia. The source of the population was all pregnant women who visited five selected health centres. All pregnant women attending ANC clinics within the study period were considered as the study population. Randomly selected pregnant women who attend ANC clinics at five randomly selected functional selected health centres, who gave informed consent and were resident in the study area were included in the study. While, those who had been undergoing anti-helminth treatment for the last one month, and those who were seriously ill during the study period were excluded from the study.\n\nThe required sample size (n) was 365, calculated by using single population proportion formula with the assumptions: by taking previously conducted prevalence of IPIs (p) (31.5%) among pregnant women attending ANC at Felege Hiwot Hospital, northwest Ethiopia9, with 95% CI (1.96), 5% margin of error (d), and addition of 10% contingency:\n\n\n\nThe final sample size was adjusted as follows:\n\nSample size = n (sample size) + (10% non-respondent)\n\nThus, final sample size (n) was calculated as n= 331.57 + 33.2 = 364.77 ≈ 365.\n\nThe random sampling technique was used to select five public health facilities among 12 functional health facilities with ANC service. The desired number of clients for each health facility was selected based on proportional sampling. The study participants were selected from each facility were by the random arrival of the pregnant women to an ANC clinic.\n\nThe dependent variable of the study was the magnitude of IPIs, it was determined by a combination of direct microscopy and the formol-ether concentration technique (diagnosed at least one or more parasitic infections). Independent variables measured in the study were socio-demographic and economic variables (such as; age, sex, marital status, educational level, occupation, income), environmental and behavioural characteristics (housing condition, ownership and type, waste refuse, drinking water availability, living with domestic animal and pets, and latrine utilization, hand washing practice, and utilization of the lake water).\n\nData was collected by using a pre-tested questionnaire to obtain socio-demographic information and pregnancy-related factors (available as Extended data)13. The English questionnaires were translated to local language by another expert using properly designed and pretested questionnaire. The questionnaires were pre-tested and validated two weeks before in the study time on 5% of ANC attendants at a health center which was outside of the study area. Finally, some modifications on sequence or arrangement of multiple answer questionnaire and missed information were edited. The data were collected by five trained nurses educated to diploma level, five Laboratory technicians and supervised by two nurses educated to BSc level. Data was collected after explaining the objective of the study, rights and responsibilities of giving information and ascertain their confidentiality to minimize information bias. Midwifery nurses who can speak the local language were trained for data collection procedures to attain standardization and maximize interviewer reliability. In addition to this, stool specimens were collected with clean plastic containers. Experienced and trained laboratory technologists assessed the samples to ensure the quality of the investigation. A combination of direct microscopy and the formol-ether concentration technique was used as soon as the specimen collected. The data collection, application of the standard procedure, accuracy of test results was supervised by principal investigators. Some specimens were taken for cross-checking of the accuracy of laboratory results. Filled questionnaires were collected after checking for consistency and completeness.\n\nEthical clearance was obtained from Hawassa University College of Medicine and Health sciences and conducted in accordance with the Declaration of Helsinki and was approved by an institutional review board or ethics committee. Support letter was obtained from the Hawassa city health department. All participants were informed about the purpose, risks, benefit and confidentiality issues related to the study. Participation was on voluntary basis and written informed consent (verbal consent for who cannot read and write respondent) was obtained from each participant. All identified parasites were treated according to the guidelines of the NTD program of Ethiopia10.\n\nData entry, cleaning, and analysis were done in SPSS v.23. Descriptive analysis including frequency distribution and the percentage was made to determine the magnitude of IPIs, to describe socio-demographic and clinical characteristics. All factors with a p-value <0.25 in the bivariate logistic regression analysis were a candidate to the multivariable model to control confounding effects. The Hosmer -Lemeshow goodness-of-fit statistic was used to assess whether the necessary assumptions for the application of multiple logistic regression are fulfilled. Odds ratios (OR) with 95% confidence intervals (CI) were calculated. A p-value<0.05 indicated a significant association.\n\n\nResults\n\nA total of 365 pregnant women attending ANC clinic were enrolled in the study. A total of 351 participants were interviewed, yielding a response rate of 96.16%. The age range of the participants was 18–39 years, with the mean (±SD) age of 29.72 (±6.329). Majority of study the participants 313 (89.2%) were married. Regarding educational status, 53 (15.1%) had no formal education whereas 166 (47.3%) had high school education or above. Considering the occupational status of the participants, 140 (39.9%) were unemployed. For the monthly income of the households, 200 (57.0%) earned between 300–1920 ETB, and 151 (43.0%) earned more than 1920 ETB (Table 1). Individual-level results are available, see Underlying data13.\n\nIncome based on (HCE, 2016) Exchange rate 1 USD to 29.3673ETB.\n\nA majority of participants (270, 76.9%) were living in a mud-floored house. A total of 188 (53.6%) had a latrine at a household level, and a further 163 (46.4%) shared a latrine with neighbours. The major source of drinking water was pipe water for 339 participants (96.6%). About, 92 (26.2%) still living with domestic animals and pets in the same room. More than half 189 (53.8%) had no handwashing facility to use after using the toilet and 241 (68.7%) were in the habit of eating raw vegetables (Extended data, Table S1)13.\n\nThe overall number of pregnant women with one or more IPIs was 45.9%, [95% CI: 40.7-51.1] (Extended data, Figure S1)13. The major identified IPs were A. lumbricoides (n=98, 27.9%), Schistosoma species (n=48, 13.7%), T. trichiura (n=18 5.1%), Hookworm (n=17, 4.8%), and Taenia species (n=5, 1.4%) (Figure 1).\n\n(n=351).\n\nIn multivariate analysis, the education status of respondents, average monthly income, ownership of the latrine, using lake water for washing clothes and habit of eating raw vegetables remained as the determinant of IPIs. The prevalence of IPIs was associated with having no formal education [AOR=2.19, 95% CI: 1.05-4.57] and elementary school-level education [AOR=1.90, 95% CI: 1.11-3.27] as compared with who had high school and above educational status. Having a household monthly income of less than 1920 ETB [AOR=2.06, 95% CI: 1.28-3.31], sharing a latrine with neighbours [AOR=1.83, 95% CI: 1.14-2.93], using lake water for washing clothes [AOR=2.24, 95% CI: 1.34-3.74] and being in the habit of eating raw vegetables [AOR=2.26, 95% CI: 1.30-3.92] were associated with having IPIs when compared to their counterparts (Table 2).\n\n*p<0.05. COR: crude odds ratio, AOR: adjusted odds ratio, CI: confidence interval, 1: reference.\n\n\nDiscussions\n\nThis facility-based cross-sectional study revealed that the magnitude of intestinal parasitic infection (IPIs) was 45.9%, [95% CI: 40.7 - 51.1] among pregnant women attending Ante-natal care clinic (ANC). The major identified IPs were A. lumbricoides (27.9%), Schistosoma spp. (13.7%), T. trichiura (5.1%), Hookworm (4.8%) and Taenia spp. (1.4%). This study result indicates that 45.9% were found to be infected by one or more IPIs among pregnant women attending ANC. This study result was consistent with the study findings in Nigeria (48.3% infected)14, Bogota, Colombia (41%)7 and in Lalo Kile district, Oromia, Western Ethiopia (43.8%)15. This result was higher when compared to a figure of 13.8% in Kenya16, 12% in another study in Osun state, Nigeria17, 38.7% in Southern Ethiopia5, 21.1% in Northwest Ethiopia18, 31.5% in Northwest Ethiopia9 and 24.7% in the Oromia Region, Ethiopia19. The value in the present study was lower when compared to 70% in a study on the Thai-Burmese border8, 76.2% in Kenya20 and 65% in Gabon21. The difference in findings among various studies could be explained variations in geography, socio-economic condition and cultural practices of study participants.\n\nThis study result revealed that the pregnant women who had no formal education were more likely to be exposed to IPIs when compared with those that had high school education and above. This finding was also similar to in Kampala, Uganda22 and northwest Ethiopia9. This may due to a lack of health-related information about prevention, early symptoms and health benefits. In addition to this, those with a household monthly income, less than 1920 ETB was more exposed to IPIs than to their counterparts. This result agrees with another from Western Ethiopia15. This might due to household income directly related to the nutritional status and health status of the individuals.\n\nThe prevalence of IPIs was associated with sharing a latrine with neighbours, Habit of eating raw vegetables were more exposed to IPIs when compared to their counterparts. This finding also agreed with that of studies conducted in Kampala, Uganda22, in Northwest Ethiopia9 and in Lalo Kile district, Oromia, Western Ethiopia15. This may due to the sharing latrine with neighbours, no handwashing facility or the absence of proper utilization of latrine and eating uncooked vegetables increases the exposure of the IPIs. This implies that strengthen health education on the proper health education schedule for the ANC attendants and for the community on the proper utilization of the latrine.\n\nThis study result shows that using lake water for washing clothes was associated with parasitic infections among pregnant women. A study from Tanzania's Lake Victoria region showed similar results23. Furthermore, Hawassa city was surrounded by lake and most of the low-income pregnant women mainly use for laundry and bathing. It was also used as a source of drinking water in few rural areas. This may lead to greater exposure to schistosomiasis. As such, needs education to change the habits or safe utilization of the lake water.\n\nThis study result shows, there was a high prevalence of IPIs; this may lead to causation or aggravation of anaemia via multiple interacting mechanisms24. Hookworms, schistosomiasis and Trichuris trichiura lead to intestinal blood loss and reduce appetite and compromise nutrient intake15,25. Further helminthiasis-inducted intestinal inflammation may limit the absorption of nutrients. This may due to the fact there is an endemic intestinal parasite in the area which exposes the patient to daily activities. The pregnant women had greater roles in the household, which exposes them to waste refuse, gutters and sewage handling. Which aggravated by low income status or economic dependency, low educational status, living condition, and other behavioural related factors and due to the poor level of health-seeking behaviour of the study participants.\n\nThis study result indicates that prevention and control activities need to address early detection of IPIs for better prevention, evaluation, and management. In addition to this, health officials and providers needs to collaborate to implement health education and promotion to minimize the impact of the IPIs. This study result also indicates that implementing and strengthening community-based pre-pregnancy care and preparation programs as health extension packages may improve the health of the pregnant women. Government bodies and other stakeholders should work together to improve the household wealth status, create job opportunities for women and maintain affirmative actions for women’s employment. It also needs better strategies to strengthen women's education and employment in the study area. The integration of qualitative research may explore more factors for the future with studies of mixed design at community level.\n\nThis study had some potential limitations that might have led to information bias on the part of the respondents on self-reportable risk factors. The study may not establish a causal relationship as we have implemented a cross-sectional study design.\n\n\nConclusions\n\nThis study result shows that nearly half of the pregnant women with IPIs. This indicates that it needs intervention to minimize maternal morbidity and their children. The most common identified IPs were Ascaris lumbricoides, Schistosoma spp., Trichuris trichiura, Hookworm, and Taenia spp. The prevalence of IPIs was associated with having no formal education and education to elementary school level as compared with those who had high school and above educational status. Those with a household monthly income less than 1920 ETB, that shared a latrine with neighbours, used lake water for washing clothes and were in the habit of eating raw vegetables were more exposed to IPIs when compared to their counterparts.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Magnitude of Parasitic Infections and Associated Factors among Pregnant Women at Health Facilities in Hawassa, Southern Ethiopia. https://doi.org/10.7910/DVN/III5HA13.\n\nThis project contains the following underlying data:\n\nS2- Additional file 1 Data.csv (raw data associated with this study).\n\nHarvard Dataverse: Replication Data for: Magnitude of Parasitic Infections and Associated Factors among Pregnant Women at Health Facilities in Hawassa, Southern Ethiopia. https://doi.org/10.7910/DVN/III5HA13.\n\nThis project contains the following extended data:\n\nS1-Additional file Table.doc (Table S1).\n\nS3 Additional file English qoestionner.doc (questionnaire used in this study; English version).\n\nS4- Additional file Figure 1.docx (Figure S1).\n\nThe prevalence of intestinal parasitic output on Sheet 2 and Multivariable logistic regration output on Sheet 1.tab (descriptive statistics derived from raw data).\n\nHarvard Dataverse: STROBE checklist for ‘Magnitude of parasitic infections and associated factors among pregnant women at health facilities in Hawassa, Southern Ethiopia’. https://doi.org/10.7910/DVN/III5HA13.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Authors’ contributions\n\nDW wrote the proposal, participated in data collection, analyzed the data and drafted the paper and manuscript writing. FB, BT, and SH approved the proposal with some revisions, participated in data collection, analysis. authors read and approved the final manuscript. All authors have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.\n\n\nAcknowledgments\n\nThe authors would like to thank Hawassa University, College of Medicine & Health Science for ethical approval. We would like to thanks all selected health Centers & Hawassa city administration health bureau for their cooperation on providing, information and support letter. The authors are also grateful to all data collectors and study participants for their valuable contributions.\n\n\nReferences\n\nTorgerson PR, Devleesschauwer B, Praet N, et al.: World Health Organization Estimates of the Global and Regional Disease Burden of 11 Foodborne Parasitic Diseases, 2010: A Data Synthesis. PLoS Med. 2015; 12(12): e1001920. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsoka-Gwegweni JM, Ntombela NP: A double load to carry: parasites and pregnancy. S Afr J Infect Dis. 2014; 29(2): 52–5. Publisher Full Text\n\nAranzales AFE, Radon K, Froeschl G, et al.: Prevalence and risk factors for intestinal parasitic infections in pregnant women residing in three districts of Bogota, Colombia. BMC Public Health. 2018; 18(1): 1071. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoel M, Carrara VI, Rijken M, et al.: Complex Interactions between soil-transmitted helminths and malaria in pregnant women on the Thai-Burmese border. PLoS Negl Trop Dis. 2010; 4(11): e887. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDerso A, Nibret E, Munshea A: Prevalence of intestinal parasitic infections and associated risk factors among pregnant women attending antenatal care center at Felege Hiwot Referral Hospital, northwest Ethiopia. BMC Infect Dis. 2016; 16(1): 530. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFMoH: National framework for tackling neglected tropical diseases through water, sanitation & hygiene. Ethiopia: Federal Ministry of Health. 2019. Reference Source\n\nGebreegziabiher D, Desta K, Howe R, et al.: Helminth infection increases the probability of indeterminate QuantiFERON gold in tube results in pregnant women. Biomed Res Int. 2014; 2014: 364137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKebede A, Gerensea H, Amare F, et al.: The magnitude of anemia and associated factors among pregnant women attending public institutions of Shire Town, Shire, Tigray, Northern Ethiopia, 2018. BMC Res Notes. 2018; 11(1): 595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWachamo D: Replication Data for: Magnitude of Parasitic Infections and Associated Factors among Pregnant Women at Health Facilities in Hawassa, Southern Ethiopia. DRAFT VERSION ed: Harvard Dataverse. 2020. http://www.doi.org/10.7910/DVN/III5HA\n\nEgwunyenga AO, Ajayi JA, Nmorsi OP, et al.: Plasmodium/intestinal helminth co-infections among pregnant Nigerian women. Mem Inst Oswaldo Cruz. 2001; 96(8): 1055–9. PubMed Abstract \| Publisher Full Text\n\nYesuf DA, Abdissa LT, Gerbi EA, et al.: Prevalence of intestinal parasitic infection and associated factors among pregnant women attending antenatal care at public health facilities in Lalo Kile district, Oromia, Western Ethiopia. BMC Res Notes. 2019; 12(1): 735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWekesa AW, Mulambalah CS, Muleke CI, et al.: Intestinal helminth infections in pregnant women attending antenatal clinic at kitale district hospital, kenya. J Parasitol Res. 2014; 2014: 823923. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOjurongbe O, Okorie PN, Opatokun RL, et al.: Prevalence and associated factors of Plasmodium falciparum and soil transmitted helminth infections among pregnant women in Osun state, Nigeria. Afr Health Sci. 2018; 18(3): 542–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShiferaw MB, Zegeye AM, Mengistu AD: Helminth infections and practice of prevention and control measures among pregnant women attending antenatal care at Anbesame health center, Northwest Ethiopia. BMC Res Notes. 2017; 10(1): 274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMengist HM, Zewdie O, Belew A: Intestinal helminthic infection and anemia among pregnant women attending ante-natal care (ANC) in East Wollega, Oromia, Ethiopia. BMC Res Notes. 2017; 10(1): 440. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Eijk AM, Lindblade KA, Odhiambo F, et al.: Geohelminth infections among pregnant women in rural western Kenya; a cross-sectional study. PLoS Negl Trop Dis. 2009; 3(1): e370. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdegnika AA, Ramharter M, Agnandji ST, et al.: Epidemiology of parasitic co-infections during pregnancy in Lambare´ne´, Gabon. Trop Med Int Health. 2010; 15(10): 1204–9. PubMed Abstract \| Publisher Full Text\n\nFuhrimann S, Winkler MS, Kabatereine NB, et al.: Risk of Intestinal Parasitic Infections in People with Different Exposures to Wastewater and Fecal Sludge in Kampala, Uganda: A Cross-Sectional Study. PLoS Negl Trop Dis. 2016; 10(3): e0004469. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowns JA, Mguta C, Kaatano GM, et al.: Urogenital schistosomiasis in women of reproductive age in Tanzania's Lake Victoria region. Am J Trop Med Hyg. 2011; 84(3): 364–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOboth P, Gavamukulya Y, Barugahare BJ: Prevalence and clinical outcomes of Plasmodium falciparum and intestinal parasitic infections among children in Kiryandongo refugee camp, mid-Western Uganda: a cross sectional study. BMC Infect Dis. 2019; 19(1): 295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: World Health Organization recommendations on antenatal care for a positive pregnancy experience. Geneva, Switzerland: WHO Press. 2016. PubMed Abstract" }	[ { "id": "79715", "date": "12 Apr 2021", "name": "Guéladio Cissé", "expertise": [ "Reviewer Expertise Intestinal parasitic infections related research", "water quality and health", "climate change and health", "WASH and nutrition integrated approaches" ], "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\nTitle: Why the use of \"Magnitude\"? The choice could be explained in the text (Introduction), if the prevalence as the main outcome presented in Figure 1 and Table 2 was not appropriate in the title.\n\nIntroduction: The problem statement is not made in a strong logical order. The text should prepare the reader to the interest of doing this study in a region and from health facilities.\nGood to check the English: some sentences are not complete (no verb, etc...). In particular, paragraph 3 needs to be re-ordered. The first sentence does not have a verb and it seems not make sense here for the problem statement. It would be more logical to start with the second part of the second sentence. (Note: starting with a capital letter \"There is ...\" is misleading, making difficult to understand the first part).\nIt would be better to start first by setting the national level burden and then the gap of knowledge or the diversity of current results at the regional levels; this would show the interest of this regional based study. It is not only important to identify the magnitude of IPIs but to know better the situation in regions.\nMethods: Need more information on the study site /region (e.g. climate, spatial location, poverty indicators, education status, parasites sensitive environment and ecosystems in the area, etc...) - Why the study is done/relevant in this region?\nResults: 2 tables and 3 figures; main results are about \"prevalence\" and risk factors.\nDiscussion: Sometimes it could be possible to reduce the extended repetition of some results. Also, when a reference is mentioned from other studies (stating similar results or differences), it would be good to provided the exact results from the other study(ies). To avoid saying that the results are similar or different from a study and giving only the reference.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "86426", "date": "04 Jun 2021", "name": "Abdelhakam G. Tamomh", "expertise": [ "Reviewer Expertise Medical Parasitology", "Infectious diseases", "Tropical diseases", "Clinical laboratory diagnostics", "Clinical Parasitology", "Clinical immunology." ], "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 about IPIs and associated risk factors among pregnant women is interesting. However, I have the following comments:\nThe title is well defined and a little change should be considered as, ‘’Prevalence rate of intestinal parasitic infections and associated risk factors among pregnant women at health facilities in Hawassa, Southern Ethiopia’’.\nThe abstract should be revised and especially the conclusion should be more concentrated.\nThe introduction should include the burden of intestinal parasitic infections among pregnant women globally.\nPregnant women are more susceptible to parasitic infections that attributed to the immunological changes during pregnancy, therefore the intestinal coccidian parasites were missing in this study and should be mentioned in the limitation of the study section.\nIn Material and Methods, direct microscopy - please change it to direct wet preparation.\nBesides a combination of direct wet preparation and formol-ether concentration techniques, a staining technique was missed, such as the modified ZN staining technique which is used only to detect intestinal Coccidia (Cryptosporidium species, Isospora belli and Cyclospora cayetanensis), please mention it in the limitations of the study section?\nThe gestation age of the pregnant women (either in the first, second, or third trimester) was not explained.\nAdditionally, the gravidity of pregnant women not addressed (such as the multigravid women may have more risk than primigravid women).\nThe results support the prevalence and risk factors, therefore, the term prevalence rate is better than Magnitude.\nIf the information related to the age of gestation and the gravidity is addressed please added it to table 2.\nIn the discussion section, the authors discussed the results well.\nIn conclusion, after mentioning the importance of some results, please conclude the outcome of the paper.\nI think after making these corrections the paper will be approved. I have attempted to add some corrections to this manuscript. Hopefully, this will be helpful for the authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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/10-122
https://f1000research.com/articles/10-120/v1	17 Feb 21	{ "type": "Research Article", "title": "COVID-19: state population coverage by mask orders in the US", "authors": [ "Philip Jacobs", "Arvi Ohinmaa", "Arvi Ohinmaa" ], "abstract": "Background: A variety of government bodies in the US - cities and tribal councils, counties, and states - have issued mask-wearing orders to prevent the spread of COVID-19. Methods: We measured the duration of these orders and the populations covered by them in each of the governed areas. We measured the in effect days from April 3, 2020 (the date of the first mask order) to November 30, 2020; the duration of the measured period amounts to 241 days. We used data generated from local and state governments, and news organizations to measure the enactment dates and the duration of the orders; and from the US Census Bureau (dated 2019) to measure municipal, county, and state populations. Results: The average coverage over all states was 57.7% of the maximum person days of coverage. States which issued statewide orders from their governors had a total of 67.1% coverage; this includes coverage from regional government orders prior to the statewide orders. States with orders only from municipal or county councils had on average 27.1% coverage. Conclusion: Overall, state governments took leadership in the implementation of mask orders.", "keywords": [ "COVID-19", "Face masks", "Mandates", "Population covered" ], "content": "Introduction\n\nAs the COVID-19 pandemic proceeded through the US in 2020, face masks as preventive devices grew in importance1. The US Centers for Disease Control (CDC) and US President Trump first recommended the wearing of face masks on April 3, 20202,3. The World Health Organization made a interim recommendation on April 64. The first local government order in the US that we found was issued by the San Diego County Council on April 35; this order covered retail employees. New Jersey was the first state to have a mask order, issued by Governor Phil Murphy on April 86. Since then US city and county lawmakers, tribal councillors, and state governors have enacted an assortment of mask orders and, in some cases, subsequently altered them7‐9. The result is a patchwork of orders across the nation, with a mixture of types of authorities, varying start dates, change or termination dates, and opt-out rules, all of which have contributed to the amount of COVID-19 protection within each state. Given the importance of masks in slowing down the spread of the virus10, we estimated the degree of protection - measured in terms of person days of coverage - across states.\n\n\nMethods\n\nOur goal is to estimate the percentage of person days in each state that were either covered by city, tribal council, county, or state orders, or that were not covered, during the period April 3 to November 30, 2020 (a maximum of 241 days). Person days are the product of days in a region (241) and the region’s population. Mask days (days in a region during which there was a mask order in effect) were calculated for each type of government. The calculated percentage is obtained by t mask days divided by person days.\n\n\n\n(1) If the state (governor) did not issue an order during the study period - that is, only local governments issued orders - we measured mask days as the time between the order in effect dates and their termination dates, or November 308. We multiplied mask days by the region’s 2019 population11,12 to obtain person days of mask coverage. If both a city (cities) and its county simultaneously issued orders, we adjusted the populations to avoid the double counting of persons.\n\n(2) In states where governors issued state orders7, and counties were covered by the mask orders (statewide coverage), we measured state coverage as the person days covered by the mask orders multiplied by state population. In these states, there may have been additional coverage by city or county orders that were enacted prior to the states’ enacting dates and which lasted up to the day of the governor’s order. We estimated these locally generated mask days for city and county coverage9 and added them to the statewide coverage days.\n\n(3) Several states issued orders that allowed for differences in mask related policies between counties. Counties with low COVID-19 incidence rates could be exempt from the state order although, over time, if the rate in one of these counties rose to a level above the state's cut-off, the exemption would be withdrawn. We used news reports to take county specific coverage into account in Louisiana13,14, Mississippi15‐17, Ohio18, and Texas19. In Kansas, a county could opt out of the state mandate; it could replace the state's mandate with one of its own20,21. The Kansas Health Institute tracked mask policies across the state and publicly reported them22.\n\nThere are a few cities that are not in any county; they are standalone entities called “independent cities.” Baltimore, Maryland; St. Louis, Missouri; Carson City, Nevada; and 38 cities in Virginia23 are independent cities; we classify them as cities. Also, there are several cities - New Orleans, Louisiana; Anchorage and Juneau, Alaska- whose boundaries coincide with those of the counties. The Census Bureau calls them “consolidated cities”24; we consider these as counties.\n\n\nResults\n\nBy the first half of April 2020, legislators in nine states had issued mask orders (See Figure 1) and by the end of June, this number had grown to 40. Data for the initiation dates for each state are provided in a data set.\n\nWe divided time periods into 15 day increments, beginning April 1, 2020. The first mask order was on April 3.\n\nThe average degree of coverage for all states between April 3 and November 30 was 57.7%. Of the nation’s population, 48.6% received coverage under statewide mandates, 6.5% under county mandates, and 2.7% under municipal orders. During the time period covered in our study, states which enacted statewide mandates had an average coverage of 67.1% for all mask days (including those generated by municipal and county mandates); states in which the governors did not introduce statewide mandates had 27.1% coverage from municipal or county orders.\n\nThe degree of coverage and source of order by state is shown in Figure 2. New Jersey had the highest degree of coverage (97.9% of all person days) because of its early start in introducing a statewide mandate. Of those states without statewide coverage, Arizona and Florida had large county components which kept their non-coverage ratios in the mid-range. Among the states with lower levels of mask coverage, Alaska and Oklahoma had relatively high degrees of municipal coverage.\n\nPercent breakdown in each state of person days into days under mask order and days without any mask order. Days under mask orders are further broken down by source of mandate - state (green), municipal (yellow) and county (blue). Mask-days with no government orders are colored red.\n\n\nDiscussion\n\nThe proportion of person days between April 3 and November 30, 2020 that came under face mask orders varied considerably by state. South Dakota had the lowest percent coverage and New Jersey the highest. There was also a variation by the source of the order: states with only local orders exhibited an average coverage of 27.1% of all person days while those which included state orders exhibited an average of 67.1%.\n\nWe brought together mask order duration and population to measure the relative degree of coverage of mask policies by state. There are numerous lists of mask orders that indicate the dates of the orders; these were found in news reports and in state and local government web pages7‐9. However, they have not been brought together to indicate duration of coverage that was weighted by populations. Thus, the degree of population coverage of the mask policies are not indicated in any of these sources.\n\nWhile the ratio of mask days to person days for each state has been estimated, we should point out some qualifications when interpreting these statistics as indicators of COVID-19 preventive activities.\n\n• We used the total regions’ populations. All regional orders had some exceptions such as age and disability. Although these omitted persons made up small portions of the populations, their inclusion in our estimates suggests that our analysis captured the number of persons who resided in an area with mask orders, rather than the number who were ordered to wear masks.\n\n• Some legislators changed mask policies over time in response to changing COVID-19 incidence and public pressure. Over time the degree of coverage in some states changed as mask orders were initiated or terminated. We have tried to capture these changes using news reports but, among the states, only Kansas, through the Kansas Health Institute, reported mask policies in a comprehensive and systematic way. As a result, there may have been missing orders and termination dates in our estimates.\n\n• As we have indicated in a previous paper7, there have been considerable differences in enforcement across regions. Enforcement, usually done at the local level, is not documented in any statistics and is especially difficult to capture in relation to local government policies. This is a limitation which would be difficult to control for.\n\n• Masks are not the only deterrent for the pandemic. Others included social distancing in its many forms and frequent handwashing25. Mask-wearing is only a partial solution to containing the virus.\n\nIn conclusion, leadership regarding mask-wearing came from the state level of government. Among the states with the greatest percentage of mask-order person days, state leadership was prominent. In those states with the lowest level of coverage, local government leadership predominated.\n\n\nData availability\n\nUniversity of Alberta Libraries (UAL) Dataverse: First days in states of mask mandates, https://doi.org/10.7939/DVN/FJHBSS26.\n\nThis project contains the following underlying data:\n\nDistribution among states of the date of the first mask order\n\nUniversity of Alberta Libraries (UAL) Dataverse: COVID-19 Mask days as a per cent of state person days https://doi.org/10.7939/DVN/GJZKVJ27.\n\nThis project contains the following underlying data:\n\nDistribution of mask days by legislative body\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 received suggestions from Charles Hunt.\n\n\nReferences\n\nMolteni M, Rogers A: How Masks Went From Don’t-Wear to Must-Have. Wired 07.02.2020 Accessed December 3, 2020 Reference Source\n\nDwyer C, Aubrey A: CDC Now Recommends Americans Consider Wearing Cloth Face Coverings In Public. National Public Radio April 3, 2020 Accessed on December 3 Reference Source\n\nRemarks by President Trump, Vice President Pence, and Members of the Coronavirus Task Force in Press Briefing.April 3, 2020 Accessed on December 3, 2020 Reference Source\n\nWorld Health Organization: Advice on the use of masks in the context of COVID-19. interim guidance 6 April 2020 Accessed on December 3, 2020 Reference Source\n\nCounty of San Diego: Addendum 1. To order of the health officer and emergency regulations.Dated March 27, 2020 effective as of April 3, 2020 Accessed on December 3, 2020 Reference Source\n\nGovernor Phil Murphy, State of New Jersey: Executive Order 122.April 8, 2020 Accessed on December 3, 2020 Reference Source\n\nJacobs P, Ohinmaa A: The enforcement of statewide mask wearing mandates to prevent COVID-19 in the US: an overview. F1000 Res 7 September 2020 Accessed on December 3, 2020 Reference SourcePubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJacobs P, Ohinmaa A: Dataset: percent of population covered by local government mask orders in the US. F1000 Res 22 October, 2020 Accessed on December 3, 2020 Reference SourcePublisher Full Text\n\nJacobs P, Ohinmaa A: Dataset: local government mask orders preceding statewide orders by US states. F1000 Res 2021 10:8 08 January 2021 Accessed on January 15, 2020 Reference Source\n\nBai N: Still confused about masks? Here’s the science behind how face masks prevent coronavirus. Patient Care June 26, 2020 Accessed on December 1, 2020 Reference Source\n\nUS Census Bureau: City and Town Population Totals: 2010-2019. Accessed: Reference Source\n\nUS Census Bureau: Annual County and Resident Population Estimates by Selected Age Groups and Sex: April 1, 2010 to July 1, 2019. Accessed: Reference Source\n\nKarlin S: John Bel Edwards shutters bars, issues mask mandate: ‘Current restrictions are not enough’. The New Orleans Advocate July 11, 2020 Accessed on December 3, 2020 Reference Source\n\nRddad Y, Karlin S: Louisiana now has a statewide mask mandate, but where and how is it enforced? New Orleans Advocate July 13, 2020 Accessed on December 3, 2020 Reference Source\n\nBologna G: ‘Take this as an alarm’: Gov. Tate Reeves orders mask mandate for 13 Mississippi counties. Mississippi Clarion Ledger July 9, 2020 Accessed on December 3 Reference Source\n\nMagee P:MS governor extends coronavirus mask mandate and adds 10 counties to list. Sun Herald July 20, 2020 Accessed on December 3, 2020 Reference Source\n\nFox13 Memphis News Staff: Gov. extends Mississippi’s Safe Return Order and adds 8 new counties to mask mandate. Accessed on December 3, 2020 Reference Source\n\nShelley J: Ohio Implements mandatory masks orders for 7 counties. ABC WPTA July 7 2020 Accessed on December 3, 2020 Reference Source\n\nAguirre P: These Texas counties are exempted from Gov. Abbott’s face mask order. My San Antonio July 6, 2020 Accessed on December 3, 2020 Reference Source\n\nGovernor Laura Kelly: Executive Order 20-52. Requiring masks and other face coverings in public.August 2,2020 Accessed on December 4, 2020 Reference Source\n\nState of Kansas, House of Representatives: House Bill 2016. Approved by the governor on June 8, 2020. Directive 6 Accessed on Febriary 1, 2021 Reference Source\n\nKansas Health Institute: County official actions in response to COVID-19 as of August 11.Topeka Kansas: Kansas Health Institute; August 11, 2020\n\nBureau of the Census: Terms and Definitions. Accessed on December 3, 2020 Reference Source\n\nAccessed on December 3 Reference Source\n\nAccessed on January 16, 2020 Reference Source\n\nJacobs P: First days in states of mask mandates. UAL Dataverse, V1, UNF:6:M2XVpxM5+emQyMQWZxWASg== [fileUNF] 2021. Publisher Full Text\n\nJacobs P: COVID-19 Mask days as a per cent of state person days. UAL Dataverse, V1, UNF:6:v3FNmuPT8VNUrbl3CdEznw== [fileUNF] 2021. Publisher Full Text" }	[ { "id": "79689", "date": "26 Mar 2021", "name": "Hannah Clapham", "expertise": [ "Reviewer Expertise infectious disease epidemiology and modeling" ], "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 summary of mask mandates is useful. A few comments:\n\nThe methods could have some more detail on where the information on mask mandates was found\n\nIt is unclear whether the only variation is time of introduction or has been introduced and lifted in some places. This would be helpful for understanding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "81376", "date": "20 Apr 2021", "name": "Shirley Girouard", "expertise": [ "Reviewer Expertise Health services researcher", "policy analyst and nurse" ], "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\nWell designed and executed study and description of numbers of people in US who live where mask policies are in place. Title and text could be rephrased a bit to make it clear that this is what the findings refer to. Would be helpful to relate these findings to COVID-19 actual mask wearing (there are some data re this) and rates of disease/test positivity rates. Compliance and impact of having policies is, of course, what is important.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/10-120
https://f1000research.com/articles/9-1392/v1	03 Dec 20	{ "type": "Method Article", "title": "Isolation and primary culture of Galleria mellonella hemocytes for infection studies", "authors": [ "Nicola J. Senior", "Richard W. Titball", "Nicola J. Senior" ], "abstract": "Galleria mellonella larvae are increasingly used to study the mechanisms of virulence of microbial pathogens and to assess the efficacy of antimicrobials. The G. mellonella model can faithfully reproduce many aspects of microbial disease which are seen in mammals, and therefore allows a reduction in the use of mammals. The model is now being widely used by researchers in universities, research institutes and industry. An attraction of the model is the interaction between pathogen and host. Hemocytes are specialised phagocytic cells which resemble neutrophils in mammals and play a major role in the response of the larvae to infection. However, the detailed interactions of hemocytes with pathogens is poorly understood, and is complicated by the presence of different sub-populations of cells. We report here a method for the isolation of hemocytes from Galleria mellonella. A needle-stick injury of larvae, before harvesting, markedly increased the recovery of hemocytes in the hemolymph. The majority of the hemocytes recovered were granulocyte-like cells. The hemocytes survived for at least 7 days in culture at either 28°C or 37°C. Pre-treatment of larvae with antibiotics did not enhance the survival of the cultured hemocytes. Our studies highlight the importance of including sham injected, rather than un-injected, controls when the G. mellonella model is used to test antimicrobial compounds. Our method will now allow investigations of the interactions of microbial pathogens with insect hemocytes enhancing the value of G. mellonella as an alternative model to replace the use of mammals, and for studies on hemocyte biology.", "keywords": [ "Galleria mellonella", "hemocytes", "3Rs", "infection model" ], "content": "\n\nAllows the interactions of microbial pathogens with host insect hemocytes to be investigated ex vivo.\n\nAllows the biology of different types of hemocytes to be investigated ex vivo.\n\nAllows comparative studies of the interaction of hemocytes and mammalian phagocytes with pathogens.\n\nG. mellonella larvae are increasingly used as alternatives to mice and other mammals for studies on the mechanisms by which microbial pathogens cause disease and to test pre-treatments and therapies for disease.\n\nThe protocol described allows the isolation of higher yields of hemocytes from G. mellonella for experimental studies using a simple technique.\n\nThe protocol described establishes the optimum time window for using isolated hemocytes in experimental studies.\n\nInfectious disease research to understand mechanism of virulence of microbial pathogens.\n\nTesting the ability of isolated hemocytes to support the growth of intracellular pathogens including Burkholderia sp, Coxiella burnetii, Mycobacterium sp. Salmonella enterica.\n\nUsing the hemocyte model to assess the ability of drugs to target intracellular pathogens.\n\nUsing the hemocyte model to compare the virulence of different isolates and different mutants of microbial pathogens.\n\n\nIntroduction\n\nImproving our understanding of infectious disease, developing new pre-treatments and therapies for diseases and testing the safety of biological and chemical materials often requires the use of regulated vertebrate animals. Typically, mammals are used for these studies. There is a need to replace, refine and reduce the use of regulated animal species in the UK and one approach is to develop alternative test systems and models for infectious disease.\n\nA wide range of alternatives have been proposed and developed, and these have different benefits and drawbacks (Table 1). Larvae of the greater waxmoth Galleria mellonella are an attractive alternative because they can be injected with precise doses of pathogen or chemical compound, and incubated at 37°C to mimic conditions in a mammalian host1. The model is becoming well developed and the late-stage larvae, which are used for research and testing, do not require food or water and are easy to maintain. Compared to regulated animals, the larvae are ethically more acceptable and more cost effective2, and their use can contribute to the target to reduce and replace the use of regulated animals. G. mellonella have been used to study virulence of pathogens, as part of the drug discovery pipeline and in chemical and drug toxicity testing1–5. The model is now used widely in academia and increasingly used by pharmaceutical industries drug and contract research organisations. Although G. mellonella larvae can never replace mammalian models completely, the rise in usage over the past decade, with 275 publications using G. mellonella in 2019, indicates how valuable the larvae have become to researchers. This work reported here is part of a project to develop the G. mellonella model to reduce the number of mammals used in research associated with insect-vectored viral pathogens. Regulated vertebrate models are frequently used to study viral disease, and to develop and evaluate therapeutics. To assess the use of mammals for virus research in the UK we carried out a PubMed search using the key words “virus+UK+mice”. This returned 118 relevant publications in 2018. We selected the first five publications for which we could obtain journal access at the University of Exeter, and found that the total number of mice used in these five publications was 235. In addition eight rabbits and six marmosets had been used in these studies. Assuming that these numbers are representative for all publications in 2018, we calculate that in the UK at least 5546 mice were used for studies in viral diseases in 2018. In addition at least 189 rabbits and 141 non-human primates were used in 2018. The procedures are judged to be severe. For example, in one laboratory almost 1000 mice were used to study viral pathogenesis and to evaluate therapies. We envisage that up to 50% of these animals could be replaced by G. mellonella larvae if a suitable infection model was available. Although studies carried out in G. mellonella larvae will never completely replace studies in mammals, they will allow any subsequent work using regulated mammals to be better designed, providing refinement of experiments.\n\nOne of the advantages of G. mellonella is that they provide a whole animal model, rather than being cell culture-based. Additionally, G. mellonella possess an innate immune system, involving cellular and humoral responses6–8. The cellular response involves hemocytes that can engulf pathogens, and these cells share a high degree of structural and functional similarity with mammalian neutrophils9. The humoral response involves activation of Toll and Imd pathways, resulting in the production of antimicrobial peptides10, the production of prophenoloxidase and the generation of melanin10.\n\nThe similarities between insect hemocytes and mammalian neutrophils are well-documented8–10. Like neutrophils, hemocytes use a respiratory burst to generate reactive oxygen species and kill pathogens11. This respiratory burst is triggered by the translocation of p47phox and p67phox proteins in neutrophils, and by the translocation of 47 and 67 kDa proteins in hemocytes11. Like neutrophils, hemocytes possess Toll-like receptors, β-glucan and interleukin-1 receptors11 and signalling occurs via NFκB pathways11. The antimicrobial peptides produced by neutrophils and hemocytes, in response to signalling, are similar11 and include lysozyme, transferrin and defensins. Finally, hemocytes and neutrophils can produce extracellular traps (NETs) to immobilize and kill pathogens12,13.\n\nIn G. mellonella four types of hemocytes have previously been identified: plasmatocytes, granulocytes, spherulocytes and oenocytoids14. A fifth type, prohemocytes15 may be stem cells that differentiate into other hemocyte types16 In larval stage Lepidoptera, granulocytes and plasmatocytes are considered to be the main phagocytic hemocytes, and they are also the most numerous cells in circulation9.\n\nPathogens that have intracellular lifestyles in mammalian hosts, such as Coxiella burnetii, Burkholderia thailandensis, Francisella tularensis and Mycobacterium tuberculosis, have previously been shown to invade hemocytes and cause death in a dose-dependent manner17–19. However, the interactions of pathogens with hemocytes are poorly understood. This information is required to allow an understanding of the similarities and differences in the ways in which pathogens interact with hemocytes from insects or phagocytes in mammals, and therefore to provide an understanding of the utility of the G. mellonella infection model. Here we report the development of a protocol for the isolation and culture of hemocytes from G. mellonella to allow the interactions of pathogens with hemocytes to be investigated. The method we have developed will be of interest to researchers using G. mellonella as a model to study infectious disease.\n\n\nMethods\n\nExperimental design. The experimental unit in this study was a single Galleria mellonella larva. The number of larvae per group was estimated using a Resource Equation method20; the assignation of 10 larvae per group across four groups gave an E value of 36, which is greater than the recommended figure of 10–20. Since the use of larvae does not breach ethical guidelines, this could be justified as we had no previous experience regarding the number of hemocytes that could be obtained from a single larva. Treatment was randomised: all treatments were started at the same time; the larvae were selected from a pool of healthy individuals within the weight parameters of 0.18–0.35 g; they were assigned to treatment groups by taking larvae from the pool with no visual reference to compare them to other larvae. All experimental groups were equal in size. There was no blinding of samples for hemocyte counts.\n\nG. mellonella larvae. TruLarv™ final (6th) instar larvae were purchased from BioSystems Technology, Exeter, UK. TruLarv are reared without antibiotics or hormones which are normally added to feedstuffs, and are 0.18 g–0.35 g final instar stage larvae. The larvae were stored at 15°C for up to one week before using in the experiments reported. During storage the larvae do not require food or water.\n\nPre-treatment of larvae. Groups of ten larvae (TruLarv™) were used in these studies to allow the collection of sufficient hemolymph for the studies outlined below. Typically, we recovered 20–30 µl of hemolymph from each larva. We tested whether pre-dosing with antibiotics would minimise the bacterial flora on the larvae and therefore minimise the possibility of bacterial infection of the isolated hemocyte cultures. Where indicated, the larvae were treated with a scaled human dose of the broad-spectrum antibiotic doxycycline or ciprofloxacin prior to the extraction of the hemolymph. Larvae were swabbed at the injection site with 70% ethanol and the antibiotic (10 μl) injected 2–3mm deep into a proleg (Figure 1). In some studies the larvae were wounded by piercing the proleg with a 21- or 22-gauge needle. These treatments were repeated daily for up to 7 days, using a different proleg at each time. Larvae were stored in Petri dishes lined with filter paper and kept in the dark at 15°C for the duration of the pre-treatment.\n\nHemolymph extraction. The main cell population in insect hemolymph is circulating hemocytes, and few other cell types are present. Before hemolymph extraction, larvae were swabbed with 70% ethanol, then positioned over a 1 ml pipette tip. A proleg was pierced 2–3 mm deep with a 21- or 22-gauge needle, and the hemolymph that emerged was collected using a pipettor set to 50 μl. Typically, the concentration of hemocytes in the pooled hemolymph was 2×106 to 4×106 cells per ml. Since we typically harvested 200–300 µl of hemolymph from 10 larvae, in total we isolated 4×105 to 1.6×106 cells. The hemolymph was transferred into a microcentrifuge tube containing 500 μl of insect physiological saline (IPS; 150 mM NaCl, 5 mM KCl, 100 mM Tris/HCl, 10 mM EDTA, 30 mM sodium citrate, pH 6.9) on ice. The hemolymph from each group of larvae was pooled and centrifuged at 500 × g, 4°C, for 5 minutes. Pellets were washed twice in ice-cold IPS, then re-suspended in 1 ml ice-cold IPS. A total of 10 µl were withdrawn from suspended pellets and mixed with 10 μl of trypan blue to enable cell enumeration in a hemocytometer. The concentration of hemocytes was adjusted as required and the cells used immediately.\n\nMaintenance of hemocytes. The required amount of cell suspension (at 2 × 105 cells/ml) was placed into the relevant number of wells in a 24-well plate, and made up to 1 ml with Grace’s Insect Medium supplemented with L-glutamine and sodium bicarbonate + 2% penicillin-streptomycin + 2.5 μg/ml amphotericin B + 10% heat-inactivated foetal bovine serum (FBS) at room temperature. The plates were placed in a lidded vented box lined with damp blue roll to reduce evaporation and incubated at 28°C under normal atmospheric conditions. Overall, 50% of the medium was replaced every 3–5 days. The plates were observed under 10 × magnification on a microscope regularly for evidence that the cells were intact and remained attached to the bottom of the wells.\n\nHemocyte quantification. Hemocytes were quantified after dissociating them from the wells as detailed below. The medium was removed and the cells washed twice in Dulbecco’s PBS. Next, 200 μl of trypsin + 0.25% EDTA, pre-warmed to 37°C, was added to each well; the plate was incubated at 37°C for 3 minutes. The trypsin was inactivated by the addition of 400 μl of the culture medium, which was pipetted slowly over the well surface at least twice. The base of each well was tapped sharply. If observations under the microscope suggested that the cells still had not dissociated, the base of the well was gently scraped with a 10 μl sterile disposable plastic inoculation loop to release any cells attached to the well bottom. Ten microliters were withdrawn from the cell suspension and mixed with 10 μl of trypan blue to enable viable cell enumeration in a hemocytometer.\n\nDifferential hemocyte counts. Hemocytes were examined to ascertain the proportion of different cell types (granulocytes, spherulocytes, plasmatocytes, prohemocytes or oenocytoids). They were collected from larvae injected with doxycycline or sham injected by inserting a needle into a proleg 3 days before harvesting hemolymph. The hemolymph was pooled from 10 larvae per treatment, and aliquots dispensed into three wells on each of two plates. One set of plates was observed for the presence of intact and attached cells under 20 × magnification with the cells unstained at T0. The other set of plates was incubated at 28°C for seven days and then observed under the same conditions.\n\nHemocytes were also observed by staining in situ. Medium was removed from wells and the cells were washed with Dulbecco’s PBS. They were briefly allowed to air-dry and 200 μl of Giemsa Stain was added to the wells to visualise the hemocytes and left for 2 minutes before being removed and replaced with 200 μl of deionized water for 3 minutes. Giemsa stain is a widely used histological stains, colouring nuclei dark blue and the cytoplasm blue or pink depending on the acidity of the cytoplasm. The wells were rinsed with deionized water; as much liquid as possible was removed using a pipette, and the wells were left to air-dry. The wells were observed under 20 × magnification on a microscope and images taken using a camera.\n\nStatistical analysis of data. Statistical analysis was carried out using the GraphPad Prism 8 program (GraphPad Software, LA Jolla California USA). The means and standard deviations were calculated for each set of results.\n\nHere we describe the step by step procedure used to extract hemocytes and their use as an ex vivo model. Reagents are listed in Table 2.\n\nStep 1: Preparation of antibiotic stocks (Day 1). We use doxycycline hyclate to make up a stock solution of doxycycline (8.5 mg in 1 ml) of sterile milliQ water. This is diluted to 60 µg in 1 ml of sterile milliQ water to ensure a scaled human dose (on a weight for weight basis) will be given to larvae (equivalent to a dose of 0.6 µg in 10 µl), × 3. We use ciprofloxacin to prepare a stock solution (10 mg in 1 ml of 0.1N hydrochloric acid). This is diluted to 250 µg in 1 ml of sterile milliQ water (equivalent to a dose of 2.5 µg in 10 µl), × 3. The stock solutions are stored at -20°C and scaled human doses are used on the day of dilution and also frozen in aliquots.\n\nStep 2: Pre-treatment of larvae (Day 1). We use TruLarv™ (BioSystems Technology, Exeter, UK), weight 0.18–0.35 g, 10 larvae per treatment. These are maintained in an incubator at 15°C before and during treatment. An infection station is set up comprising a 90 mm filter paper taped to the bench with a 1-ml pipette tip taped across it (Figure 2). Larva are held over the pipette tip, underside uppermost, and swabbed with 70% ethanol. Next, 10 µl of the doxycycline dilution is drawn into a 22-gauge cemented-needle Hamilton syringe; this is injected into one of the larval prolegs. The syringe is lifted with the larvae still attached and held over a Petri dish lid lined with a 90-mm filter paper; the larva is allowed to free itself into the lid. This is repeated for the other nine larvae, and the base of the petri dish used as the lid. The ciprofloxacin solution is injected into another 10 larvae. Another 10 larvae are wounded by piercing a proleg with the syringe needle, but without corresponding injection, and these are held in a third Petri dish. A further 10 larvae are put into a fourth Petri dish as the untreated controls. All dishes are then stored in the incubator at 15°C for 24 hours.\n\nStep 3: Pre-treatment of larvae (Day 2). Aliquots of diluted antibiotic are removed from -20°C storage and thawed. The larvae from the previous day are removed from the incubator, and an infection station is prepared as detailed above. The larvae are swabbed with 70% ethanol before their respective treatments, which are injected into a different proleg to that used on the previous day. The larvae are returned to the 15°C incubator for 24 hours.\n\nStep 4: Pre-treatment of larvae (Day 3). An aliquot of diluted antibiotic is removed from -20°C and thawed. The larvae in dishes are removed from the incubator, and an infection station is prepared. The larvae are again swabbed with 70% ethanol prior to their respective treatments, which are injected into a third proleg. The larvae are returned to the 15°C incubator for at least 1 hour.\n\nStep 5: Extraction of hemolymph (Day 3). The larvae are removed from the 15°C incubator. Next, 500 µl of insect physiological saline (IPS; 150 mM NaCl, 5 mM KCl, 100 mM Tris/HCl, 10 mM EDTA, 30 mM sodium citrate, pH 6.9) is added into each of four microcentrifuge tubes that are placed on ice. A 1-ml pipette tip is placed into the lid of a Petri dish. In turn, each larva from the doxycycline set is swabbed with 70% ethanol and held backwards over the pipette tip. A different proleg is pierced with a 21-gauge needle, and the hemolymph that emerges is drawn into a pipette tip on a 200 µl pipettor set to 50 µl. The hemolymph is pooled into the microcentrifuge tube containing ice-cold IPS. In the same way, the hemolymph extracted from the ciprofloxacin, wounded and untreated sets are pooled into separate microcentrifuge tubes on ice. The pools are centrifuged at 500 × g, 4°C, for 5 minutes, then supernatants are discarded. Pellets are washed in 1 ml ice-cold IPS and re-centrifuged under the same conditions. There is a second wash step, after which the pelleted hemocytes are re-suspended in 500 µl of ice-cold IPS.\n\nStep 6: Quantification of hemocytes (Day 3). We mix 10 µl of suspended hemocytes with 10 µl of 0.4% trypan blue dye, then load 10 µl of the mixture beneath the cover slip of a hemocytometer cleaned with 70% ethanol. Live cells do not stain with trypan blue dye. The hemocytometer is observed under 10 × magnification on a microscope. Cell counts are made in the four outer squares, and the average of the four is calculated, then doubled to account for the dilution with trypan blue strain. This value is used to adjust the concentration of the hemocytes to 2 × 105 cells/ml in the culture plate.\n\nStep 7: Seeding of hemocytes (Day 3). We use 24-well plastic cell culture plates. The required volume of hemocyte suspension is added to three wells in a row, one row per treatment. The suspension is made up to 1 ml using Grace’s Insect Medium supplemented with L-glutamine and sodium bicarbonate + 2% penicillin-streptomycin solution + 2.5 µg/ml amphotericin B + 10% heat-inactivated foetal bovine serum at room temperature. Separate plates are made up for different time points as testing is generally destructive. The plates are placed in a vented lidded box containing damp blue roll, and incubated in a non-CO2 incubator at the appropriate temperature – either 28°C (the usual temperature for the culture of insect cells) or 37°C (the mammalian body temperature).\n\nStep 8: Viability counts (Day 3). Once seeded, the T0 plate is used for viability counts. A total of 10 µl of suspension are withdrawn from each well and mixed with 10 µl of 0.4% trypan blue stain, then 10 µl of the mixture is loaded beneath the cover slip on a 70% ethanol-cleaned hemocytometer. Cells in the four outer corners are counted after trypan blue staining and approximately 95% of the cells should exclude trypan blue strain indicating they are viable.\n\nStep 9: Maintenance of hemocytes (Day 5–7). We check the hemocytes under 10 × magnification on the microscope to make sure that they are intact and attached to the bottom of the plate. We carry out a 50% media change using Grace’s Insect Medium supplemented with L-glutamine and sodium bicarbonate + 2% penicillin-streptomycin solution + 2.5 µg/ml amphotericin B + 10% heat-inactivated foetal bovine serum at room temperature as before.\n\nStep 10: Viability counts (Day 10). We pre-warm trypsin + 0.25% EDTA in a 37°C water bath. The plate is removed from the incubator and the medium withdrawn from each well. We use 1 ml of Dulbecco’s PBS to wash each well, × 2. We add 200 µl of the warmed trypsin + 0.25% EDTA to each well, and incubate the plate in an incubator at 37°C for 3 minutes. We inactivate the trypsin by addition of 400 µl of room temperature Grace’s Insect Medium supplemented with L-glutamine and sodium bicarbonate + 2% penicillin-streptomycin solution + 2.5 µg/ml amphotericin B + 10% heat-inactivated foetal bovine serum, pipetting the mixture slowly across the base of each well at least twice. The bases of the wells are also tapped sharply. The plate is observed at 10 × magnification under the microscope to ascertain whether the hemocytes are detaching from the surface of the well; if not, they are gently scraped with a 10 µl plastic loop. Once hemocytes are sufficiently detached, 10 µl from each well is mixed with 10 µl of trypan blue stain, and viability counts are undertaken as before. Typically, 94–97% of the cells are viable at this stage.\n\nStep 11: Differential hemocyte counts (Days 3 – 10). Seeded wells are examined under 20 × magnification on the microscope, and counts are made across five views of the three wells per treatment of granulocytes, spherulocytes, plasmatocytes, prohemocytes or oenocytoids according to their morphology. Giemsa staining facilitates the identification of the different types of hemocytes because the cell and nuclei shape are more easily observed.\n\nStep 12: Giemsa staining (Days 3 – 10). If we are undertaking Giemsa staining, we set up extra wells in the 24-well plate as this is a destructive process. The medium is removed and the wells are washed with 1 ml of room temperature Dulbecco’s PBS. Once this is removed, they are allowed to air dry briefly. We add 200 µl of undiluted Giemsa stain to each well and leave the plate for 2 minutes at room temperature. The stain is removed as thoroughly as possible. We add 200 µl of room temperature sterile deionized water and leave the plate for 3 minutes before removing as much water as possible. The wells are rinsed with a further 200 µl of deionized water, which is pipetted off as thoroughly as possible; the plate is then left to air dry before being examined at 20 × magnification on the microscope.\n\n\nResults\n\nInjection promotes the mobilisation of hemocytes. We initially investigated whether dosing larvae with an antibiotic affected the recovery of hemocytes compared to sham injected, PBS dosed or untreated control larvae. Pre-treating larvae with doxycycline, ciprofloxacin or PBS increased by 2–3-fold the number of hemocytes we recovered in hemolymph, compared to untreated controls (Figure 3). However, sham injection by the insertion of a needle without any injection also increased the numbers of hemocytes recovered. The significance of these results could not be tested as hemolymph from each experimental unit was pooled into treatment sets and we thus do not have separate results for each larva.\n\nThe results shown are the average of four counts made on hemolymph extracted from groups of 10 larvae per treatment. Error bars = SD.\n\nSurvival of recovered hemocytes. We collected the hemocytes from hemolymph by centrifugation, and suspended them in Grace’s Insect Medium supplemented with L-glutamine, sodium bicarbonate, penicillin, streptomycin, amphotericin and FBS. The hemocytes were plated at a density of 2 × 105 cells/ml into 24 well cell culture plates at 28°C. We found (Figure 4) that the proportion of cells which excluded trypan blue dye (i.e. live cells) immediately after plating (93–98%) was similar to the proportion of cells which excluded trypan blue dye 7 days after plating (94–97% of T0 cells), indicating that we could maintain the cells for 7 days without loss of viability. Pre-treatment of the larvae with doxycycline or ciprofloxacin, before harvesting the hemocytes, did not affect the 7-day survival of hemocytes. When we cultured the hemocytes for 14 days after plating, we found a reduction in the proportion of cells which excluded trypan blue dye (33–38%).\n\nViability of hemocytes incubated at 28ºC at T0 (A) or T7 (B). Average across three replicate wells per treatment, derived from the pooled hemolymph from 10 larvae, and seeded at 2 × 105 cells/ml. Hemocytes dissociated with trypsin and stained with trypan blue. Error bars = SD. Mean percentage (and SD) of hemocytes live at T0: doxycycline treated, 93% (±3.2%); ciprofloxacin treated, 98% (±2.5%); wounded, 96% (±3.5%); untreated, 93% (±3.6%). Mean percentage (and SD) of hemocytes live at T7: doxycycline treated, 95% (±4.7%); ciprofloxacin treated, 95% (±2.5%); wounded, 96% (±2.1%); wounded, 94% (±3.5%).\n\nThe total number of hemocytes, observed by microscopy, was similar at T0 and T7 (Figure 5) with mean cell counts of 68 (standard deviation ±32) and 62 (standard deviation ±13), respectively. This suggests that we did not see replication of the hemocytes. However, it is also possible that there was balanced growth and division and corresponding death of hemocytes.\n\nAverage across three replicate wells, derived from the pooled hemolymph from 10 larvae and seeded at 2 × 105 cells/ml. Hemocytes stained with trypan blue and dissociated with trypsin at T7 only. Errors bars = SD. Mean (and SD) cell counts of hemocytes at T0; 68 (±32) or at T7; 62 (±12.5).\n\nHemocyte survival at 37°C. We next repeated the study outlined above, but incubated the hemocytes at 37°C (Figure 6). We found that 71%–81% of the hemocytes excluded trypan blue day 7 days after isolation from larvae compared to 85%–95% at T0. The dosing of larvae with doxycycline or ciprofloxacin before harvesting the hemocytes did not affect their subsequent survival at T7, compared to the survival of hemocytes isolated from larvae dosed with PBS or sham injected. Overall, our results indicate reduced survival of hemocytes maintained at 37°C compared to 28°C. However, the numbers of cells that survive for 7 days at 37°C were sufficient to allow experimental studies during this time.\n\nViability of ex vivo hemocytes incubated at 37°C at T0 (A) and T7 (B). Average across 3 replicate wells per treatment, derived from the pooled hemolymph from 10 larvae and seeded at 2 × 105 cells/ml. Hemocytes were dissociated with trypsin and stained with trypan blue. Error bars = SD. Mean percentage (and SD) of hemocytes live at T0: doxycycline treated 93% (±3); ciprofloxacin treated, 85% (±6.9); wounded, 95% (±6.2); untreated, 93% (±2.1). Mean percentage (and SD) of hemocytes live at T7: doxycycline treated 71% (±2.5); ciprofloxacin treated, 81% (±4.2); wounded, 79% (±4.4); untreated, 77% (±6.7).\n\nDifferential hemocyte counts. Unstained hemocytes isolated from larvae that were sham injected were assigned into different groups depending on their appearance by microscopy. We identified five forms of cells which we termed granulocyte-like, spherulocyte-like, plasmatocyte-like, prohemocyte-like or oenocytoid-like (Table 3). The representation of the different hemocyte types was broadly similar at the time of isolation (T0) or after culture for 7 days (T7). However, there was a small reduction in the total number of hemocytes recorded (Figure 6) and there was a reduction in the proportion of spherulocyte-like cells and an increase in the proportion of prohemocyte-like cells. Cell counts for all experiments, in addition to the raw image files used to generate figures, are available as Underlying data21.\n\nDifferential hemocyte counts were based on morphological differences of unstained cells under 20 × magnification. Percentages shown are the averages across five views per well from triplicate wells of hemolymph pooled from 10 larvae.\n\n\nDiscussion\n\nWe report here a method for the reliable isolation of hemocytes for further studies on the biology of these cells. The method is easily transferred to other laboratories and requires no specialist techniques or equipment. Since the hemocytes survive for at least 7 days as primary cultures, even at 37°C, it should be possible to study the uptake, growth and survival of a range of pathogens within these phagocytes. This will allow the behaviour or pathogens in insect hemocytes to be compared with the behaviour of pathogens in mammalian phagocytes. In the longer term, an increased understanding of the behaviour of G. mellonella hemocytes will increase our understanding of the limitations of using insects to model infectious diseases of mammals.\n\nPreviously it has been reported that dosing with a sub-lethal dose of pathogen or a microbial component can evoke immune priming in G. mellonella larvae, where hemocytes which are normally bound to the inner surface of the cuticle become activated and mobilised into the hemolymph22. A similar response can be elicited by some antimicrobials and by physical stresses such as physical agitation of the larvae or temperature changes22. Our findings show that injury is another stress that can cause hemocyte mobilisation. Our findings highlight the importance of including a sham-injected control group in infection studies.\n\nWe did not see evidence of growth and replication of hemocytes in our study. It is likely that hemocytes require the presence of additional factors in order to replicate23. Yamashita and Iwabuchi16 reported that prohemocytes from Bombyx mori were more likely to divide if larval hemolymph was included in the culture medium. Further work should investigate how the replication of hemocytes can be encouraged ex vivo.\n\nThe hemocyte types that we found are similar to the types previously reported by Arteaga Blanco et al.15 and Gwokyalya and Altuntaş24 though other workers have reported different sub-populations of hemocytes25–27. Granulocytes are considered to be the most abundant type of hemocyte and one of the main types of phagocytic cell14. They would be expected to be mobilised following injection or wounding. Plasmatocytes are the other main type of phagocyte14. Whilst our findings broadly reflect those of Arteaga Blanco et al.15 and Gwokyalya and Altuntaş 24, there are some differences in the proportions of different hemocyte types. These previous studies concluded that granulocytes and plasmatocytes together formed the majority of hemocytes (81.6–87.8% and 93.8–96%, respectively), whereas our predominant sub-populations at T0 were granulocyte-like and spherulocyte-like cells (81.6%). The method we have reported here will allow further work to investigate whether pathogens interact in different ways with the different sub-populations of hemocytes and to study whether some sub-populations are more able to eliminate pathogens than others. This information might provide new insight into the different roles of the hemocyte sub-populations. One important goal of any future research should therefore be to develop methods for the selective enrichment of the individual sub-populations of hemocytes.\n\nIn summary, we have optimised a protocol for the extraction of hemocytes from G. mellonella and the maintenance of these hemocytes ex vivo for a period of at least 7 days. The method we report here will now allow other investigators to isolate hemocytes and study the interactions of different types of hemocytes with pathogens. Our work also raises the possibility that protection against infection seen after the administration of drugs may be a consequence of the mobilisation of hemocytes as a consequence of the traumatic injury suffered.\n\n\nData availability\n\nOpen Science Framework: Isolation and primary culture of Galleria mellonella hemocytes for infection studies. https://doi.org/10.17605/OSF.IO/C97DT21.\n\nThis project contains the following underlying data:\n\nHemocyte_counts_treated_untreated_larvae_sets (numbers of hemocytes recovered from pooled hemolymph drawn from treated and untreated larvae sets)\n\nT0_hemocyte_viability_28C (numbers of live and dead hemocytes recovered from pooled hemolymph, incubated at 28°C and assessed at T0)\n\nT0_hemocyte_viability_37°C (numbers of live and dead hemocytes recovered from pooled hemolymph, incubated at 37°C andassessed at T0)\n\nT7_hemocyte_viability_28C (numbers of live and dead hemocytes recovered from pooled hemolymph, incubated at 28°C and assessed at T7)\n\nT7_hemocyte_viability_37°C (numbers of live and dead hemocytes recovered from pooled hemolymph, incubated at 37°C and assessed at T7)\n\nTotal_hemocyte_count_wounded (total number of hemocytes in 10 µl of cell suspension from sham injected larvae, incubated at 28°C, assessed at T0 and T7)\n\nFigure_3_hemocyte_counts_treated_untreated (bar chart of numbers of hemocytes recovered from pooled hemolymph drawn from treated and untreated larvae sets)\n\nFigure_4A_T0_hemocyte_viability_28C (bar chart of percentage of live and dead hemocytes recovered from pooled hemolymph, incubated at 28°C and assessed at T0)\n\nFigure_4B_T7_hemocytes_viability_28C (bar chart of percentage of live and dead hemocytes recovered from pooled hemolymph, incubated at 28°C and assessed at T7)\n\nFigure_5_total_hemocyte_count_wounded (bar chart of total number of hemocytes in 10 µl of cell suspension from sham injected larvae, incubated at 28°C, assessed at T0 and T7)\n\nFigure_6A_T0_hemocyte_viability_37C (bar chart of percentage of live and dead hemocytes recovered from pooled hemolymph, incubated at 37°C and assessed at T0)\n\nFigure_6B_T7_hemocyte_viability_37C (bar chart of percentage of live and dead hemocytes recovered from pooled hemolymph, incubated at 37°C and assessed at T7)\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 roles\n\nTitball R: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Validation, Visualization, Writing – Review & Editing; Senior, N: Experimental work, Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing", "appendix": "References\n\nChampion OL, Wagley S, Titball RW: Galleria mellonella as a model host for microbiological and toxin research. Virulence. 2016; 7(7): 840–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai CJY, Loh JMS, Proft T: Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing. Virulence. 2016; 7(3): 214–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCutuli MA, Petronio GP, Vergalito XX, et al.: Galleria mellonella as a consolidated in vivo model hosts: New developments in antibacterial strategies and novel drug testing. Virulence. 2019; 10(1): 527–541. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKavanagh K, Sheehan G: The Use of Galleria mellonella Larvae to Identify Novel Antimicrobial Agents against Fungal Species of Medical Interest. J Fungi (Basel). 2018; 4(3): 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllegra E, Titball RW, Carter J, et al.: Galleria mellonella larvae allow the discrimination of toxic and non-toxic chemicals. Chemosphere. 2018; 198: 469–472. PubMed Abstract \| Publisher Full Text\n\nKavanagh K, Reeves EP: Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens. FEMS Microbiol Rev. 2004; 28(1): 101–12. PubMed Abstract \| Publisher Full Text\n\nPereira TC, de Barros PP, de Oliveira Fugisaki LR, et al.: Recent Advances in the Use of Galleria mellonella Model to Study Immune Responses against Human Pathogens. J Fungi (Basel). 2018; 4(4): 128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWojda I: Immunity of the greater wax moth Galleria mellonella. Insect Sci. 2017; 24(3): 342–357. PubMed Abstract \| Publisher Full Text\n\nLavine MD, Strand MR: Insect hemocytes and their role in immunity. Insect Biochem Mol Biol. 2002; 32(10): 1295–309. PubMed Abstract \| Publisher Full Text\n\nSheehan G, Garvey A, Croke M, et al.: Innate humoral immune defences in mammals and insects: The same, with differences? Virulence. 2018; 9(1): 1625–1639. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrowne N, Heelan M, Kavanagh K: An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes. Virulence. 2013; 4(7): 597–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrinkmann V, Reichard U, Goosmann C, et al.: Neutrophil extracellular traps kill bacteria. Science. 2004; 303(5663): 1532–5. PubMed Abstract \| Publisher Full Text\n\nAltincicek B, Stötzel S, Wygrecka M, et al.: Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects. J Immunol. 2008; 181(4): 2705–12. PubMed Abstract \| Publisher Full Text\n\nWu G, Liu Y, Ding Y, et al.: Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity. Tissue Cell. 2016; 48(4): 297–304. PubMed Abstract \| Publisher Full Text\n\nArteaga Blanco LA, Crispim JS, Fernandes KM, et al.: Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae. Cell Tissue Res. 2017; 370(1): 153–168. PubMed Abstract \| Publisher Full Text\n\nYamashita M, Iwabuchi K: Bombyx mori prohemocyte division and differentiation in individual microcultures. J Insect Physiol. 2001; 47(4–5): 325–31. PubMed Abstract \| Publisher Full Text\n\nNorville IH, Hartley MG, Martinez E, et al.: Galleria mellonella as an alternative model of Coxiella burnetii infection. Microbiology (Reading). 2014; 160(Pt 6): 1175–81. PubMed Abstract \| Publisher Full Text\n\nThomas RJ, Hamblin KA, Armstrong SJ, et al.: Galleria mellonella as a model system to test the pharmacokinetics and efficacy of antibiotics against Burkholderia pseudomallei. Int J Antimicrob Agents. 2013; 41(4): 330–6. PubMed Abstract \| Publisher Full Text\n\nAperis G, Fuchs BB, Anderson AA, et al.: Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain. Microbes Infect. 2007; 9(6): 729–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharan J, Kantharia ND: How to calculate sample size in animal studies? J Pharmacol Pharmacother. 2013; 4(4): 303–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenior N, Titball R: Isolation and primary culture of Galleria mellonella hemocytes for infection studies. 2020. http://www.doi.org/10.17605/OSF.IO/C97DT\n\nSheehan G, Farrell G, Kavanagh K: Immune priming: the secret weapon of the insect world. Virulence. 2020; 11(1): 238–246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNardi JB, Pilas B, Ujhelyi E, et al.: Hematopoietic organs of Manduca sexta and hemocyte lineages. Dev Genes Evol. 2003; 213(10): 477–91. PubMed Abstract \| Publisher Full Text\n\nGwokyalya R, Altuntas H: Boric acid-induced immunotoxicity and genotoxicity in model insect Galleria mellonella L. (Lepidoptera: Pyralidae). Arch Insect Biochem Physiol. 2019; 101(4): e21588. PubMed Abstract \| Publisher Full Text\n\nJones JC: CHANGES IN THE HEMOCYTE PICTURE OF GALLERIA MELLONELLA (LINNAEUS). Biol Bull. 1967; 132(2): 211–221. PubMed Abstract \| Publisher Full Text\n\nBrowne N, Surlis C, Maher A, et al.: Prolonged pre-incubation increases the susceptibility of Galleria mellonella larvae to bacterial and fungal infection. Virulence. 2015; 6(5): 458–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltuntas H, Kılıç AY, Uçkan F, et al.: Effects of gibberellic acid on hemocytes of Galleria mellonella L. (Lepidoptera: Pyralidae). Environ Entomol. 2012; 41(3): 688–96. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75815", "date": "18 Dec 2020", "name": "Lauren Ames", "expertise": [ "Reviewer Expertise Medical mycology", "invertebrate models of fungal infection", "yeast genetics", "antifungal drugs." ], "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 paper details a novel method for the extraction and culture of hemocytes from the wax moth larva, Galleria mellonella. This invertebrate host has become routinely used for studying the virulence of microbial pathogens and antimicrobial drug efficacy. However, current G. mellonella infection models are mostly limited to measuring larval survival as the main data output. In this study, the authors found that hemocytes can be extracted from G. mellonella larvae and maintained ex vivo for seven days post isolation with high viability. These findings are important because they broaden the G. mellonella infection model toolbox to include the study of pathogen-immune cell interactions. The authors also note increased hemocyte recovery from needle stick injured larvae, highlighting the importance of including relevant controls in routine G. mellonella survival experiments.\nThe method in this paper is described with high attention to detail and the results are clearly presented. Conclusions drawn are strongly supported by data from carefully thought-out experiments. The work contributes to the development of invertebrate infection models which is vital for reducing the use of mammalian hosts. My only suggestion is that a figure showing representative images of the hemocyte subpopulations identified could be useful to a researcher using this method for the first time.\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": [ { "c_id": "6344", "date": "16 Feb 2021", "name": "Nicola Senior", "role": "Author Response", "response": "We thank the reviewer for the time they have invested in improving our manuscript. We agree that images would be useful. We do not have publication quality images to hand but will now aim to collect these images." } ] }, { "id": "77334", "date": "20 Jan 2021", "name": "Daniel R. Neill", "expertise": [ "Reviewer Expertise Infection models and host-pathogen interactions. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact." ], "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 by Senior and Titball describes a novel method for isolation and culture of hemocytes from Galleria mellonella larvae. These larvae are, as the authors point out, increasingly used as a cheap, reliable and more ethically acceptable alternative to mammalian species, for the study of infection and in therapeutic development. Despite this, methodologies for working with G. mellonella are not standardised and many aspects of the larvae's biology are not well described. This paper sets out to address some of these knowledge gaps, and makes several important contributions to the field.\nFirstly, a method for reliable isolation of hemocytes is described. Sufficient detail is given for others to be able to adopt the method and we expect that it will be widely used. The authors highlight a number of potential applications in the fields of immunology, infection and therapeutics and there is clear potential for exploitation of the model in all these areas.\nSecondly, the authors describe the effect of needle injury on hemocyte recovery and highlight the implications this has for experiments performed with G. mellonella. We agree with the authors that the increased recovery of immune cells following needle injury (in the absence of substance administration) demonstrates the importance of including sham injected controls in experiments.\nThe manuscript is clear and well-written throughout and represents an important step forward for the field, with obvious 3Rs implications. We have a few minor comments that might help readers better understand and utilise the model:\nOne use of this model would be to look at hemocyte responses in infected larvae. Such experiments would likely be performed at 37C. The authors provide nice data on hemocyte survival ex vivo at 37C but can they comment on how recovery differs when the larvae are maintained at 37C, rather than 15C, prior to harvesting the hemocytes?\n\nTo the uninitiated, the terms used for the hemocyte sub-types will likely mean little. Microscopy images would be useful to enable users to distinguish between subtypes in their cultures.\n\nThe authors state that using pooled hemocyte cultures prevents statistical analysis when the individual larvae is treated as the experimental unit, which is fine, but this doesn't seem practical for most users, who will want to statistically compare cultures. Can the authors comment on sample size determination for future studies that treat individual culture wells as the experimental unit? The mean and S.D. of recovery and viability per larvae is given, but are there other factors that need to be considered, such as the rate of bacterial contamination of recovered cells?\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? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6345", "date": "16 Feb 2021", "name": "Nicola Senior", "role": "Author Response", "response": "We thank the reviewers for the time they have invested in improving our manuscript. Response to point 1. This is an interesting point raised by the reviewers. For infection studies using G. mellonella larvae we normally maintain them at 15C, and then transfer them to a higher temperature after challenge. This is a protocol used by most other researchers. We have used this protocol in the studies reported here, where hemocytes are harvested from larvae at 15C, but then incubated at 37C (or 28C where indicated). We do not have any data on the yield or properties of hemocytes taken from larvae which had been held at 37C before harvesting the cells. This is certainly a study that other investigators might want to undertake. Response to point 2. We agree that images would be useful. We do not have publication quality images to hand but will now aim to collect these images. Response to point 3. It would be feasible to include 3 or 4 groups of 10 larvae in each experimental unit, allowing statistical analysis to be undertaken. We did not observe any bacterial contamination in the studies we undertook, over the time period we reported, and we would not expect this to be a problem." } ] } ]	1	https://f1000research.com/articles/9-1392
https://f1000research.com/articles/10-116/v1	16 Feb 21	{ "type": "Research Article", "title": "Analysis of diagnostic product portfolios using the Portfolio-To-Impact modelling tool", "authors": [ "Maël Redard-Jacot", "Devy M. Emperador", "Eva Junyent", "Mickey Urdea", "Rich Thayer", "Rangarajan Sampath", "Devy M. Emperador", "Eva Junyent", "Mickey Urdea", "Rich Thayer", "Rangarajan Sampath" ], "abstract": "Background: The Portfolio-To-Impact version 2 (P2I v.2) financial forecasting tool estimates funding requirements for development of portfolios of candidate health products (drugs, biologics, vaccines or diagnostics). The assumptions and archetypes relating to diagnostics in P2I v.2 are based on limited data and may not accurately describe research and development costs, timelines and probability of success. This study aimed to revise the P2I v.2 tool by modifying the diagnostic assumptions to improve accuracy of predictions for diagnostic portfolios. Methods: Data from expert interviews and historical information on development of 26 existing diagnostics were used to determine approximate research and development costs, timelines and probability of success for development of diagnostics, and to revise diagnostic archetypes and development phases. To compare the revised tool with P2I v.2, data on 27 candidates from the Foundation for Innovative New Diagnostics (FIND) tuberculosis and pandemic preparedness portfolios were input into both versions. Results: The number of diagnostic archetypes increased from two in P2I v.2 to three in the revised tool. Total estimated costs to move the 27 candidates along the pipeline to launch were US$641.62 million with P2I v.2 and US$274.00 million with the revised model. The number of expected launches was 21.65 over five years with P2I v.2 and 11.48 over eight years with the revised model. Development timelines were extended and probability of success was lower with the revised model compared with P2I v.2. Conclusions: Outputs from the revised tool were in line with expert experience, suggesting that the proposed revisions improve the accuracy of the tool for estimating research and development costs, timelines and probability of success relating to diagnostic portfolios. Additional improvements to the tool could include further refinement of archetypes, incorporation of a measure of potential public health impact, and addition of a commercialization phase for diagnostics.", "keywords": [ "diagnostics", "forecasting", "archetypes", "costs", "probability of success", "portfolio", "P2I" ], "content": "Introduction\n\nThe Portfolio-To-Impact (P2I) model is a financial forecasting tool developed by the Special Programme for Research and Training in Tropical Diseases (TDR)1. The model enables users to estimate funding requirements for development of a portfolio of candidate health products, from late stage preclinical studies to phase III clinical trials, and estimates the number of potential product launches over time, as well as the probability of success of health products. The P2I v.2 model allows a range of different types of medical products to be assessed, including drugs, biologics, vaccines, and diagnostics. These are described as ‘archetypes’ and have further subdivisions within the model depending on the complexity of the product1,2. The model is intended to be continually refined and improved, and users are encouraged to provide inputs to update key parameters and assumptions. In line with this, a second iteration of the model (P2I v.2) that includes additional archetypes and has modified cost assumptions was subsequently developed and used to estimate research and development costs associated with a pipeline of candidates for 35 neglected diseases2. The model has also been used to assess the vaccine portfolio of the European Vaccine Initiative (EVI), which includes vaccine candidates for various diseases of poverty and emerging infectious diseases3.\n\nThe development pathway for diagnostics differs considerably from that of pharmaceuticals and vaccines, as they do not follow the traditional pathway from preclinical to phase III studies. Rather, after initial concept and research and feasibility planning, diagnostics move from the design and development phase directly to clinical validation, approval and launch4,5. However, there is a lack of academic literature available on development characteristics for diagnostics, thus the archetypes and assumptions used within the P2I v.2 model were based solely on interviews with diagnostics developers and other diagnostic experts. Based on the experience of the Foundation for Innovative New Diagnostics (FIND), a product development partnership (PDP) focusing on diagnostics for diseases of poverty6, the diagnostic assumptions in the P2I v.2 model tend to underestimate the research and development costs and timelines for diagnostic discovery and early stage development. Additionally, due to the complex nature of the diagnostic landscape, the archetypes and assumptions in P2I v.2 may not accurately describe all diagnostics. A range of factors can impact diagnostic research and development costs, timelines and probability of success, including the underlying technology of an assay, the maturity of the manufacturing company and their expertise and experience (in clinical, regulatory, quality management and infectious disease areas, as well as in assay development), the disease area, ease of obtaining samples for validation, and the Stringent Regulatory Authority (SRA) being pursued.\n\nFIND has a large diagnostics pipeline across six disease areas (antimicrobial resistance, hepatitis C and human immunodeficiency virus [HIV], malaria and fever, neglected tropical diseases, tuberculosis [TB] and pandemic preparedness), and plans to use the P2I v.2 model as a complementary tool to existing portfolio management methods for prospective scenario planning, to inform funding decisions, refine technology assessments, and predict product launches and estimated research and development costs. The objective of this study was to propose revisions to the P2I v.2 diagnostic archetypes and assumptions, to improve accuracy of predictions for diagnostic portfolios, and to compare outputs of the revised model with the original P2I v.2 tool.\n\n\nMethods\n\nThis work was carried out between April and December 2019. Data to inform the refinement of the P2I v.2 tool were collected through two methods: a) expert interviews and b) collection of historical data on development of existing diagnostics. The expert interviews were performed by the FIND project team. Seven experts with experience in diagnostic development in both high-income countries (HICs) and in low- and middle-income countries (LMICs) were asked if they were willing to be interviewed; all experts were either employees of FIND, consultants, or FIND external partners (Table 1). We obtained consent orally before the beginning of the interviews. Interviewees were provided with background information about the P2I v.2 tool, and were asked to give feedback on the tool’s diagnostic assumptions in relation to at least one product that they had supported through development. Data ranges for research and development costs, development timelines, and probability of success per product development phase were collected.\n\nHCV, hepatitis C virus; HIV, human immunodeficiency virus; TB, tuberculosis.\n\nThe historical data for existing diagnostics (ideally commercially available tests) were retrieved from the database housed by Halteres Associates (San Ramon, CA, USA), a consulting company with extensive experience in diagnostic development. A set of 26 diagnostic products for which sufficient data were available were selected from a broad group of product types and product development stages (Table 2). Research and development costs were approximate, and were based either on information from the companies themselves or from persons with knowledge of the companies; additionally, a number of the products were not yet marketed, thus final numbers for research and development costs and timelines were not available. As such, ranges for research and development costs and timelines were used except where otherwise stated.\n\naIncluding instrument; bincluding manufacturing system; cwith new organization; dincluding instrument and disposable or cartridge; eassay only; fno regulatory; gCLIA LDT; hFDA. Products are not ascribed to a specific company due to confidentiality agreements. Ag, antigen; AMR, antimicrobial resistance; BC, breast cancer; CAA, circulating anodic antigen; CBC, complete blood count; CLIA, Clinical Laboratory Improvement Amendments; CMOS, complementary metal-oxide semiconductor; CrAg, Cryptococcal Antigen; FDA, United States Food and Drug Administration; HAI, haemagglutination inhibition assay; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; hs, high-sensitivity; IA, immunoassay; LAM, lipoarabinomannan; Lat, latent; PC, pancreatic cancer; RA, rheumatoid arthritis; RDT, rapid diagnostic test; ST, self-test; TB, tuberculosis; Tni, troponin I; TSH, thyroid stimulating hormone; VL, viral load.\n\nDiagnostic archetypes were determined by identifying clusters among the 26 products with similar estimates for research and development costs, timelines and probability of success. This was done by calculating a composite ‘level of risk’ score that comprised factors thought to have most impact on these three assumptions. Initially, the following factors were included: target product profile, biomarker status, assay technology, instrumentation, per assay development costs, system development costs, regulatory requirements, preclinical/clinical studies, team competence, financial strength, payment/reimbursement model, product line extension, voice of customer, partner options, market development and commercialization plan/budget, entity stability, number of tests needed, and market demand/development. From this list, 15 factors were extracted that were believed to have the most impact (resulting in the removal of entity stability, number of tests needed, and market demand/development from the list), and the list was then further reduced for practicality of scoring, to obtain a set of eight criteria. Each of the 26 products was then assessed using the eight criteria, scored on a scale of 0 to 3 (Table 3). Approximate groupings were then determined based on the level of risk score.\n\nTPP, target product profile.\n\nIn order to compare the two models, data on the products from two of FIND’s portfolios, TB and pandemic preparedness, were input into the P2I v.2 model, using the original diagnostic assumptions and archetypes (Table 4 and Table 5) and using the revised diagnostic assumptions and archetypes collected from the expert interviews and the historical database of 26 existing diagnostics. The research and development cost and timelines to move the candidates from the two FIND portfolios through the pipeline, and to estimate the likely number of launches per disease, was estimated. The model assumed a 2019 start, with all diagnostic candidates inputted based on their development phase at the time of review.\n\nThis table is adapted from references 1 and 2 under a CC-By 4.0 license.\n\nThis table is adapted from reference 2 under a CC-By 4.0 license.\n\nIt can be challenging to compare diagnostic opportunities when they have comparable levels of risk. In these cases, the potential public health impact of a diagnostic can be a valuable differentiating factor. Factors that can influence the potential public health impact of a diagnostic product were identified from the historical database of 26 diagnostic products. Ten factors were identified; these were: increase in number of patients/year receiving appropriate treatment, cost per result, improvement in guideline adherence, time to intervention, improved access to critical data, support for diagnostic, surveillance or outbreak programmes (as intended), impact metrics, change in disease incidence or prevalence, reduction in loss to follow-up, and change in morbidity or mortality. The list was reduced to seven factors with the most influence, and a potential impact score for ranking was developed, scored on a scale of 0 to 3 (Table 6). Each of the 26 products from the historical database was scored according to this system.\n\nMDA, Medical Devices Agency; TPP, target product profile.\n\n\nResults\n\nDiagnostic archetypes. To determine the optimal diagnostic archetypes, clusters of diagnostic products that depicted similar estimates for research and development cost, timelines and probability of success were identified from the historical dataset of 26 diagnostic products (Figure 1). Approximate groupings of products that aligned with three archetypes emerged:\n\nProducts are not ascribed to a specific company due to confidentiality agreements. Ag, antigen; AMR, antimicrobial resistance; BC, breast cancer; CAA, circulating anodic antigen; CBC, complete blood count; CMOS, complementary metal-oxide semiconductor; CrAg, Cryptococcal Antigen; HAI, haemagglutination inhibition assay; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; hs, high-sensitivity; IA, immunoassay; LAM, lipoarabinomannan; Lat, latent; PC, pancreatic cancer; RA, rheumatoid arthritis; RDT, rapid diagnostic test; ST, self-test; TB, tuberculosis; Tni, troponin I; TSH, thyroid stimulating hormone; VL, viral load.\n\n1. “New or existing entity, discovery” archetype: a research or commercial stage entity in the discovery phase of a new technology\n\n2. “Existing entity, new product/technology” archetype: an established commercial company that is creating a new product that is not a product line extension\n\n3. “Existing entity, product extension” archetype: an established commercial company that is creating a new product as an extension of an existing product line\n\nDiagnostic assumptions. The expert interviewees proposed three development phases for diagnostics: “discovery”, “design and development” and “clinical validation and launch readiness”. These were fitted to the phases in the P2I v.2 model2 by encompassing both “concept and research” and “feasibility and planning” from the original P2I v.2 tool into the “discovery” phase. The remaining two development phases were the same as those in the P2I v.2 tool (“design and development” and “clinical validation and launch readiness”). A fourth phase, “commercialization”, was recommended by the interviewees, as this can be costly for diagnostics. Data for this phase are presented, but it was not included in the comparison analysis with the P2I v.2 tool.\n\nData from the expert interviews and the historical database of 26 existing diagnostics were used to identify the revised research and development cost, timeline and probability of success assumptions per product phase. Where the database included data ranges, averages were calculated for the purposes of inputting into the P2I model. Data were weighted at 0.67 for “concept and research” and 0.33 for “feasibility and planning”, based on expert experience suggesting that greater time and cost is placed at the concept stage.\n\nTable 7 lists the revised diagnostic assumptions. The average costs per phase were higher in the updated assumptions compared with the original P2I v.2 tool assumptions (“discovery”: US$1.1–24.6 million versus 3.0 million (combined “concept and research” and “feasibility and planning”); “clinical validation and launch readiness”: US$6.9–10.0 million versus 3.5 million), with the exception of the cost of the “design and development” phase, which was significantly lower than the initial assumptions (US$3.3–6.9 million versus US$2.0 to 100.0 million). As it was not possible to calculate specific probability of success scores for each archetype with the available data, an average across all archetypes was used for each phase. The probability of success was significantly lower in the updated diagnostic assumptions compared with the initial assumptions.\n\naPhase based on recommendation from experts and combines “concept and research” and “feasibility and planning” in P2I v2 model. bTotal up to “clinical validation and launch readiness”. cProbability of success in “concept and research”: 30%; probability of success in “feasibility and planning”: 60%.\n\nFIND’s current TB and pandemic preparedness portfolios contained a total of 27 diagnostic candidates under development (Table 8). This included three products for Lassa fever, two on multi-disease fever-causing pathogens, one on yellow fever, and 12 products for TB. Using the P2I v.2 model, 12 products were classified as the “simple technical platform development” archetype and 15 were categorized as the “assay development” archetype. All four development phases were covered. Using the revised model, nine products were classified as the “existing entity, new product/technology” archetype; 10 as the “new or existing entity, discovery” archetype; and eight as the “existing entity, product extension” archetype.\n\nTB, tuberculosis.\n\nUsing the P2I v.2 model, the total estimated cost to move the 27 candidates along the pipeline to launch was US$641.62 million, with the majority of costs (77%) incurred by the development of the TB diagnostic candidates, followed by the multi-disease fever candidates (20%) (Figure 2A). Using the revised model, total estimated costs to move the 27 candidates along the pipeline to expected launch were around US$274.00 million, substantially lower than the cost estimate from the initial model. However, the costs of developing the yellow fever and Lassa fever diagnostics were higher compared with the initial model due to increased costs during early development (Figure 2B).\n\nCost by disease area using A) P2I v.2 model and B) revised model.\n\nThe total expected number of launches across all 27 candidates using the P2I v.2 model was 21.65, with the majority of launches for the TB candidates (17.82), followed by the Lassa fever candidates (2.42) (Figure 3A). Using the revised model, the total launch probability for all 27 candidates was reduced to 11.48 (Figure 3B), a little over 50% lower than the P2I v.2 model projected probability, most likely due to the lower probability of success rates.\n\nNumber of launches by disease area using A) P2I v.2 model and B) revised model.\n\nThe P2I v.2 model predicted that the majority of costs would fall in the first three years of development; development time for all diagnostic candidates was predicted to be completed by 2024 (Figures 4A and 4B). Using the revised model, development timelines were extended, with development time for all diagnostic candidates predicted to be completed by 2028 (Figures 4C and 4D).\n\nTotal portfolio cost per year (A and B: P2I v.2; C and D: revised model).\n\nLevel of risk scores versus potential impact scores for each product are shown in Figure 5. A number of products had the same level of risk score but substantially different potential impact scores.\n\nProducts are not ascribed to a specific company due to confidentiality agreements. Ag, antigen; AMR, antimicrobial resistance; BC, breast cancer; CAA, circulating anodic antigen; CBC, complete blood count; CMOS, complementary metal-oxide semiconductor; CrAg, Cryptococcal Antigen; HAI, haemagglutination inhibition assay; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; hs, high-sensitivity; IA, immunoassay; LAM, lipoarabinomannan; Lat, latent; PC, pancreatic cancer; RA, rheumatoid arthritis; RDT, rapid diagnostic test; ST, self-test; TB, tuberculosis; Tni, troponin I; TSH, thyroid stimulating hormone; VL, viral load.\n\n\nDiscussion\n\nThe P2I v.2 tool predicted that we would require US$642 million to move the 27 products from the FIND TB and pandemic preparedness portfolios forward through the pipeline, with a launch probability of 21.65 launches over five years. Using our revised P2I model with updated diagnostic archetypes and assumptions, costs were reduced to US$274 million, with a launch probability of 11.48 launches over eight years. While the diagnostic archetypes and assumptions in P2I v.2 were solely based on interview feedback, our revised archetypes and assumptions were partially based on an analysis of the development history for 26 existing diagnostics, thus our revised tool incorporated actual data as well as expert feedback. The revised outputs are more in line with FIND’s experience compared with the outputs of the P2I v.2 model, especially for early stage products. The considerable differences between the outputs of the two models suggest that the current P2I v.2 tool may not accurately capture the research and development costs, timelines, and probability of success for diagnostics.\n\nWe found that the previous P2I diagnostic archetypes, “assay development” and “simple technical platform development”, were too confining given the breadth of assay and platform technologies used for the 26 products we analysed from the historical database. For example, some products were being developed on existing platforms and others on new platforms, but both would have been categorized under the “assay development” archetype even though the costs and development times differed significantly. While some products that improve upon current technologies with simple modifications, such as readers for lateral flow assays, fit the “simple technical platform” archetype, others were new systems with novel disposables, chemistry and instruments, which were too complex in terms of the technology used to fit under this heading. However, while the updated diagnostic archetypes may better capture the complexity of diagnostics product development, they do not necessarily represent the optimal and complete view of the reality. We recommend continued collection of additional data in order to identify more precisely the archetypes and factors that have the most impact on the assumptions examined, and allow more effective prioritization of investment in diagnostic companies and products.\n\nThe reduction in cost using the revised model compared with the P2I v.2 tool is most likely due to the reduced cost for clinical validation in the updated assumptions, as well as the lower probability of success rates compared with the original assumptions. Notably, the lower probability of success assumptions are in line with previous work on this subject7. As expected, our analysis and the feedback from the expert interviewees showed that developing newer products tends to require more investment and development time compared with menu expansion and product extension. This reflects FIND’s experience in diagnostic development for LMIC settings. In addition, there is a significant development burden in the discovery stage, particularly under the “concept and research” phase, as successful products must not only be technically sound, but must reflect their intended use.\n\nEvaluating the public health impact of a product in addition to the probability of success is valuable, as products with a high public health impact but low probability of success may still be worth investing in; although riskier than products with higher success probability, they are likely to improve the health of a greater number of people if successful. We assessed the potential impact of a new diagnostic product at the same time as assessing its level of risk, as a way to prioritize investment in diagnostic products or technologies that, on the surface, display similar cost requirements. We believe that it is important to incorporate this notion into decision-making when estimating funding needs to move candidate health products through the pipeline. Understanding the potential impact of a product can inform decisions on whether to fund or include a high-risk product in a portfolio, as well as supporting advocacy and fundraising efforts.\n\nThe interviewees recommended the addition of average research and development costs, timelines and probability of success of commercialization to the model, by inserting a new development phase. Once a product has launched, understanding the costs associated with access and sustainability, especially in LMIC markets, is important, as the probability of success of a new diagnostic test depends not only on the development of the product itself but also on the way it is delivered to the target patient populations. Even when they are available, diagnostic tests are not necessarily affordable, appropriate for use in LMICs, and/or adopted in these settings. To maximize the access to a diagnostic product, a sizeable level of human and financial resources needs to be invested in marketing, sales, manufacturing, distribution and in-country registration, which can lead to higher costs. For more accurate estimates of overall funding needs for diagnostics, future iterations of the tool could incorporate the commercialization phase.\n\nIn addition to the limitations of the P2I model already described1,2, there are some further limitations specific to our revised model that should be noted. First, the research and development costs and timelines for product development phase used in the revised P2I model correspond to averages of the historical data collected, as we were only able to obtain ranges of data rather than unique data points, from which averages were calculated to allow for inclusion into the tool. The averages may not effectively capture the large differences that were observed between products within the same archetype, which stem from a complex and multifaceted environment of the diagnostics industry. Secondly, like the developers of the original P2I tool, we faced challenges in data collection for diagnostic development, as the information needed was either not publicly available or not systematically recorded. For FIND and other PDPs with interest in diagnostics, we recommend improving organizational processes and systems for tracking, storing and sharing this type of information and/or dedicating human resources, such as a Portfolio Manager, to this task. Similarly, the number of diagnostic products used to generate new archetypes and development assumptions was limited, thus more data are required to further validate the proposed archetypes and assumptions.\n\nIn summary, we believe that our proposed revisions to the archetypes and assumptions for diagnostic products of the P2I model improve the accuracy of the tool for estimating costs and timelines relating to diagnostic portfolios. During the preparation of this article, a corrected version of the P2I v.2. was published8. We hope the data and recommendations presented here are considered for inclusion in a future iteration of this tool, particularly with regards to the inclusion of commercialization and public health impact, which may be applicable to other health products.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required. Feedback from expert interviews is not provided due to data protection/confidentiality considerations. The diagnostic products used to inform revised assumptions are not ascribed to a specific company due to confidentiality agreements.", "appendix": "Acknowledgments\n\nThe authors would like to thank the experts who were interviewed as part of this study, and Madeleine Werner, Jon Bastow, Samuel Schumacher and Kavi Ramjeet for their contributions to the planning of the project. Medical writing and editorial assistance were provided by Rachel Wright, PhD, funded by FIND, according to Good Publication Practice guidelines.\n\n\nReferences\n\nTerry RF, Yamey G, Miyazaki-Krause R, et al.: Funding global health product R&D: the Portfolio-To-Impact Model (P2I), a new tool for modelling the impact of different research portfolios [version 2; peer review: 2 approved]. Gates Open Res. 2018; 2: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoung R, Bekele T, Gunn A, et al.: Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model [version 2; peer review: 3 approved]. Gates Open Res. 2018; 2: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGunn A, Bandara S, Yamey G, et al.: Pipeline analysis of a vaccine candidate portfolio for diseases of poverty using the Portfolio-To-Impact modelling tool [version 2; peer review: 3 approved]. F1000Res. 2019; 8: 1066. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeeflang MMG, Allerberger F: How to: evaluate a diagnostic test. Clin Microbiol Infect. 2019; 25(1): 54–9. PubMed Abstract \| Publisher Full Text\n\nTB diagnostics critical pathway. 2017. Reference Source\n\nFoundation for Innovative New Diagnostics (FIND): Vision and Mission. 2019. Reference Source\n\nUrdea M: Critical success factors for diagnostics companies competing in low- and middle income markets 2018 29 May 2020. Reference Source\n\nYoung R, Bekele T, Gunn A, et al.: Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model [version 3; peer review: 3 approved]. Gates Open Res. 2020; 2: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "124697", "date": "28 Feb 2022", "name": "Alec Morton", "expertise": [ "Reviewer Expertise I do methodological and applied research in resource allocation", "decision analysis and health economics relating to health interventions and products", "contracting for innovation in new technologies and general public health and health financing." ], "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 the opportunity to review this interesting paper. The research reported here represents an advance in an area where relatively little is known. My view would be that while this a step forward, and is publishable as such, much more remains to be done and my comments should be taken in that spirit.\n\nIn terms of the general approach, I would highlight that there is a considerable amount of uncertainty in the inputs but this uncertainty is not apparent in the output numbers (Figs 2 to 4). It is recognised in the structure of the model that managing a portfolio is about managing risk but risk is only captured through the probability of success. I would argue that managing a portfolio also requires managing the cost risk esp as Table 2 shows that cost uncertainty about particular products can be quite substantial. Squeezing out the uncertainty by averaging seems to me a missed opportunity and loses managerially relevant information.\nThe most obvious gap from the point of view of building confidence in the underlying figures is to close the validation loop, and undertake prospective research to validate these numbers. To some extent this is recognised in the conclusion but this takes the form of relatively weak recommendations to “gather more data”. This data gathering would have to be coordinated by some powerful actor in the field (such as a funder) to ensure methodological consistency and accuracy. Are the authors able to make more concrete recommendations on this validation can be undertaken and confidence built, including saying who should do this and how? I also feel that the paper could be strengthened by clearer explanation and justification of the methods in various places and this is my reason for my “partly” reply to the questions about replicability and also about source data.\nTable 2. It is possible to give a statement of what is included in the “costs”? For example, are the costs simply the direct costs of the staff working on the product, and materials required for development, or do they also include estate overheads including for shared facilities and if so how apportioned? Management overheads? Cost of capital? Etc etc. Do they include payments to third parties in which case the prices will reflect not only the cost to the third party but also the surplus which the 3rd party can command due to its market position? I understand that these are top down estimates rather than bottom up micro costed numbers but it would be good to know more about what the people giving the estimated thought they had been asked to estimate. “Research and development costs were approximate, and were based either on information from the companies themselves or from persons with knowledge of the companies; additionally, a number of the products were not yet marketed, thus final numbers for research and development costs and timelines were not available.” – is it possible to show on the table which numbers came from the companies and which from outside experts; and where the numbers are forecasts and where they are for realised actuals?\nIs it possible to say what the ranges in table 2 mean? Can they be given a probabilistic interpretation, such as an 95% credence interval (“I am 95% sure that the true value lies within this interval”).\nTable 3. Are the individual criterion level risk scores simply added up to come to a composite risk score? In that case has the assumption that the criteria all contribute equally been validated? What is intended profile of the person who is making this assessment, as many of the criteria would require quite a bit of contextual knowledge to assess (e.g experience with the intended customer), and what is the process by which this assessment would be made? Conceptually isn’t there an overlap between financial strength and market development criteria?\nProbability of success How was the information from the different interviews aggregated to come up with a probability of success number? Is there a reason why the product are grouped into classes and then a probability of success is defined for the class rather than having the probability success defined as a function of the risk score, which would make better use of all of the information in the risk scoring? Also, trivially but also usefully for the avoidance of unnecessary confusion would it not be more appropriate to call the risk score a “confidence score” as a high score means low risk?\nPotential impact I can see the rationale for the criteria. However, I would like to know about how performance against these criteria can be robustly assessed in practice as for many if not most of the criteria this may be quite challenging to do. I’d also argue that some of the criteria are instrumental (e.g. improvement in guideline adherence) and some relate to more health gain which is a more fundamental objective (eg. the change in disease incidence and change in mortality and morbidity). Given the differing nature of the criteria, just summing up scores may not much sense (Morton, 2017). I’m also puzzled that on fig5 the discovery archetype products seem to be low impact and the extension archetype products are high impact – shouldn’t it be the other way around? Maybe it would be good to label the quadrants of the chart as high/ low risk and high/ low impact to make the message clearer.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "125480", "date": "27 Apr 2022", "name": "Célia Lemaire", "expertise": [ "Reviewer Expertise Areas of expertise: costing in health care organizations", "key performance indicators and management control systems in health and social care sectors." ], "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 entitled \"Analysis of diagnostic product portfolios using the Portfolio-To-Impact modelling tool\". In this article, the authors intend to revise an existing tool: The Portfolio-To-Impact version 2 (P2I v.2) financial forecasting tool in order to make the results more accurate and useful for prospective scenario planning (for the initial model, cf. Terry RF, Yamey G, Miyazaki-Krause R, et al.: Funding global health product R&D: the Portfolio-To-Impact Model (P2I), a new tool for modelling the impact of different research portfolios [version 2; peer review: 2 approved]. Gates Open Res. 2018; 2: 24.).\nThe target readers are heads of programs such as FIND how try to \"inform funding decisions, refine technology assessments, and predict product launches and estimated research and development costs\". The topic is important and the design approach is original. Considering the article as an improving proposition in progress, it is indexable as such. My comments aim to provide for ideas and warnings to strengthen the analysis for the future.\nThe purpose of the article: The article argues that the initial model lacks precision, notably due to the complexity of the diagnoses studied. However, if the study carried out presents a notable progress, some uncertainty remains, notably because of the newness of the purpose and the questions related to the methods. This is probably due to a lack of explanation rather than to the inadequacy of the study, but it would be relevant to underline more precisely the advances made in the model and the methodological choices that underlie them.\nThe methods: The approach combining the interviews and the retrospective data is fruitful and appropriate for the study. The use of feedback from the 26 products makes the analysis of the tool more refined. My suggestions concerning this part of the study first regards the improvement of the traceability of the results by giving more details on the data collected (1), and more details on the choices made (2). I also propose to deepen the elements concerning the cost structure (3) and to envisage a collection of data as the project progresses (4).\nTo improve traceability of results, provide more detail on the data collected:\nDetails about the seven experts interviewed should be provided: if the type of diagnosis they are experts in and the archetype in the diagnosis is specified, the reader lacks an understanding of their more precise field of expertise, their function, it should be understood from what precise point of view these experts are speaking, as they are making recommendations that are decisive in the revision of the model (e.g., the allocation of costs between the development phases).\n\nBeyond that, the authors state that the experts provided information on research and development costs, development times and probabilities of success per development phase. I do not question the expertise or the interpretation, but more details would have been appreciated: at this stage, the content of the information and the data analysis process needs to be detailed in order to understand the logic of the demonstration in the article.\n\nMore details on the choices made:\nRegarding the choice of the 26 products studied, it would be interesting to specify the reasons for abandoning the other products for the study.\nConcerning the criteria for the level of risk, the authors propose 15 and then 8 criteria. Here, the reasons for the choices are not substantiated, and more clarity is needed on this point. In addition, some criteria were rejected even though they seemed central, for example, the team’s competences. It should be specified why they were removed from the analysis.\nSimilarly, the impact criteria are reduced from 10 to 7 without explanation. This should be corrected to make the study more robust.\n\nScope of costs:\nThis topic is central to the article, and thus deserves special attention. Five difficulties are identified:\n\nRegarding the impact analysis, costs per result are calculated. These are in fact production costs, which are partial costs. While they are useful for comparisons between several organizations, these costs are insufficient for internal analysis in terms of impact. The authors make this point when they suggest that it would have been better to include the commercialization costs.\n\nThere is little support for the 67%/33% choice in the allocation of costs to phases. The authors suggest that the interviewees indicated that a large proportion was dedicated to design, but this is not sufficient to understand the choice of allocation.\n\nThe authors show a much lower valuation with their revised model, but the traceability of the process leading to the determination of the costs lacks precision to be totally credible. In particular, the extension in time of the development necessarily induces higher indirect costs for the organizations, but these costs do not seem to have been taken into account in the model (figure 4 p12).\n\nNo mention is made of the notion of opportunity costs, which correspond to the cost of abandoning certain products. However, determining these costs is essential in a management decision, because the abandonment of certain products, even expensive ones, can destabilize the overall cost structure.\n\nFinally, as the authors point out in the discussion “The averages may not effectively capture the large differences that were observed between products within the same archetype”, averages are too encompassing measures, they smooth out the differences. It would be interesting to use medians and to have more precise data, based on a collection over time.\n\nLimitations of retrospective data without measurement over time:\nThe data on research and development costs are based on declarative data. This seems to be problematic because it is a difficult item to evaluate, even from within the organization and retrospectively, if data has not been collected as the project progresses. In addition, different organizations do not have the same cost structures, which can skew the results.\n\nOther recommendations: First, in a portfolio analysis, costs and probabilities of success are important, but marketability and associated prices are also central. Further studies on these points could be considered.\nSecondly, the analysis in terms of impact deserves more investigation, and it would be interesting to specify, here again, the perspective from which the authors are working (public authorities, patients, investors, etc.). It would also be appropriate to consider the competition of other organizations in the model. Indeed, in a competitive dynamic, it is sometimes interesting to propose a ‘niche’ product in order to differentiate oneself and have a greater impact on a given pathology.\nThirdly, the authors propose that time and resources be devoted to improving organizational processes. This proposal is very relevant, and should be incorporated for analysis. In the same way, the lack of consideration of marketing costs is a pitfall to be overcome in future studies. Fourthly, since the study was conducted in 2019, does the pandemic change the findings?\nMinor Notes:\n\nThe paragraph that states 'developing newer products tends to require more investment and development time compared with menu expansion and product extension' seems unhelpful.\n\nTo improve clarity, I suggest that different terms be used for different underlying realities. In particular, the use of the word archetype poses a problem for the reader since it refers to different realities as the text progresses; a specific term should be used for each element described. For example, in the introduction, the archetypes refer to “drugs, biologics, vaccines, and diagnostics”. Here, the diagnostic part is thus one of the archetypes but is itself composed of archetypes. Then in table 4, the 'archetypes' refer to \"Assay development\" and \"Simple technical platform development\". Then, in the results, they are \"New or existing entity, discovery\", \"Existing entity, new product/technology\" and \"Existing entity, product extension\". The perspectives adopted to distinguish these three kinds of archetypes are very different and should be made explicit in the text, so different terms should be used for each and the term archetype should be dedicated to the authors' contribution (currently in the results).\n\nThe initial model could be explained with more details. For this, the recently published article with a revision of the model is very clear about the expected inputs and outputs of the model. It would be interesting to draw on it to make the tool's approach more explicit. (Young R, Bekele T, Gunn A, et al: Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model [version 3; peer review: 3 approved]. Gates Open Res. 2020; 2: 23.).\n\nRegarding the notion of risk, it would be necessary to specify which risk is involved (financial, reputational, organizational, etc.) and from which point of view (program coordinator), as this would allow for a better understanding of why a higher development cost leads to a higher risk (Table 3).\n\nFigure 5 should specify the reading direction to be clearer (where are the products with the highest risk?)\nThank you for your interesting work, I am looking forward to reading the next version 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?\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-116
https://f1000research.com/articles/10-115/v1	15 Feb 21	{ "type": "Research Article", "title": "Defining and validating regenerative farm systems using a composite of ranked agricultural practices", "authors": [ "Tommy L.D. Fenster", "Claire E. LaCanne", "Jacob R. Pecenka", "Ryan B. Schmid", "Michael M. Bredeson", "Katya M. Busenitz", "Alex M. Michels", "Kelton D. Welch", "Jonathan G. Lundgren", "Tommy L.D. Fenster", "Claire E. LaCanne", "Jacob R. Pecenka", "Ryan B. Schmid", "Michael M. Bredeson", "Katya M. Busenitz", "Alex M. Michels", "Kelton D. Welch" ], "abstract": "Background: Ongoing efforts attempt to define farms as regenerative to aid marketers, policymakers, farmers, etc. The approach needs to balance precision with function, and must be transparent, simple, scalable, transferable, incorruptible, and replicable. Methods: We developed practice-based scoring systems to distinguish regenerative cropland and rangeland, and validate them based on whether these scores scaled with regenerative goals on actual farm operations. Study systems included cornfields of the Upper Midwest, almond orchards of California, and rangeland systems of the Northern Plains. Response variables included soil carbon and organic matter, soil micronutrients, water infiltration rates, soil microbial communities, plant community structure, invertebrate community structure, pest populations, yields, and profit. Results: Regenerative outcomes were strongly correlated with our approach to farm scoring. Soil organic matter, fine particulate organic matter, total soil carbon, total soil nitrogen, phosphorous, calcium and sulfur all increased alongside regenerative matrix scores in one or both of the cropping systems. Water infiltration rates were significantly faster in more regenerative almond orchards. Soil bacterial biomass and Haney soil health test scores were higher as cropland incorporated more regenerative practices. Plant species diversity and biomass increased significantly with the number of regenerative practices employed on almonds and rangelands. Invertebrate species diversity and richness were positively associated with regenerative practices in corn, almonds, and rangelands, whereas pest populations and almond yields were unaffected by the number of regenerative practices. Corn yields were negatively associated with more regenerative practices, while almond yields were unaffected by the number of regenerative practices. Profit was significantly higher on more regenerative corn and almond operations. Conclusions: Our scoring system scaled positively with desired regenerative outcomes, and provides the basis for predicting ecosystem responses with minimal information about the farming operation. Natural clusters in the number of regenerative practices used can be used to distinguish regenerative and conventional operations.", "keywords": [ "Biodiversity", "Haney Test", "Invertebrates", "Regenerative Agriculture Label", "Soil Carbon", "Soil Health", "Soil Microbiology" ], "content": "Introduction\n\nThe term “regenerative agriculture” was first coined by Robert Rodale (1983). The name highlighted how industrialized agriculture was severely reducing its natural resource base, and that without rebuilding that natural resource base, “sustainable agriculture” and “conservation agriculture” were insufficient for supporting the food and natural resource needs of a growing human population. Regenerative agricultural systems increase soil health and promote biodiversity while producing nutritious food profitably. Some outcomes of a regenerative operation are improved greenhouse gas relationships, balanced water relationships, reduced pollution from agrichemicals, increased resiliency of farms, more nutritionally robust foods, increased ecosystem services, etc. (Elevitch et al., 2018; Francis et al., 1986; Sherwood & Uphoff, 2000). A central challenge for researchers, policy makers, consumers, etc. is defining what a regenerative farm is and having a standardized method for discerning them from conventional farms (Newton et al., 2020; Schreefel et al., 2020).\n\nThe principles and philosophy that define regenerative systems have been well formulated by practitioners and educators (Lal, 2020; Schreefel et al., 2020; UnderstandingAG, 2020; USDA-NRCS, 2020). Essentially, regenerative practices can be distilled down to two central principles: 1) reduce uniform disturbance (such as tillage and agrichemical use), and 2) increase diversity (biodiversity, and the diversity of revenue streams from an operation). The first working formula for these principles was proposed by LaCanne & Lundgren (2018) and included four principles: 1) reduce or eliminate tillage, 2) never leave bare soil, 3) maximize plant diversity and productivity on a farm, and 4) integrate livestock and cropping operations. Here, we add a fifth principle: 5) reduce or eliminate synthetic agrichemicals. In croplands, practices such as no-till, diverse crop rotations lasting more than four seasons, cover cropping, intercropping and interseeding, and livestock integration all contribute to a regenerative farming model (LaCanne & Lundgren, 2018; Rhodes, 2017). In rangelands, practices that actualize regenerative principles include reduction/elimination of synthetic agrichemicals and adaptive multi-paddock grazing systems that allow a rangeland to rest following the punctuated disturbance of grazing (Teague & Kreuter, 2020; Wagner et al., 2020). Regenerative farms are also diverse and complementary in their enterprises, and adaptive in their management choices, ensuring that a farm is resilient and profitable in the face of adversity. These practices are dependent upon one another within a system for them to be optimally successful, and it is the system, not the individual practices, that drives the success of an operation.\n\nClassifying regenerative farming operations has to balance precision with function. Adaptability and innovation are hallmarks of operations that are guided by regenerative principles. Therefore, while a matrix of responses or practices must have sufficient nuance to accurately reflect a regenerative system, it cannot be a rigid formula that inhibits that critical adaptability and ongoing innovation. Nor should a scoring system be so complex as to exclude adoption by regenerative farmers. Classification schema have been developed for other movements within ecological agriculture, including conservation agriculture (Hendrix et al., 1986; Kassam et al., 2012), organic agriculture (USDA-AMS, 2020), holistic management (Alfaro-Arguello et al., 2010), mob grazing (Gurda et al., 2018), etc. In these systems, the tangible manifestations of core principles are represented by a series of standards or practices. An inherent risk is that regenerative agriculture will follow the same pattern, and come to be defined by specific standards or practices, rather than by the principles and goals of a regenerative system. There are a growing number of certification programs and definitions for regenerative farming that capture key themes of regenerative agriculture and assemble various practices that may produce intended outcomes (AGW, 2020; Carbon_Underground, 2020; HMI, 2020; ROC, 2020; TerraGenesis, 2020). In the end, any labelling or certification system must be validated empirically to determine whether it results in the intended outcomes.\n\nRegenerative agriculture needs to be defined by principles and supported by empirical data from farms displaying a wide range of management practices to ensure the implemented principles are achieving regenerative farming goals. The approach to defining a regenerative system must be transparent, simple, scalable, transferable, incorruptible, and replicable. Our team has created a series of questions for land managers that encapsulate the principles that drive regenerative agriculture as defined by the farmers and ranchers (Fenster et al., 2021; LaCanne & Lundgren, 2018; Pecenka & Lundgren, 2019; Schmid & Lundgren, 2021). Each answer receives a score, and higher scores are indicative of more regenerative operations. A regenerative matrix score is attained by summing the scored results to these questions. The first goal of this paper is to determine whether key response variables pertaining to soil health, biodiversity, ecosystem function, and farm performance (Elevitch et al., 2018; Schreefel et al., 2020) scale with the assignment of scores from our regenerative matrix. Essentially, does a higher regenerative score in cropland and rangeland consistently scale positively with the desired outcomes of regenerative farming? The second goal of this paper is to determine whether there is a threshold score that can be used to distinguish between regenerative and conventional systems? If this matrix performs these functions, then this approach could be used to advance the field of regenerative agriculture by providing a clear, scalable and transferable approach to characterizing a regenerative farm.\n\n\nMethods\n\nTwo independent scoring systems based on a narrow suite of practices were used to define regenerative cropland and rangeland systems. Three food systems were examined in this project, corn in the Upper Midwest (LaCanne & Lundgren, 2018), almond orchards in California (Fenster et al., 2021), and rangelands of the Northern Plains (Pecenka & Lundgren, 2019; Schmid & Lundgren, 2021). Farms included in these studies represented successful, established systems and were in their respective farming philosophies for at least three years. Some of the farms were targeted for their reputation as being leaders in regenerative farming, but the resulting fields and ranches in reality represented a wide continuum from very conventional to very regenerative farming systems.\n\nWe examined different suites of response variables that are considered representative of a regenerative system. In croplands (Table 1), the elimination of tillage, maintaining ground cover through planting cover crops or fostering resident vegetation, planting hedgerows, and use of organic amendments (compost, manure, mulch, compost teas), and grazing, were all considered regenerative (LaCanne & Lundgren, 2018; Soto et al., 2021). Tillage, maintaining bare soil, and spraying synthetic insecticides, herbicides, fungicides, use of chemical fertilizers were all considered conventional practices. Regenerative practices were scored as 1, and conventional practices were scored as 0. In rangelands (Table 2), management systems were defined by their stocking density, rotation frequency, the duration that the pasture rested following grazing, and use of ivermectin products. Cattle operations were categorized based upon their combination of these practices to form the different systems (Colley et al., 2019; Teague & Barnes, 2017). Use of ivermectin in the ranches was categorized as high (multiple applications during a year; scored as 0), low (single annual use, not applied during grazing period; scored as 1) and no ivermectin (scored as 2). Operations’ stocking densities (animal units [AU] per ha), were categorized as fewer than 5 animal units (AU) per ha (scored as 0), 5–10 AU per ha (scored as 1), and more than 10 AU per ha (scored as 2). Operations were categorized as having a rotation frequency of 30 d or more (scored 0), between 10–30 d (scored as 1), and less than 10 d (scored as 2). Finally, rest periods on ranches were scored as continuously grazed (no rest; scored as 0), allowed to rest 1 > and < 30 d (1), or >30 d (2) over a growing season. A simple questionnaire to obtain the necessary information to populate our matrices can be found as in Extended data (Lundgren, 2021).\n\nShaded rows refer to corn farms in the Upper Midwest, and unshaded rows refer to almond orchards in California. Fungicide use, organic amendments, and field margins/hedgerows were not described in the cornfields. A ‘1’ was assigned for each practice if it was considered regenerative and ‘0’ if it was considered conventional. Eliminating synthetic fertilizers, herbicides, fungicides, insecticides, and tillage were considered regenerative. Including cover crops, organic amendments (e.g., composts, compost teas, etc), livestock integration, and diversified field margins and hedgerows were also considered regenerative practices.\n\nUnshaded rows refer to ranches that were included in the dung invertebrate assessment. Shaded rows refer to ranches that were examined in the fecal parasite and plant community study.\n\nManagement practices of cattle operations were scored 0-2 based, with higher numbers reflecting practices that promote biodiversity and soil quality. Ivermectin application frequency was divided into multiple applications (0), single application not during grazing season (1), and no ivermectin use (2). Stocking density (animal units; AU) was divided into <5 AU/ha (0), 5-10 AU/ha (1), and >10 AU/ha (2). Rotation frequency was divided into >30 d rotation (0), 10-30 d rotation (1), and <10 d rotation (2). Rest period was considered as continuously grazed during the growing season (0), a rest period of 1-30 d (1), and a rest of > 30 d (2).\n\nCorn was examined on 20 farms (each with four fields) in North Dakota, South Dakota, Minnesota, and Nebraska in 2015 and 2016. Fields were a minimum of 4 ha in size, and the sampling area in each field was 61 × 61 m. All samples were taken at least 12 m into the field to minimize border effects. Only three of these farms were certified organic. Genetically modified (Bt hybrid) corn was universally treated with neonicotinoid seed treatments and was regarded as insecticide-treated. In corn, we examined soil organic matter (SOM), fine particulate organic matter (fPOM), soil bulk density, water infiltration, invertebrate communities in the soil, on the soil surface and in the plant canopy, pest abundance, yields, and profits.\n\nSixteen California almond orchards were studied in 2018 and 2019. Replicate plots (n = 4; 40 × 40 m) were established in each orchard. Plots were established 20 m into the field to avoid field margin effects. The orchards in the study ranged from the Northern half of the San Joaquin Valley through the Capay Valley to Chico. Orchards were 3–38 y old. Almond orchards are generally planted with at least two different varieties, with over-lapping bloom periods to promote pollination. Therefore, all of the orchards in the study contained at least two varieties, and almond varieties varied among the orchards. In each orchard, we examined a full range of soil and water characteristics, soil microbial, plant, and invertebrate community characteristics, pest damage, yields, and economics.\n\nDung invertebrate communities were examined on 16 ranches in a 7,935 km2 region in eastern South Dakota during 2015 and 2016. All sites were grazed by cattle for at least 5 y, but annual grazing intensity and grazing period varied. Herds ranged from 20 to 120 individuals, and the cattle differed in size, breed, and administered ivermectin products. Focal pastures were at least 4 ha in size. The systems were ranked from regenerative to conventional based on several practices (Table 2). In our study, the designation of management systems was based upon the grouping of several management practices that were occasionally all present on the same cattle operation.\n\nA second study of cattle ranches examined plant communities and helminth fecal parasites. Selected sites (n = 20 per grazing treatment) focused on rangelands in the Dakotas during 2019 and 2020. Grazing treatments were assigned based on a ranch management character matrix, which consisted of rancher-defined best management practices for regenerative and conventional ranching systems (Table 2). This character matrix was adapted from a similar study conducted in the region by Pecenka & Lundgren (2019). Grazing treatments were paired within each region and year. Each grazing system had been practiced on the site for at least 4 years.\n\nSoil physical and chemical properties were assessed in corn fields and in almond orchards. SOM and bulk density (BD) were assessed in both of these systems. In cornfields and almonds, four soil cores (8.5 cm deep, 5 cm in diam) were randomly collected from a single field on each of 20 farms; cores were collected at locations that were at least 10 m apart. On each sample, vegetative material was removed from 60 g of soil by hand, and the resulting samples were stored in aluminum containers at 105°C overnight. SOM in the soil sample was measured using the weight loss on ignition (LOI) technique (Davies, 1974). To measure bulk density, four soil cores of known volume per field were weighed before and after drying at 100°C for 55 h.\n\nIn cornfields, we also measured fine particulate organic matter (fPOM). Approximately 30 g of soil was weighed in sterile aluminum pans. The soil was soaked in 90 mL of hexametaphosphate for 24 h, mixed for 5 min with a stainless-steel mixer, and were then sieved through screens with 500 μm and then 53 μm holes; the finer screen isolated the fPOM fraction. The isolated sample was then weighed, dried for 24 h at 105°C. Samples were then cooled in a desiccator cabinet, weighed, baked in a furnace for 4 h at 450°C. The samples were then cooled in a desiccator cabinet and weighed a final time.\n\nIn almond orchards, soil samples were collected at random locations in the inter-row area of each plot to determine total soil carbon (TSC) and total soil nitrogen (TSN). The probe (2.54 cm × 91.44 cm) was inserted 60 cm deep. Each core was placed into a plastic bag that was stored on ice until it could be transferred to a paper bag in the laboratory. Samples were weighed, dried, and weighed again. All visible pieces of rock and organic matter were removed from the samples. The samples were ground and were then passed through a sieve with 0.180 mm openings. Elemental analysis was conducted on three subsamples (12–15 mg) that were housed on tin capsules (5 × 9 mm) (ECS 8020, NC Technologies, Milan, Italy). To control for the relative compaction and other circumstances, the mass (Mg) of TSC per ha was assessed using the Equivalent Soil Mass (ESM) method, in which a cubic spline of Mg of TSC per depth layer was calculated (Wendt & Hauser, 2013). This resulted in the assessment of carbon as Mg of TSC/ha at 6,000 Mg (59.2 cm deep).\n\nSoil macro- and micronutrients were also quantified in the almond orchards. The samples were ground to pass a 2 mm sieve and divided into three subsamples (two were 4 g each and one weighed 40 g). The 40 g soil sample is analyzed with a 24 h incubation test at 24oC. This sample is wetted through capillary action by adding 20 mL of deionized water to a 237 mL glass jar and then capped. After 24 h, the gas inside the jar was analyzed using an infrared gas analyzer (IRGA) (Li-Cor 840A, LI-COR Biosciences, Lincoln NE) for CO2-C. The two 4 g samples were extracted with 40 mL of deionized water and 40 mL of H3A to extract the NO3-N, NH4-N, and PO4-P from the samples. The samples were shaken for 10 min, centrifuged for 5 min, and filtered through filter paper (Whatman 2V, Cytiva, Marlborough, MA). The water and H3A extracts were analyzed on a flow injection analyzer (Lachat 8000, Hach Company, Loveland CO). The water extract was also analyzed on a Teledyne-Tekmar Torch C:N analyzer for water-extractable organic C and total N. The H3A extract was also analyzed on a Thermo Scientific ICP-OES instrument for P, K, Mg, Ca, Na, Zn, Fe, Mn, Cu, S and Al. The Haney soil health score combines five independent measurements of a soil’s biological properties to provide a general estimate of the overall health of a soil system (Haney et al., 2018). It is calculated as 1-d-CO2-Carbon/10 plus water extractable organic carbon (WEOC)/50 plus water extractable organic nitrogen (WEON)/10.\n\nWe used the rainfall infiltration rate kits of the Natural Resource Conservation Service (NRCS) to sample water infiltration rates in northern cornfields and California almonds. A metal ring (15 cm diam, 13.5 cm tall) was hammered 6.5 cm into the soil. Water (444 mL) was poured into the ring, and the time it required to saturate into the soil was recorded. A second container of 444 mL of water was poured into the ring, and the time to saturation was recorded again. In cornfields, this process was recorded during anthesis.\n\nMicrobial communities were only sampled in almond orchards using the Phospholipid fatty acid (PLFA) test. Soil cores (15 cm depth, 1.9 cm diam; N = 16), were taken from four replicates per site per farm during the fruiting period. The samples were taken at random locations within each replicate, at least 5 m apart, using a transect that diagonally bisected the plot. Soil cores for each orchard were combined in a sealed plastic bag and placed in a cooler with dry ice (Drenovsky et al., 2010). Soil samples were stored at -80˚C until they could be freeze-dried and ground to 2 mm particle sizes. Phospholipid fatty acid (PLFA) testing provided an index of a soil’s microbial biomass and composition (Frostegård et al., 2011). The microbial biomass and community composition were recorded as total microbial biomass, undifferentiated microbial biomass, total bacteria, Gram-positive bacteria, Actinomycetes, Gram-negative bacteria, Rhizobia bacteria, total fungi, arbuscular mycorrhizal fungi, saprophytic fungi, and Protozoa.\n\nPlant community structure was assessed solely as ground cover in the almond study. The ground cover height and composition in each of the replicates/plots was recorded during each of the three sampling periods. The percent ground cover was categorized as 0–25%, 25–50%, 50–75%, and 75–100%. Percent ground cover in the overall orchard was assessed using visual assessments of the percent ground cover in each invertebrate quadrat.\n\nPlant community diversity and green canopy cover were examined during the 2019 and 2020 grazing seasons of the rangeland study, while vegetation biomass was assessed only during the 2020 season. All three plant community metrics were measured during three periods of the grazing season; early June, mid-July, and late-August/early-September. At each pasture site, two 50 m transect lines were established 15 m apart and perpendicular to the slope of the land. Plant community diversity was assessed using the belt transect method for monitoring native prairie vegetation in the Dakotas (Grant et al., 2004). Briefly, plant community structure was recorded for a randomly selected 5 × 0.5 m2 belt along each transect line. Vegetation is classified in each 5 m segment according to a hierarchical breakdown of plant groups common to the region (Grant et al., 2004). Green canopy cover was documented using the smartphone app Canopeo, which quantifies fractal green vegetation canopy cover area per unit area (Patrignani & Oschner, 2015). Quadrats (50 × 25 cm2) were established at 0, 25, and 50 m along transect lines to quantify green canopy cover with Canopeo. Lastly, a disc pasture meter was used to estimate above-ground vegetation biomass along each transect line and within their immediate surrounding area (<15 m). Compression height of vegetation was recorded by dropping a 0.13 m2 plate weighing 1.34 kg from a height of 183 cm.\n\nInsect surveys in cornfields and almonds involved sampling the epigeal community from 0.25 m2 sheet-metal quadrats (15 cm tall) inserted into the soil (Lundgren et al., 2006). Quadrats were placed in randomly selected locations between corn rows (n = 5 per plot) during anthesis. Invertebrates were exhaustively collected from the surface of the soil and beneath plant debris using handheld mouth aspirators. In almonds, sampling of the invertebrate communities occurred during the bloom, fruit development, and harvest periods. The invertebrate communities that could be collected from the soil surface and top 2 cm of the soil with mouth-operated aspirators in 15 min were preserved in 70% ethanol.\n\nIn cornfields, we also sampled the foliar invertebrate community. The foliar invertebrate community was sampled using a destructive whole plant assessment. Corn plants (n = 25 per plot) were randomly selected, thoroughly examined, and invertebrates that were not sight-identified were collected using mouth aspirators. Plants were then severed at the ground level and were transported to the field margin where their leaves, stems, ears, whorls, and tassels were inspected and dissected on white sheets.\n\nSoil invertebrate and dung invertebrate communities were assessed using soil cores in corn fields and rangelands. In corn fields, five soil cores (10 cm diameter, 10 cm height) were collected per field in randomly selected locations within and between corn rows during anthesis. In rangelands, the same core approach was used to collect invertebrates from 2–5 d old dung pats and the soil directly beneath the pat (n = 10 per ranch). Samples were collected monthly from May to September. All cores were refrigerated for < 36 h, and then were placed into a Berlese collection system over 7 d, when they reached a constant weight.\n\nAll extracted invertebrates were identified microscopically and cataloged; each unique specimen was identified to the lowest taxonomic level possible representing a functional morphospecies. Each morphospecies was placed into a trophic guild (coprophage, predator, parasitoid, herbivore, or pest) based upon previous descriptions of the biology of arthropod community in these systems. Voucher specimens are housed in the Mark F. Longfellow Biological Collection at Blue Dasher Farm, Estelline, SD, USA.\n\nIn cornfields, pests included aphids, lepidopteran larvae, and corn rootworm adults that were identified from the foliar bioinventories. In almonds, pest incidence was assessed on 500 almonds per farm in 2018 and 600 almonds per farm in 2019 (< 20 from any one tree) (Bentley et al., 2001; Doll, 2009; Legner & Gordh, 1992). The almonds were each categorized as having no pest damage, navel orange worm damage (Amyelois transitella [Walker]; Lepidoptera: Pyralidae), ant damage (Formicidae), oriental fruit moth damage (Grapholita molesta [Busck]; Lepidoptera: Tortricidae), peach twig borer damage (Anarsia lineatella Zeller; Lepidoptera: Gelechiidae), leaf footed plant bug or stinkbug damage (Hemiptera: Coreidae, Pentatomidae), and unknown pest damage (Bentley et al., 2001; Doll, 2009; Symmes, 2018).\n\nFecal oocyst counts of coccidia and egg counts of gastrointestinal nematodes belonging to the superfamilies Trichostrongyloidea and Strongyloidea were conducted on herds from the 2019 – 2020 rangeland study. Three grams of dung was collected from 2 – 4 d old cattle dung pats present in pastures (n = 5 pats sampled/site). Fecal samples were processed according to the Modified Wisconsin Sugar Float Technique (Cox & Todd, 1962). Coccidia oocysts and eggs of Trichostrongyles and Strongyles were quantified for 1 g of fecal sample. Sampling for internal parasites in the herds was conducted twice in 2019 (mid-July and late-August/early September) and three times in 2020 (early-June, mid-July, and late-August/early September).\n\nIn cornfields and almonds, responses from producer surveys were used to determine management practices, costs, and revenues that went into the direct net profitability of each operation. Gross profit analysis included yield, return on grain, and additional revenue that included livestock grazing and production. In cornfields, yield information provided by the farmers were confirmed by yields that were hand-gathered from three 3.5-m row sections from each replicate-field. Costs associated with corn production included corn seed, cover crop seed, drying/cleaning grain, crop insurance, tillage, planting corn, planting cover crop, fertilizers, herbicides, and irrigation (additional information on profit/loss analysis can be found in LaCanne & Lundgren 2018). Operating costs in almonds included winter sanitation, sampling for tree nutrient status and soil salinity, pH, and nutrient levels, irrigation and frost protection, fertilizers, insecticides, herbicides, fungicides, disease treatment sprays, trapping vertebrate pests, cover crop seed, tillage, mowing, flamers, grazers, and harvest.). Harvest in the almonds included the hourly labor to conduct the harvest and the price paid to external contractors (additional information on the almond profit/loss analysis can be found in Fenster et al. in prep). In both systems, only direct costs and revenues were used to calculate profitability.\n\nUnless otherwise described, we used linear regressions to search for correlations between the regenerative matrix scores and response variables. In the almond study, clay percentages of the soils were considered as co-factors in all models that examined TSC. Plot values were composited across plots into single values in the almond study and linear regression analyses were used to compare regenerative scores and response variables. In instances where resampling a single farm was performed (either spatially or temporally) Linear Mixed Models were used to remove pseudoreplication and account for this dependence; field and farm were included as random factors in the corn models, and month was included as a fixed factor and farm as a random factor in the rangeland studies. All statistics were conducted using Systat 13.1 (Systat Software Inc., San Jose, CA)\n\n\nResults\n\nIn cornfields, fPOM of the soil was strongly and positively associated with regenerative matrix scores (F1, 17 = 4.61; P = 0.047) (Figure 1A), but SOM (F1, 17 = 0.11; P = 0.75), bulk density (F1, 17 = 2.32; P = 0.15), and water infiltration rates (F1, 11 = 0.47; P = 0.51) of the soil were not correlated with matrix scores. The slowest water infiltration rates in the higher regenerative scoring farms were those that practiced tillage.\n\nThe relationships of regenerative farming intensity on fine particulate organic matter in cornfields of the Upper Midwest (A) and percent soil organic matter in California almond orchards (B).\n\nIn almond orchards, higher matrix scores correlated to significantly higher levels of TSC and TSN (500–6000 ESM layers) and SOM (15 cm depth). At the 0–6000 Mg ESM layer, there was a significant correlation between the orchards’ matrix scores and TSC (model: F2, 13 = 13.77, P = 0.001; score: t = 2.26, P = 0.04; clay: t = 4.73, P < 0.001) (Figure 2). At the 6000 Mg ESM layer there was a significant correlation between the orchards’ matrix scores and TSN (model: F2, 13 = 17.73, P < 0.001; score: t = 1.94, P = 0.08; clay: t = 5.63, P < 0.001). In contrast to what we found in cornfields, in the top 15 cm of soil there was a significant correlation between the orchards’ SOM%, matrix scores, and clay percentage (model: F2, 13 = 47.56, P < 0.001; score: t = 5.50, P < 0.001; clay: t = 8.05, P < 0.001) (Figure 1B).\n\nIn almonds, higher matrix scores and soil clay percentages correlated to higher levels of WEON (F2, 13 = 9.16, P = 0.003) and WEOC (F2, 13 = 15.61, P = 0.001) in the organic matter of the soils. Total phosphorus (model: F2, 13 = 4.32, P = 0.04; score: t = 2.65, P = 0. 02, clay: t = -1.28, P = 0.23) and inorganic phosphorus (model: F2, 13 = 4.56, P = 0.03, score: t = 2.74, P = 0.02, clay: t = -1.28, P = 0.22) were significantly and positively correlated with an orchard’s matrix score. Higher matrix scores correlated to higher levels of Calcium (F2, 13 = 5.97, P = 0.01) and Sulfur (F1, 14 = 10.80, P = 0.01), but lower levels of Aluminum (F1, 14 = 6.13, P = 0.03). There were no correlations between other micronutrients and the regenerative matrix score (P > 0.05). Haney soil health test scores were positively correlated with regenerative matrix scores (model: F2, 13 = 7.82, P = 0.01, Score t = 2.99, P = 0.01, clay: t = 2.59, P = 0.02) (Figure 4). Soil respiration was unaffected by regenerative matrix scores (model: F2, 13 = 2.91, P = 0.09, Score t = 1.62, P = 0.13, clay: t = 1.77, P = 0.10).\n\nHigher regenerative-conventional matrix scores in almonds correlated to lower bulk densities (model F2, 13 = 8.95, P = 0.004; score: t = -3.91, P = 0.002, clay: t = -1.61, P = 0.13). Water infiltration rates increased alongside matrix scores for these orchards (model: F2, 5 = 11.43, P = 0.01; score: t = -4.50; P = 0.009, clay: t = 2.56, P = 0.05) (Figure 3).\n\nIn almond orchards, higher matrix scores correlated to higher amounts of bacterial biomass (F1, 14 = 6.25, P = 0.03) and higher Gram (+) biomass (F1, 14 = 9.12, P = 0.01). The percent composition in the microbial community was not affected by regenerative score (F1, 14 = 2.47, P = 0.14), indicating greater overall microbial biomass rather than bacterial dominance. The remainder of these microbial community metrics were not correlated to the regenerative conventional matrix score (P > 0.05).\n\nMore regenerative cropland and rangeland supported more diverse and robust plant communities. In almond orchards, higher matrix scores correlated to more ground cover (F1, 14 = 127, P < 0.001), more plant species (F1, 14 = 27.61, P < 0.001) (Figure 5A), and greater biomass height (F1, 14 = 9.90, P = 0. 01). In rangelands of the Northern Plains, there was a significant relationship between matrix score and plant biomass (Score: F1, 53 = 8.47, P = 0.01; month: F2, 53 = 0.45, P = 0.64) (Figure 5B), plant diversity (Score: F1, 106 = 14.22, P < 0.001; month: F3, 106 = 1.02, P = 0.39) (Figure 5C), and ground cover (Score: F1, 106 = 10.06, P = 0.002; month: F3, 106 = 14.76, P < 0.001).\n\nPlant species richness on the orchard floor in California almonds (A), and plant diversity (Shannon H; B) and biomass index (C) in rangelands of the Northern Plains as they relate to how regenerative a farm is.\n\nAcross cropland and rangeland, more regenerative farms had more robust invertebrate communities. Several aspects of invertebrate community structure in cornfields were strongly and positively correlated with how regenerative a field was scored. The abundance (F1, 55 = 6.53, P = 0.01), species richness (F1, 55 = 38.31, P < 0.001), and diversity (F1, 55 = 7.34, P = 0.01) (Figure 6A) of the epigeal invertebrate community was positively affected by matrix score. Similarly, matrix scores were positively associated with the species richness of the invertebrate community found in the soil column (F1, 56 = 12.96, P = 0.001), but the abundance was only marginally associated with matrix score (F1, 56 = 3.41, P = 0.07), and the diversity of this community was unaffected by matrix score (F1, 56 = 2.45, P = 0.12). Foliar invertebrate abundance (F1, 55 = 0.99, P = 0.32), species richness (F1, 55 = 1.35, P = 0.25), and species diversity (F1, 55 = 0.20, P = 0.66) were not related to the matrix scores.\n\nThe invertebrate species diversity (Shannon H) on the soil surface in cornfields of the Upper Midwest (A), and on the orchard floors of California almonds (B), as they relate to the number of regenerative practices implemented on farms.\n\nThe invertebrate community on the soil surface was positively affected by the number of regenerative practices on almond orchards. Invertebrate abundance (F1, 14 = 10.28, P = 0.01), biomass (F1, 14 = 70.10, P < 0.001), species richness (F1, 14 = 14.44, P = 0.002) and species diversity indices (H: F1, 14 = 12.37, P = 0.003; DS: F1, 14 = 11.11, P = 0.01) (Figure 6B) were positively correlated with matrix score. There was a positive correlation between earthworm (square rooted to normalize residuals) abundance and matrix score on these orchards (F1, 14 = 9.87, P = 0.007).\n\nIn rangelands of the Northern Plains, the invertebrate community associated with cattle dung was strongly and positively associated with the matrix score attained. The species richness (Score F1, 74 = 15.38, P < 0.001; month F4, 74 = 6.54, P < 0.001), and species diversity (Score F1, 74 = 16.10, P < 0.001; Date: F4, 74 = 1.50, P = 0.21) (Figure 7A) of the dung invertebrate community were positively associated with matrix score. Abundance of dung invertebrates was uncorrelated with matrix score (Score F1, 74 = 0.01, P = 0.99; month: F4, 74 = 4.83, P = 0.001). The abundances of two critical functional groups, dung beetles (Score F1, 60 = 10.05, P = 0. 006; month: F4, 60 = 5.70, P = 0.001) (Figure 7B) and invertebrate predators, (Score F1, 74 = 7.41, P = 0.01; month: F4, 74 = 4.09; P = 0.01) increased as a rangeland became more regenerative.\n\nInvertebrate species diversity (Shannon H) in dung pats (A) and the abundance of dung beetles (B) as they relate to regenerative intensity on ranches in the Northern Plains.\n\nIn all of these systems, pest populations were uniformly below any recognized economic thresholds. Pests were not correlated with the number of regenerative practices used in cornfields (pest abundance: F1, 55 = 2.18, P = 0.15) or in almond orchards (% damaged nuts: F1, 14 = 0.69; P = 0.42). In rangelands, there was no relationship between the matrix score and Trichostrongyles (Score: F1, 88 = 0.09, P =0.77; month: F3, 88 = 0.39, P = 0.76) or Coccidia (Score: F1, 88 = 1.78, P = 0.19; month: F3, 88 = 0.70, P = 0.56) fecal parasite loads.\n\nCorn yields were negatively correlated with the regenerative matrix score (F1, 56 = 3.88, P = 0.05). Despite this, three of the top ten yielding cornfields were regenerative, indicating that regenerative practices are not necessarily tied with yield reductions. Standard deviations became greater proportions of the mean as a farm became more regenerative (variability in yields were higher among regenerative farms) (R2 = 0.79; F1, 4 = 5.12, P = 0.11). In almonds there was no relationship between almond yield and how regenerative an orchard was (F1, 12 = 0.86, P = 0.37). On a treatment level, regenerative farms had twice the variance in yield relative to the conventional orchards.\n\nAlthough there was a positive relationship between matrix score and profitability in cornfields, this relationship was not significant (F1, 53 = 2.49, P = 0.12). In cornfields, there was a significant increase in the standard deviation of profit as a farm became more regenerative (e.g., standard deviation as a proportion of the mean; R2 = 0.72; F1, 4 = 7.68, P = 0.07). This indicates more variability in profits among the more regenerative producers. The top ten highest netting farms were all regenerative farms (scores of four and five). In almonds, profit was strongly and positively correlated with matrix scores (F1, 11 = 7.41, P = 0.02) (Figure 8). Operating costs were unaffected by matrix score (F1, 13 = 0.70, P = 0.42), but net income increased as regenerative scores increased (F1, 11 = 8.50, P = 0.01).\n\n\nDiscussion\n\nOur approach to defining farms based on a small suite of carefully chosen practices was strongly associated with several key metrics that define regenerative systems, including soil health, biodiversity promotion and profitability. With our matrix, we have distilled complex agricultural systems down to fewer than 10 practices that serve as useful indicators of regenerative operations in cropland and rangeland. These practices represent each of the five principles that define regenerative agriculture in cropland and in rangeland. We have determined that these practices are regenerative across cropping systems; the cropland matrix works in row crop systems as well as a perennial system including orchards. Also, it was noteworthy that none of the regenerative farm attributes attained an asymptote, which suggests that farms could become more regenerative if they continue to incorporate additional regenerative practices. This matrix can be used to categorize a farm as regenerative or conventional based on a threshold score.\n\nRegenerative farming practices inherently increase plant biomass and diversity in both cropland and rangeland systems, and this plant community is a central mechanism for improving the soil health, biodiversity, and resilience of these farming operations. Farmers in this study enhanced their plant diversity and biomass (Figure 5) using a variety of tools. In almonds and cornfields, many of the farmers planted annual cover crops to improve the health of their soils. In some regenerative orchards, farmers allowed the native vegetation to persist. All of the regenerative orchards stopped the use of synthetic herbicides, and this likely contributed to greater plant diversity. In rangelands, short-term, intense grazing stimulates plant communities to diversify and grow if the plant community is allowed to rest following a grazing event (Hillenbrand et al., 2019; Teague et al., 2011). Plants are a vital part of the carbon cycle, and as such they are the primary means whereby energy enters into an ecosystem (Weil & Brady, 2017). Additionally, plant communities are requisite directly and indirectly to the genesis of healthy soils (Lucas, 2001). Plant roots provide the needed polysaccharides for microbial communities to grow and perform key nutrient cycling services (Philippot et al., 2013). Physical properties of soils are also influenced by plants, including water infiltration rates, soil aggregate structure, bulk density, etc. (Gulick et al., 1994; Liu et al., 2005). Plant diversity and biomass scales positively with the diversity of nearly every other group of organisms (Bianchi et al., 2006; Saunders et al., 2013; Zak et al., 2003), and thus plant communities are a driver of biodiversity within agricultural habitats. Taken in sum, the relationships between plant communities and regenerative score illustrate an important mechanism for how regenerative farms positively enhance many intended regenerative outcomes.\n\nAnimal integration is another important tool for managing farms that improve regenerative outcomes. The most regenerative cropping operations observed in this set of studies always integrated livestock (chickens, sheep, or cattle), and these farms also had the greatest biodiversity, soil health, water infiltration rates, and economic metrics. Regeneratively-managed livestock in cropland improve soil chemical and physical properties, increase niche diversity which allows other organisms to thrive, and provides additional revenue and an additional plant management tool that can increase farm resilience (Colley et al., 2019; Delgado et al., 2011; Niles et al., 2018; Teague et al., 2016). Important logistical constraints influence the circumstances of integrating livestock into cropland, especially in standing crops like almonds. Barriers preventing farmers from integrating livestock into cropland should be removed so this important management tool is more commonly adopted.\n\nSoil chemical and physical properties were positively affected by the number of regenerative practices in cropland. Bulk density was significantly lower in the more regenerative almond orchards. Soil carbon and soil organic matter were strongly associated with more regenerative farms. Although we did not conduct a carbon life cycle assessment on corn or almonds, these results support the idea that regenerative farming systems can help our cropland sequester more carbon and help to offset greenhouse gas emissions (Lal, 2020; Soto et al., 2021). Organic and inorganic phosphorous are also enhanced as almond orchards become more regenerative. Phosphorous, a mined agricultural nutrient, is becoming increasingly rare and is available at significant economic and environmental costs (Alewell et al., 2020), so enhancing the plant-available forms of phosphorous that are present in the soils is an important outcome of regenerative agriculture. Micronutrients calcium and sulfur were also enhanced by increasing the number of regenerative practices in almonds. In almonds, water infiltration rates were significantly improved by regenerative production practices. Water is becoming more scarce (Burri et al., 2019), especially in California (Mall & Herman, 2019), as climates continue to change and ground and surface waters are exhausted from conventional agricultural practices. Absorbing water into the soil when it becomes available is a crucial step in keeping these agricultural areas productive. Similar to previous studies (Pikul & Aase, 2003; Pikul et al., 2009), tilled cornfields had lower water infiltration rates and lower bulk density. This data supports the argument that no-till practices are an indispensable core component of regenerative agriculture, and argues in favor of mandating that regenerative operations be no-till.\n\nAll organismal groups measured were enhanced as farms became more regenerative. There was greater bacterial biomass and Gram+ bacteria from the farms with higher regenerative scores. In general, bacterial dominated soils are undesirable, as they tend to drive faster decomposition and reduce soil organic matter (Hendrix et al., 1986; Lehman et al., 2015). Bacteria dominated soils are produced through tillage, and by definition, regenerative farms are not tilled. Only two of the almond orchards in this study had been tilled; and this must be considered when comparing bacterial communities in regenerative versus conventional almond systems. Despite increasing bacterial biomass in the regenerative orchard soils, the proportion of bacteria to other microbial life did not increase. Furthermore our soil health metric, the Haney Test that accounts for microbial function in a soil (Haney et al., 2018), was positively associated with the regenerative score of a farm. This suggests that although soil bacterial biomass may be increasing on regenerative almond orchards, it is not at the expense of other microbial taxa and it enhances bacterial function. Invertebrate biomass, diversity, and abundance in the soil, on the soil surface, and in the vegetation were positively affected by regenerative practices in all three study systems. This was particularly true for the epigeal communities. We hypothesize that promotion of the invertebrate community on the soil surface is a product of enhancing the plant community in this same stratum. Herbicides (Bohnenblust et al., 2016; Morton et al., 1972), fungicides (Johnson et al., 2013), insecticides (Bredeson & Lundgren, 2018; Pecenka & Lundgren, 2019; Seagraves & Lundgren, 2012), and synthetic fertilizers (Cuesta et al., 2008; Kromp, 1999) disrupt invertebrate communities. Regenerative farms that abandon these chemicals may experience enhanced invertebrate communities and biological diversity, which is important for farmers because it provides valuable services, including pest suppression.\n\nPest populations of rangelands and croplands were below economically damaging levels, but these populations were suppressed using very different means in regenerative and conventional systems. In conventional systems, pests are kept low through genetically modified crops and insecticide applications. Conventional almond orchards in this study experienced up to five insecticide applications annually. All of the conventional corn farms are planted with genetically modified, insect resistant (i.e., Bt) varieties that were treated with neonicotinoids. All of the most conventional ranches applied ivermectins in their animals to reduce fecal parasite loads. Our research shows that regenerative practices produce the same low pest populations as when cattle were treated with ivermectins, but with lower input costs. Other work in agricultural ecosystems has shown that invertebrate diversity is essential to mitigating pest populations (Crowder et al., 2010; Letourneau et al., 2009; Lundgren & Fausti, 2015; Lundgren & Fergen, 2014). This diversity within agricultural systems results in biological control performed by invertebrates and pathogens keeping pest populations at sub-economically damaging levels. We will test whether this same pattern was true in dung pats. Predation isn’t the only mechanism at work, and we also hypothesize that host plants and livestock are healthier in regenerative systems, which contributes to fewer pests (Barbosa et al., 2009). Also, in almond orchards the more robust epigeal invertebrate community or livestock may be playing a key role in breaking down fallen mummy nuts in which navel orange worm larvae overwinter. There also are likely synergisms among organisms in regenerative systems that balance communities and resist outbreaks of specific pest populations. But in the end, the complexity of biological network interactions in complex ecosystems prevent us from understanding all of the mechanisms whereby regenerative systems consistently suppress pests without pesticide inputs.\n\nImproved soil health and biodiversity on the studied farms was associated with increased profitability. In almonds, there was a clear relationship between more regenerative practices and the profitability of the orchards. In cornfields, the most regenerative farms were also the most profitable, but there was substantially more variance in this relationship. These higher net profits were the result of lowering seed costs, reducing chemical premiums, and value-added marketing of the most regenerative products. Yields did not follow the same pattern as the profitability with regards to matrix scores. Almond yield was not correlated with how regenerative a farm was, and corn yields were significantly reduced as regenerative practices were added to cornfields. It is important to interpret these results recognizing that regenerative agriculture is still in its infancy in many production systems. Three of the top 10 yielding corn fields were regenerative, meaning regenerative corn production methods do not necessarily lead to yield reductions. Given the low reliance on corn as a human food source, and the lack of yield reductions in almonds, concerns over regenerative food systems’ ability to feed a growing human population are not supported by our research. All of this is to say that regenerative farming practices are correlated with increased profitability of a farm, thus supporting the notion that we can improve farm resilience while promoting the natural resource base of a habitat (Schipanski et al., 2016).\n\nIn looking at the data on these established farm systems, there were consistently two distinct clusters of farms based on regenerative score that could be used to categorize regenerative and conventional farming operations. The natural groupings could be divided at scores of 5 and 3 for cropland and rangeland, respectively (in cornfields, this dividing line was around 3, since we did not ask all of the questions to corn farmers in that study). One explanation for this may be that farms have trouble staying in business when they only employ an intermediate number of regenerative practices. Farms in the “regenerative” and “conventional” categories are not all created equal, since changing the number of regenerative or conventional practices can produce a variance in farm performance within each category. Also, the range of scores in the regenerative ranches were greater than that for in cropland, and could indicate a transition in the adoption of regenerative practices by ranchers of the Northern Plains. The duration that a farm is in its respective system also will likely affect the observed regenerative outcomes, and this temporal factor should be considered in further interpretations of regenerative scores. Uses for this matrix might include regulation based on carbon sequestration, water use efficiency, or pollution. Certification of regenerative operations and marketing of regenerative labelling might also employ the matrix approach as we have laid out. In the Extended data, we provide a survey for producers to generate their regenerative scores. We hope that this work will be built upon and tested as we confront the challenge of how to empirically define regenerative farming systems.\n\n\nData availability\n\nOpen Science Framework: Matrix paper, https://doi.org/10.17605/OSF.IO/G697Y (Lundgren, 2021) (registered 15th January 2021 https://osf.io/7v4z2).\n\nThis project contains the following underlying data:\n\n- Corn\n\n- Regenerative Rangeland 1\n\n- Almond\n\n- Regenerative Rangeland 2\n\nOpen Science Framework: Matrix paper, https://doi.org/10.17605/OSF.IO/G697Y (Lundgren, 2021) (registered 15th January 2021 https://osf.io/7v4z2).\n\nThis project contains the following extended data:\n\n- CROPLAND Regenerative Score Calculator\n\n- RANGELAND Regenerative Score Calculator\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 thank the dozens of technicians who helped us to collect and enter the data from these projects. 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Ecology. 2003; 84(8): 2042-2050. Publisher Full Text" }	[ { "id": "79606", "date": "09 Mar 2021", "name": "John M. Holland", "expertise": [ "Reviewer Expertise Agroecologist", "entomologist", "soil scientist" ], "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 regenerative agriculture matrix scoring system is proposed that can be used to evaluate the extent to which a farming system has adopted regenerative practices. This is an important study as it defines a standard for regenerative systems in a similar way to those for other farming systems such as organic using simple scoring matrix that can be used by farmers. This has the advantage that they can monitor the progress of their system over time and adjust accordingly.\nThis paper represents a substantial amount of work, covering a large number of sites, fully replicated studies with a lot of samples and appropriate experimental designs. It includes three food production systems. The analysis was relevant and is described in good detail, and the statistics employed are robust. The paper uses a novel approach to overcome the problems of comparing the overall outcomes from the regenerative agriculture system between farms, with many different variables. The authors focus on methods that underly the principles of regenerative agriculture rather than defining it as a set of practices. This was achieved by defining a matrix scoring system compiled from sums of regenerative and conventional practices and looking for correlations between the matrix scores with detailed biological and physical measurements. This enabled them to compare a range of systems that exhibit few or many of the regenerative agriculture practices, and regress these with a large number of soil health; plant, insect and microbial diversity; pests and profitability. Much useful information is provided on the different methodologies and their use in farming systems research.\nThere are some limitations to the approach, especially with respect to farms that may be in transition to regenerative agriculture and are achieving this through slowly reducing reliance on agrochemicals. Therefore, with such farms their matrix scores may remain high despite achieving substantial decreases in agrochemicals because 4 of the 9 points depending on not using inorganic fertilisers, herbicides, fungicides and insecticides. Nevertheless, the paper provides a contribution to scientific knowledge by allowing some broad comparisons to be made on the overall benefits of the regenerative agriculture approach.\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": "79612", "date": "10 Mar 2021", "name": "Richard W. Teague", "expertise": [ "Reviewer Expertise I am a research scientist in the Texas A&M AgriLife Research organization working on documenting and testing causal mechanisms in comparing results from soil", "vegetation", "livestock", "watershed health", "and ecological economics. I have published widely on these aspects of regenerative agriculture with many co-authors for 5 decades." ], "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 of regenerative agriculture in many parts of US is the most comprehensive and thorough I have yet seen on the subject. It covers a wide array of biological disciplines to assess the various biodiversity elements required to assess the efficacy of regenerative agriculture relative to conventional agriculture.\nThe authors are to be particularly commended for the solid statistical analyses that provide an excellent foundation for the results and conclusions they present. This is a very welcome and solid contribution to the multiple co-benefits evident from those practicing regenerative agriculture as it is conducted by the agricultural community.\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/10-115
https://f1000research.com/articles/10-112/v1	15 Feb 21	{ "type": "Research Article", "title": "Safe abortion services during the COVID -19 pandemic: a cross-sectional study from a tertiary center in Nepal", "authors": [ "Shreyashi Aryal", "Samata Nepal", "Sagun Ballav Pant", "Samata Nepal", "Sagun Ballav Pant" ], "abstract": "Background: Abortion is an essential service, the need for which has increased during the coronavirus disease 2019 (COVID-19) pandemic. Because of the lockdowns at several periods, these services were hampered. This study analyzed the pattern of Safe Abortion Services (SAS) at a tertiary healthcare center during the first six months of the COVID-19 pandemic in Nepal. Methods: This is a cross-sectional analytical study. We compared the pattern of safe abortion services between the first three months of the pandemic when a lockdown was implemented and the second three months when the lockdown was eased. Demographic and obstetric profile of women, their abortion choices, method of termination, difficulty in accessibility, and level of psychological distress were studied. Results: A total of 52 women were provided SAS during the study period. The number of women coming for SAS during lockdown was 47.1% less than that after easing of the lockdown. During the lockdown, women came at a later period of gestation with a mean of 9.5 weeks compared to 7.5 weeks in the later three months. Because of fear of COVID-19, 19.2% (n=10) women opted for termination of pregnancy. Increased need of contraception was felt but 40% (n=12) had problems of accessibility. More women had probable serious mental illness during the lockdown period (p=0.008). Conclusion: Lockdown during the pandemic decreased the number of women coming for SAS due to barriers in accessibility. Contraceptive needs are also increased but access is difficult. The need for safe abortion services and contraception has increased during the pandemic but the lockdown caused inaccessibility. Psychological distress is prevalent, and fear of COVID-19 has become a common reason for termination of pregnancy. This pandemic can be taken as an opportunity to provide and improve contraception and abortion accessibility, and quality with integration of mental health support.", "keywords": [ "abortion", "contraception", "lockdown", "pandemic", "psychological distress", "Nepal" ], "content": "Introduction\n\nSafe Abortion Services (SAS) in Nepal include pre and post counseling on abortion and contraception methods, termination of pregnancy, diagnosis, and treatment of existing reproductive tract infection, providing contraceptive methods as per informed choice, and then follow up for post abortion complication management (National Safe Abortion Policy, 2003). SAS are essential healthcare services for women, but the COVID-19 pandemic caused all elective procedures to be suspended in hospitals which includes SAS as well. The Royal College of Obstetricians and Gynaecologists (RCOG) and International Federation of Gynecologists and Obstetricians (FIGO) guidelines suggest that abortion be kept under emergency services during this pandemic and be provided to all women in need (Poon et al., 2020; RCOG, 2020). During this crisis, the need for these services are higher because women are unable to make free reproductive health choices due to quarantine travel bans, lack of access to health facilities, fewer healthcare providers and loss of income. The need for contraception is higher as regular employment is interrupted and migrant workers have returned home in large numbers. There is additional risk of rape and domestic violence. In Nepal, the unintended pregnancy rate was 68 per 1,000 women of reproductive age in 2014 (Puri et al., 2016). If SAS are not available, these women may resort to unsafe abortion practices. Marie Stopes International (MSI) predicts 2.7 million unsafe Abortions in 2020 due to the closure of its services in 37 countries (Cousins, 2020). This study was done with the aim of analyzing the pattern of SAS at a tertiary health center during the first six months of the COVID-19 pandemic in Nepal.\n\n\nMethods\n\nThis is a cross-sectional analytical study done at Lumbini Medical College Teaching Hospital (LMCTH) which is a teaching hospital situated in hilly western Nepal and is a referral center for the surrounding districts like Gulmi, Syangja and Rupandehi.\n\nAll women attending Gynecology outpatient clinic of LMCTH for first trimester abortion services during the first six months of the COVID-19 pandemic in Nepal were included in the study after informed consent.\n\nEthical approval was taken from the Institutional Review Board of Lumbini Medical college (IRC-LMC 08-C/020) before the commencement of the study.\n\nNepal reported its first case of COVID-19 in January 2020 and with an increasing number of cases, the government declared the first phase lockdown beginning from 24 March 2020. This lockdown was eased at different time periods after July 2020, as per the decision of the provincial government. In the first three months, a lockdown was implemented, and all non-essential services were shut down, including out-patient services in hospitals and essential services were open for limited hours only. After the first three months, the lockdown was eased allowing restricted use of non-essential services. Private vehicles were allowed to be used for essential services, but public transport was not operational. This study compared the pattern of SAS between the first three months of 2020 (April–June) and the successive three months (July-September).\n\nThe demographic and obstetric profile of these women about their abortion choices included the reason for termination of pregnancy and method of termination, difficulty they are facing relating to access to these services, and their level of psychological distress were studied.\n\nA self-designed proforma was used for data collection and interview technique was used. Interviews were taken by the attending gynecologist who underwent an orientation program before data collection. The Kessler psychological distress tool, “K-6 tool”, which is a self-administered tool validated for Nepal, translated by experts in Nepali language (Kessler et al., 2003; Kessler et al., 2010) [6,7]. This tool was used to find the level of psychological distress. The K-6 tool is a six-item self-report measure of psychological distress used to assess risk for serious mental illness in the general population answerable in five point scale. Using a 30-day reference period, respondents rated how often they felt nervous, hopeless, restless, or fidgety, so sad that nothing could cheer them up, that everything was an effort, and worthless.\n\nThe number of women coming for SAS three months prior to the beginning of the lockdown was analyzed using hospital medical records.\n\nData was entered and analyzed using Statistical Package for Social Sciences (SPSS) v16. Results are presented in mean and SD, percentages. Relationship between categorical data was analyzed using chi square test. P value of <0.05 was considered as significant.\n\n\nResults\n\nA total of 52 women were provided SAS during the study period. Three months prior to the lockdown (January–March 2020), 69 women were provided with SAS. Since the beginning of the lockdown, women coming for SAS were less by 25.7%.\n\nEighteen women came for SAS during the first three months and 34 cases came in the second three months. The number of women coming for SAS during lockdown was 47.1% less than that after easing of the lockdown.\n\nThe mean age of women was 26.1 years (SD=4.8), and the mean period of gestation was 8.23 (SD=1.98) completed weeks. Table 1 shows that more multigravidas came for SAS during both the periods. Employed women came for abortion more after the easing of lockdown (p=0.002).\n\nDuring the lockdown, women came for abortion at a later period of gestation with a mean of 9.5 weeks compared to 7.5 weeks in the latter half of the study. 19.2% (n=10) women came for abortion because of fear of COVID-19. There was no significant difference in the method chosen for abortion (p=.378) in the two groups. Satisfaction towards services was found more after easing of the lockdown but this was not statistically significant (p=0.690) (Table 2).\n\nFisher exact.\n\nIncreased need of contraception was felt by 48.07% (n=25) women during the study period but there was no significant difference in the need of contraception during or after the lockdown. Out of 57.6% (n=30) women who did not use contraception, 40% (n=12) stated the reason of non-use to be inaccessibility due to lockdown. Injectable contraception was the most common family planning method chosen after abortion in both groups (Table 3).\n\nTable 4 shows that women who lived more than five hours drive away from the hospital by public transport did not come for treatment during the lockdown period. 25% (n=13) women had to use an ambulance to reach the hospital. 21.1% (n=11) women had difficulty finding vehicles to reach the hospitals and 25% (n=13) women paid double the normal price to these vehicles. It was noted that transport costs double for some women (5 out of 16), even after the lockdown was eased. 17.3% (n=9) women had tried to get abortion pills from the local pharmacy before coming to the hospital.\n\nFisher Exact.\n\n*Independent sample t test.\n\nThe mean score K-6 score for all women coming for SAS during the study period was 17.4 (SD=6.7). The score was significantly more (p=.008) in women coming for SAS during the lockdown period. More women had probable serious mental illness during the lockdown period compared to those coming after the easing of lockdown (p=0.008) (Table 5).\n\n\nDiscussion\n\nThe COVID-19 pandemic has had a profound effect on sexual and reproductive health (SRH), and the findings of this study suggest that abortion and contraceptive services, which form a major component of SRH, have been affected to a great extent.\n\nThe findings of this study show that, because of travel bans, number of women coming for first trimester SAS was decreased by over 25% since the beginning of the lockdown. These women either have continued their pregnancy unwillingly or must have resorted to unsafe measures. The Guttmacher institute estimates that if 10% women who would normally come for SAS resorted to an unsafe method, an additional 3.3 million unsafe abortions would occur in low-middle income countries over the course of a year. This would in turn result in an additional 1,000 maternal deaths (Riley et al., 2020). With 25% women not coming for services in a referral center, as shown by this study, we can estimate that abortion related maternal deaths will increase.\n\nAbortion was legalized in Nepal in 2002. It is available up to 12 weeks’ gestation on request, up to 18 weeks’ gestation in cases of rape or incest, and at any time if the pregnancy poses a danger to the woman’s life or physical or mental health or if there is a fetal abnormality (National Safe Abortion Policy, 2003). Medical abortion was introduced in 2009 which increased access to abortion care. This was achieved after a long battle, and since then, Nepal has come a long way in providing SAS and reducing maternal mortality rate due to abortion complications. This pandemic seems to affect this hard-earned good outcome (Wu et al., 2017).\n\nAfter easing of the lockdown, the number of women coming for SAS increased by just over 50% compared to the lockdown period. Women who came for abortion during the lockdown period had higher mean gestational age because they had problems of accessibility. Abortion is time sensitive as the higher the period of gestation, there are more chances of complications. They were not allowed to walk, had no means of transport to reach the hospital and, even if they hired a vehicle, they had to pay a higher price than normal even after the easing of lockdown.\n\nFear of COVID-19 is prevalent among people of all age groups. This study shows that 19.2% women wanted termination of pregnancy due to fear of COVID-19. There was no significant difference in women wanting termination during the lockdown or thereafter. Studies have shown that many pregnant women fear of COVID-19 and think it might affect their fetus (Hossain et al., 2020). This could be the main reason for women wanting termination during this time.\n\nSome women chose termination because of financial reasons. Many women live under the poverty line in Nepal and more who did not hold an office job came for SAS in this study. Eighteen women were not working because of the pandemic and this was one of the reasons for pregnancy termination.\n\nIncidence of gender-based violence has increased during this pandemic (Raj et al., 2020). During this study, there was one woman who came during the lockdown period who wanted termination of pregnancy occurring as a result of sexual assault. There were two women who wanted termination of pregnancy because of out of wedlock pregnancy. Both sexual assault and extra marital pregnancy are associated with social taboos in Nepal and can lead to increased psychological distress.\n\nThe findings of this study show that more women preferred medical abortion (MA) rather than surgical abortion during the pandemic, both during the lockdown and the period after that. The reason for this being less patient to service provider interaction and shorter hospital stays which was understood by both parties. Some women opted for surgical abortion because MA required a compulsory hospital follow up after two weeks. Abortion pills are easily available in local pharmacies in Nepal, but abortion has been de-medicalized legally for the purpose of easing accessibility to MA during the pandemic from May 2020. COVID-19 guideline for Maternal, Neonatal and Child Health (MNCH) has also been endorsed which allows home-based MA through outreach model and telemedicine. This will further increase the number of women choosing MA since pregnancy can be terminated without having to come to the hospital. This in turn will help mitigate accessibility problems and will be a relief for the over-exhausted health facilities concentrating mainly on the facilities concentrating mainly on the treatment of COVID-19 patients. Prescribing MA without an internal pelvic examination or an ultrasonogram would pose a risk of missing extra uterine pregnancies, so strict guidelines have been developed which needs to be strictly adhered to (Raymond et al., 2020).\n\nMore women who came for SAS during the post lockdown period were satisfied with the service and service provider when compared to the early lockdown period. Studies have shown that health care providers have moderate fear and burnout due to the pandemic (Hu et al., 2020), this could be the reason for the inability to provide satisfactory care to women coming for SAS. Especially during the initial days of the pandemic, when there was inadequate and low quality personal protective equipment (PPE), which was one major reason for inability of service providers to give satisfactory care.\n\nContraception is a major component of SAS, and the key area of concern is the increased need for contraception and its inaccessibility. Many women in this study felt an increased need for contraception and more so after the easing of the lockdown. One reason for this being the return of migrant workers from different countries and, at the same time, inaccessibility to contraception. Injection Depot Medroxyprogesterone was still the most popular choice among women coming for SAS which is the same as the national data of 2016 (Pant et al., 2019). Women did choose long-acting reversible contraceptives (LARC) as they could insert it in the same setting. Women have not been able to go for follow up visits and for refilling of their contraceptive pills. In this study, out of all women who did not use contraception, 40% had problems of inaccessibility.\n\nNepal is a hilly country with difficult terrain and lack of access for easy commutes, where some women have to walk for hours to days to reach a health facility even when they are not facing the pandemic. Accessibility to abortion and contraception becomes even difficult when there are travel bans. For women who are within reach, the closing of health facilities and pharmacies has posed a problem. Many women walked because transport was not easily available, and they had to pay more than the regular price. During the times of financial burden, this weighed them down more. The financial burden on the transport operators was passed down to the consumers and women coming to hospitals have to bear this brunt.\n\nA study in pregnant women and their husbands showed that fear of COVID-19 was present in many couples which lead to depression and suicidal ideation in pregnant women. The findings of this study highlight that psychological distress was more during the lockdown period when compared to the time when lockdown was eased. Women having probable serious mental illness was high during the first three months (Ahorsu et al., 2020). During the time when the pandemic was announced, there was uncertainty about COVID-19, its pathogenesis, and complications. This caused increased mental pressure on many women, especially pregnant ones. As the situation was understood, the stress level decreased, and people began to normalize the situation. This could be the reason this study shows decreased levels of psychological distress in the later months.\n\nIncreased psychological distress could have a serious effect on the long run on maternal mental health. Abortion itself is a traumatizing experience which has a devastating long-term effect on the woman and her family. Unsatisfactory SAS for a woman who has a chance of mental illness could be detrimental. Many women lost their follow-ups in this study; such loss of follow-up would compound the mental health effects of the pandemic and of SAS.\n\nThe limitation of this study is its small sample size so the significant results may have been due to chance alone. This study also does not address the issue of second trimester abortion. Due to inaccessibility, the need of second trimester abortions will be more than ever. A further analysis of second trimester SAS can be a scope for future studies so that the gestational limit for termination of pregnancy can be increased at least for the duration of the pandemic. Another issue to be analyzed in the coming months would be the use of MA through local pharmacies or through outreach means before concluding that women have resorted to unsafe measures by not coming to the health care center for SAS.\n\nWomen continue to become pregnant during this pandemic so guaranteeing resources to SAS is critical in preventing maternal deaths. It is a commendable effort by the government to recognize SRH services as essential and allow its operation but other areas must be strengthened like increasing supply chains, improving access, and using innovations like tele-health for medical abortion. Access of proper PPE and incentives may also help in tackling fear and exhaustion of service providers so that they can give satisfactory abortion services.\n\nThis pandemic can be taken as an opportunity to provide and improvise contraception and abortion accessibility and quality with an integration of mental health support. This can make SAS a dignified experience for women and their families.\n\n\nConclusions\n\nThe lockdown during the pandemic has decreased the number of women coming for SAS due to barriers in accessibility. Contraceptive needs are increased but access is difficult. Medical abortion has been the more sought-after method for those who are undergoing SAS treatment. Psychological distress is prevalent, and fear of COVID-19 has become a common reason for the termination of pregnancy.\n\n\nConsent\n\nWritten informed consent for collection of data and publication of the participants’ details was obtained from the participants.\n\n\nData availability\n\nDryad: Safe abortion services during COVID pandemic in Nepal. https://doi.org/10.5061/dryad.3tx95x6fc (Aryal et al., 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\nAhorsu DK, Imani V, Lin CY, et al.: Associations Between Fear of COVID-19, Mental Health, and Preventive Behaviours Across Pregnant Women and Husbands: An Actor-Partner Interdependence Modelling. Int J Ment Health Addict. 2020; 1–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAryal S, Nepal S, Pant SB: Safe abortion services during COVID pandemic in Nepal, Dryad, Dataset, 2021. http://www.doi.org/10.5061/dryad.3tx95x6fc\n\nCousins S: COVID-19 has \"devastating\" effect on women and girls. Lancet. 2020; 396(10247): 301–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFSRH, BSACP, Royal College of Midwives: Coronavirus (COVID-19) infection and abortion care: information for healthcare professionals. Royal College of Obstetricians and Gynaecologists. 2020; (published 31 July 2020). Reference Source\n\nHossain N, Samuel M, Sandeep R, et al.: Perceptions, Generalized Anxiety and Fears of Pregnant women about Corona Virus infection in the heart of Pandemic. PREPRINT (Version 1). Research Square. 2020. Publisher Full Text\n\nHu D, Kong Y, Li W, et al.: Frontline nurses' burnout, anxiety, depression, and fear statuses and their associated factors during the COVID-19 outbreak in Wuhan, China: A large-scale cross-sectional study. EClinicalMedicine. 2020; 24: 100424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKessler RC, Barker PR, Colpe LJ, et al.: Screening for serious mental illness in the general population. Arch Gen Psychiatry. 2003; 60(2): 184–9. PubMed Abstract \| Publisher Full Text\n\nKessler RC, Green JG, Gruber MJ, et al.: Screening for serious mental illness in the general population with the K6 screening scale: results from the WHO World Mental Health (WMH) survey initiative. Int J Methods Psychiatr Res. 2010; 19 Suppl 1(Suppl 1): 4–22. Erratum in: Int J Methods Psychiatr Res. 2011; 20(1): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Safe Abortion Policy. Kathmandu: Ministry of Health and Population, Department of Health Services, Family Health Division, 2003. Reference Source\n\nPant PD, Pandey JP, Bietsch K: Unmet need for family planning and fertility in Nepal: Levels, trends, and determinants. DHS Further Analysis Reports No. 119. Rockville, Maryland, USA: ICF. 2019. Reference Source\n\nPoon LC, Yang H, Kapur A, et al.: Global interim guidance on coronavirus disease 2019 (COVID-19) during pregnancy and puerperium from FIGO and allied partners: Information for healthcare professionals. Int J Gynaecol Obstet. 2020; 149(3): 273–286. PubMed Abstract \| Publisher Full Text\n\nPuri M, Singh S, Sundaram A, et al.: Abortion Incidence and Unintended Pregnancy in Nepal. Int Perspect Sex Reprod Health. 2016; 42(4): 197–209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaj A, Johns NE, Barker KM, et al.: Time from COVID-19 shutdown, gender-based violence exposure, and mental health outcomes among a state representative sample of California residents. EClinicalMedicine. 2020; 26: 100520. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaymond EG, Grossman D, Mark A, et al.: Commentary: No-test medication abortion: A sample protocol for increasing access during a pandemic and beyond. Contraception. 2020; 101(6): 361–366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiley T, Sully E, Ahmed Z, et al.: Estimates of the Potential Impact of the COVID-19 Pandemic on Sexual and Reproductive Health in Low-and Middle-Income Countries. Int Perspect Sex Reprod Health. 2020; 46: 73–76. PubMed Abstract \| Publisher Full Text\n\nWu WJ, Maru S, Regmi K, et al.: Abortion Care in Nepal, 15 Years after Legalization: Gaps in Access, Equity, and Quality. Health Hum Rights. 2017; 19(1): 221–230. PubMed Abstract \| Free Full Text" }	[ { "id": "79657", "date": "09 Mar 2021", "name": "Suresh Kayastha", "expertise": [ "Reviewer Expertise obstetrics and gynecology" ], "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 has addressed one of the important aspects of reproductive health i.e. safe abortion care in Nepal during the COVID-19 pandemic. As the pandemic and subsequent lockdown had significantly hampered the health care delivery process, safe abortion care service was also interrupted in Nepal. This article has tried to address change in the pattern of abortion care seeking behaviour, obstacles faced by clients during lockdowns and pattern of chance on abortion care services in two phases of lockdown. Though the sample size and study population is small, the results found are quite significant for the policymakers as government should make proper arrangement to deliver one of the essential health care services in Nepal This article is well written though some grammatical corrections and minor changes need to be made.\n1) ABSTRACT The conclusion that improving safe abortion care services along with contraception and mental health support during the pandemic is quite true given the data and statistical analysis. It would have been better if the type of study (e.g. prospective or retrospective study) has been clearly mentioned in the methodology section. Some of the points in the conclusion section are a repetition of the findings in the results section (decreased the number of women coming for SAS, Contraceptive needs are also increased, Psychological distress is prevalent, and fear of COVID-19 has become a common reason for termination of pregnancy.)\n2) INTRODUCTION\nThe link of the effect of the pandemic in Safe abortion care, contraception and the possible threat of unsafe abortion during the pandemic is quite impressive. It would have been better if the national guidelines from the government (if available) and national society (Nepal society of obstetricians and gynecologists -NESOG) regarding abortion care during pandemics has also been mentioned (http://www.nesog.org.np/images/resources/nesog_guideline.pdf).\n\n3) METHODs 3a) Study design – better to mention prospective/retrospective study.\n3b) Duration – it would be better if we remove unnecessary paragraphs and better to focus on study duration only (no need to mention-the demographic and obstetric profile of these women about their abortion choices included the reason for termination of pregnancy and method of termination, the difficulty they are facing relating to access to these services, and their level of psychological distress were studied.)\n3c) Study tool – some of the points made have no link with the study tool e.g. the number of women coming for SAS three months prior to the beginning of the lockdown was analyzed using hospital medical records.\n4) RESULT\n\nThe comparison made for women seeking abortion care before the commencement of lockdown and first 6 months of lockdown (69 vs 52) does not seem to be true. Point to be noted is that duration or time frame is different (3 months vs 6 months). Had we taken the same time frame, the result would have been different (see below at discussion portion) Table 1- Only mean age has been shown. It would have been more informative if age groups and numbers were also mentioned. Only limited demographic and obstetric profiles have been shown. For better result and interpretation, more parameters should be explored (e.g-marital status/ religion/ occupation /education status). These factors have a significant effect on abortion care in Nepal.\n\nTable 2- Only mean age has been shown. It would have been more informative if gestational age groups and numbers were also mentioned. Reason for termination- there is a single case in section “OTHERS”. It would have been more clear if that “other cause” was mentioned.\n\n5) DISCUSSION\nThe finding that number of women coming for first trimester SAS was decreased by over 25% since the beginning of the lockdown seems to be largely underestimated. There is difference in duration taken before (i.e. 3 months) and after ( i.e 6 months ) lockdown for this comparison . Provided that we compare 3 month before lockdown with initial 3 month of the lockdown, this difference would be 73.9% ( (69-18)/69x100).\n\nParagraph 4, 6 and partly in paragraph 5, 7 are just description of what is seen in result. There is no need to restate the finding of result section at discussion portion. This is seen in almost all paragraphs.\n\nNeed reference for – Paragraph 2- The Guttmacher institute estimates that if 10% women who would normally come for SAS resorted to an unsafe method, an additional 3.3 million unsafe abortions would occur in low-middle income countries over the course of a year. Paragraph 3- Medical abortion was introduced in 2009 which increased access to abortion care. Paragraph 4- Abortion is time sensitive as the higher the period of gestation, there are more chances of complications. Paragraph 8-Abortion pills are easily available in local pharmacies in Nepal, but abortion has been de-medicalized legally for the purpose of easing accessibility to MA during the pandemic from May 2020. COVID-19 guideline for Maternal, Neonatal and Child Health (MNCH) has also been endorsed which allows home-based MA through outreach model and telemedicine. Paragraph 12-A study in pregnant women and their husbands showed that fear of COVID-19 was present in many couples which lead to depression and suicidal ideation in pregnant women\n\n6) CONCLUSION – It is once again a repetition of the finding of the result section.\n\nI would like to thank the author to bring this topic in highlight as many women suffered during pandemics. This paper will definitely make policymakers aware of the problems faced by Nepalese women and make proper arrangements for good services during pandemics and lockdown in the future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "82476", "date": "12 Apr 2021", "name": "Evelyn Eisenstein", "expertise": [ "Reviewer Expertise Pediatrics and Adolescent Health and Medicine" ], "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 paper and again highlighting the importance of prevention and health education during adolescence, The best time to change some of these sexual behaviours that all over the world was extrapolated during the Covid pandemics.\nThe accessibility of free contraception is a MUST everywhere and should be part of public campaigns.\nSAS abortion in Nepal is a plus, here in Brazil abortion is still illegal due to religious and political reasons, notwithstanding women's rights.\nAs the mean age was 26 y, they are not anymore young, but are they married (?) whose responsibility it is for the SAS decision? The couple? The husband? Free women's choice?\n\nIf I could just suggest adding in the discussion > the UN references 2014 > Operational Guidance for Comprehensive Sexuality Education and Chandra Mouli et al. 20 years after the International Conf on Population and Development Where are we with adolescent sexual and reproductive health rights? Journal of Adolescent Health 56 (2015) S1-S6 > available at: http://dx.doi.org/10.1016/j.jadohealth.2014.09.015.1\nIt would be cheaper and safer to have contraceptives available and accessible and free (even during a pandemic) than safe abortions/ or even unsafe abortions and again divide the culprit between men and women, as they (men) also need sexual education, for sure.\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": [] } ]	1	https://f1000research.com/articles/10-112
https://f1000research.com/articles/8-1675/v1	23 Sep 19	{ "type": "Brief Report", "title": "Methane emissions in triple rice cropping: patterns and a method for reduction", "authors": [ "Masato Oda", "Huu Chiem Nguyen", "Huu Chiem Nguyen" ], "abstract": "The Mekong Delta paddies are known as hotspots of methane emission, but these emissions are not well studied. We analyzed methane emission patterns based on monitoring data from typical triple rice cropping paddies collected over 5 years. We found that the total emissions in a crop season doubled in the second crop, tripled in the third crop, and reset after the annual natural flood of the Mekong River. The emission peaks occurred around 0 to 3 weeks after starting irrigation, then gradually decreased. This suggests that methane was generated by the soil organic matter, because the small rice plants provide little carbon for methanogenesis. In general, the main source of emitted methane is rice-derived carbon by current-season photosynthates and the emission peaks at the rice heading stage. However, the contribution of the rice-derived carbon is negligible in the hotspot paddies while total emission is high. The increase in emission levels from the first to the third crop can be explained by the accumulation of rice residue from the preceding crops, especially rice straw incorporated into the soil. The reset of emission levels after annual flood means that the rice straw is decomposed without methanogenesis in water with dissolved oxygen. Thus, the annual emission pattern shows that decomposing rice straw in paddy surface-water is an effective method to reduce methane emissions.", "keywords": [ "Greenhouse gases", "Mekong Delta", "Methanogenesis inhibition", "Rice straw", "Flooding", "Methane reduction" ], "content": "Introduction\n\nThe Mekong Delta produces 23.8 million tons of rice (General Statistics Office of Vietnam 2016). The climate of tropical monsoon (Am) enables high productivity by triple rice cropping (cropping three times a year). Rice paddies are a methane emission source, and the Mekong Delta is a hotspot (Arai et al., 2018; Werner et al., 2016). However, this has not been well studied (Vo et al., 2018).\n\nThe Mekong's natural flood of two months (starting from around late September to late October) limits the rice cultivation period. The 1st crop (winter-spring) begins after the natural flood, then after harvesting the rice straw is incorporated into the soil. The 2nd (spring-summer) and the 3rd crop (summer-autumn) follows without interval. Just after the 3rd crop, the natural flood starts so the straw is left on the paddies and decomposes under the floodwater. Then, the 1st crop begins again without incorporation of the straw in the soil (field leveling only), because they are sufficiently decomposed by that time.\n\nCan Tho University (CTU) and the Japan International Research Center for Agricultural Sciences (JIRCAS) conducted joint research and monitored methane emissions in typical triple rice cropping paddies for 5 years (for a total of 15 crops). The present study is a specific analysis of a part of the data set from this project. The results show that the strategy of decomposing rice straw on the surface water effectively reduces methane emission from the paddies.\n\n\nMethods\n\nThe data were obtained from a farmer’s paddies (three fields) in Thuan Hung village (10°22' N, 105°58' E), Thot Not district, Can Tho city, Vietnam from 2011 to 2016. Normally, from May to October is the rainy season. The farmer managed the water with continuous flooding using a dike system. The rice variety Jasmine was used for the 1st crop, and OM501 was used for the 2nd and 3rd crop every year. The average number of growth days per crop were 103, 89, and 92, for the 1st, 2nd, and 3rd crops, respectively. The average intervals between the 1st and the 2nd crop and the 2nd and the 3rd crop were 5.6 and 6.6 days, respectively. This study was conducted with the approval of the farmer.\n\nWe used the closed chamber method, described previously (Oda & Chiem, 2019). In periods of natural flood, chambers with attached Styrofoam floats were used. Measurements were taken once a week throughout the rice growing stage, but every 3 days for 2 weeks after seeding, heading stage, and around draining.\n\nDescriptive statistics were calculated using Microsoft Excel 2016.\n\n\nResults\n\nAccording to the IPCC guidelines, standard methane emissions over 100 days of continuously flooding rice cropping are 130 kg ha−1 crop−1. Wassmann et al. (1996) reported very high emissions (160–240 kg ha−1 crop−1) from double cropping rice paddies in the Philippines after organic matter incorporation. However, we observed larger emissions (710–1845 kg ha−1 crop−1). The emission level doubled in the 2nd crop, and tripled in the 3rd crop, then reset after the natural flood (Figure 1). Furthermore, the total emissions during the flood period and the 1st crop was lower than that of the 3rd crop (Figure 1). The total emission should be higher than that of the 3rd crop; because, the absence of rice plants doubles the methane emission from the field (Oda & Chiem, 2019). Note, we confirmed that no rice straw (the source of methanogenesis) was lost to the floodwater. Raw results are available as Underlying data (Oda, 2019a).\n\nFive-year average of CH4 emissions of triple-cropped rice paddies in the Mekong Delta (2011–2016). Error bars are standard deviation (n = 15).\n\nThe previous study (Oda & Chiem, 2019) indicated three types of methane emission patterns during the rice growth period. Generally, the emissions peak at the heading stage due to the methanogenesis substrate provided by the present rice. Another pattern can occur with an additional peak at the early stage of rice growth if organic matter was incorporated beforehand. The third is the pattern in the triple rice cropping. The emission peaks at the early stage of rice growth, then gradually decreases; the peak at the heading stage is undetectable because of the high emission levels. This means the contribution of the rice-derived carbon is small. In the present study, the pattern was the same as the previous study (Oda & Chiem, 2019). The emissions began with irrigation, reached peaks from 0 to 3 weeks after the start of irrigation (see Extended data, Supplemental figure; Oda, 2019b), and gradually decreased, and the peak at the heading stage was undetected. Furthermore, the emissions during the natural flood appeared to be a continuation of the emissions of the 3rd crop (Figure 2).\n\nCH4 emissions of triple crop rice paddies in the Mekong Delta (2011–2016). Data are the mean of three replications.\n\n\nDiscussion\n\nThe total emissions in a crop season doubled in the second crop, tripled in the third crop, and reset after the natural flood. This can be explained by the accumulation of rice residue from the preceding crops, especially by the rice straw incorporated into the soil, because the contribution of the present rice-derived carbon is small.\n\nThe reset of emission levels after the annual flood means that the rice straw is decomposed without methanogenesis in water because the water includes dissolved oxygen. The fact that the emissions under natural flood appeared to be a continuation of the emissions of the 3rd crop suggests that the rice straw on the paddy surface contribute to no methane emission. A portion of emission in the first crop will be caused by incorporation of the remaining rice straw related to the leveling of the field.\n\nOur results indicate that the main cause of the increase in methane emissions was the incorporation of rice straw into the soil. In contrast, decomposing rice straw in paddy surface-water generated less methane. Thus, decomposing rice straw in paddy surface-water is an effective method to reduce methane emissions.\n\n\nConclusion\n\nWe analyzed the methane emission patterns of triple rice cropping paddies in the Mekong Delta. Methane emissions increased with rice straw incorporation into the soil. The natural flood resulted in decomposition occurring in the water, leading to less methane emission. Therefore, the annual emission pattern suggests that decomposing rice straw in paddy surface-water is an effective method to reduce methane emissions. The development of practical technology to attain this reduction is a subject for a future study.\n\n\nData availability\n\nFigshare: Methane emission from triple cropping rice field. https://doi.org/10.6084/m9.figshare.9757934.v1 (Oda, 2019a).\n\nFigshare: Methane flux of days after transplanting. https://doi.org/10.6084/m9.figshare.9746006.v1 (Oda, 2019b).\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 our former colleagues for the use of legacy data.\n\n\nReferences\n\nAnon: Impact of climate change on future rice production in the Mekong River Delta. CCAFS: CGIAR research program on Climate Change, Agriculture and Food Security [Online]. [Accessed: 30 August 2019]. Reference Source\n\nArai H, Takeuchi W, Oyoshi K, et al.: Estimation of Methane Emissions from Rice Paddies in the Mekong Delta Based on Land Surface Dynamics Characterization with Remote Sensing. Remote Sens. 2018; 10(9): 1438. Publisher Full Text\n\nOda M: Methane emission from triple cropping rice field. figshare. Dataset. 2019a. http://www.doi.org/10.6084/m9.figshare.9757934.v1\n\nOda M: Methane flux of days after transplanting. figshare. Figure. 2019b. http://www.doi.org/10.6084/m9.figshare.9746006.v1\n\nOda M, Chiem NH: Rice plants reduce methane emissions in high-emitting paddies [version 3; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2019; 7: 1349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWassmann R, Neue HU, Alberto MC, et al.: Fluxes and pools of methane in wetland rice soils with varying organic inputs. Environ Monit Assess. 1996; 42(1–2): 163–173. PubMed Abstract \| Publisher Full Text\n\nWerner C, Kraus D, Mai TV, et al.: CH4 and N2O Emissions from Rice Paddy Soils in Vietnam - Identifying Regional Hotspots and Quantifying the Total Emission Strength Using a Biogeochemical Model. AGU Fall Meeting Abstracts. 2016. Reference Source\n\nVo TBT, Wassmann R, Tirol-Padre A, et al.: Methane emission from rice cultivation in different agro-ecological zones of the Mekong river delta: seasonal patterns and emission factors for baseline water management. SSPN. 2018; 64(1): 47–58. Publisher Full Text" }	[ { "id": "54183", "date": "18 Oct 2019", "name": "Arika Bridhikitti", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe idea of estimating methane emissions from rice paddy fields is not novel. Many publications, including those conducted in SE Asia, have been widely accepted, such that collected and reported in the 1996 IPCC Guidelines for National Greenhouse Gas Inventories: Reference Manual. The study on triple rice cropping in the Mekong River Delta is quite interesting since few studies carried on such the cropping system, but usually estimating the total flux based upon one single cropping experiment.\nMy first significant concern in this work is involved with the methodology of methane sampling and analysis. The closed chamber method used in this study (Oda and Chiem, 2019) had not yet been validated with other traditional closed chamber studies. Furthermore, methane emission using this method seems to return significantly higher methane emissions compared to those emissions reported worldwide in the IPCC report. Since very much high emission rates can result in significantly overestimating methane emissions from the SE Asia region, I suggest the authors carefully discuss the reliability of the proposed method in comparison to the traditional method.\nMy last concern is on the suggestions given by the authors. They claim that decomposing rice straw in paddy surface-water (nonrice period) is an effective method to reduce methane emissions. This suggestion is not conforming to the fact that the wetland system is a significant source of methane emission. I think the author could mean lower methane emission during the nonrice flooding period, but the conclusion written in the manuscript is not clear that way. Although, I think the conclusion given by the authors is not feasible since promoting long-term nonrice flooding instead of rice cropping could mean lower rice yield.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5003", "date": "12 Nov 2019", "name": "Masato Oda", "role": "Author Response", "response": "Thank you for giving valuable comments for improving our manuscript. 1. The methodology of methane sampling and analysis.Our methodology is the traditional method and there are high emission data in An Giang province. The water management conditions are better than our site because of the full-dike system. Thus, we think our data are reliable. We added the explanations. 2. Suggestion by authorsWe agree with you that the wetland system is a significant source of methane emission. We also agree with you that the methane emission in the paddy field is extremely high compared to the simple wetland. Our finding is that the cause of the high emission in the paddy field is the incorporation of rice straws. The recent spread of combine harvesting decreases the need for rice straw burning. The methane emission will increase much more. Therefore, decreasing the methane emission of the paddy field to the levels of the simple wetland is still significant according to the data. Our suggestion is directly led by the results of the present study; although there are many other indirect options. For the yield of rice, we think by the profit. A recent study reported that the profit of triple rice is only 6% higher than double cropping (J Environ Manage. 2018). However, utilizing rice straw enables organic farming and that brings much profit by the high unit price. We are trying to establish cultivation practice. Furthermore, using the ratooning triple cropping is also possible." } ] }, { "id": "54181", "date": "23 Oct 2019", "name": "Tran Dang Hoa", "expertise": [ "Reviewer Expertise Low carbon rice production" ], "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 measurement of GHG emission from paddy fields in Mekong Delta have been carried out by several authors. However, understanding of GHG emission from paddy field by triple seasons isn't well known. This study contributed some good information on GHG emission, focusing on CH4 to understanding how to reduce GHG emission from the paddy field.\nMy commends are below:\nIntroduction:\nIntroduction should be given a general information on rice production in Mekong Delta in order to explain this study is a representative.\nMethod:\nSite description should be more clear, such as, what about the field practices of the farmer on their fields? Were the three observed fields similar in these practices? What about the water management in the fields? The author said the fields was continuously flooding, but how about water level/regime in three observed fields? How about straw management? What about the height of stubble for each season? How was about fertilization?...\n\nMethane measurement: The author said the closed chamber method described by Oda &Chien, 2019. However, a brief description should be added. What time of the day were GAS samples take? How was CH4 calculated?\n\nStatistical analysis: A one-way ANOVA should be analysed to compare the means among season?\n\nDiscussion:\nThe authors just explained the difference of CH4 emission among seasons was due to straw incorporation into the soil. However, I did not see any information on straw incorporation in the observed fields (as above commented). Other factors such as rice varieties, weather, fertilization... should be discussed. In this case, these factors differed among seasons, why did only straw incorporation influence on CH4 emissions?\n\nConclusion:\n\nAs above commented, the conclusion on CH4 emission increase being due to straw incorporation is not 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? 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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5004", "date": "12 Nov 2019", "name": "Masato Oda", "role": "Author Response", "response": "Thank you very much for giving valuable comments. 1. Introduction: a general information in order to explain this study is a representativeThank you. We added. 2. Methods2-1. Site description:The sentence was improved to clarify that the present study was an observation of typical farmer’s paddies mentioned in the introduction (including rice straw treatment). 2-2. Farmers practices:Thank you. We added the reference. The height of the stubble is about 30 cm. The daily average water levels were monitored with water level loggers at the corner of the fields; the average levels were 2.0 cm (−0.6 to 6.1 cm) until drained (the data will be published in another paper). 2-3. Methane measurement:Thank you. We added. 3. Statistical analysis:Figure 1 is clear enough to omit ANOVA (see the standard deviation (not SE)). 4. Discussion:The information on straw incorporation in the observed fields is mentioned in the introduction. Rice straw incorporation enhances methane emission. We added a referred to our previous study that is a kind of short review of the effect of straw incorporation." } ] } ]	1	https://f1000research.com/articles/8-1675
https://f1000research.com/articles/9-1274/v1	26 Oct 20	{ "type": "Research Article", "title": "Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally, Caranx ignobilis", "authors": [ "Firdus Firdus", "Samadi Samadi", "Abdullah A. Muhammadar", "Muhammad A. Sarong", "Zainal A. Muchlisin", "Widya Sari", "Siska Mellisa", "Satria Satria", "Boihaqi Boihaqi", "Agung Setia Batubara", "Firdus Firdus", "Abdullah A. Muhammadar", "Muhammad A. Sarong", "Zainal A. Muchlisin", "Widya Sari", "Siska Mellisa", "Satria Satria", "Boihaqi Boihaqi", "Agung Setia Batubara" ], "abstract": "Background: Research on supplementing feed with rice husk activated charcoal was carried out to determine the effect of variations in the concentration of rice husk activated charcoal on the growth and histological features of the Caranx ignobilis intestine. Methods: This study used an experimental method with a completely randomized design consisting of six treatments and four replications, including adding activated charcoal to feed at concentrations of 0%, 1%, 1.5%, 2%, 2.5%, and 3% for 42 days. The measured parameters included daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE), survival rate (SR), length of foveola gastrica, width of foveola gastrica, length of intestinal villi, and width of intestinal villi. Data were analyzed statistically using one-way analysis of variance and Duncan’s test. Results: The results showed that supplementing fish feed with rice husk activated charcoal at different concentrations significantly affected the values of DGR, AGR, FCR, FE, SR, length of the foveola gastrica, length of the villous intestine, and width of the villous intestine, but did not significantly affect SGR or foveola gastrica width. Conclusion: The 2% rice husk activated charcoal treatment showed the best results for all parameters.", "keywords": [ "foveola gastrica", "villi", "ratio", "biometrics" ], "content": "Introduction\n\nGiant trevally (Caranx ignobilis) is a commercially valuable fish species widely distributed in Indonesian waters1–3. This fish is a top predator in coral reef ecosystems with a relatively long predicted age of 25 years and can grow to a size of 165 cm and 87 kg, making C. ignobilis the largest species in the Carangidae family4. However, fishing continues to increase without conservation efforts, causing the fish population to decline in the last few decades5–7. Therefore, efforts towards fish domestication are needed to reduce wild fishing. However, several obstacles have been encountered during domestication, such as slow growth of fish due to underdeveloped feed technology in this species.\n\nThe development of C. ignobilis culture is dependent on trash fish (fish considered to have little value and therefore typically discarded whenever caught) because the fish grow faster than when using commercial feed. However, the costs incurred to provide trash fish are relatively expensive and not balanced with the selling price of fish at harvest. In addition, trash fish are not always available and can carry diseases. Therefore, C. ignobilis feed technology needs to be assessed immediately, through the compilation of appropriate feed ingredients so that it can support the nutritional needs of the fish. One of the technologies that has the potential to be applied is supplementing the feed with activated charcoal.\n\nActivated charcoal is a non-nutritional ingredient that binds toxic substances during digestion to increase intestinal absorption of food that enters the digestive tract8. Activated charcoal can be produced with a variety of ingredients, such as coconut shells9,10, pine wood powder11, banana peels12, corn stalks13,14, peanut shells14, rice straw and rice husk14,15, oil palm stems and shells16,17, wine stalks18, bamboo19, almond stems and bark20, and durian peel21. Therefore, activated charcoal has good application potential because the resource is easily found and the feed is supplemented with a particular concentration according to the needs of the cultured fish.\n\nHigh-carbon activated charcoal has the best function, which includes rice husk. Jasman22 reported that rice husk activated charcoal contains 85–95% carbon. In addition, rice husk activated charcoal has an Iod value of 527.8 mg/g, indicating a good quality of absorbent activated charcoal23. Furthermore, rice husks contain 13%–39% ash, 34%–44% cellulose, and 23%–30% lignin24, where the higher the content of hemicellulose, cellulose, and lignin, the higher the amount of activated carbon and the better the quality of the charcoal25.\n\nSeveral studies have reported applying activated charcoal in fish, such as supplementing activated charcoal in feed on the growth of Takifugu rubripes26, Paralichthys olivaceus27, Pangasiaodon28, Oreochormis niloticus8, Clarias gariepinus29, Sparus aurata30, and Huso huso31. A study on supplementing coconut shell activated charcoal, mangrove wood, rice husk, and oil palm shell in feed on intestinal growth of C. ignobilis indicated that 2% activated rice husk charcoal had an optimal effect on growth of intestinal tissues32. However, the appropriate concentration to supplement rice husk activated charcoal in feed for C. ignobilis has not been reported.\n\n\nMethods\n\nThis study was conducted from April to September 2019 at the Ujung Batee Brackish Aquaculture Fisheries Center, Ministry of Maritime Affairs and Fisheries, Aceh Besar. The activated charcoal was characterized at the Laboratory and Integrated Testing of Gadjah Mada University, Yogyakarta. The gastric and intestinal histological samples were evaluated at the histology laboratory of the Faculty of Mathematics and Natural Sciences, Syiah Kuala University, Banda Aceh Indonesia.\n\nThis study used a completely randomized design method with six treatments and four replications. The fish were fed experimental food containing 50% protein twice a day, at 7 am and 5 pm, at 3% of body weight.\n\nA = Treated feed without activated charcoal (control/0%)\n\nB = Feed treated to contain 1% rice husk activated charcoal\n\nC = Feed treated to contain 1.5% rice husk activated charcoal\n\nD = Feed treated to contain 2% rice husk activated charcoal\n\nE = Feed treated to contain 2.5% rice husk activated charcoal\n\nE = Feed treated to contain 3% rice husk activated charcoal\n\nA total of 360 C. ignobilis juveniles (average weight, 16.47 ± 4.69 g; average length, 9.61 ± 0.71 cm) were collected from local fishermen in Ulee Lheue Village, Banda Aceh City, Indonesia. The fish was acclimatized in the maintenance pond at Ujung Batee Brackish Aquaculture Fisheries Center for 2 weeks.\n\nJuvenile C. ignobilis were chosen randomly, and the total weight and length of the fish were measured. The fish were distributed into 24 plastic containers (48 × 43 × 70 cm) with a water volume of 75 liters (15 fish/container). The fish were fed the experimental food twice daily at 7 am and 5 pm for 42 days.\n\nRice husk was ground into flour until smooth, and about 500 g was placed in an iron container that was coated with aluminum foil, and burned in a furnace at 400°C for 1 hour. Nitrogen gas was flowed into the furnace to remove oxygen. The temperature was gradually reduced to 30°C for 1 hour. The charcoal was removed from the furnace and filtered through a 40-mesh sieve, and stored in a bottle before activating. A total of 100 g of charcoal was mixed with 400 ml of 0.2 M citric acid. The solution was stirred for 24 hours and filtered through filter paper. The filtered charcoal was washed with distilled water and dried in an oven at 110°C for 24 hours. The activated charcoal was stored in a desiccator before use.\n\nThe treated feed was prepared from fish flour, rebon shrimp flour, tapioca flour, coconut oil, CaCO3, isoleucine, L-tryptophan, and DL-methionine, and premixed with 50% protein feed content. All ingredients were mixed and analyzed for protein content. Subsequently the feed was added to the rice husk activated charcoal at 1%, 1.5%, 2%, 2.5%, and 3% (see Table 1 for feed makeup).\n\nGastric and intestinal samples were excised at the end of the study. Four fish were taken randomly for each replication of the treatments. Fish were anesthetized in 30 ppm clove oil, and the belly of the fish was dissected following the procedure of Purushothaman et al.33. The stomach and intestines were removed with tweezers, and placed in a bottle containing 4% formalin for 1 week. Histological sampling was carried out using the paraffin method based on Osman and Caceci34. Samples were dehydrated through a gradient alcohol series (ethanol solutions of increasing concentration in 70%, 80% and 90%) and dehydrated in xylol. The stomach and intestinal samples were embedded in paraffin blocks. The paraffin blocks were cut 6-mm thick and stained with hematoxylin and eosin. The length and width of the histological preparations were measured using a binocular microscope (Zeiss Primo Star, Carl Zeiss Suzhou Co., Ltd., Beijing, China) which was projected onto a screen. Treatment of the experimental animals followed the guidelines for the use of animals in research at Syiah Kuala University.\n\nThe parameters measured in this study were the daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE), survival rate (SR), length of the foveola gastrica, width of the foveola gastrica, and length of the intestinal villi. The biometric measurements, including the length and width of the foveola gastrica and intestinal villi, were made following the methods of Amiri et al.35. DGR and SGR were analyzed according to the formula by Muchlisin et al.36,37. AGR was analyzed based on the formula of Jones38:\n\n\n\nWhere Wo is initial fish weight (g); Wt is fish weight at the end of the study (g); t is the duration of the study (days). L1 is the initial length of the fish (cm), L2 is the final length of the fish (cm), Δt is the rearing period (days). Nt is the number of fish at the end of maintenance, No is the number of fish at the beginning of maintenance.\n\nThe research parameters were analyzed statistically using one-way analysis of variance to detect differences followed by Duncan’s multiple range test. P < 0.05 was considered significant. All data were analyzed using SPSS software version 20 (SPSS Inc., Chicago, IL, USA). Qualitative (histological) data of the stomach and intestine were analyzed descriptively.\n\n\nResults\n\nThe active rice husk charcoal supplement had a significant effect on DGR and AGR, but did not significantly affect SGR (Table 2). The highest average DGR value was observed in the 2% rice husk activated charcoal treatment (0.359 g/day), followed by 1.5% rice husk activated charcoal (0.289 g/day), the control treatment (0.235 g/day), 1% rice husk activated charcoal (0.221 g/day), 3% rice husk activated charcoal (0.220 g/day), and 2.5% rice husk activated charcoal (0.210 g/day). Furthermore, the highest average SGR value was detected in the 2% rice husk activated charcoal treatment (1.492%/day), followed by 1.5% (1.408%/day) rice husk activated charcoal, the control treatment (1.122%/day), 3% rice husk activated charcoal (1.060%/day), 1% rice husk activated charcoal (1.046%/day), and 2.5% rice husk activated charcoal (1.009%/day). The highest average AGR value was observed in the 2% rice husk activated charcoal treatment (0.092 cm/day), followed by the 1.5% rice husk activated charcoal (0.081 cm/day), the control treatment (0.068 cm/day), 3% rice husk activated charcoal (0.058 cm/day), 1% rice husk activated charcoal (0.054 cm/day), and 2.5% rice husk activated charcoal (0.043 cm/day).\n\nNote: Numbers followed by different letter superscripts are significantly different (P < 0.05).\n\nThe results showed that adding activated rice husk charcoal to feed significantly affected the FCR (P<0.001), FE (P<0.001), and SR (P=0.002) of C. ignobilis juveniles. The best FCR was observed in the 2% rice husk activated charcoal treatment with a value of 1.257, indicating that 1.257 kg of feed is required to increase fish weight 1 kg. The highest FCR value was detected in the 2.5% rice husk activated charcoal treatment at 1.922. Furthermore, the best FE and SR values were also observed in the of 2% rice husk activated charcoal treatment with 80.264% and 88.33%, respectively, while the lowest FE and SR values were observed in the 2.5% rice husk activated charcoal treatment at 52.114% and 65%, respectively (Table 3).\n\nNote: Numbers followed by different letter superscripts are significantly different (P < 0.05).\n\nAdding activated rice husk charcoal to the feed had a significant effect (P < 0.05) on the length of the foveola gastrica in C. ignobilis juveniles. The highest average length and width of the foveola gastrica were observed in the 2% rice husk activated charcoal treatment with values of 171.574 µm and 120.409 µm. respectively, while the shortest average length of the foveola gastrica was detected in the control treatment (122.287 µm) and the shortest foveola gastrica width was observed in the 1.5% rice husk activated charcoal treatment (89.134 µm) (Table 4 and Figure 1).\n\nNote: Numbers followed by different letter superscripts are significantly different (P < 0.05).\n\n(A) Feed without added activated charcoal (control). (B) Feed with 1% rice husk activated charcoal. (C) Feed with 1.5% rice husk activated charcoal. (D) Feed with 2% rice husk activated charcoal. (E) Feed with 2.5% rice husk activated charcoal. (F) Feed with 3% rice husk activated charcoal. M, Tunica mucosa; e, epithelium lamina; p, lamina propria; SM, submucosal tunica; Mc, muscular tunica; S, serous tunica; lm, longitudinal muscles.\n\nSupplementing with rice husk activated charcoal significantly affected (P < 0.05) the length and width of the C. ignobilis intestinal villi. The longest mean length and width of intestinal villi were observed in the 2% rice husk activated charcoal treatment at 64.027 µm and 16.672 µm, respectively. The shortest mean length of intestinal villi was detected in the 2.5% rice husk activated charcoal (47.204 µm), while the shortest mean width of intestinal villi was observed in the 1% rice husk activated charcoal treatment (11.668 µm) (Table 5 and Figure 2).\n\nNote: Numbers followed by different letter superscripts are significantly different (P < 0.05).\n\n(A) Feed without added activated charcoal (control). (B) Feed with 1% rice husk activated charcoal. (C) Feed with 1.5% rice husk activated charcoal. (D) Feed with 2% rice husk activated charcoal. (E) Feed with 2.5% rice husk activated charcoal. (F) Feed with 3% rice husk activated charcoal. M, Tunica mucosa; SM, tunica submucosa; Mc, tunica muscularis; S, serous tunica; e, epithelial lamina; p, lamina propria; gc, goblet cell.\n\n\nDiscussion\n\nSupplementing fish feed with different concentrations of rice husk activated charcoal had significant effects on DGR, AGR, FCR, FE, SR, length of the foveola gastrica, length of the villous intestine, and width of intestinal villi, but had no significant effect on SGR or the width of the foveola gastrica. The 2% activated charcoal treatment revealed the best results for all parameters, including DGR (0.359 g/day), SGR (1.492%/day), AGR (0.092 cm/day), FCR (1.257), FE (80.264%), SR (88.33%), foveola gastrica length (171.574 µm), foveola gastrica width (120.409 µm), villous bowel length (64.027 µm), and intestinal villous width (16.672 µm). These results indicate that supplementing with 2% rice husk activated charcoal was a catalyst for absorption of feed nutrients by C. ignobilis juveniles, which enhanced the immune system, leading to a high SR. According to Prescott et al.39, activated charcoal functions as a bacterial endotoxin absorbent, which inhibits absorption of nutrients. In addition, Mulyono and Wibisono40 reported that activated charcoal absorbs ammonia (NH3), which is a toxic substance. Kutlu et al.41 stated that adding activated charcoal to feed accelerates the healing process of the mucosa by eliminating intestinal pathogenic bacteria. Furthermore, Thu et al.27 reported that activated charcoal plays a role reducing intestinal surface pressure by removing and absorbing gases and poisons that occur along the intestine, so that nutrient absorption is optimal.\n\nAdding more or less than 2% rice husk activated charcoal did not change the DGR, SGR or AGR values from the control treatment, indicating that increasing the rice husk activated charcoal concentration more or less than 2% is no better than control feed. However, the 1.5%–2% rice husk activated charcoal treatment increased feed protein absorption (FE) better than the other treatments, thereby reducing the quantity of feed (FCR) given to the fish. This shows that the 1.5%–2% rice husk activated charcoal treatment functioned effectively and efficiently, resulting in higher fish weights with less feed compared to the other treatments. In addition, the SR was maximum in this treatment, with a value of 83.33% in the 1.5% rice husk activated charcoal treatment and 88.33% in the 2% rice husk activated charcoal treatment. However, different results were reported by Ademola et al.29 who found that adding 2.5% rice husk activated charcoal increases the growth and survival of catfish. This difference was likely due to the different test species, which are physiologically different.\n\nAdding activated rice husk charcoal to the feed significantly affected the length of foveola gastrica, but did not affect the width of the foveola gastrica in C. ignobilis juveniles. The length of the foveola gastrica was positively correlated with increasing concentrations of rice husk activated charcoal in feed from 0%–2%, but the size decreased at concentrations of 2.5% and 3%. Although there was a decrease in the length of the foveola gastrica in the 2.5% and 3% activated rice husk charcoal treatments, the decrease was not significantly different from the longest foveola gastrica value in the 2% rice husk activated charcoal treatment. Pirarat et al.8 reported that exceeding the optimum concentration of activated charcoal in feed does not have a positive effect on the development of digestive organs and interferes with absorption of nutrients from feed.\n\nFigure 1 shows that the tunic muscularis forms two thin layers of muscle called the circular and the longitudinal muscles. Latania et al.42 explained the presence and degree of muscular cooperation between the circular and longitudinal muscles indicated whether the type of feed consumed by the fishes was relatively good. This results in better absorption by the lamina epithelium and facilitates the channeling of nutrients by the lamina propria so that the foveola gastrica reacts positively by increasing in size. Furthermore, the results of histological analysis showed that C. ignobilis fed 2% activated charcoal developed an epithelial surface layer covering the entire foveola gastrica. This reinforces the conclusion that growth of the foveola gastrica occurred optimally with the 2% active rice husk charcoal supplement.\n\nTable 5 and Figure 2 show that the average length and width of intestinal villi were optimum in the 2% rice husk activated charcoal treatment, indicating that activated charcoal in feed contributes to increase the length and width of the intestinal villi. Kuperman and Kuz'mina43 and Mekbungwan et al.44 reported that adding activated charcoal to feed increases the growth and intestinal function of land and aquatic animals and improves FE. Thus, growth of intestinal villi is one of the determinants of nutrient uptake during fish digestion.\n\n\nConclusion\n\nThe results showed that supplementing the feed with different concentrations of activated rice husk charcoal significantly affected the values of DGR, AGR, FCR, FE, SR, length of foveola gastrica, length of villous intestine, and width of the intestinal villi, but had no significant effect on SGR or width of the foveola gastrica in C. ignobilis. The 2% active rice husk charcoal treatment revealed the best results for all research parameters, including DGR (0.359 g/day), SGR (1.492%/day), AGR (0.092 cm/day), FCR (1.257), FE (FE) 80.264%), SR (88.33%), foveola gastrica length (171.574 µm), foveola gastrica width (120.409 µm), villous bowel length (64.027 µm), and villous bowel width (16.672 µm).\n\n\nData availability\n\nFigshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis, https://doi.org/10.6084/m9.figshare.12859973.v145.\n\nThis project contains the raw data for the faveola gastica and villi of all fish examined in this study.\n\nFigshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis, https://doi.org/10.6084/m9.figshare.12860033.v146.\n\nThis project contains uncropped, unprocessed images of the faveola gastrica of giant trevally.\n\nFigshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis, https://doi.org/10.6084/m9.figshare.12860054.v147.\n\nThis project contains uncropped, unprocessed images of the intestinal villi of giant trevally.\n\nFigshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis, https://doi.org/10.6084/m9.figshare.12901784.v148.\n\nThis project contains the daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), and feed efficiency (FE) of Caranx ignobilis.\n\nFigshare: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis, https://doi.org/10.6084/m9.figshare.13094930.v249.\n\nThis project contains the feed conversion ratio (FCR) and survival rate (SR) of Caranx ignobilis.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThanks to all of the staff at the Ujung Batee Brackish Aquaculture Fisheries Center who assisted with the research. Special thanks go to Mr. Marbawi, Yessi Iman Sari, Amalia, and Humaira Armi for their participation during the research.\n\n\nReferences\n\nBatubara AS, Muchlisin ZA, Thamren MY, et al.: Check list of marine fishes from Simeulue Island waters, Aceh Province, Indonesia. Aceh Journal of Animal Science. 2017; 2(2): 77–84. Publisher Full Text\n\nTimorya Y, Abdullah A, Batubara AS, et al.: Conservation and economic status fishes in the Krueng Sabee River, Aceh Jaya District, Aceh Province, Indonesia. IOP Conference Series: Earth and Environmental Science. 2018; 216: 012044. Publisher Full Text\n\nPurba GY, Haryono E, Manan J, et al.: Jellyfish Lakes at Misool Islands, Raja Ampat, West Papua, Indonesia. Biodiversitas. 2018; 19(1): 172–182. Reference Source\n\nAbdussamad EM, Kasim HM, Balasubramanian TS: Distribution, biology and behaviour of the giant trevally, Caranx ignobilis - a candidate species for mariculture. Bangladesh Journal of Fisheries Research. 2008; 12(1): 89–94. 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Reference Source\n\nMozammel HM, Masahiro O, Bhattacharya SC: Activated charcoal from coconut shell using ZnCl2 activation. Biomass Bioenergy. 2002; 22(5): 397–400. Publisher Full Text\n\nJain A, Tripathi SK: Fabrication and characterization of energy storing supercapacitor devices using coconut shell based activated charcoal electrode. Materials Science and Engineering: B. 2014; 183: 54–60. Publisher Full Text\n\nQiu KQ, Yang SW, Yang J: Characteristics of activated carbon prepared from Chinese fir sawdust by zinc chloride activation under vacuum condition. J Cent South Univ T. 2009; 16(3): 385–391. Publisher Full Text\n\nMopoung S: Surface image of charcoal and activated charcoal from banana peel. Journal of Microscopy Society of Thailand. 2008; 22: 15–19. Reference Source\n\nTsai WT, Chang CY, Wang SY, et al.: Cleaner production of carbon adsorbents by utilizing agricultural waste corn cob. Resources, conservation and recycling. 2001; 32(1): 43–53. Publisher Full Text\n\nAbdul A, Aberuagba F: Comparative study of the adsorption of phosphate by activated charcoal from corncobs, groundnut shells and rice-husks. Australian Journal of Technology. 2005; 9: 59–63. Reference Source\n\nAi N, Zeng G, Zhou H, et al.: Co-production of activated carbon and bio-oil from agricultural residues by molten salt pyrolysis. BioResources. 2013; 8(2): 1551–1562. Reference Source\n\nAdinata D, Daud WMAW, Aroua MK: Preparation and characterization of activated carbon from palm shell by chemical activation with K2CO3. Bioresour Technol. 2007; 98(1): 145–149. PubMed Abstract \| Publisher Full Text\n\nAlothman Z, Habila M, Ali R: Preparation of activated carbon using the copyrolysis of agricultural and municipal solid wastes at a low carbonization temperature. Carbon. 2011; 24: 67–72. Publisher Full Text\n\nDeiana AC, Sardella MF, Silva H, et al.: Use of grape stalk, a waste of the viticulture industry, to obtain activated carbon. J Hazard Mater. 2009; 172(1): 13–19. PubMed Abstract \| Publisher Full Text\n\nChoy KK, Barford JP, McKay G: Production of activated carbon from bamboo scaffolding waste—process design, evaluation and sensitivity analysis. Chem Eng J. 2005; 109(1–3): 147–165. Publisher Full Text\n\nGonzález JF, Román S, Encinar JM, et al.: Pyrolysis of various biomass residues and char utilization for the production of activated carbons. J Anal Appl Pyrolysis. 2009; 85(1–2): 134–141. Publisher Full Text\n\nJun TY, Arumugam SD, Latip NHA, et al.: Effect of activation temperature and heating duration on physical characteristics of activated carbon prepared from agriculture waste. EnvironmentAsia. 2010; 3: 143–148. Publisher Full Text\n\nJasman J: Uji coba arang aktif sekam padi sebagai media filtrasi dalam menurunkan kadar Fe pada air sumur bor di asrama jurusan kesehatan lingkungan Manado. Jurnal Kesehatan Lingkungan. 2011; 1(1): 49–53. Reference Source\n\nJamilatun S, Setyawan M, Salamah S, et al.: Pembuatan arang aktif dari tempurung kelapa dengan aktivasi sebelum dan sesudah pilorisis. Seminar Nasional Sains dan Teknologi. 2015. Reference Source\n\nMahvi AH, Maleki A, Elslami A: Potential of rice husk and rice husk ash for phenol removal from aqueous systems. Am J Appl Sci. 2004; 1(3): 321–326. Publisher Full Text\n\nMaria NSL, Banu ASS: Activated carbon from rice husk for treating dye waste water. International Journal of Green Chemistry. 2015; 1(1): 1–9. Reference Source\n\nThu M, Koshio S, Ishikawa M, et al.: Effects of dietary bamboo charcoal on growth parameters, apparent digestibility and ammonia nitrogen excretion of tiger puffer fish, Takifugu rubripes. Aquaculture Science. 2009; 57(1): 53–60. Publisher Full Text\n\nThu M, Koshio S, Ishikawa M, et al.: Effects of supplementation of dietary bamboo charcoal on growth performance and body composition of juvenile Japanese ﬂounder, Paralichthys olivaceus. J World Aquac Soc. 2010; 41: 255–262. Publisher Full Text\n\nJahan R, Abdul MQ, Jahan N, et al.: Dietary added bamboo charcoal can evoke pangasiaodon growth and can reduce ammonia from culture medium. International Journal of Fisheries and Aquaculture. 2014; 6(7): 87–93. Publisher Full Text\n\nAdemola ZA, Muyideen LO, Toheeb AT: Effects of graded activated charcoal in rice husk diets for mud catfish, Clarias gariepinus juveniles (Teleostei: Clariidae). Iran J Ichthyol. 2015; 3(3): 203–209. Publisher Full Text\n\nMichael FR, Helal AM: Rule of dietary activated wood charcoal on the growth and biochemical composition of Gilthead Seabream (Sparus aurata) reared under different stocking densities. Life Sci J. 2018; 15(4): 79–86. Publisher Full Text\n\nSamadaii S, Bahrekazemi M: The effect of diets containing different levels of active charcoal on growth performance, body composition, haematological parameters and possibility of heavy metals detoxification in big sturgeon (Huso huso). Aquac Res. 2020; 51(1): 91–101. Publisher Full Text\n\nFirdus F, Samadi S, Muhammadar AA, et al.: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPurushothaman K, Lau D, Saju JM, et al.: Morpho-histological characterisation of the alimentary canal of an important food fish, Asian seabass (Lates calcarifer). PeerJ. 2016; 4: e2377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsman AH, Caccemi T: Histology of stomach of Tilaphia nitolica (Linnaeus, 1758) from the river Nile. J Fish Biol. 1991; 38: 211–223. Publisher Full Text\n\nAmiri BM, Rafiee GR, Abedi M, et al.: Comparative study on the gastrointestinal tract of mature and maturing female and male of Caspian lamprey, Caspiomyzon wagneri. (Kessler, 1870) in the southern Caspian Sea (Cephalaspidomorphi: Petromyzontidae). Iran J Ichthyol. 2016; 3(2): 140–149. Publisher Full Text\n\nMuchlisin ZA, Arisa AA, Muhammadar AA, et al.: Growth performance and feed utilization of keureling (Tor tambra) fingerlings fed a formulated diet with different doses of vitamin E (alpha-tocopherol). Fisheries & Aquatic Life. 2016; 24(1): 47–52. Publisher Full Text\n\nMuchlisin ZA, Afrido F, Murda T, et al.: The effectiveness of experimental diet with varying levels of papain on the growth performance, survival rate and feed utilization of keureling fish (Tor tambra). Biosaintifika. 2016; 8(2): 172–177.Publisher Full Text\n\nJones CM: Age and growth. Fishery Science: The unique contributions of early life stages. Blackwell Publishing. USA. 2002.\n\nPrescott LM, Harley JP, Klein DA: Microbiology 5th Edition. Boston: McGraw-Hill, USA. 2002. Reference Source\n\nMulyono P, Wibisono W, et al.: Kinetika adsorpsi amonia dalam air dengan karbon aktif. Media Teknik. 2007; 2: 26–42. [In Indonesian]. Reference Source\n\nKutlu HR, Unsal I, Gorgulu I: Effect of providing directory wood (oak) charcoal to broiler chicks and laying hens. Anim Feed Sci Technol. 2001; 90: 213–226. Publisher Full Text\n\nLatania SF, Zainuddin Z, Winaruddin W, et al.: Gambaran histologi esofagus, lambung dan usus ikan banyak Kabupaten Lima Puluh Kota Provinsi Sumatera Barat. Jurnal Ilmiah Mahasiswa Veteriner. 2018; 2(1): 124–129. [In Indonesian]. Reference Source\n\nKuperman BI, Kuz’mina VV: The ultrastructure of the intestinal epithelium in fishes with different types of feeding. J Fish Biol. 1994; 44(2): 181–193. Publisher Full Text\n\nMekbungkwan A, Yamakuchi K, Sakaida T: Intestinal villus histological alterations in piglets fed dietary charcoal powder including wood vinegar compound liquid. Anat Histol Embryol. 2004; 33(1): 11–16. PubMed Abstract \| Publisher Full Text\n\nBatubara AS: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis. Figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12859973.v1\n\nBatubara AS: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis. Figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12860033.v1\n\nBatubara AS: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis. Figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12860054.v1\n\nBatubara AS: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis. Figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12901784.v1\n\nBatubara A: Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestines from giant trevally Caranx ignobilis. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13094930.v2" }	[ { "id": "77124", "date": "25 Jan 2021", "name": "Karun Thongprajukaew", "expertise": [ "Reviewer Expertise Aquatic animal nutrition", "digestive enzymes in aquatic animals", "aquafeed chemistry" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBased on my opinion, the manuscript entitled “Supplementation of rice husk activated charcoal in feed and its effects on growth and histology of the stomach and intestine from giant trevally, Caranx ignobilis” provides basic information for feed development and aquaculture of giant trevally. However, the manuscript writing and the presentation of data are not enough to index in its current form. I suggest the authors to use the broken-line analysis for predicting the optimal level of rice husk activated charcoal. More details and considering comments of this manuscript are listed below.\nGeneral comments:\nPlease provide common name of fish species, followed by its scientific name in parenthesis. The scientific name of species must be used the full name in the first mention and then use the abbreviations of generic name in the other places.\n\nThere are several errors relating to the use of capital/lowercase letters, italics, units, symbol, brackets and scientific names. Please carefully check the consistency of writing throughout the manuscript.\n\nSome sections contained errors from the use of abbreviations. If all parameters were already abbreviated, so that the subsequent part must be explained using abbreviations. Please carefully check throughout the manuscript.\n\nPlease replace “%” by “g/kg” for feed ingredients.\nSpecific comments:\nIntroduction: lack of hypothesis and objective.\n\nMethodology section should be re-categorized as time and site, charcoal preparation and activation, feed preparation, feeding trial (grouped experimental design, experimental fish and research parameters together), histological investigation and data analysis. In addition, it is suitable to replace the treatment titles from “A-F” to “0-3%”.\n\nPlease replace fish flour and shrimp flour by “fish meal” and “shrimp meal”, respectively.\n\nMethods to collect uneaten feed and water management (as well as water quality) should be given.\n\nHow to prepare the fish before sampling? Were they starved?\n\nPlease provide ID or reference number for animal ethics.\n\nFor parameters indicating growth and feed utilization, it is necessary to provide the references for each equation; these equations are fact and all literatures are not original.\n\nTable 1: 1) delete the column “protein content ingredients (%)”; 2) provide detail for the premix composition; and 3) provide proximate chemical composition of basal diet. In addition, this table must be re-written since only one feed formulation was used for all six experimental feeds. The authors should explain the formulation of “basal feed” and then included with varying levels of rice husk activated charcoal (0-3%).\n\nFor data analysis: Did you test the normality and homogeneity of each of your datasets? Were the data transformed before conducting the statistical analysis? In addition, I suggest the authors to use the broken-line analysis for predicting the optimal level of rice husk activated charcoal.\n\nTables 2 and 3 should be grouped as only one table, as well as for Tables 4 and 5.\n\nThe results section should be re-written and shortened. It is not necessary to add any value in text again since the readers can access all the values directly from tables or figures. Moreover, some result explanations were not supported by the data in the tables; please carefully check.\n\nThe references were not well checked and contain many errors, in both content and format. Please carefully check the references 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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1274
https://f1000research.com/articles/9-1042/v1	25 Aug 20	{ "type": "Research Article", "title": "Optimising retention success: a research team’s experience of following-up participants recruited to a pilot trial through community pharmacies in England", "authors": [ "Michelle Watson", "Anne van Dongen", "Catherine Hewitt", "Laura Mandefield", "Duncan Stewart", "Judith Watson", "Jim McCambridge", "Anne van Dongen", "Catherine Hewitt", "Laura Mandefield", "Duncan Stewart", "Judith Watson", "Jim McCambridge" ], "abstract": "Background: Community pharmacies support a range of patients and medical conditions, and form an important part of comprehensive, holistic healthcare services. The role of a community pharmacist has changed significantly over recent years, developing to include research activities. The CHAMP-1 (Community pharmacy: Highlighting Alcohol use in Medication aPpointments) pilot trial aimed to explore an intervention discussing alcohol during medication consultations. It presented various challenges regarding patient retention, and various actions were taken to address these, which are discussed in this manuscript. Methods: Community pharmacists recruited patients aged 18 and over, attending a Medicine Use Review (MUR) or New Medicine Service (NMS) consultation, and drinking alcohol at least twice per week. Pharmacies were randomised to conduct their consultations as usual (control), or to incorporate the Medicines and Alcohol Consultation (MAC) intervention. All participants were followed-up by a researcher after two months to complete data collection via telephone or post. Results: Forty-seven of 51 participants (92%) completed the two month follow-up. Thirty-eight (81%) responses were provided by telephone and nine (19%) by post. Of the 38 follow-up calls completed by telephone, 17 (45%) participants were reached at first attempt; 16 (42%) at second attempt; and five (13%) at the third attempt. Conclusions: The results suggest that patients recruited to a trial by community pharmacists are willing to take part in data collection activities, and follow-up can be successfully conducted by researchers. The techniques employed to encourage high levels of retention should be investigated further in a larger study.", "keywords": [ "Pharmacy services and practice", "Trials/Randomised controlled trials", "Health services research" ], "content": "Introduction\n\nCommunity pharmacies are a dynamic environment with professionals who are keen to provide care and support for a wide range of healthcare service users. There are around 11,600 community pharmacies in England, and 89% of the population are able to reach such facilities within a 20-minute walk1, allowing pharmacies to be at the core of communities and patient care. Over time, the role of a community pharmacist has expanded beyond the traditional dispensing duties2, including taking on wider public health roles and research activities. Research in this setting is, however, not without its challenges, with time constraints and remuneration3 having been reported as difficulties previously. More widely, difficulties in retaining research participants recruited to trials is a common issue and represents a significant risk to the statistical power and analysis of trial results4.\n\nThe CHAMP-1 (Community pharmacy: Highlighting Alcohol use in Medication aPpointments) pilot cluster trial is part of a programme of work which aims to collaborate with the pharmacy profession and patients, to produce an intervention discussing alcohol within routine medication consultations. The design of the trial was informed by pre-trial studies conducted by the CHAMP-1 research team, including observational and interview work with patients and pharmacists5,6. Outcome data collection in the trial provided a challenge as participants were recruited by their community pharmacist but followed-up by a trained researcher who the participant had no prior contact with. Full results of the pilot trial will be reported elsewhere. This paper focuses specifically on our experiences of contacting participants and the techniques used in an attempt to maximise our follow-up rate.\n\n\nMethods\n\nEthical approval for the CHAMP-1 pilot trial was provided by South West - Frenchay Research Ethics Committee (19/SW/0082). The trial was registered with the ISRCTN registry (ISRCTN57447996) on 17th June 2019. All participants provided written informed consent to participate in the trial as described in the Participant Information Sheet7.\n\nAt least four clusters per arm are recommended for cluster pilot randomised, controlled trials8. Assuming an average of eight participants per pharmacy are recruited, we planned to recruit 80 participants from 10 successfully recruiting pharmacies (equivalent to 70 participants in an individually randomised trial, assuming an intracluster correlation coefficient (ICC) 0.02). A trial of this size will allow a completion rate of 80% to be estimated within a 95% confidence interval of ±9%9. If we identify 160 eligible subjects, then we will be able to estimate a participation rate of 50% to within a 95% confidence interval of ±8%.\n\nRandomisation for the pilot trial was undertaken by an independent York Trials Unit statistician using minimisation. Minimisation was undertaken in minimPy using naïve minimisation with base probability 1.0 (i.e. deterministic minimisation) using marginal balance as the distance measure and with minimisation factors having a weighting of 1. Randomisation was at the level of the community pharmacy (with one practitioner per pharmacy). A separate randomisation sequence investigating the methodological feasibility of sending text messages to participants and their effect on retention was generated using block randomisation stratified by pharmacy.\n\nAnalyses for the pilot trial were conducted in R (R Development Core Team and R Core Team, 2011) following the principles of intention-to-treat with participant outcomes analysed according to their original, randomised group, where data are available, irrespective of deviations based on non-compliance. Analyses regarding the text message aspect of the trial were conducted in Microsoft Excel (2016).\n\nTwenty-seven community pharmacies in Yorkshire, England expressed an interest to be involved in the pilot trial, of which four were excluded (two had previous CHAMP-1 involvement and two did not respond) and 23 were assessed for eligibility. Of these, two were found to be ineligible and 11 were excluded for varying reasons. Ten pharmacies were deemed to be eligible and were randomised to conduct their Medicine Use Review (MUR) or New Medicine Service (NMS) consultations as usual (control), or to incorporate the Medicines and Alcohol Consultation (MAC) intervention. Five pharmacies were randomised to the control group, and five were randomised to provide the intervention. The median cluster size was five in the control arm, and four in the intervention arm. One pharmacist from each of the pharmacies received training in the recruitment and study procedures for the trial; with those randomised to the intervention arm receiving additional training on the MAC. The MAC intervention is intended to help patients to think through whether drinking alcohol affects their medication use, the conditions for which they are prescribed, and their health more broadly; and to enable pharmacists to skilfully engage with these issues in a person-centred manner. The intervention involved pharmacists attending two practice development training days; using a MAC guide which summarised the structure of the intervention and core content within medication consultations; engaging with a range of learning support resources; receiving individually tailored weekly practice development support site visits or telephone calls by the MAC support team; and an invitation to engage in peer support.\n\nParticipant recruitment was conducted by community pharmacists, and patients were eligible for the trial if they were aged 18 and over, attending for a MUR or NMS consultation with their pharmacist, and drinking alcohol at least twice per week. Patients were excluded from the trial if they had received alcohol treatment in the past 12 months.\n\nAll participants were followed-up with a telephone call from a trained researcher two months after entering the trial and having their consultation with the pharmacist. During the telephone call, the trained researcher collected outcome data using a Case Report Form. The research team considered the potential challenges to successful follow-up based on pre-trial feasibility work, such as the two month time lapse between the consultation and follow-up call, and the willingness of participants to speak about their health and wellbeing to someone they did not know. To facilitate trial follow-up, the study team established various procedures to address these such as: asking participants for more than one telephone number if available (e.g. a landline and mobile telephone number); asking for the participant’s preferred days and times for contact; sending a text message to remind them that a researcher would be contacting them in the near future to conduct their follow-up telephone call; attempting to contact participants three times before sending the follow-up questionnaire7 (the same form as that completed by telephone) by post; calling from one single telephone number to enable participants to recognise the number if they were unable to be contacted at the first attempt; and leaving voicemail messages where possible requesting that participants return the call. Participants who completed follow-up were given a £10 ‘thank you’ gift card. This was explained in the patient information sheet7 provided during recruitment and mentioned early in the conversation during the follow-up telephone call.\n\n\nResults\n\nThe CHAMP-1 pilot trial recruited 51 participants from 10 pharmacies. Of these, 55% were men and 45% were women7. The mean age of those involved was 66.5 years. Forty-seven (92%) participants were successfully followed up at two months. Thirty-eight (81%) of the 47 responses were provided by telephone and nine (19%) participants completed the follow-up questionnaire after being sent it by post, having not responded to the telephone calls. Four participants did not respond to the telephone calls or return the follow-up questionnaire by post and therefore their outcome data was not collected.\n\nAll participants provided at least one telephone number and 34 (67%) participants provided a mobile telephone number, and therefore were possibly more likely to be contactable if they were not at the location of their landline telephone. Forty-eight participants (94%) also consented to receiving a text message that would remind them about the follow-up telephone call; however, of these, 15 (31%) did not provide a mobile number to enable this to occur. Due to technical difficulties, only seven of the text messages were sent as planned.\n\nOf the 38 follow-up calls completed by telephone, 17 (45%) participants were successfully reached at the first attempt of contact by the trained researcher; 16 (42%) at the second attempt; and five (13%) at the third attempt. If no contact had been made after the third attempt, the follow-up questionnaire, which was the same as that completed by telephone, was posted to the participant. Having made contact, nine of the participants requested that the follow-up call be arranged for a more convenient time and this was scheduled accordingly. Eight of these calls were completed successfully as arranged, with five participants requesting this at the first attempt at contact, and three asking during the second. The ninth participant arranged their follow-up call after one attempt at contact; however, they did not engage with the re-arranged or subsequent telephone calls, and their data collection was completed by post.\n\n\nDiscussion/conclusions\n\nThe trial involved participants with a range of ages and medical conditions and therefore is broadly representative of the type of patients that use community pharmacies. Whilst this was a small pilot cluster trial, it describes the initiatives used to encourage a successful follow-up rate in potentially challenging circumstances. The results suggest that patients recruited within community pharmacies are willing to complete further data collection activities which do not involve their pharmacy or pharmacist. Repeated efforts to make contact were required for over half of participants.\n\nIt is important to ensure that all necessary information is collected whilst completing recruitment procedures, as approximately a third of participants consented to receive a text message reminder about their follow-up telephone call, however did not provide a mobile number for this to be sent to.\n\nFuture research is needed with larger samples and longer follow-up periods to examine other potential mechanisms that contribute to successful follow-up of trial participants recruited in this clinical setting.\n\n\nData availability\n\nOpen Science Framework: CHAMP-1 Pilot Retention Data. https://doi.org/10.17605/OSF.IO/KCPHQ7.\n\nThis project contains the following underlying data:\n\n- CHAMP-1 Pilot Retention Data.csv\n\n- Participant demographic data.csv\n\nOpen Science Framework: CHAMP-1 Pilot Retention Data. https://doi.org/10.17605/OSF.IO/KCPHQ7.\n\nThis project contains the following extended data:\n\n- CHAMP1_FUpLong_v2 (49582 – Activated, VersiForm)_Reference.pdf (follow-up questionnaire)\n\n- CHAMP1_FUpShort_v2 (13283 – Activated, VersiForm)_Reference.pdf (follow-up questionnaire)\n\n- REFERENCE 2A CHAMP-1 Pilot Patient Consent Form Version 2.0 07.05.2019.pdf\n\n- REFERENCE 2A CHAMP-1 Pilot Patient Information Sheet Version 2.0 07.05.2019.pdf\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\nCommittee BMAGP and Committee PSN: The community pharmacy – a guide for general practitioners and practice staff. BMA and PSNC. 2019. Reference Source\n\nSabater-Galindo M, de Maya SR, Benrimoj SI, et al.: Patients' expectations of the role of the community pharmacist: Development and testing of a conceptual model. Res Social Adm Pharm. 2017; 13(2): 313–320. PubMed Abstract \| Publisher Full Text\n\nCrilly P, Patel N, Ogunrinde A, et al.: Community pharmacists’ involvement in research in the United Kingdom. Pharmacy (Basel). 2017; 5: 48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKearney A, Daykin A, Shaw ARG, et al.: Identifying research priorities for effective retention strategies in clinical trials. Trials. 2017; 18(1): 406. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadden M, Morris S, Atkin K, et al.: Patient perspectives on discussing alcohol as part of medicines review in community pharmacies. Res Soc Admin Pharm. 2020; 16(1): 96–101. PubMed Abstract \| Publisher Full Text\n\nMorris S, Madden M, Gough B, et al.: Missing in action: Insights from an exploratory ethnographic observation study of alcohol in everyday UK community pharmacy practice. Drug Alcohol Rev. 2019; 38(5): 561–568. PubMed Abstract \| Publisher Full Text\n\nWatson M: CHAMP-1 Pilot Retention Data. OSF. 2020. http://www.doi.org/10.17605/OSF.IO/KCPHQ\n\nHayes RJ, Moulton LH: Cluster randomised trials. CRC press, 2017. Publisher Full Text\n\nHertzog MA: Considerations in determining sample size for pilot studies. Res Nurs Health. 2008; 31(2): 180–191. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72701", "date": "22 Oct 2020", "name": "Christopher Partlett", "expertise": [ "Reviewer Expertise Medical Statistics & Trial Methodology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting case study exploring the feasibility of conducting follow-up for a pilot randomised trial within the community pharmacy setting. While I think this article is certainly worthy of indexing, I have some recommendations to improve the article.\nFirstly, and most importantly, I found that the methods section (of both the abstract and the main article) read similarly to the methods section of a main trial publication. Since the article is focussing on the experience of following-up participants, the methods section should focus on this aspect of the trial. For instance, the final paragraph of the methods section is most pertinent to this article. Other important aspects of trial design would be better placed in the background, while less relevant aspects of the trial design could be omitted completely (and perhaps replaced with a reference to the trial protocol). This change would significantly improve the overall readability of the article. I have listed some other minor comments below:\nThe authors state in the final paragraph of the introduction that loss to follow-up represents a significant risk to statistical power and analysis. I would add that it also presents a potential risk to the generalisability of the trial findings.\n\nA limitation of the study is a lack of geographical spread, which may hinder generalisability of the findings within this article. This should be noted in the discussion.\n\nIdeally, the reasons for exclusion and ineligibility of pharmacies should be listed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6337", "date": "15 Feb 2021", "name": "Michelle Watson", "role": "Author Response", "response": "The authors of the manuscript thank the peer reviewers for their comments and have made the following changes based on their suggestions: Abstract We have updated the abstract to reflect the changes made to the manuscript based on peer review comments. Introduction We have added that loss to follow-up presents a risk to generalisability and may introduce bias. We have added a statement advising that the pre-trial work guided development and implementation of the intervention, and did not investigate follow-up procedures; therefore standard procedures were followed. The paper references the pre-trial work publications should readers wish to look into this further. Methods We have removed the methodology for the pilot trial and included a reference to the publication describing this instead. We have added the reasons for excluding pharmacies. We have added further information about the randomisation methods. We have stated the measures which were included in the follow-up questionnaire. We added details about the follow-up questionnaire and measures used. Results We have presented data regarding telephone or postal completion of the follow-up questionnaire. Discussion We have again provided a reference to the pilot trial publication as this covers many of the points raised during review. We have reflected further on the factors that may have affected retention and the limitations of our work, including geographical spread, amongst others. We have discussed the data regarding telephone or postal completion of the follow-up questionnaire. We have discussed potential follow-up and retention strategies that future research could focus on." } ] }, { "id": "74512", "date": "18 Dec 2020", "name": "Roberta R. Littleford", "expertise": [ "Reviewer Expertise Clinical Trial 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\nThe article’s aim is to present the research team’s experience of following-up participants recruited into a pilot trial assessing an intervention discussing alcohol consumption habits during routine medication consultations within a community pharmacy environment. There are several issues the authors should consider addressing.\nThe manuscript’s narrative focuses on the overarching main study, thereby omitting details required to describe the presented articles aims. The premise of optimising retention strategies and therefore retention success are not systematically studied or quantified. The authors present the important role that community pharmacists had in the main study's intervention, but pharmacists are out with the retention and follow-up scope and the authors do not postulate if intervention delivery had an impact on retention. Standard follow-up strategies were employed. The abstract will require to be amended if the recommendations provided below are addressed.\nIntroduction: The introduction mainly addresses the challenges related to pharmacists being researchers, but their role is limited within the scope of the retention and follow-up strategies employed, which were conducted by University researchers. This section would benefit from a description of the literature that was considered when designing the follow-up strategies, and report upon the outcomes from the pre-trial work that were implemented into the design.\n\nMethods: This section concentrates on the overarching main study, providing significant detail related to sample size, randomisation, and analysis (which can be addressed separately in the main study manuscript), thereby limiting information required to address the retention and follow-up methodology. The primary outcome of the main study could be presented to provide some context. This section requires to be rewritten to incorporate a robust methods section of the retention and follow-up strategies employed. Including a clarification of the randomisation methods employed for SMS, consent and an outline of the actual method(s) used - automated manual by pharmacists or researcher. Further details here would assist in describing the limitations in the discussion. The methods section does not currently describe the validation status of the questionnaires used, including self/researcher completion and how the visualisation of the units of alcohol and VAS were addressed during telephone follow-up calls versus in-person completion (the results/discussion section should address any noted differences). The tense within the section varies throughout; this section should use past tense.\nDiscussion The optimisation and limitations of the project should be addressed in more detail. It may be prudent for the authors (with access to the main study results) to consider and discuss elements that may have impacted retention and follow-up; intervention delivery, impact of tokens, number of medicines, co-morbidities, usual pharmacy/new pharmacy, location within-GP practice or independent. The authors should consider expanding on possible future strategies for trials, for example including pharmacists as part of the follow-up and retention success strategies, self-completion contact details, web/app questionnaire completion. The rationale of failing to recruit the required sample size (63%) for the main study should be addressed, and also providing the reasons for pharmacy ineligibility and criteria for exclusion, leading to 13/23 (56%) of pharmacies being excluded. These factors may impact future success strategies. There appears to have been significant training employed with weekly follow-up at pharmacies by the research team. However, the manuscript does not report if data monitoring was conducted, which may have identified the 33.3% missing mobile numbers. The authors should consider adding the project timelines to give context to trial efficiency.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6338", "date": "15 Feb 2021", "name": "Michelle Watson", "role": "Author Response", "response": "The authors of the manuscript thank the peer reviewers for their comments and have made the following changes based on their suggestions: Abstract We have updated the abstract to reflect the changes made to the manuscript based on peer review comments. Introduction We have added that loss to follow-up presents a risk to generalisability and may introduce bias. We have added a statement advising that the pre-trial work guided development and implementation of the intervention, and did not investigate follow-up procedures; therefore standard procedures were followed. The paper references the pre-trial work publications should readers wish to look into this further. Methods We have removed the methodology for the pilot trial and included a reference to the publication describing this instead. We have added the reasons for excluding pharmacies. We have added further information about the randomisation methods. We have stated the measures which were included in the follow-up questionnaire. We added details about the follow-up questionnaire and measures used. Results We have presented data regarding telephone or postal completion of the follow-up questionnaire. Discussion We have again provided a reference to the pilot trial publication as this covers many of the points raised during review. We have reflected further on the factors that may have affected retention and the limitations of our work, including geographical spread, amongst others. We have discussed the data regarding telephone or postal completion of the follow-up questionnaire. We have discussed potential follow-up and retention strategies that future research could focus on." } ] } ]	1	https://f1000research.com/articles/9-1042
https://f1000research.com/articles/9-1248/v1	15 Oct 20	{ "type": "Software Tool Article", "title": "Fiji plugins for qualitative image annotations: routine analysis and application to image classification", "authors": [ "Laurent S. V. Thomas", "Franz Schaefer", "Jochen Gehrig", "Franz Schaefer" ], "abstract": "Quantitative measurements and qualitative description of scientific images are both important to describe the complexity of digital image data. While various software solutions for quantitative measurements in images exist, there is a lack of simple tools for the qualitative description of images in common user-oriented image analysis software. To address this issue, we developed a set of Fiji plugins that facilitate the systematic manual annotation of images or image-regions. From a list of user-defined keywords, these plugins generate an easy-to-use graphical interface with buttons or checkboxes for the assignment of single or multiple pre-defined categories to full images or individual regions of interest. In addition to qualitative annotations, any quantitative measurement from the standard Fiji options can also be automatically reported. Besides the interactive user interface, keyboard shortcuts are available to speed-up the annotation process for larger datasets. The annotations are reported in a Fiji result table that can be exported as a pre-formatted csv file, for further analysis with common spreadsheet software or custom automated pipelines. To facilitate and spread the usage of analysis tools, we provide examples of such pipelines, including a complete workflow for training and application of a deep learning model for image classification in KNIME. Ultimately, the plugins enable standardized routine sample evaluation, classification, or ground-truth category annotation of any digital image data compatible with Fiji.", "keywords": [ "ImageJ", "Fiji", "KNIME", "image annotation", "image classification", "ground-truth labelling", "qualitative analysis", "bioimage analysis" ], "content": "Introduction\n\nA common requirement of most imaging projects is to qualitatively describe images, either by assigning them to defined categories or by selecting a set of descriptive keywords. This routine task is shared by various scientific fields, for instance in biomedical research for the categorization of samples, in clinical imaging for image-based diagnostics, or in manufacturing for the description of object-properties.\n\nQualitative descriptors, or keywords, can correspond to the presence or discrete count of features, the evaluation of quality criteria, or the assignment of images to specific categories. While automated methods for such qualitative description may exist, they usually require substantial effort for their implementation and validation. Therefore, for simple tasks like routine image data analysis and inspection, the qualitative description is usually performed manually.\n\nFor small datasets of a few dozen images, manual description of images can be performed by reporting the image and qualitative descriptors in a simple spreadsheet. However, for larger datasets or large number of descriptors, this becomes quickly overwhelming and error prone as one needs to inspect a multitude of images while appending information to increasingly complex tables. Although solutions for qualitative annotations of large datasets exist (Westhoff et al., 2020), there is surprisingly no widespread solution available in common user-oriented image analysis software, for the assignment of multiple descriptive keywords to images. Therefore, we developed a set of plugins for Fiji (Schindelin et al., 2012), to facilitate and standardize routine qualitative image annotations. We also illustrate possible applications of the resulting standardized qualitative description for the exploration of data-distribution, or the training of supervised image classification algorithms.\n\n\nMethods\n\nWe developed a set of Fiji plugins for the assignment of multiple pre-defined keywords to images. This set of keywords is entered by the user at the beginning of the annotation session. The image annotation is then carried out using an intuitive graphical user interface consisting of either buttons, checkboxes, or dropdown menus, which are automatically generated from the user-defined keywords (Figure 1–Figure 3). In addition to the pre-defined set of keywords, arbitrary image-specific comments can also be entered via a text input field. Furthermore, if run Measure is selected in the graphical interface, quantitative measurements as defined in Fiji’s menu Analyze>Set Measurements are reported in addition to the selected keywords. By default, the annotations and measurements are assigned to the entire image but can also describe image-regions outlined by regions of interest (ROI). The latter is simply achieved by drawing a new ROI on the image or selecting existing ROIs in the ROI Manager before assigning the keywords (Figure 3). Newly drawn ROIs are automatically added to the ROI Manager upon annotation with the plugins.\n\n(A) Example of multi-dimensional image stack used to annotate mitotic stages in time-lapse data (source: ImageJ example image “Mitosis” – image credit NIH). (B) Graphical interface of the single class (buttons) plugin configured for annotation of 4 mitotic stages. (C) Results table with annotated categories (column category) generated by the plugin after selecting the single category column option in the plugin configuration window (not shown). (D) Alternative results table output using 1-hot encoding after selecting the option 1 column per category. The resulting 1-hot encoding of categories can be used for the training of classification algorithms.\n\n(A) Example images of transgenic zebrafish larvae of the Tg(wt1b:egfp) transgenic line after injection with control morpholino (upper panel) or with ift172 morpholino (lower panel) inducing pronephric cysts. In this illustration, the plugin is used to score overall morphology and cyst formation. It could also be used to mark erroneous images (such as out-of-focus or empty wells). Images are from (Pandey et al., 2019). (B) Graphical interface of the checkbox annotation plugin configured with 2 checkboxes for overall morphology, 2 checkboxes for presence of pronephric cysts, and checkboxes to report out-of-focus and empty wells. Contrary to the single class (button) plugin, multiple categories can be assigned to a given image. (C) Resulting multi-category classification table with binary encoding of the annotations (True/False).\n\n(A) ImageJ’s sample image “embryos” after conversion to grayscale using the command Image > Type > 32-bit (image credit: NIH). The embryos outlined with yellow regions of interest were annotated using the “multi-class (dropdown)” plugin. The insets at the top shows the annotation of overlapping ROIs, here corresponding to embryos with phenotype granular texture, dark pigmentation and elliptic shape. The inset at the bottom shows other embryos with different phenotypes (10: smooth/clear/circular, 12: granular/clear/elliptic, 14: smooth/dark/circular). (B) Graphical interface of the multi-class (dropdown) plugin. Three exemplary features are scored for each embryo: texture (granular, smooth), shape (circle, ellipse) and pigmentation (dark, clear). Quantitative measurements as selected in the Analyze > set Measurements menu (here Mean, Min and Max grey level) are also reported for each embryo, when the run Measure option is ticked. (C) ROI Manager with ROIs corresponding to annotated regions. (D) Resulting classification table with the selected features, qualitative measurement and associated ROI identifier for the outlined embryos.\n\nThe selected keywords, comments, measurements, and the image filename and directory are reported in a Fiji result table window with one row per annotation event (Figure 1C, D). An annotation event is triggered when a button is clicked, or a keyboard shortcut is pressed. With ROIs, the identifiers of the ROIs are also reported in the table, and the descriptors are saved as properties of the ROI objects. The information can then be retrieved from the ROIs using the Fiji scripting functions. The result table is updated row by row, as the user progresses with the annotations and can be saved as a csv file at any time. The csv file can be edited in any spreadsheet software or text editor, for instance to update the image directory column when images have been transferred to a different location or workstation.\n\nThree plugins are provided to accommodate for different annotation modalities. The single-class (buttons) plugin (Figure 1) allows the assignment of a single keyword per image by clicking the corresponding button. With this plugin, the user can decide if the result table should contain a single category column containing the clicked category keyword for each image (Figure 1C), or one column per category with a binary code (0/1) depicting the assignment (Figure 1D). The latter option is particularly suitable for the training of supervised classification algorithms, which typically expect for their training an array of probabilities with 1 for the actual image-category and 0 for all other categories (also called 1-hot encoding, see (Müller & Guido, 2016)).\n\nThe multi-class (checkboxes) plugin (Figure 2) allows multiple keywords per image, which are selected via associated checkboxes (Figure 2B). This yields a result table with one column per keyword, and a 0/1 code if the keywords apply or not (Figure 2C). In this case, the table structure is similar to Figure 1D except that multiple keywords might be selected for a given image (i.e. multiple 1 for a given table row, as in row 1).\n\nThe multi-class (dropdown) plugin (Figure 3) allows to choose keywords from several lists of choices using dropdown menus (Figure 3B). The labels and choices for the dropdown menus are defined by the user in a simple csv file (Extended data, Supplementary Figure 1) (Thomas, 2020). This is convenient if multiple image features should be reported in separate columns (content, quality, etc.) with several options for each feature.\n\nThe plugins run on any system capable of executing Fiji. Executing one of the plugins will first display a configuration window requesting the user-defined keywords. Upon validation of the configuration, the actual annotation interface as in Figure 1–Figure 3 is displayed and ready to use for annotation. For the single-class (buttons) plugin, annotations can be recorded by clicking the corresponding category button, or by pressing one of the F1-F12 keyboard shortcuts. The shortcuts are automatically mapped to the categories in the order of their respective buttons, e.g. pressing F1 is equivalent to clicking the leftmost button. For the multi-class plugins, the annotations can be recorded by clicking the Add button or pressing one of the + keys of the keyboard.\n\nFor every plugin, an annotation event updates the results table as described in the implementation section, stores any newly drawn ROI into Fiji’s ROI Manager, and if the corresponding option is selected in the graphical user interface, the next image slice is displayed when a stack is annotated. The annotation table can be saved to a csv file at any time like a regular Fiji table. Moreover, the annotations can be appended to an existing result table in Fiji, provided the associated window is entitled “Annotations” or “Annotations.csv”.\n\n\nUse cases\n\nThe described plugins allow the rapid and systematic description of images or image regions with custom keywords. Additionally, by activating the measurement option, the qualitative description can be complemented by any of ImageJ’s quantitative measurements. The annotation tools can be used for routine image evaluation e.g. for the assignment of predefined categories, to identify outliers or low-quality images, or to assess the presence of a particular object or structure. Examples annotations are illustrated in Figure 1 (single-cell mitotic stage), Figure 2 (pronephric morphological alterations in transgenic zebrafish larvae (Pandey et al., 2019) and Figure 3 (phenotypic description of multi-cellular embryos). Images and annotations are available as Underlying data (Pandey et al., 2020).\n\nFor the annotation of ROIs, the presented plugins can be used in combination with our previously published ROI 1-click tools (Thomas & Gehrig, 2020), which facilitate the creation of ROIs of predefined shapes, and the automated execution of custom commands for these ROIs. The generated ROIs can then be described with qualitative features using the hereby presented plugins, either for one ROI at a time, or by simultaneously selecting multiple ROIs. Besides facilitating qualitative annotations, the plugins have the advantage to generate tables with standardized structures that can potentially facilitate the visualization and analysis of the annotations by automated workflows.\n\nTo illustrate and expand on the potential of such annotations, we provide a set of generic KNIME workflows which directly operate on tables generated by one of the presented plugins. The workflows do not require advance knowledge of KNIME and can be readily used without major adaptation. KNIME (Berthold et al., 2009) is particularly suitable for the analysis of data involving both images and tabular data, in an intuitive graphical environment. Especially it allows users to design complex analysis workflows with no or little coding skills. The example workflows demonstrate classical scenarios for the exploitation of qualitative descriptors: (i) the visualization of data-distribution using interactive sunburst chart (Extended data, Supplementary Figure 2) (Thomas, 2020), and (ii) the training of a deep learning model for image classification (Figure 4). By providing those examples, we also wish to facilitate and spread those advanced data-processing tools, by drastically reducing the need for custom development. We also provide detailed documentation about the workflows and other software dependencies on the GitHub repository (https://github.com/LauLauThom/Fiji-QualiAnnotations).\n\n(A) Example images representing the two classes the deep-learning model was trained to recognize. The images show fluorescently-labelled pronephroi in larvae of the Tg(wt1b:EGFP) transgenic zebrafish line showing 2 common morphologies (Left: Normal, Right: Cystic), observed in a previously published chemical screen (Pandey et al., 2019). (B) Screenshot of the KNIME workflow for the training of the model. A subset of the annotated data (training and validation set) is used for the model training (upper part of the workflow outlined in yellow). The remainder of the annotated data (test set) is used for the evaluation of the model performance after training (lower part of the workflow, outlined in blue). The parameters for the training can be adjusted via the settings panel of the Keras network learner node (outlined with dashed black line - See Extended data, Supplementary Figure 3). (C) Confusion matrix as shown by the Scorer node (rightmost side of the workflow, highlighted with red dashed line) depicting the accuracy of the classification by the trained model for the test set. After training for only 5 epochs with 116 training images as explained in the main text, the model achieved already a very good classification accuracy with only 1 image misclassified, out of 145 test images.\n\nSunburst charts offer an intuitive option for the visualization of the distribution of multiple qualitative features in a dataset. In these charts, the distributions of distinct features are represented as concentric “pies”, which help to identify relations between features. Supplementary Figure 2B (see extend data, Thomas, 2020) shows an example of such a sunburst chart generated with the provided workflow (Sunburst-chart-workflow) for the visualization of three qualitative features (texture, shape, and pigmentation) describing samples annotated in Figure 3. To generate such visualization, the workflow takes as input a table as in Figure 3 and counts the occurrence for each unique combination of qualitative features appearing in the table. The resulting occurrence-table (Extended data, Supplementary Figure 2A) (Thomas, 2020) is then represented as a sunburst chart. The order of the concentric circles is defined by the order of the columns in the annotation table, which can be configured in the workflow. In KNIME, the visualization is rendered as an interactive figure, allowing high-lighting specific sections of the plot, or reducing the number of displayed levels to inspect the distribution of a sub-population (e.g. samples with a smooth texture only). Finally, the provided workflow can be adapted for custom analysis and visualization, using any functionality available in KNIME.\n\nThe plugins can also be used to generate the ground-truth annotation for the training of supervised image classification algorithms, such as deep learning models. For this purpose, we adapted an existing KNIME example workflow designed for the training of a convolutional neural network (CNN) by transfer learning, to directly use as input the annotation table provided by the single-class (buttons) plugin (Figure 4). The workflow generates a hybrid model architecture made of a pre-trained VGG16 base (Simonyan & Zisserman, 2015) used for feature-extraction from the images, completed by newly initialized fully-connected layers for the classification with custom classes. The workflow reads the image-locations and image-categories from the annotation table generated by the plugin, and performs classical data pre-processing for the training of CNNs: splitting the annotated dataset between training, validation and test sets, loading and pre-processing of the images and finally training and evaluation of the model. The trained model can be exported as a h5 file, suitable for the prediction of classes for new images. A second workflow is provided for this purpose. As an example of application, the workflow was used to train a model for the identification of two distinct morphological phenotypes of fluorescently-labelled pronephroi (normal and cystic) in transgenic zebrafish larvae as observed in a drug discovery screen (Pandey et al., 2019) (Figure 2A and Figure 4A). For this particular dataset and classification problem, the trained model readily achieved high classification accuracy (>99%, see confusion matrix Figure 4C) on a test set of 145 images (69 normal, 76 cystic) after training for only 5 epochs with a distinct training set of 116 images (57 normal, 59 cystic).\n\nUsually training a custom deep learning model requires substantial programming skills, and a good comprehension of deep learning architecture and parameters. Recent efforts have aimed at facilitating the access of deep learning solutions to non-expert users (Gómez-de-Mariscal et al., 2019; Kräter et al., 2020; von Chamier et al., 2020); however, they usually do not cover the full pipeline from ground-truth annotation to model training, evaluation and application. Here we propose a complete pipeline for image classification that does not require major coding skills and is pre-configured with widely applicable default parameters. The workflow is expected to yield a model with satisfactory classification performance with the default parameters, such that non-expert users can directly get familiar with the method. For better performance, parameters for data pre-processing and model training (e.g. number of epochs, batch-size, learning rate) can be adapted in the configuration window of the Keras network learner node (Extended data, Supplementary Figure 3A). Importantly, using transfer-learning and only training the newly initialized fully-connected layers, the size of the training set and the time to train the network until it reaches a decent accuracy can be dramatically reduced compared to a training from scratch. For the classification of zebrafish pronephroi described above, the training by transfer learning took about 1 minute on a workstation with a Nvidia GTX760 GPU with 2 GB dedicated memory. In contrast, reaching a similar accuracy with the same model trained from scratch would likely require a few hours even on more modern and high-end hardware. Regarding hardware requirement, a CUDA-capable GPU is advised to achieve rapid model training, but the training can also be carried out with CPU only, on any machine supporting KNIME and python. Readers can refer to the GitHub repository (see section Software availability) for detailed descriptions of installation and requirements.\n\n\nConclusion\n\nHere, we propose a set of plugins for the qualitative annotations of images or image regions, designed for the popular image analysis software Fiji. The annotations comprise user-defined keywords, as well as optional quantitative measurements as available in Fiji. The keywords can describe categorical classification, the evaluation of quality metrics or the presence of particular objects or structures. The plugins are easy to install and to use via an intuitive graphical user interface. In particular, the tools facilitate tedious qualitative annotation tasks, especially for large-datasets, or for the evaluation of multiple features. The annotations are recorded as standardized result tables, to facilitate automated analysis by generic workflows. To this extent, example workflows for data-visualization and supervised data classification are provided, which can be directly executed with the resulting annotation table without further customization effort. Finally, video tutorials about the plugins and analysis workflows are available on YouTube.\n\n\nData availability\n\nZenodo: Fluorescently-labelled zebrafish pronephroi + ground truth classes (normal/cystic) + trained CNN model. https://doi.org/10.5281/zenodo.3997728 (Pandey et al., 2020).\n\nThis project contains the following underlying data:\n\nAnnotations-multiColumn.csv. (Ground-truth category annotations.)\n\nAnnotations-singleColumn.csv. (Ground-truth category annotations.)\n\nimages.zip. (Images of fluorescently labelled pronephroi in transgenic Tg(wt1b:EGFP) zebrafish larvae used for the training and validation of the deep learning model for classification.)\n\ntrainedModel.zip (Pretrained model.)\n\nZenodo: Qualitative image annotation plugins for Fiji - https://doi.org/10.5281/zenodo.4064118 (Thomas, 2020).\n\nThis project contains the following extended data:\n\nSuppl.Fig.1: Detail of the input for the multi-class (dropdown) plugin.\n\nSuppl.Fig.2: Supplementary Figure 2: Visualizing data-distribution using sunburst charts in KNIME.\n\nSupplementary Figure 3: Detail of the Keras network learner node.\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 codes, documentation and example workflows are available at: https://github.com/LauLauThom/Fiji-QualiAnnotations.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.4064118 (Thomas, 2020).\n\nLicense: GNU General Public License v3.\n\nThe plugins can be installed in Fiji by simply activating the Qualitative Annotations update site. Then the plugins are listed under the menu Plugins > Qualitative Annotations.\n\nA pre-configured Fiji installation bundle for windows is also archived in the release section of the repository.\n\nThe following KNIME workflows and associated documentation README files are available in the subdirectory KNIMEworkflows under a Creative Commons Attribution 4.0 International License (CC-BY):\n\n- View-Images-And-Annotations workflow\n\n- Sunburst-chart-workflow (with csv of annotations for multi-cellular embryos)\n\n- Deep-Learning – binary classifier – training workflow\n\n- Deep-Learning – binary classifier – prediction workflow\n\n- Deep-Learning – multi-class classifier – training workflow\n\n- Deep-Learning – multi-class classifier – prediction workflow", "appendix": "Acknowledgements\n\nWe thank Gunjan Pandey (ACQUIFER and Children’s Hospital) for generating images and Jens Westhoff (Children’s Hospital, Heidelberg) for general support.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nBerthold MR, Cebron N, Dill F, et al.: KNIME - the Konstanz information miner: version 2.0 and beyond. ACM SIGKDD Explor Newsl. 2009; 11(1): 26–31. Publisher Full Text\n\nGómez-de-Mariscal E, García-López-de-Haro C, Donati L, et al.: DeepImageJ: A user-friendly plugin to run deep learning models in ImageJ (preprint). Bioengineering. 2019. Publisher Full Text\n\nKräter M, Abuhattum S, Soteriou D, et al.: AIDeveloper: deep learning image classification in life science and beyond (preprint). Bioinformatics. 2020. Publisher Full Text\n\nMüller AC, Guido S: Introduction to machine learning with Python: a guide for data scientists. First edition. ed. O’Reilly Media, Inc, Sebastopol, CA. 2016.\n\nPandey G, Gehrig J, Thomas L: Fluorescently-labelled zebrafish pronephroi + ground truth classes (normal/cystic) + trained CNN model (Version 1.0) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3997728\n\nPandey G, Westhoff J, Schaefer F, et al.: A Smart Imaging Workflow for Organ-Specific Screening in a Cystic Kidney Zebrafish Disease Model. Int J Mol Sci. 2019; 20(6): 1290. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimonyan K, Zisserman A: Very Deep Convolutional Networks for Large-Scale Image Recognition. ArXiv14091556 Cs. 2015. Reference Source\n\nThomas L: Qualitative image annotation plugins for Fiji (Version 1.0.2bis). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4064118\n\nThomas LS, Gehrig J: ImageJ/Fiji ROI 1-click tools for rapid manual image annotations and measurements. 2020. Publisher Full Text\n\nvon Chamier L, Jukkala J, Spahn C, et al.: ZeroCostDL4Mic: an open platform to simplify access and use of Deep-Learning in Microscopy (preprint). Bioinformatics. 2020. Publisher Full Text\n\nWesthoff JH, Steenbergen PJ, Thomas LSV, et al.: In vivo High-Content Screening in Zebrafish for Developmental Nephrotoxicity of Approved Drugs. Front Cell Dev Biol. 2020; 8: 583. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "73675", "date": "13 Nov 2020", "name": "Jan Eglinger", "expertise": [ "Reviewer Expertise Image Processing", "Software Development", "Microscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce a new set of Fiji plugins that allows user-friendly annotation of images in two ways: 1) tagging entire images with one or several keywords/classes, and 2) tagging selected regions within an image. Thanks to the integration with the Fiji/ImageJ framework (installation via an ImageJ update site, graphical user interface using the built-in functionality provided by Fiji), the plugins are indeed simple to install and use, and simplify the process to get tabular data from a manual annotation task.\nIn the introduction, the authors state that there is \"no widespread solution available [...] for the assignment of multiple descriptive keywords to images\". In my opinion, this statement demonstrates a lack of research, as a simple online search for \"image annotation tool\" reveals many solutions used in the field of Computer Vision to annotate ground truth for machine learning datasets (both for image classification and for bounding box annotations). While some of these tools might be more commonly used than others, I believe they should have at least included a few of them in a thorough comparison, such as e.g. LabelMe [1], MakeSense.AI [2], LabelImg [3], VGG Image Annotator [4], or Scalabel [5]. The only other available tool they cite is ACQUIFER Manual Annotation Tool, a software created by (partially) the same authors, available on request only.\nNevertheless, the set of plugins introduced here certainly fills a gap for users of Fiji/ImageJ in particular, which means a lot of scientists using it to answer biological questions with (microscopy) image data, who will welcome a way to generate annotation data from within a framework that they already use and know.\nSpecifically for biological applications however, I see a potential limitation of the use of this plugin. With modern microscopy datasets often being multi-dimensional, region annotations (bounding-box or pixel-wise) would benefit from being 3-dimensional to reflect a volume of interest. By being tied very much to the legacy user interface of Fiji (built-in ROI support is 2D only), the Qualitative Annotations plugin will be of very limited use for true 3D applications, since any annotation can only be done slice-wise, in the current state. Other plugins, such as the 3D ImageJ Suite, offer support for 3-dimensional object annotations, but lack an easy way to add keywords/class annotations.\nAnother current limitation of the Qualitative Annotations plugin is the absence of ways to review and/or refine an annotation after it has been added to the results table.\nLastly, since the authors include KNIME workflows in their article to illustrate further processing of the annotation data (e.g. for training of machine learning models), I am surprised that they didn't also explore ways to do the manual annotation within a KNIME workflow, in order to include the annotation process in their \"complete pipeline for image classification\".\n\nThe KNIME Image Processing [6] extension offers the 'Interactive Annotator' and 'Interactive Labeling Editor' nodes that can be used to annotate image regions. For interactive sequential classification of images, the 'Active Learning Loop' nodes offer an excellent alternative. These options should be mentioned for comparison when discussing the performance of the Qualitative Annotations tools introduced here.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6322", "date": "12 Feb 2021", "name": "Laurent Thomas", "role": "Author Response", "response": "Dear Jan, thank you for your feedback. The tools are indeed mostly targeting the annotation of 2D images, or from image slices in multi-dimensional images. They are not designed to address qualitative annotations of 3D volumes, which as you pointed out have limited support in ImageJ/Fiji. In the introduction, the authors state that there is \"no widespread solution available [...] for the assignment of multiple descriptive keywords to images\". In my opinion, this statement demonstrates a lack of research, as a simple online search for \"image annotation tool\" reveals many solutions used in the field of Computer Vision to annotate ground truth for machine learning datasets (both for image classification and for bounding box annotations). While some of these tools might be more commonly used than others, I believe they should have at least included a few of them in a thorough comparison, such as e.g. LabelMe [1], MakeSense.AI [2], LabelImg [3], VGG Image Annotator [4], or Scalabel [5]. Regarding other image annotations solutions mentioned above, most of them target RGB colour images and thus are not well adapted to scientific images or file format (e.g 16-bit multi-dimensional tiff). Besides, they mostly target single-category annotations of images or images regions for classification, object-detection or semantic segmentation and usually do not allow the assignment of multiple qualitative keywords to a single image instance. Moreover, these are often standalone software, and thus for non-experts likely not as accessible as integrated solutions in bioimage analysis software such as ImageJ/Fiji. We have reformulated the introduction to include a brief comparison with existing solutions and their limitations. Although the plugins presented in this article can indeed be used for the tasks mentioned above, we still believe that the annotations with multiple keywords was a truly missing functionality and can be of major interest to describe complex biological phenotypes. Another current limitation of the Qualitative Annotations plugin is the absence of ways to review and/or refine an annotation after it has been added to the results table. Regarding this limitation, again due to its tight integration in ImageJ/Fiji, individual table cells of the result table cannot be edited interactively. However, if a mistake was done while annotating, the concerned table rows can be selected and deleted, and the annotation repeated for the images. We have added a sentence in the paragraph implementation mentioning this possibility. The table could also be edited using the macro language, although it is likely not a probable use-case. After annotations, the csv file containing the results can be edited in any software supporting csv such as spreadsheet software or text editors. Lastly, since the authors include KNIME workflows in their article to illustrate further processing of the annotation data (e.g. for training of machine learning models), I am surprised that they didn't also explore ways to do the manual annotation within a KNIME workflow, in order to include the annotation process in their \"complete pipeline for image classification\". The KNIME Image Processing [6] extension offers the 'Interactive Annotator' and 'Interactive Labeling Editor' nodes that can be used to annotate image regions. For interactive sequential classification of images, the 'Active Learning Loop' nodes offer an excellent alternative. These options should be mentioned for comparison when discussing the performance of the Qualitative Annotations tools introduced here. KNIME indeed offers powerful functionalities for ground-truth annotations of image regions or image category, however the annotations are here also limited to a single category per ROI or image. Besides, despite its advanced data and image-analysis functionalities, we believe that it is not the most intuitive software for interactive data-annotation, especially for users not already familiar with KNIME. Still, we have included KNIME in the list of existing annotation solutions compatible with bioimages, in the introduction." } ] }, { "id": "74544", "date": "26 Nov 2020", "name": "Christian Tischer", "expertise": [ "Reviewer Expertise Bioimage 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 authors present an ImageJ plugin suite for the annotation of image planes and regions of interest (ROI) within image planes, using standard ImageJ technology such as results tables and the ROI manager. We think this is a very valuable addition to the ImageJ ecosystem as it is easy to use and very well integrated with current functionality that is already known to many users and developers.\nBelow we have some specific comments which we hope could be helpful to further improve the publication and the plugins.\nWe thank the authors for their efforts in implementing this great addition to the ImageJ ecosystem and hope that below suggestions are helpful!\nWith kind regards, Christian Tischer and Aliaksandr Halavatyi\nSuggestions for the publication\nWe think the publication is nicely written and describes their plugins very well.\nThe authors describe how the output of their plugins could be consumed by a machine learning workflow in KNIME. While the workflow in KNIME is very interesting and relevant we feel at the same time that it is slightly out of scope and we thus suggest moving this to the supplemental material. The reason is that we would expect the main readers of this publication and also the main users of the plugins to be familiar with the ImageJ ecosystem, but not necessarily with KNIME. In addition, if the final aim of the users is to execute a workflow in KNIME there may be other annotation tools available and the authors should clarify how their tool is superior to potential other solutions for image annotation.\nAlong those lines, it would be great to also present (maybe also in the supplemental material) a follow up workflow that can be executed entirely within ImageJ. Specifically, it would be great to see an example of how the annotated ROIs could be used, e.g. to train a machine learning model. However, we don’t know how feasible this is and it is really just a suggestion.\n\nTechnical issues and suggestions for the plugins\nThere are hotkeys (F1, F2, …) available for the single class plugin, which is great as it speeds up annotation. However, we did not manage to have them working on MacOS (10.14.6). In addition, if one has many classes one would have to count the buttons to figure out which hotkey belongs to which class. We thus suggest that the hotkeys are either written on the category buttons or appears as pop-up messages when a user hovers over the corresponding button.\nCurrently, one cannot add additional categories during the labelling process. We think it would be great to be able to (e.g., via a new button: [ Add category ] ), because images may contain phenotypes that one did not think about upfront.\nOne can annotate the same image, slice and ROI multiple times, where each annotation will add another row to the table. As a consequence, the table contains conflicting annotations for the same image region. We think it would be (much) better if the annotation was replaced, at least optionally.\n\nThe possibility to continue an annotation by loading a table and renaming it to “Annotations” is a useful feature, but we think the current implementation has a limitation in a sense that the class names are not read from the table, but rather taken from the last annotation session. This can be an issue if users work on several annotations. We suggest adding an option to fetch the set of category names from the table.\nThe logic of jumping to the next image slice in case of hyperstacks (4D or 5D data) is not completely obvious. We suggest adding a drop-down menu next to the “auto next slice” checkbox, where the user can select the dimension (z, c, or t) along which to automatically progress.\n\nCurrently, annotating a set of images that cannot be loaded into a hyperstack, e.g. because they may have different sizes or dimensionality, is possible but a bit tedious. We suggest adding a modality where the user would select one folder on the disk and then the plugin would automatically open and close the images in the folder one-by-one, allowing them to be annotated one-by-one. The additional advantage of this modality also is that loading different images into a hyperstack is not something that every user may know how to achieve. We suggest adding [ previous ] and [ next ] buttons to this modality, allowing one to correct a previous annotation in case a mistake has been made.\nImageJ ROIs are not a “cross platform standard”. In order to enable follow up workflows outside ImageJ it may be thus useful to store the bounding box coordinates for rectangular ROIs in the table.\nWhen clicking the Help button on one of the annotation UIs the UI was closing, which we think is probably a bug (tested on Mac).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6323", "date": "12 Feb 2021", "name": "Laurent Thomas", "role": "Author Response", "response": "We thank the reviewers for their constructive criticism and helpful suggestions. Since the first manuscript version we implemented most of the previous suggestions to improve the plugins, which are readily available by updating the plugins in Fiji. We further address each point in details below. The authors describe how the output of their plugins could be consumed by a machine learning workflow in KNIME. While the workflow in KNIME is very interesting and relevant we feel at the same time that it is slightly out of scope and we thus suggest moving this to the supplemental material. The reason is that we would expect the main readers of this publication and also the main users of the plugins to be familiar with the ImageJ ecosystem, but not necessarily with KNIME. In addition, if the final aim of the users is to execute a workflow in KNIME there may be other annotation tools available and the authors should clarify how their tool is superior to potential other solutions for image annotation. Following this observation, we have simplified the article such that the examples of visualization and analysis (including the deep learning workflows) are mentioned in the “Use cases” and summarized in the new Figure 4. Details about the deep learning implementation and individual workflow requirements is covered in Supplementary Figures (available on the Zenodo repository) and in the online documentation of the GitHub repository. Regarding annotation tools, there are several software packages available for this specific purpose of ground-truth annotations for supervised machine learning. However, as now explained in introduction they are sometimes not adapted to scientific image formats, while most of them are not integrated into user-oriented scientific image analysis software, and thus require additional installation or configuration. The qualitative annotations plugins instead can be easily installed in ImageJ/Fiji, which supports a large variety of scientific image formats, and is a familiar software environment for most life-scientists. Another advantage of the presented plugins is the support for multiple descriptive keywords per image instance, which is not systematically available with other software packages. Ground-truth category annotations for image-classification or object-detection is one possible application of the qualitative annotation tools. We chose KNIME to illustrate the training of an image-classifier, because it is a relatively accessible data-analysis platform for non-expert users, compared to a programming language. It supports advanced image-processing functionalities, was readily used for bioimage analysis and allows rapid prototyping and customization thanks to its interactive graphical user interface. However, more advanced users can surely reproduce this workflow in a programming language of their choice. Along those lines, it would be great to also present (maybe also in the supplemental material) a follow up workflow that can be executed entirely within ImageJ. Specifically, it would be great to see an example of how the annotated ROIs could be used, e.g. to train a machine learning model. However, we don’t know how feasible this is and it is really just a suggestion. It is potentially possible to train a machine learning or deep learning model directly in ImageJ/Fiji e.g. thanks to respectively the Weka or TensorFlow integration in Fiji. However, this would represent a significative amount of work, and would not offer as much flexibility as the reported KNIME workflows for deep learning, which can be rapidly adapted in KNIME’s graphical user-interface. As an illustration of use case in Fiji, we propose instead an additional plugin for data-visualization relying on the JFreeChart library (available with Fiji). This new plugin generates a pie chart from a table column, to visualize the category distribution. It is available in the same menu “Qualitative Annotation” under “PieChart from category column”– (see Figure 5.B and supplementary figure 2). The plugins support any column of data from a table opened in Fiji and is macro recordable. It is thus not limited to annotation tables generated with the presented plugins. To our knowledge, the pie chart visualization was not previously available to users via the Fiji menus or plugins. Therefore, we believe that it can be an interesting complement to the annotation plugins. Technical issues and suggestions for the plugins There are hotkeys (F1, F2, …) available for the single class plugin, which is great as it speeds up annotation. However, we did not manage to have them working on MacOS (10.14.6) At the time of the fist manuscript version, the plugins were only tested on windows but are expected to run similarly across platforms. Hotkeys might be an exception, for instance on Linux we also observe a different behaviour (see Issue #9 · LauLauThom/Fiji-QualiAnnotations (github.com)). We don’t have a MacOS system at hand for testing, but we invite the reviewers to follow up in the dedicated issue thread on GitHub mentioned above. The hotkeys are sometimes not responsive also on Windows. Clicking the plugin window or one of the buttons usually allows reactivating the hotkey functionality. That said, we believe this is not a major issue preventing the use of the plugins, and we hope to fix it in later versions of the plugins. [..] if one has many classes one would have to count the buttons to figure out which hotkey belongs to which class. We thus suggest that the hotkeys are either written on the category buttons or appears as pop-up messages when a user hovers over the corresponding button. We have updated the button plugin with buttons from the java swing package (previously java awt buttons), which support pop-up messages. The associated hotkey is thus now displayed when the mouse is hovered over a category button. With this new button class, the layout of the GUI might be slightly impaired when a new button is added by clicking the “Add new category”. When this happens, the window can be resized manually to make sure all GUI elements fits in the window. We will fix this issue in later plugin versions if we can identify the source of the problem. Currently, one cannot add additional categories during the labelling process. We think it would be great to be able to (e.g., via a new button: [ Add category ] ), because images may contain phenotypes that one did not think about upfront. We added a “Add new category” button to the single class (button) and multi-class (checkboxes) plugins, which allows updating the plugin interface with new categories on the fly. We did not add this functionality to the dropdown plugin, which would require providing multiple items (label and list of choices). However, with any of the annotation plugins, the plugin interface can be closed and restarted while keeping the current annotation table opened in ImageJ/Fiji. Although not as straightforward as the “Add new category” option, the annotation can thus be interrupted to update the graphical interface and resumed at a later time without losing information. One can annotate the same image, slice and ROI multiple times, where each annotation will add another row to the table. As a consequence, the table contains conflicting annotations for the same image region. We think it would be (much) better if the annotation was replaced, at least optionally. We have considered the possibility to add an option (checkbox) to replace previous annotations of identical image or ROI (see dedicated branch on GitHub). However, when “run Measure” is selected, a table row is anyway added to the result table with the quantitative measurements, before the descriptive keywords are added to the table row. Accounting for this alternative behaviour would further complicate the code (see function defaultActionSequence() ). A more elegant solution would be to have a way to store the measurement into a variable first, which is apparently not the case with the current ImageJ1 package. We thus might address this issue in later releases of the tools. The possibility to continue an annotation by loading a table and renaming it to “Annotations” is a useful feature, but we think the current implementation has a limitation in a sense that the class names are not read from the table, but rather taken from the last annotation session. This can be an issue if users work on several annotations. We suggest adding an option to fetch the set of category names from the table. We added an option to fetch the categories names from a table currently opened in ImageJ/Fiji. If the table contains a column name “Category” then the set of categories is initialized from this column. Otherwise, the set of categories is initialized from the column headers, by excluding the headers for the measurement and file information columns. However, the number of categories will still be taken into account. Besides, with ImageJ versions above 1.53g, the annotations will be appended to/read from any active table window. Below this version, the annotations are appended to/read from table windows entitled “Annotations” or “Annotations.csv”. The logic of jumping to the next image slice in case of hyperstacks (4D or 5D data) is not completely obvious. We suggest adding a drop-down menu next to the “auto next slice” checkbox, where the user can select the dimension (z, c, or t) along which to automatically progress. As suggested, we added a dropdown menu next to the “Auto next slice” checkbox to specify the dimension to explore with hyperstacks. This dropdown menu is shown when “stack” is selected as browsing mode in the initial configuration window (see below). Currently, annotating a set of images that cannot be loaded into a hyperstack, e.g. because they may have different sizes or dimensionality, is possible but a bit tedious. We suggest adding a modality where the user would select one folder on the disk and then the plugin would automatically open and close the images in the folder one-by-one, allowing them to be annotated one-by-one. The additional advantage of this modality also is that loading different images into a hyperstack is not something that every user may know how to achieve. We suggest adding [ previous ] and [ next ] buttons to this modality, allowing one to correct a previous annotation in case a mistake has been made. We added a new setting “Browsing mode” in the initial configuration window, which can be set to “stack” or “directory”. The “stack” mode corresponds to the previously available modality and is well adapted to the annotations of image slices in stacks or hyperstacks. With the “directory” mode, ticking the “Auto next slice/image file” will switch to the next image file in the directory of the currently opened image file. This allows annotating a list of files in a directory as proposed. In “directory” browsing mode the annotation interface also has “previous” and “next image file” buttons, as suggested. ImageJ ROIs are not a “cross platform standard”. In order to enable follow up workflows outside ImageJ it may be thus useful to store the bounding box coordinates for rectangular ROIs in the table. Bounding box coordinates for ROIs can be readily recovered by activating the “Bounding Rectangle” option in Analyze > Set measurements and activating the “run Measure” option in the plugin configuration. For other ROI shapes, such as polygon and free shapes, the number of summits/coordinates is variable. There is thus the option to have on column per coordinates, which is not ideal as it can potentially yield lots of columns. The second option is to have a single column containing the coordinates as a list or another type of collection. However, there is no real standard convention for polygons and other free ROI shapes coordinates encoding. Some further discussion would thus be needed before implementing such functionality (in a dedicated GitHub issue for instance). When clicking the Help button on one of the annotation UIs the UI was closing, which we think is probably a bug (tested on Mac). It is expected that clicking the help button should close the UI, but it should also open the page of the GitHub repository in the default browser. If this is not the case this might be an OS-dependant error, like for the hotkeys. We have opened a dedicated issue thread on GitHub (Issue #22 · LauLauThom/Fiji-QualiAnnotations (github.com)." } ] } ]	1	https://f1000research.com/articles/9-1248
https://f1000research.com/articles/10-104/v1	11 Feb 21	{ "type": "Systematic Review", "title": "Association between metabolic syndrome components and the risk of developing nephrolithiasis: A systematic review and bayesian meta-analysis", "authors": [ "Ilham Akbar Rahman", "Ilham Fauzan Nusaly", "Syakri Syahrir", "Harry Nusaly", "Makbul Aman Mansyur", "Ilham Fauzan Nusaly", "Syakri Syahrir", "Harry Nusaly", "Makbul Aman Mansyur" ], "abstract": "Background: There is increasing evidence that nephrolithiasis is a systemic disease, as opposed to an isolated urinary metabolic problem, after considerable links were found between nephrolithiasis and systemic diseases such as hypertension, obesity, dyslipidemia, and insulin resistance. The interplay between these four factors defines metabolic syndrome (MetS). In this review we aim to clarify the associations of MetS and its components to kidney stone incident. Methods: Online databases of EMBASE, MEDLINE, and Google Scholar were searched from January 1998 up to October 2020 to identify observational studies examining the association between metabolic syndrome components and kidney stone incident. Bayesian random-effects meta-analysis and meta-regression were performed to observe the association. Linear dose-response analysis was conducted to shape the direction of the association. Data analysis was performed using STATA, and R statistics. Results: A total of 25 potentially relevant studies (n = 934,588 participants) were eventually identified. The pooled results suggested that metabolic syndrome was associated with an increased risk of nephrolithiasis with an odds ratio (OR) of 1.769 (95% CI: 1.386 – 2.309). The summary OR of hypertension and dyslipidemia for developing nephrolithiasis were 1.613 (95% CI: 1.213 – 2.169) and 1.586 (95% CI: 1.007 – 2.502) respectively. The presence of diabetes mellitus and obesity had an OR of 1.552 (95% CI: 1.027 – 2.344) and 1.531 (95% CI: 1.099 – 2.109) respectively. Our results revealed that the increasing number of MetS traits will increase the risk of developing nephrolithiasis, the higher the fasting plasma glucose, and body mass index, the higher the risk of kidney stones incident. Conclusions: Our results suggest that hypertension, diabetes, obesity and dyslipidemia are associated with increased risk of developing nephrolithiasis. Linear significant association between MetS components and nephrolithiasis were revealed in our study which reinforced the notion that should be considered a systemic disorder.", "keywords": [ "Metabolic syndrome", "Hypertension", "Obesity", "Dyslipidemia", "Diabetes", "Nephrolithiasis" ], "content": "Introduction\n\nKidney stone disorder, characterized by abnormal urine composition and dehydration, is an increasingly common condition that is most common in older men (Shoag et al., 2015). Its incidence and occurrence has grown progressively and globally for decades. It has been reported that nephrolithiasis was found in all ages, peaking between the ages of 20 and 60 where it rapidly increases between the ages of 40 to 59 (Hiatt et al., 1982).\n\nIn recent years, nephrolithiasis is hypothesized to be a systemic disorder which requires more attention and evaluation in primary health care. The prevalence of kidney stones has increased and been associated with the components of metabolic syndrome including obesity, elevated blood pressure, dyslipidemia, and glucose intolerance (Obligado & Goldfarb, 2008; Sakhaee, 2008; Sakhaee, 2009; Sakhaee & Maalouf, 2008). As opposed to an isolated urinary metabolic problem, research showed that it was related to an increased risk of several comorbidities such as coronary heart disease, stroke, renal cell carcinoma and end-stage renal disease (ESRD) (Dhondup et al., 2018; Peng & Zheng, 2017; van de Pol et al., 2019). In addition to being a prevalent and costly problem, recent evidence also showed that urinary stones are associated with several metabolic traits such as hypertension, diabetes mellitus (DM), and obesity (Kovshilovskaya et al., 2012; West et al., 2008). Comprehensive evidence shows that the association seems to be reciprocal: either stone formers seem to induce MetS or ones with MetS induce the increasing risk of kidney stones (Reiner et al., 2011). Several meta-analyses have found an association between hypertension and diabetes mellitus in nephrolithiasis patients (Aune et al., 2018; Shang et al., 2017). However, there are no previous studies that have compared all possible metabolic syndrome traits in determining which traits have the most significant influence for the risk of nephrolithiasis and to determine the direction of the association. Moreover, due to sample size problems individual studies may not have enough - statistical power, therefore we performed a systematic review and meta-analysis to determine the association between all possible metabolic syndrome traits to assess the most significant risk for developing nephrolithiasis from all the evidence. Meta-regression and dose response analysis were performed to assess the direction of association. All these analyses were implemented in a Bayesian framework so that we could provide the results with more confidence (O’Hagan, 2004; Spiegelhalter, 2004). Our study outcome could clarify the association between metabolic syndrome and its components to the risk of developing nephrolithiasis.\n\n\nMethods\n\nRelevant articles were retrieved from online databases including EMBASE, MEDLINE and Google scholar from January 1998 until October 2020. The search strategy consisted of two parts (metabolic syndrome and nephrolithiasis) using specific keywords combined with medical subject heading (MeSH) terms and words: \"metabolic syndrome \", \"hypertension\", \"diabetes mellitus\", \"insulin resistance\", \"dyslipidemia\", \"obesity\", “waist circumferences”, “hypertriacylglycerolemia”, “hypertriglyceridemia”, “kidney stones”, “nephrolithiasis\". The full texts and abstracts were originally and independently selected by two reviewers. Full texts and abstracts were scored according to the inclusion and exclusion criteria. The studies which did not fit with the inclusion criteria were excluded. Discrepancies between the two reviewers were resolved through a discussion with a third reviewer. Ethical approval was not required because the data did not contain individual patient information.\n\nThe studies included in this article met the following criteria: (1) the subjects were nephrolithiasis patients; (2) research was conducted as observational study to show causal-effect relationship (case control or cohort studies); (3) the study showed association as risk with 95% CI; (4) the language of the article was published in English. Two reviewers (IAR and IF) separately extracted and analyzed the data based on study selection criteria using standardized, structured and pilot extraction forms. The results were reviewed and discussed by IAR and IF to complete the included studies. Any discrepancies were resolved in a discussion with a third reviewer. For each included study, several pieces of information were extracted including author name, year of publication, number of sample sizes, mean age, study design, countries, number of participants and cases, study period, predictors and outcomes. Table 1 comprehensively shows all of the above data. Newcastle Ottawa Scale (NOS) tool was used to assess the relevance of the included studies and the strength of the evidence. Figure 1 shows the detailed literature search and selection process as PRISMA guideline was followed. Publication bias was investigated by the Begg and Egger tests.\n\nPrimary endpoint was the occurrence of kidney stones with the predictors assessed as metabolic syndrome traits. Secondary endpoint was the direction of association assessing between the dose of the predictors and the response of kidney stones occurrence.\n\nWe performed an empirical Bayesian random effect meta-analysis and meta regression to understand the association between predictors and outcome of kidney stones. All data were analyzed using R language (version 1.2.1335), STATA (version 12.0; Stata Corp LP, College Station, TX) and R. An odds ratio (OR) with a 95% confidence interval (CI) used for each association was generated using Bayesian meta-analysis. The comparison of risk factors with each other was conducted based on OR, where HRs (Hazard Ratio) and RRs (Risk Ratio) were both directly considered as OR (Greenland, 1987), for kidney stones outcome by performing the simulation with 16,000 iterations and with the burned out of 10,000 iterations in the Bayesian meta-analysis model. Chi-squared based on Q-statistic test (P<0.10) and I2 statistic quantification were used to assess heterogeneity of studies. I2 values of heterogeneity were interpreted as insignificant (0–25%), low (26–50%), moderate (51–75%) and high (>75%) (Higgins et al., 2003). Empirical Bayesian random effects meta-regression was further conducted to explore the impact of moderators in the study effect size. Linear dose response analysis was conducted to shape the direction of association. Sensitivity analyses were conducted to explore various levels of heterogeneity between studies and observe how the results will change for different values of between study variance τ2 and the heterogeneity statistic I2. Publication bias and small study effect were evaluated by funnel plots, Egger’s regression test and Begg’s rank correlation test.\n\n\nResults\n\nThis meta-analysis study included a total of 207 studies found using the search strategy. A full text review and abstract selection were then performed and 138 studies were excluded due to reviews, case reports, duplicates, and not in English. Finally, 25 studies met the inclusion criteria of 934,588 patients, which were eligible and added for further analysis. A block diagram of the procedure for searching and selecting studies is shown in Figure 1.\n\nAs demonstrated in Figure 2, prior metabolic syndrome was significantly associated with the mean risk of nephrolithiasis (OR: 1.769; 95% CI, 1.386-2.309) in the meta-analysis of 6 studies. There was low heterogeneity (Q test; I2 = 66.84%). Also, publication bias and small study effect were not found based on the symmetry of the funnel plot (Supplementary material 1a (Rahman et al., 2021b)) and the Begg’s rank correlation test (P = 0.469) and Egger’s regression test (P= 0.3093) tests. The pooled ORs remained significant after 3 more studies were added to both meta-analyses using the trim-and-fill method (Supplementary material 1b (Rahman et al., 2021b)). Sensitivity analyses were consistent for different values of study variance and heterogeneity (Supplementary material 1c (Rahman et al., 2021b)).\n\nAs demonstrated in Figure 3, prior HTN (hypertension) was significantly associated with the risk of nephrolithiasis (RR: 1.613; 95% CI, 1.213-2.169) in the meta-analysis of 14 observational studies. There was high heterogeneity (Q test, P < 0.001; I 2 = 98%). Also, publication bias and small study effect were not suggested based on the symmetry of the funnel plot and the Begg’s (P = 0.233) and Egger’s (P= 0.1386) tests (Supplementary material 2a (Rahman et al., 2021b)). The pooled ORs remained significant after 6 more studies were added to both meta-analyses using the trim-and-fill method (Supplementary material 2b (Rahman et al., 2021b)). Sensitivity analyses were consistent for different values of study variance and heterogeneity. (Supplementary material 2c (Rahman et al., 2021b)).\n\nThe results of meta-analyses that assessed the relationship between diabetes and nephrolithiasis were shown in Figure 3. DM was significantly linked to increased risk of nephrolithiasis formation (RR: 1.552; 95% CI, 1.027-2.344). For incident nephrolithiasis with Begg’s (P= 1.000) and Egger’s (P = 0.3106) tests revealed no publication bias however funnel plots were asymmetrical with a few dots outside the intervals (Supplementary material 3a (Rahman et al., 2021b)). There were no new studies added by the trim-and-fill method and the results of sensitivity analyses remained consistent in different values of study variances and heterogeneity (Supplementary Item 3b and 3c (Rahman et al., 2021b)). High heterogeneity existed in the analyses of nephrolithiasis incident (Q test, I2 = 97%)\n\nThe pooled analysis of OR of 10 individual studies showed that patients with obesity had overall RR to the risk of nephrolithiasis of 1.531 [95%CI: 1.099–2.109]. Publication bias and small study effect were not found based on symmetry of the funnel plot (Supplementary material 4a (Rahman et al., 2021b)) and the results of Begg’s (P = 0.8618) and Egger’s (P = 0.9990) test. After four more studies added to meta analyses by using the trim-and-fill method, the pooled ORs remained significant (Supplementary material 4b (Rahman et al., 2021b)). Despite high heterogeneity existed (Q test, I2 = 96%) between studies, the results of sensitivity analyses remained consistent in different values of study variances (Supplementary material 4c (Rahman et al., 2021b)).\n\nThere were significant associations between dyslipidemia and nephrolithiasis as demonstrated in Figure 3. Specifically, patients with dyslipidemia were 1.586 times more likely to develop nephrolithiasis (95% CI, 1.007 -2.502). In this pooled analysis, the heterogeneity was substantial with I2 = 97%. The funnel plot of studies assessing dyslipidemia and the risk of nephrolithiasis indicated potential publication bias that was however not supported by the result of Begg’s (P = 0.9195) and Egger’s (P = 0.5712) tests (Supplementary Item 5a (Rahman et al., 2021b)). The pooled ORs remained significant after 1 new study added with the trim and fill analysis. Sensitivity analyses yielded consistent results (Supplementary Item 5b and 5c (Rahman et al., 2021b)).\n\nAs demonstrated in Figure 4, the effect of metabolic syndrome traits on kidney stone formation was also assessed by using Bayesian meta regression in six reports on the study of MetS and nephrolithiasis. Stratification for hypertension, dyslipidemia, obesity, and diabetes were conducted. Moreover, linear dose response analysis was also conducted as shown in Figure 5. Our analyses revealed that the incident of nephrolithiasis was significantly associated (p value <0.05) with the increasing of age. Fasting plasma glucose and body mass index were also reported to significantly associated (p value <0.05) with incidence of kidney stones. The more increased the fasting glucose and body mass index then the more increased the risk of kidney stones incident. Linear correlation was also found in metabolic traits where the increasing number of MetS traits will increase the risk of developing nephrolithiasis. For the effect of blood pressure, there is a trend that the more increased blood pressure then the more increased risk of kidney stones, however the result was not significant (p value > 0.05). HDL (high density lipoprotein) level revealed no trend and significance (p value > 0.05) for the association with kidney stone incident.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first systematic review study to confirm the associations between nephrolithiasis and metabolic syndrome components using Bayesian meta-analysis with meta regression and dose response analysis. All studies selected for meta-analysis were of high quality with NOS ≥7. Our results indicated significant relationships between kidney stones and metabolic syndrome traits such as hypertension, diabetes, obesity, and dyslipidemia. Although heterogeneity existed across most analyses, the results were consistent in all sensitivity analyses and trim-and-fill tests, suggesting these relationships were relatively robust and unlikely to be the consequence of confounding.\n\nOur results indicate that hypertension has been associated as a risk factor in the development of nephrolithiasis. Our result is also in coherent with (Cappuccio et al., 1990) where we found that the risk of kidney stones development was increased stepwise with the severity of hypertension that the higher the blood pressure, the higher the risk in developing nephrolithiasis. The relationship could be clarified by many possible mechanisms. Hypercalciuria or increased renal calcium excretion was identified as the first theory concerning the relation between hypertension and nephrolithiasis. This hypothesis was also supported in previous studies that HTN has increased the occurrence of kidney stones, by its association with hypercalciuria, hyperoxaluria, and hypocitraturia (Borghi et al., 1999; Coe et al., 2010). This hypothesis was strengthened by research (Coe & Bushinsky, 1984; Pak et al., 1975; Resnick et al., 1986) showing that hypertensive and stone-forming patients often have certain hormonal correlations, such as elevated levels of 1,25(OH)2D and parathyroid hormone, which may justify the finding of hypercalciuria in large subgroups of both subjects. This hypothesis is linked for stones containing calcium, including calcium oxalate and calcium phosphate (Coe et al., 2010). A primary renal tubular defect (the 'renal calcium leak' hypothesis) or the result of central volume expansion observed in hypertension (the 'central blood volume' hypothesis) may be attributed to increased urinary calcium excretion. This disorder is well evident in the Italian cohort studied by Borghi et al. (Borghi et al., 1999), where urinary calcium excretion was higher in hypertensive patients, both men and women, than in normotensive patients. Previous authors also advance the hypothesis that a pathogenic relation between hypertension and nephrolithiasis may be caused by the increased renal calcium excretion (Devynck et al., 1984; McCarron, 1985; Resnick, 1987). Previous research also revealed that there is an existence of calcium metabolism alterations, including increased renal excretion both in hypertensive animals (Hsu et al., 1986; Wexler & McMurtry, 1981) and humans (Gennari et al., 1986; Strazzullo et al., 1983). Furthermore, it is understood that these conditions are aggravated by a high dietary intake of sodium chloride. High dietary salt intake will in particular, facilitate hypertension by volume expansion and stone forming through increasing urinary calcium excretion and reducing citrate (Canessa et al., 1980; Postnov et al., 1979). This finding was also confirmed by research (Borghi et al., 1999) showing that in many hypertensive patients, the effect of NaCl intake appears to be important in inducing high blood pressure and causing high calcium excretion. It is well known that a small increase in urinary oxalate increases the lithogenic risk of calcium oxalate, while hyperuricosuria can encourage both uric oxalate production (Robertson & Peacock, 1980). Genetic theory also describes how genetics is associated with calcium metabolism alterations in hypertension and kidney stone incident. A previous study (Cirillo et al., 1989) also reported that alteration in renal calcium handling in hypertensive rats led to higher urinary calcium excretion. Other epidemiological studies tend to indicate that hypertensive subjects and stone formers have common dietary patterns, distinguished above all by a low consumption of calcium (McCarron & Morris, 1987; McCarron et al., 1984; Menon, 1993), which, as is well known, can contribute to increased oxaluria due to increased intestinal oxalate absorption. This theory is born out of the fact that the reduction of arterial pressure levels and reduction of the likelihood of renal stone development appear to be supported by calcium-rich diet (Curhan et al., 1997).\n\nA significant association of DM with the risk of nephrolithiasis was reported in our research. It is well known that by deranging ammoniagenesis and increasing sodium and bicarbonate reabsorption, insulin resistance, as the central feature of DM, could cause decreased urine pH (Spatola et al., 2018). Insulin resistance causes high levels of plasma-free fatty acids to reach the proximal tubule cells and interfere with the use of glutamine in ammonium production (Bagnasco et al., 1983; Lemieux et al., 1977; Vinay et al., 1976). Furthermore, insulin resistance may directly affect ammoniagenesis at the level of the kidney. This could lead to lower urinary PH which is a major risk factor for uric acid nephrolithiasis (Asplin, 1996; Riese & Sakhaee, 1992). However, a lower level of urinary citrate also resulted in DM, which further induced hypercalciuria due to reduced citrate binding (Spatola et al., 2018). In addition, through isonatric inhibition of proximal tubular reabsorption of calcium, hyperglycemia and concurrent glycosuria acted separately to elevate urinary calcium excretion. These pathways, working together, facilitated the development of uric acid and calcium stone in diabetic patients (Daudon et al., 2006). Our results also provide evidence that there is a trend that the risk of developing nephrolithiasis was correlated with the higher the fasting plasma glucose. The links between distinct glycemic status and kidney stone disorder have been explored in recent research. Positive correlations between prediabetes and diabetes with the risk of nephrolithiasis were seen in the results and with insulin resistance, a greater risk of kidney stones was found (Lien et al., 2016; Weinberg et al., 2014). Thus it is possible that kidney stones are developed after prediabetes, but before DM progresses. As a consequence of diagnostic chronology, this may partially explain the greater incidence of DM in kidney stone patients.\n\nInsulin resistance may also play role in the link between obesity and nephrolithiasis which was reported in our research. In previous studies, urinary calcium excretion and body mass index (BMI) were found to be positively correlated (Ekeruo et al., 2004; Powell et al., 2000; Siener et al., 2004). It is well understood that one of the characteristics of obesity is insulin resistance. In patients with high BMI, it is found that increased uric acid excretion, low urinary pH both cause urinary supersaturation (Maalouf et al., 2004; Powell et al., 2000). Insulin resistance with urinary pH <5.5 through the activation of exchanger NH3 in the proximal renal tubule thus reduces the synthesis and urinary excretion of ammonium (Abate et al., 2004; Chobanian & Hammerman, 1987; Klisic et al., 2002). In acid urine, uric acid is found in an undissociated, readily precipitable state such that the deposition of uric acid stone can easily take place (Pak et al., 2001). Excess uric acid in the urine can also by a heterogeneous nucleation process, causing the precipitation of calcium oxalate dehydrate (Coe et al., 1980). The process of insulin resistance is not influenced by diet and lifestyle (Kovesdy et al., 2017).\n\nOur study reveals that there was a 1.586 -fold increased risk of nephrolithiasis in participants with dyslipidemia. There has been previous meta-analysis that supports the correlation between dyslipidemia and nephrolithiasis (Besiroglu & Ozbek, 2019). Our result was also supported by the fact that consuming statin medications can reduce stone genesis compared to those not consuming statin medications (Inci et al., 2012; Sur et al., 2013). The study from Toricelli et al. (Torricelli et al., 2014) also reported low HDL and high TGs (triglycerides) are associated with lower urinary pH, and uric acid stones. Possible linking mechanism was explained that dyslipidemia and similar diseases are often concerned with systemic inflammation and oxidative stress (Gorbachinsky et al., 2010). They concluded that oxidative stress tends to be a key cytotoxic activity of the monohydrate calcium oxalate that can harm or destroy renal cells, and by unknown cellular physiological mechanisms could also contribute to stone forming. The oxidative stress cascade, which is the leading factor in cell damage, may be caused by dyslipidemia. Ultimately, the area of oxidatively stressed may lead to crystallization by the contribution of high calcium and phosphate or oxalate, and low urine citrate or magnesium. Moreover, lipid accumulation in the kidney which is defined as lipotoxicity caused changes in renal structure and function (Gorbachinsky et al., 2010). Lipotoxicity can induce an increased acid extraction, with decreased ammonia synthesis and ammonium excretion, resulting in a lower urinary pH. Bobulescu et al. (Bobulescu et al., 2008) in the rats study also revealed that TG steatosis around the tubular portions of the kidney is associated with low urinary pH and NH4+ and high titratable acidity. They demonstrated that the decrease in NH4+ excretion might be attributed to a lipotoxicity-induced reduction in NHE3 activity. In a follow up study of Bobulescu and colleagues, the reduction of renal steatosis in rats improved urinary NH4+, increased pH, reduced titratable acidity and finally increased citrate and brush border NHE3 levels and activity was demonstrated (Bobulescu et al., 2009).\n\nThe current findings have important public health implications in light of the current epidemics of obesity, diabetes, hypertension and dyslipidemia and suggest that these four factors play important roles in the development of nephrolithiasis. With this knowledge, determining common modifiable risk factors for the development of kidney stones might uncover new strategies enabling improved patient management and treatment of stone disease. Finally, there are several strengths of this review. First, the implementation of Bayesian framework in meta-analysis and meta regression could provide better confidence in the results; therefore, it gives clarity in the associations between nephrolithiasis and metabolic syndrome traits. Second, the results were tested using sensitivity analysis, trim-fill analysis, funnel plot, Begg’s rank correlation and Egger’s regression test to ensure that the results were relatively consistent and robust without the influence of confounders. Third, all studies selected for meta-analysis were of high quality, which report the association between the traits and nephrolithiasis. However, there are several limitations in this study. Firstly, the number of studies that can be pooled due to the fact that not many primary study have been conducted; secondly, not all studies included stone analysis therefore it is difficult to observe the association between metabolic syndrome components and different type of kidney stones; and thirdly, there are not many individual studies on metabolic syndrome and further updated meta-analyses need to be performed.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nDryad: Data from: Association between metabolic syndrome components and the risk of developing nephrolithiasis: Bayesian meta-analysis and meta-regression with dose-response analysis, https://doi.org/10.5061/dryad.76hdr7svg (Rahman et al., 2021b).\n\nThis project contains the following extended data:\n\n- Supplementary material 1. Funnel plot (a), trim-fill (b) and sensitivity analysis (c) of the association between metabolic syndrome and nephrolithiasis\n\n- Supplementary material 2. Funnel plot (a), trim-fill (b) and sensitivity analysis (c) of the association between hypertension and nephrolithiasis\n\n- Supplementary material 3. Funnel plot (a), trim-fill (b) and sensitivity analysis (c) of the association between DM and nephrolithiasis\n\n- Supplementary material 4. Funnel plot (a), trim-fill (b) and sensitivity analysis (c) of the association between obesity and nephrolithiasis\n\n- Supplementary material 5. Funnel plot (a), trim-fill (b) and sensitivity analysis (c) of the association between dyslipidemia and nephrolithiasis\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: PRISMA checklist for ‘Comparison of major bleeding in patients with acute coronary syndrome that underwent coronary artery bypass grafting treated with clopidogrel or ticagrelor: a systematic review and meta-analysis, https://doi.org/10.6084/m9.figshare.13585607 (Rahman et al., 2021a).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbate N, Chandalia M, Cabo-Chan Jr AV, et al.: The metabolic syndrome and uric acid nephrolithiasis: Novel features of renal manifestation of insulin resistance. Kidney Int. 2004; 65(2): 386–392. PubMed Abstract \| Publisher Full Text\n\nAsplin JR: Uric acid stones. 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PubMed Abstract \| Publisher Full Text\n\nMcCarron DA: Calcium in the pathogenesis and therapy of human hypertension. Am J Med. 1985; 78(2B): 27–34. PubMed Abstract \| Publisher Full Text\n\nMcCarron DA, Morris CD, Henry HJ, et al.: Blood pressure and nutrient intake in the United States. Nutrition Today. 1984; 19(4): 14–23. Publisher Full Text\n\nMenon M: A prospective study of dietary calcium and other nutrients and the risk of symptomatic kidney stones. J Urol. 1993; 150(2 Pt 1): 563–564. PubMed Abstract\n\nO’Hagan A: Bayesian statistics: principles and benefits. Bayesian Statistics and Quality Modelling in Agro-Food Production Chain. (Wageningen UR Frontis Series). 2004; 31–45. Reference Source\n\nObligado SH, Goldfarb DS: The association of nephrolithiasis with hypertension and obesity: A review. Am J Hypertens. 2008; 21(3): 257–264. PubMed Abstract \| Publisher Full Text\n\nPak CY, Kaplan R, Bone H, et al.: A Simple Test for the Diagnosis of Absorptive, Resorptive and Renal Hypercalciurias. N Engl J Med. 1975; 292(10): 497–500. PubMed Abstract \| Publisher Full Text\n\nPak CY, Sakhaee K, Peterson RD, et al.: Biochemical profile of idiopathic uric acid nephrolithiasis. Kidney Int. 2001; 60(2): 757–761. PubMed Abstract \| Publisher Full Text\n\nPeng JP, Zheng H: Kidney stones may increase the risk of coronary heart disease and stroke: A PRISMA-Compliant meta-analysis. Medicine (Baltimore). (United States). 2017; 96(34): e7898. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPostnov YV, Orlov SN, Pokudin NI: Decrease of calcium binding by the red blood cell membrane in spontaneously hypertensive rats and in essential hypertension. Pflugers Arch. 1979; 379(2): 191–195. PubMed Abstract \| Publisher Full Text\n\nPowell CR, Stoller ML, Schwartz BF, et al.: Impact of body weight on urinary electrolytes in urinary stone formers. Urology. 2000; 55(6): 825–830. PubMed Abstract \| Publisher Full Text\n\nRahman IA, Nusaly IF, Syahrir S, et al.: PRISMA 2009 checklist.doc. figshare. Figure. 2021a. http://www.doi.org/10.6084/m9.figshare.13585607\n\nRahman IA, Nusaly IF, Syahrir S, et al.: Data from: Association between metabolic syndrome components and the risk of developing nephrolithiasis: Bayesian meta-analysis and meta-regression with dose-response analysis, Dryad. 2021b. http://www.doi.org/10.5061/dryad.76hdr7svg\n\nReiner AP, Kahn A, Eisner BH, et al.: Kidney stones and subclinical atherosclerosis in young adults: The CARDIA study. J Urol. 2011; 185(3): 920–925. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResnick LM, Müller FB, Laragh JH: Calcium-regulating hormones in essential hypertension. Relation to plasma renin activity and sodium metabolism. Ann Intern Med. 1986; 105(5): 649–654. PubMed Abstract \| Publisher Full Text\n\nResnick LM: Uniformity and diversity of calcium metabolism in hypertension. A conceptual framework. Am J Med. 1987; 82(1B): 16–26. PubMed Abstract \| Publisher Full Text\n\nRiese RJ, Sakhaee K: Uric acid nephrolithiasis: Pathogenesis and treatment. J Urol. 1992; 148(3): 765–771. PubMed Abstract \| Publisher Full Text\n\nRobertson WG, Peacock M: The cause of idiopathic calcium stone disease: Hypercalciuria or hyperoxaluria? Nephron. 1980; 26(3): 105–110. PubMed Abstract \| Publisher Full Text\n\nSakhaee K: Nephrolithiasis as a systemic disorder. Curr Opin Nephrol Hypertens. 2008; 17(3): 304–309. PubMed Abstract \| Publisher Full Text\n\nSakhaee K: Recent advances in the pathophysiology of nephrolithiasis. Kidney Int. 2009; 75(6): 585–595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSakhaee K, Maalouf NM: Metabolic Syndrome and Uric Acid Nephrolithiasis. Semin Nephrol. 2008; 28(2): 174–180. PubMed Abstract \| Publisher Full Text\n\nShang W, Li Y, Ren Y, et al.: Nephrolithiasis and risk of hypertension: A meta-analysis of observational studies. BMC Nephrol. 2017; 18(1): 344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShoag J, Tasian GE, Goldfarb DS, et al.: The New Epidemiology of Nephrolithiasis. Adv Chronic Kidney Dis. 2015; 22(4): 273–278. PubMed Abstract \| Publisher Full Text\n\nSiener R, Glatz S, Nicolay C, et al.: The role of overweight and obesity in calcium oxalate stone formation. Obes Res. 2004; 12(1): 106–113. PubMed Abstract \| Publisher Full Text\n\nSpatola L, Ferraro PM, Gambaro G, et al.: Metabolic syndrome and uric acid nephrolithiasis: insulin resistance in focus. Metabolism. 2018; 83: 225–233. PubMed Abstract \| Publisher Full Text\n\nSpiegelhalter DJ: Incorporating Bayesian ideas into health-care evaluation. Statist Sci. 2004; 19(1): 156–174. Publisher Full Text\n\nStrazzullo P, Nunziata V, Cirillo M, et al.: Abnormalities of calcium metabolism in essential hypertension. Clin Sci (Lond). 1983; 65(2): 137–141. PubMed Abstract \| Publisher Full Text\n\nSur RL, Masterson JH, Palazzi KL, et al.: Impact of statins on nephrolithiasis in hyperlipidemic patients: A 10-year review of an equal access health care system. Clin Nephrol. 2013; 79(5): 351–355. PubMed Abstract \| Publisher Full Text\n\nTorricelli FCM, De SK, Gebreselassie S, et al.: Dyslipidemia and kidney stone risk. J Urol. 2014; 191(3): 667–672. PubMed Abstract \| Publisher Full Text\n\nvan de Pol JAA, van den Brandt PA, Schouten LJ: Kidney stones and the risk of renal cell carcinoma and upper tract urothelial carcinoma: the Netherlands Cohort Study. Br J Cancer. 2019; 120(3): 368–374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVinay P, Lemieux G, Cartier P, et al.: Effect of fatty acids on renal ammoniagenesis in in vivo and in vitro studies. Am J Physiol. 1976; 231(3): 880–887. PubMed Abstract \| Publisher Full Text\n\nWeinberg AE, Patel CJ, Chertow GM, et al.: Diabetic severity and risk of kidney stone disease. Eur Urol. 2014; 65(1): 242–247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWest B, Luke A, Durazo-Arvizu RA, et al.: Metabolic Syndrome and Self-Reported History of Kidney Stones: The National Health and Nutrition Examination Survey (NHANES III) 1988-1994. Am J Kidney Dis. 2008; 51(5): 741–747. PubMed Abstract \| Publisher Full Text\n\nWexler BC, McMurtry JP: Kidney and bladder calculi in spontaneously hypertensive rats. Br J Exp Pathol. 1981; 62(4): 369–374. PubMed Abstract \| Free Full Text" }	[ { "id": "80876", "date": "22 Mar 2021", "name": "Joseph philipraj", "expertise": [ "Reviewer Expertise Endourogy", "Urolithiasis", "Andrology and Male Infertility", "Uro-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\nThis systematic review is appropriate for the journal with a global problem of Mets and Urolithiasis. The introduction part clearly explains the motivation. The manuscript is clear and balanced. The manuscript stays focused on the subject. Authors have gone through the process of searching relevant articles from all websites and of sufficient duration. The inclusion and exclusion criteria in the analysis have been clearly stated. The impact of the analysis is clearly stated.\nThe statistical analysis supports the paper well. The interpretation of the results, visualisation are well presented. The tables and figures are clear, relevant and correct.\nThe authors demonstrate the knowledge of basic composition skills, including word choice, sentence structure, paragraph development and grammar.\nLimitations:\n\nThe studies included in the meta-analysis have cross-sectional nature and hence ascertainment of temporal association is not possible which also dictates need for further prospective studies. The specific type of stone formation is not correlated with studies. Despite these limitations all studies included in the meta-analysis showed the same directionality in the association between urolithiasis and Mets.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "96149", "date": "08 Nov 2021", "name": "Muhammad Faruk", "expertise": [ "Reviewer Expertise General Surgery", "Urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study assessed the association between metabolic syndrome and its components with the risk of developing nephrolithiasis by conducting systematic review, Bayesian random-effects meta-analysis, meta-regression and dose-response analysis. This study was done appropriately based on PRISMA flowchart. Risk of bias was also conducted of the included studies. This study has successfully presented the proper meta-analysis for this design. However, to complete this study for indexing, I personally recommended several revisions:\n1. Abstract:\nIntroduction section: It is better to address meta-regression as the analysis to assess the correlation of association along with dose-response analysis\n\nConclusion section: In reporting the association between predictors and nephrolithiasis, state only the predictors in which its coefficient was statistically significant\n2. R language was not considered as a statistical software for data analysis. The software for data analysis should be written as \"R\" (Please refer to methods section in statistical analysis subsection).\n3. Please update the PRISMA flowchart (refer to PRISMA guideline 2009).\n4. Give the numbering of each Forrest plot in Figure 3 and numbering of each meta-regression plot in Figure 4. Design these figures so that it could be well presented.\n5. Uniformly decide the word choice of \"traits\" or \"components\", choose whether to use traits or components in the whole text, use one of these words consistently to avoid any misunderstanding.\n6. It is better to provide the meta-regression of hypertension in systolic blood pressure and diastolic blood pressure as it is important to explain the relationship to nephrolithiasis in differentiation for these two types of blood pressure.\n7. Meta-regression of body mass index was sufficient in this study thus waist circumference meta-regression was not necessary to be included.\n8. Provide the value of coefficient and confidence interval of each meta-regression analysis in the result section so that better understanding of predictors-outcome relationship could be reached clearly.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-104
https://f1000research.com/articles/10-102/v1	11 Feb 21	{ "type": "Study Protocol", "title": "Guidance for ensuring fair and ethical broad consent for future use. A scoping review protocol.", "authors": [ "Lauren Maxwell", "Regina Gilyan", "Sayali Arvind Chavan", "Laura Merson", "Abha Saxena", "Rob Terry", "Regina Gilyan", "Sayali Arvind Chavan", "Laura Merson", "Abha Saxena", "Rob Terry" ], "abstract": "Introduction: Broad consent for future use is the reuse of data and/or samples collected by a study by researchers who may not be affiliated with the original study team for purposes that may differ from the objectives of the original study. Sharing participant-level data and samples collected from research participants facilitates reuse and transparency and can accelerate drug or vaccine development, research findings, and translation. Data reuse and synthesis help prevent unnecessary research, thereby respecting research participants time and efforts and building their trust in the research process. Despite these myriad benefits, data and sample sharing represent a significant investment of time for the team that collected the data or samples, and may present additional risks for research participants, including that of re-identifiability and incidental findings, or for the source community. This scoping review will summarize existing guidance on broad consent for future use and highlight evidence gaps related to the ethical, equitable implementation of broad consent for future use. Methods and analysis: We will apply the Arskey and O’Malley scoping review methodology and best practice as outlined in the Joanna Briggs scoping review guidelines. The research questions have been identified through a literature review and consultation with subject-matter experts. The systematic search will be conducted in three databases using a tailored search strategy. We will search the reference lists of included articles or related systematic reviews for additional citations. The title-abstract and full text screening and charting the data will be conducted independently by two reviewers. Discrepancies will be resolved by a third reviewer. Results will be summarized in narrative form. Ethics and dissemination: This scoping review summarizes findings from existing publications and grey literature rather than primary data and, as such, does not require ethics review. Findings will be disseminated through an open access publication and webinar.", "keywords": [ "scoping review", "data sharing", "broad consent for future use", "community engagement", "benefit sharing" ], "content": "Background\n\nResearch funders1, regulatory agencies, and journals2,3 are increasingly advocating for and requiring that participant-level data obtained from various forms of health research are shared to maximize their utility. The advantages of sharing data include improving research transparency, facilitating innovation and the conduct of subgroup analyses, and reducing the burden and costs of unnecessary duplication of research4,5. The public health benefits of sharing data for purposes other than those for which the data were collected, i.e. future use of data, must be balanced with the risk to research participants and the team that collected the data and legal, equity, and ethical concerns, including re-identifiability, incidental findings, benefit sharing, and respecting community values6–8. These concerns are especially important for researchers and communities in low-and-middle-income countries (LMIC) who may experience a disproportionate proportion of the burden related to data sharing without the same level of benefits as those received by research participants and their communities and researchers in higher income countries7,8.\n\nInformed consent speaks directly to the core principle of respect for persons and is of central importance to the ethical conduct of human subjects research9. Broad consent for future use is a type of informed consent whereby participants are asked to prospectively agree to the use of data or samples derived from them by unspecified groups for unspecified purposes that are not necessarily related to the objectives of the original study, pending the development or establishment of ethically sound governance for said sharing10,11. The wording used for broad consent explicitly states or implies that participants will not be re-contacted when sharing their data or samples. While ensuring that the benefits of data or sample sharing outweigh the risks to research participants, researchers and ethics review committees (ERCs) must also take care to ensure that participants understand and agree to data and sample sharing12.\n\nData sharing can both engender and undermine participants’ trust in the research process. For example, data sharing may accelerate innovations that directly benefit research participants or lead to improved treatments that are only accessible to high income populations13. While the Council for International Organizations of Medical Sciences’ (CIOMS) published guidance for the application of broad consent in 2016, there is a need to supplement this guidance through understanding best practice as it relates to ensuring research participants and their communities understand broad consent for future use and define and realize the benefits of data and sample sharing and that different stakeholders’ interests, risks, and benefits are recognized and addressed in the application of broad consent for future use.\n\n\nStudy rationale and objectives\n\nIn this scoping review, we will summarize empirical findings from primary research and other forms of guidance from international stakeholder groups related to: 1) recommendations around when broad consent should or should not be applied, 2) how to ensure ethical and equitable implementation of broad consent; and 3) and recommended language for broad consent in the health research context. Several prior evidence synthesis efforts have summarized attitudes and best practice related to data sharing. This rapid evidence synthesis will allow for the identification of existing resources for helping ERCs and research teams and communities understand and implement best practice for broad consent for future use and to identify priority areas for future research. This research responds to the challenges faced by researchers and ERCs who must ensure the voluntary and informed participation in the research that they conduct or review and balance the risks and benefits that data sharing represents to different stakeholders while responding to the public health imperative or journal or funder requirements to share data and biological samples derived from clinical trial participants. Findings will be used to develop a research agenda for better addressing key concerns in the ethical and equitable application of broad consent for future use, including re-identifiability, incidental findings, community and research team engagement, and benefit sharing.\n\n\nReview methods\n\nScoping reviews provide a broad overview of existing literature and may be used to identify priority areas for further exploration in a subsequent systematic review14. The key scoping review questions are based on a review of the literature and feedback from subject matter experts and will be updated as needed based on the scoping review findings. This scoping review will follow the Arksey and O’Malley approach to scoping reviews15 and will present the results in keeping with the PRISMA Extension for Scoping Reviews guidance (PRISMA-ScR)16. We will undertake the following steps, from Levac, et al.’s recommendations for the conduct of scoping reviews:17\n\n1. clarify and link the purpose and research question (balancing)\n\n2. apply an iterative approach to developing and evaluating the comprehensiveness of the scoping process\n\n3. clarify approach to study selection\n\n4. clarify approach to data extraction\n\n5. qualitative thematic analysis, summarizing results, and clarifying policy, practice, and research implications\n\n6. stakeholder consultation to summarize results and facilitate research translation\n\nThis scoping review protocol was developed in keeping with the PRISMA-P guidelines18.\n\n\nStage I: Identifying the research question\n\nThe initial review questions were developed with feedback from members of the Reconciliation of Cohort Data in Infectious Disease (ReCoDID) and COVID-19 Clinical Data Research Consortia and the Special Programme for Research and Training in Tropical Diseases (TDR), research groups and an international organization that work on public health data sharing with a focus on ethical and equitable sharing of data and samples collected by research teams in LMIC.\n\n1. What guidelines exist for when investigators should include or not include broad consent for future use in research protocols and informed consent forms?\n\n2. (How) do community members, research participants, research teams, ethics committee members, and regulators understand broad consent and what it means for data, privacy, and the rights of research participants and their communities?\n\n3. When and how should community engagement to ascertain understanding and acceptance of broad consent for future use and meaningful benefit sharing be undertaken?\n\n4. What are the knowledge, attitudes, and practices (KAP) of ethics committees in relation to broad consent?\n\n5. What are the challenges faced by researchers when requesting ethics approval for broad consent for future use of samples and data?\n\n6. How do ERCs evaluate whether investigators have adequately assessed community preferences related to broad consent for future use and benefit sharing?\n\n7. What (ongoing) role should the community/research participants play in the development of governance for sharing different data types (genetic data, identifiable or de-identified clinical-epidemiological data, samples)?\n\n8. What are the recommendations for developing and assessing participants’ understanding of language around broad consent for future use?\n\n9. What options should be provided when presenting broad consent for future use to research participants (tiered, opt in/out)?\n\n10. Should there be a consistent approach to seeking broad consent for future use or does each research project need to determine the best process/language for their research protocol/population? Why/why not?\n\n11. What considerations inform views about whether broad consent is needed for sharing de-identified data/data that are not re-identifiable?\n\n12. How can researchers balance funder and journal requirements for data sharing and institutional requirements around the language for broad consent for future use with community values and preferences for data and sample reuse?\n\n13. What are the competing or conflicting requirements, ethical concerns, risks/benefits, and preferences from different stakeholder groups when considering broad consent for future use? What guidance exists for resolving conflicts or power imbalances in responding to stakeholder priorities?\n\n14. How do researchers navigate the interaction between broad consent for future use, the public health rationale or funder/journal requirements to share data, and research participant and research team member trust?\n\n\nStage II: Identifying relevant studies\n\nIn order to identify studies relevant to the subject of this review, we will systematically search the following electronic databases: Ovid (Medline), Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Web of Science. Search strategies include a combination of text and MeSH terms, tailored for each database. Searches will be limited to published papers from the year 2000 onward in order to capture recent broad consent-related studies but will not be restricted by geography or language of publication. We will also search Google and the websites of national or international bodies whose work focuses on data sharing for guidance documents (e.g. Wellcome Trust, Bill and Melinda Gates Foundation, CIOMS). Search results from each database will be imported to EndNote X9 and duplicates will be removed. We will apply snowball sampling wherein we search the reference lists of included studies or related systematic reviews for additional eligible studies. If warranted, the search will be updated and reconducted to further explore emerging research questions. The search strategy for each database is presented in Table 1.\n\nCINAHL=Cumulative Index to Nursing and Allied Health Literature.\n\n\nStage III: Study selection and eligibility criteria\n\nCitations will be exported to EndNote for deduplication. Title and abstract screening will be conducted independently by two reviewers. Review of full texts for inclusion will be conducted independently by two reviewers and may result in changes to the inclusion criteria, in accordance with the iterative nature of a scoping review. Title-abstract screening, full text screening, and data extraction will be managed in Covidence19. Inter-reviewer disagreements in the title-abstract or full text screening will be resolved by a third reviewer.\n\nIncluded studies will be published from 2000 onwards and will report on primary research with research participants or stakeholders (e.g. policy makers, researchers, ERC committee members) or guidance documents from international bodies and guidelines development groups, e.g., the WHO, the Pan American Health Organization, CIOMS, Wellcome Trust, the Global Research Collaboration for Infectious Disease Preparedness, Canadian Institutes for Health Research, Bill and Melinda Gates Foundation, and National Institutes of Health. Studies may apply qualitative, quantitative, or mixed methods approaches to understanding broad consent for future use. Quantitative studies may include interventions or observational studies.\n\nSystematic reviews, commentaries, and editorials or other forms of research that do not present primary research or reflect the opinions of a larger stakeholder group will be excluded. Editorials and commentaries represent the opinions of individual authors rather than an internationally recognized group or guidelines development organization. Primary studies that report on data sharing preferences or practices but that do not specifically address broad consent for future use and guidance or research from fields that are not related to public health will be excluded. Lastly, manuscripts and guidance published prior to 2000 be excluded.\n\n\nStage IV: charting the data\n\nWe will develop and pilot a charting form to facilitate the descriptive synthesis of scoping review findings prior to beginning the charting process. We will extract the key data detailed in Table 2 from included studies to facilitate the summary of available guidance and existing gaps in the literature. We will pilot the data extraction forms using two-three full text articles. Data extraction and charting will completed independently by two reviewers, in keeping with best practice. We will not include an assessment of the quality of included studies. The full text of included studies will be exported to MAXQDA 202020 for thematic analysis using deductive codes and inductive codes that emerge from the review of full text articles.\n\n\nStage V: collating, summarising and reporting results\n\nResults from the data charting work will be presented in narrative form. A PRISMA flow diagram will be used to report the final number of included studies in the review and basic study information will be presented in table format. The results summary will present both quantitative and qualitative aspects of included studies.\n\n\nStage VI: consultation, patient & public involvement\n\nA group of experts in the field of data sharing and broad consent for future use will inform the analysis and summary of findings and assist with the dissemination of any recommendations. Research findings will be shared publicly through a webinar and through the websites of partner institutions.\n\n\nEthics and dissemination\n\nNo ethical approval is required for this systematic review as published articles, rather than primary data, will be used in the analyses. Findings will be presented in an Open Access publication and disseminated on the COVID-19 Clinical Data Consortium’s website and through Twitter.\n\n\nStudy status\n\nThe current study status and anticipated timeline are presented in Table 3.\n\n\nConclusion\n\nThe global coronavirus disease 2019 (COVID-19) pandemic has foregrounded the public health importance of sharing participant-level data and samples. In response to the urgent need for supplementary guidance to support the inclusion of broad consent for future use in research protocols and informed consent forms, we will review and summarize empirical research and guidance related to broad consent for future use of data or samples collected in health-related research. By applying best practice to a rapid scoping review, we hope to facilitate the ethical and equitable application of broad consent for future use in the public health research response to COVID-19.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nKnight G: Funder requirements for data management and sharing. 2012. Publisher Full Text\n\nTaichman DB, Sahni P, Pinborg A, et al.: Data sharing statements for clinical trials: a requirement of the International Committee of Medical Journal Editors. Lancet. 2017; 389(10086): e12–e14. PubMed Abstract \| Publisher Full Text\n\nChristian TM, Gooch A, Vision T, et al.: Journal data policies: Exploring how the understanding of editors and authors corresponds to the policies themselves. PLoS One. 2020; 15(3): e0230281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPisani E, Aaby P, Breugelmans JG, et al.: Beyond open data: realising the health benefits of sharing data. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrady C: Enduring and Emerging Challenges of Informed Consent. N Engl J Med. 2015; 372(9): 855–62. PubMed Abstract \| Publisher Full Text\n\nGrady C, Eckstein L, Berkman B, et al.: Broad consent for research with biological samples: workshop conclusions. Am J Bioeth. 2015; 15(9): 34–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n(CIOMS) CfIOoMS: International ethical guidelines for health-related research involving humans. Geneva, Switzerland. 2016. Reference Source\n\nCheah PY, Jatupornpimol N, Hanboonkunupakarn B, et al.: Challenges arising when seeking broad consent for health research data sharing: a qualitative study of perspectives in Thailand. BMC Med Ethics. 2018; 19(1): 86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPisani E, AbouZahr C: Sharing health data: good intentions are not enough. Bull World Health Organ. 2010; 88(6): 462–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMunn Z, Peters MDJ, Stern C, et al.: Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Med Res Methodol. 2018; 18(1): 143. PubMed Abstract \| Publisher Full Text \| Free Full Text\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\nTricco AC, Lillie E, Zarin W, et al.: Prisma extension for scoping reviews (prisma-scr): Checklist and explanation. Ann Intern Med. 2018; 169(7): 467–473. PubMed Abstract \| 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\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015; 4(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCovidence: Covidence Systematic Review Software. Melbourne, Australia; 2017.\n\nPlus M: MAXQDA. Berlin, Germany: VERBI Software GmbH; 2020." }	[ { "id": "79603", "date": "01 Mar 2021", "name": "David Osrin", "expertise": [ "Reviewer Expertise Public health", "ethics of data sharing" ], "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 protocol for a scoping review is concise, clear, and timely. The authors will follow recommended guidelines for scoping reviews.\nGeneral comments\nOne thing that strikes me — and will certainly have been discussed by the authors— is the inclusivity of the search terms. Adding search terms sequentially reduced the returned list to as few as 2 or 0 publications, whereas keeping the search broad returned manageable numbers under 100.\nSpecific comments:\nAbstract\nThe first sentence doesn’t quite work: “Broad consent for future use is the reuse of data and/or samples collected by a study by researchers who may not be affiliated with the original study team for purposes that may differ from the objectives of the original study.” The sentence is saying what is meant by future use, not broad consent for it.\n\n“…participants time and efforts…” Apostrophe “participants’”\nBackground\nP3 column 2 line 1: there is an extra “and”\n\nP5 Stage 4: “Data extraction and charting will completed:” missing “be”\n\nP5 Stage 4: “We will not include an assessment of the quality of included studies.” I’d suggest a sentence saying why not.\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": "81730", "date": "16 Apr 2021", "name": "Limbanazo Matandika", "expertise": [ "Reviewer Expertise My expertise is in research ethics. Areas include", "biobanking", "informed consent (communication strategies and various models of Informed consent)." ], "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 this scoping review that’s aims at summarizing existing guidance on broad consent for future use and highlight evidence gaps related to the ethical, equitable implementation of broad consent for future use is reasonably designed and a potentially important contribution to the literature. That said, there is a shortcoming in one of the strategies for data collection stage VI of the protocol there is no clarification whether this is part of the methods or a procedure. The author needs to clearly state if the consultation, patient and public involvement aspect is aimed at collecting data as part of confirming the findings or not? There is need for a more comprehensive outline of how this will be achieved.\nRevisit the definition of Broad consent in the first sentence of the abstract. It seems there is a mix-up on what broad consent is and its benefits.\nThe strongest points in this article is the use of the PRISMA-P guidelines that has outlined 5 stages for data collection in the scooping exercise. The methods do not only outline literature review aspects but incorporate engagement with relevant stakeholders in the practice. Involvement of stakeholders as part of data collection will allow the validation of the findings from literature and outline the grey areas in the use of Broad Consent in research.\nThe paper though could have been strengthened much better if the data collection method included exploring of perspectives from sample donor’s themselves especially in times of a global pandemic like COVID-19. Literature on sample use may provide insights on regional epidemic/pandemics. COVID-19 is a global pandemic hence it's very critical to understand how perspectives on use of sample could differ. Therefore, the researcher may need to consider how insights from a wider perspective would be relevant to the global pandemic.\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/10-102
https://f1000research.com/articles/10-98/v1	11 Feb 21	{ "type": "Research Article", "title": "Limited containment options of COVID-19 outbreak revealed by regional agent-based simulations for South Africa", "authors": [ "Andreas Bossert", "Moritz Kersting", "Marc Timme", "Malte Schröder", "Azza Feki", "Justin Coetzee", "Jan Schlüter", "Andreas Bossert", "Moritz Kersting", "Marc Timme", "Malte Schröder", "Azza Feki", "Justin Coetzee" ], "abstract": "Background: COVID-19 has spread from China across Europe and the United States and has become a global pandemic. In countries of the Global South, due to often weaker socioeconomic options and health care systems, effective local countermeasures remain debated. Methods: We combine large-scale socioeconomic and traffic survey data with detailed agent-based simulations of local transportation to analyze COVID-19 spreading in a regional model for the Nelson Mandela Bay Municipality in South Africa under a range of countermeasure scenarios. Results: The simulations indicate that any realistic containment strategy, including those similar to the one ongoing in South Africa, may yield a manifold overload of available intensive care units. Only immediate and the most severe countermeasures, up to a complete lock-down that essentially inhibits all joint human activities, can contain the epidemic effectively. Conclusions: As South Africa exhibits rather favorable conditions compared to many other countries of the Global South, our findings constitute rough conservative estimates and may support identifying strategies towards containing COVID-19 as well as any major future pandemics in these countries.", "keywords": [ "COVID-19", "South Africa", "Countermeasure", "Agent-based Simulations", "Public Health" ], "content": "Introduction\n\nThe first cases of the coronavirus disease COVID-19 were confirmed on December 29, 2019 in China1. Due to high contagiousness2, the disease has spread rapidly3,4 and by April 2020 the World Health Organization (WHO) had confirmed5 about 1.6 million cases worldwide. Consequently, the COVID-19 epidemic was declared a pandemic as it started to impact economies worldwide. However, epidemiological expertise lagged behind the exponential spread in this specific situation. In an attempt to contribute to the scientific assessment of the COVID-19 pandemic, we quickly derived first results and were able to make first predictions for the South African COVID-19 development. The significance of timely countermeasures could be seen in the development of infections in the United States, where cases surged from 1,678 to 307,318 between March 16, and April 6, 20206,7. Therefore, our research applied agent-based simulations to assess the effectiveness of countermeasures at an early stage of the COVID-19 pandemic and the following paper displays the state of literature and infection figures for the period of March and April 2020.\n\nMost countries of the Global South were affected as well, often with reported case numbers just beginning to surge8. However, the reported low numbers in April, e.g. about 9000 cases in all of Africa, may be biased as only few individuals have been tested, see9 for the example of South Africa, and also10. Furthermore, such countries operate under vastly different socio-economic conditions, with larger social inequality, distinct transportation options and weaker health care systems compared to those of most countries in the global north11,12,13, such that core characteristics of potential COVID-19 spreading dynamics and thus the effectiveness of specific countermeasures remain largely unknown for countries of the Global South.\n\nThe transmission of COVID-19 is currently thought to occur through direct inter-person droplet-based infections through coughing and sneezing, with possible additional infection paths through aerosols and via contaminated surfaces. Symptoms often occur only after an incubation period of several days, with many infected responding entirely asymptomatic. Contagion may also occur via such symptom-free carriers, posing a challenge for tracing and estimating spreading patterns14. Transmission predominantly occurs through interactions while being at home, being at work, using shared transportation modes or during group-based leisure activities. In many countries of the Global South, most of these social activities occur under conditions drastically different from those in, for example, Europe or the U.S.. For instance, these countries feature, on average, more people per household, higher unemployment rates, more manual and lower payed work15,16 and much of publicly available transportation services used by the middle and low-income population are offered as paratransit shared-mobility services17,18. Moreover, public health care services are different and often less well equipped than in countries of the Global North13,19.\n\nAs a paradigmatic region offering large-scale data availability, we consider the Nelson Mandela Bay Municipality (NMBM) in South Africa to study scenarios of COVID-19 spreading dynamics and the impact of countermeasures. We combine socio-economic and travel survey data from more than 100,000 people20, about 10% of the local population, based on employment status, household size, age group and income level together with a detailed 24-hour travel diary component integrated into an agent-based traffic simulation (see Methods). The resulting contact network forms the basis for extended Susceptible-Infected-Recovered (SIR) model dynamics with parameters adapted to COVID-1921. Such agent-based simulations capture the inhomogeneity among the agents and are capable of modeling intricate nonlinear dynamic relationships between them, including a spreading rate that depends on the individual agent’s detailed activities, their modes of transportation used, and their distance to each other22.\n\nTo evaluate the impact of various policy measures on the course of the disease, we systematically compare several scenarios by varying simulation parameters accordingly (see Methods). First, a baseline scenario without any countermeasures; second, a default scenario in line with the current measures implemented in South Africa as of early April 202023. These include the shutdown of childcare and educational institutions, the prohibition of leisure activities of any kind and cutting shopping activity options by about 70%. Moreover, work related and “other” activities (for example trips to health care facilities and visits to public institutions) and travelling as passenger in a car are reduced by 80% and formal and informal public transport is reduced by 30%. Third, as a harsher variation of the lockdown that may be achievable in principle, we study the effect of a realistic lockdown scenario, increasing the restriction of activities related to work, shopping, leisure, and “other” by 90% while childcare, educational activities as well as formal and informal public transport are completely shut down. Finally, we consider a theoretical complete lockdown where all travel and outside activities are prohibited. All countermeasures come into effect 7 days after an initial infection of 100 people. We furthermore investigate additional simulations that start with the (currently enacted) default measures and introduce the realistic lockdown scenario after a number of days. Our results may inform and complement the ongoing discussion around tightening, loosening, introducing or repealing certain countermeasures.\n\n\nMethods\n\nFor the transport simulation, we employ MATSim version 12.0-2019w48-SNAPSHOT24. The analysis relies on the population data file provided by Joubert20. It processes travel diaries from the 2004 Travel Survey to compute a synthetic population sample of the Nelson Mandela Bay Municipality (NMBM). Table 1 outlines the descriptive statistics of the synthetic agents and the modal split. It becomes apparent that the minibus taxis are the backbone of the region’s transport system. Private transport such as passenger cars play a rather marginal role. This difference underlines the interdependence of geographical, historical and economic characteristics of the region.\n\nThe 114,346 agents represent a 10% sample of the total population of NMBM. The trip distribution by transport mode shows the high prevalence of low cost transport modes (walk and minibus taxi) compared to private car usage. (Data from20,25).\n\nWe employ the Demand Responsive Transport (DRT) framework for MATSim26 to include the informal minibus taxi transit. In the underpinning MATSim, agents walk to a bus stop and request a DRT vehicle (in this case a minibus taxi). Their ride is pooled with rides of other agents with similar destinations. The simulated routing is more flexible than in reality, since the minibus taxis operate on a stop-based system with routes. Due to a lack of data, this mode is implemented based on a door-to-door based operating scheme and pick ups waiting passengers from the stops. Consequently on the one hand the vehicles are less frequented but on the other hand travel longer distances with passengers on board to pick up customers in the city. This reduces the likelihood of being on a minibus with an infected person, but increases the contact time if an infected person is actually on board. The formal bus transport services in NMBM are provided by Algoa Bus Company27. Currently, all formal bus operations are suspended in NMBM. It can therefore be assumed that customers are substituting regular bus services with minibus taxis. The MATSim population file is modified accordingly.\n\nDue to the 10% population sample, the total capacity of the minibus taxis vehicles must be adapted to reflect the proportions in reality. In 2014, a total of 2,374 minibus taxis operated in NMBM with an average capacity of 15 persons25. As scaling the capacity of minibus taxis to 1.5 passengers would strongly underestimate the infections during minibus taxi trips, several test scenarios with different fleet sizes and passenger capacities were carried out.\n\nDue to the operating scheme of door-to-door DRT a reduction either in fleet size and/or passenger capacity of the vehicles would lead to a high rejection rate and accordingly high infection numbers at home facilities that could bias the simulation results. For this reason, the number of vehicles is chosen with respect to the trade-off between capacity utilisation and the rejection rate. A fleet of 2,374 vehicles with a capacity of 15 optimises these criteria and is introduced into the model. The vehicles are placed randomly in the area at the beginning of the simulation, although in reality they wait for customers at designated places at the beginning of the day and start their tour when a certain degree of occupancy is reached.\n\nPublic minibus taxis are assumed to take an important role in the epidemic simulation, as both the probability and the intensity of contact are assumed to be high. Moreover, people with different places of work and residence mix up at this small space.\n\nThe model relies on the MATSim-based Episim-framework to simulate the epidemic spreading in the research area. The following briefly summarises both the functionality of the default Episim configuration and the parametric adaptions to the NMBN. It is important to note that the package is still in a early stage of development. For the following simulations, we used the latest version dated April 11, 2020 from the master branch (see https://github.com/matsim-org/matsim-episim/tree/85ca164a472b7f60117a51a365daed1eaf376199).\n\nEpisim is based on a traditional SIR model, which is a common model for the analysis of epidemics28 and has been continuously improved, e.g. by29,30,31. The basic mechanism is that people go through different stages during an epidemic, and have different characteristics with each transition. In short, initially all persons are susceptible for a disease and, over time, become infected with a given probability, partly influenced by individual characteristics. Later on, they recover. The states and transitions are usually extended to a more complex framework and include quarantine, seriously sick and critical patients in order to account for policy measures and the need for either hospital beds or ICUs.\n\nThe infection process is based on a probabilistic model and occurs in “containers”. These containers represent locations where several agents may interact, such as households, workplaces or transport vehicles, and are computed based on the information from NMBM MATSim simulation output event data. These chronicle all trajectories covered by agents during the day and the vehicles and facilities they visited and stayed at. Once a susceptible and a contagious agent stay in the same container, an infection occurs with a certain probability, which is described by equation 1 (see below).\n\nAt the beginning of the simulation, 10 randomly selected agents (corresponding to 100 people) are initially infected. After 7 days of uncontrolled spread, various countermeasures are implemented. We consider the dynamics until the countermeasures are in effect for 60 days.\n\nIn Episim, infected agents undergo several state transitions before their infection is terminated. Since not all humans suffer equally from COVID-19, the agents follow different paths from infection to recovery. In the beginning of the simulation, all agents except the ”Patients Zero” are initially healthy and thus susceptible. After an infection, an agent’s state changes to infected but not contagious, due to incubation time. With the beginning of the fourth day after an infection, the agent’s state changes to contagious. From this step on, the agent’s differ in their behaviour. In the default scenario, 20% of the agents put themselves into quarantine. The idea of the simulation model is that only a certain share of those suffering from COVID-19 notices symptoms. In addition, it is to be expected that some infected persons will go into public despite symptoms. The self-quarantine lasts 14 days and is assumed to mitigate social contacts completely, even within the household. 4.5% of all infected agents become seriously sick on day 10 and of these, 25% become critical the following day. In this way, a distinction is made between patients who require regular medical care and those who are dependent on ICUs. Contagious agents recover 16 days after infection, whereas the infection of patients with severe conditions terminates after 23 days.\n\nIn reality, an infection would end by recovery or death. For usability reasons, every infected agent recovers in Episim. As recovered agents are assumed to be immune and no longer contagious, omitting death does not bias the further course. The same mechanism is applied for self-quarantined, who remain mobile in the simulation but are neither contagious nor susceptible.\n\nThe probability of agent n becoming infected at time t when leaving a container is given by\n\nMost of the contact intensities were left at their default since it is reasonable to assume that the contact intensities of most activities do not differ significantly from the values determined by21. The activities work, shopping, dropby and other were introduced so that the first two have the same contact intensity as leisure (5) and the other two take the values 7 and 3. It is assumed that due to the available space and seating arrangements in the minibus taxis, the contact between passengers in the minibus taxis is much higher than in formal public transport in Berlin. Since no reliable data on the contact intensity of passengers are available, we take the interaction intensity to be 20, compared to 10 in the default setting. Likewise, the contact intensity of being at home is doubled from 3 to 6 due to limited space, larger households and general living conditions.\n\nThe countermeasures in the default setting, realistic lockdown and complete lockdown are described by modifiers to the activity rates of the agents, describing the reduction in the frequency of the respective activity. Table 2 lists all parameters for the scenarios studied.\n\nAllowed share per activity in four scenarios. Episim allows to prohibit or restrict the practice of certain activities in order to simulate social distancing. Four sets of restrictions were implemented in the simulations. While in the baseline scenarios no measures are in power, a complete lockdown does not allow the population to leave their homes. Between those extreme cases, two different partial lockdowns are set up.\n\n\nResults and discussion\n\nDirect model simulations in the four scenarios (Figure 1) reveal that a large fraction of the population becomes infected, not only for the baseline scenario without countermeasures (as expected), but also with the default countermeasures active as of this writing (April 11 2020), see Figure panels 1A and 1B. Even if scenarios of harsh but realistically possible or complete lockdown were enacted only seven days after reaching 100 infected people, the outbreaks would still become macroscopic, with tens of thousands of infected people (Figure 1C and 1D) in the NMBM alone. Across all scenarios enacted, outbreak dynamics also causes overload of the approximately ICU beds estimated as baseline for NMBM (Figure 1E,F). As a precondition for successful containment, lockdown needs to be implemented sufficiently early on (in particular less than 7 days after the first 100 infected, not the first 100 confirmed infected).\n\nDynamics for the Nelson Mandela Bay Municipality, Eastern Cape, South Africa, with a total population of 1.15 million people. (A-D) Evolution of the number of infected (dotted lines), seriously sick (dashed) and critical (solid) patients in four different scenarios: (A) baseline without any countermeasures, (B) with default countermeasures in line with current conditions in South Africa, (C) with harsh realistic countermeasures shutting down 90% of all activities, and (D) with complete lockdown prohibiting any movement and group activities (infections within a household are still possible, see Supplementary Material for details). All countermeasures are initiated 7 days (grey shading) after the an initial infection of 100 people. (E) and (F) Number of critical cases requiring intensive care compared to the available ICU capacity (horizontal dashed line) for all four countermeasure scenarios. None of the scenarios reduces the number of critical cases sufficiently to guarantee intensive care (ICU) treatment for all critical patients.\n\nIn all four scenarios, the outbreak strongly overloads the available intensive care unit (ICU) beds, with ten- to 100-fold overload in default and baseline scenario, respectively (see Figure 2A). Out of the total 267 ICU beds available in (public and private) hospitals in the whole Eastern Cape Province (data from 2008,32), only about 50 would be available for critical cases from the NMBM (scaled proportional to the relative population counts in 201133,34). We additionally quantify the sustained pressure on the health care system by computing the cumulative overload λ of the health system as the total number of person days critical patients go without intensive care (Figure 2B),\n\nThe potential severity of the outbreak in NMBM even with strong countermeasures, is especially evident when compared to that of the United Kingdom (UK) (Figure 2C). In the UK, ICU capacity would be exceeded by a factor of about 35 without any countermeasures, by a factor of about 2 with social distancing (similar to default in our simulations) and not at all with measures currently enacted35. In our NMBM scenario simulations, ICU capacity would be exceeded by a factor of more than 200 without any countermeasures, by factor of about 20 with default measures, by a factor of 5 with realistic lockdown and still by a factor of 3 even with complete lockdown. These numbers underline the strong fragility of the health care services by example of the NMBM. We expect similar orders of magnitude of capacity exceedance throughout South Africa and in many countries of the Global South.\n\n(A) Maximum number of concurrent critical patients during the peak of the outbreak. The available ICU capacity (dashed line) is widely exceeded in all scenarios. (B) The measure λ for the sustained overload, counting the total number of person days that critical patients are without intensive care. (C) Relative exceedance of the ICU capacity at the peak of the outbreak. The potential consequences of COVID-19 in countries of the Global South becomes clear from the comparison to UK estimates35 with about 35-fold overload without interventions (UK baseline, upper dotted line) and no overload with UK lockdown (lower dotted line) enforcing school and university closure, case isolation and general social distancing. Note the logarithmic vertical axes in all panels to make vast case number differences visible simultaneously with the capacity.\n\nTherefore, all realistic scenarios, including the default scenario currently enacted in South Africa, hardly seem capable of containing the COVID-19 pandemic without substantially overloading ICU availability. Our results suggest that such overload could even be expected if all the 267 ICU beds in both private and public hospitals within the entire Eastern Cape Province would be exclusively available for critical COVID-19 patients in the Nelson Mandela Bay Municipality only.\n\nRealistic or complete lockdown offer options of greatly reducing the pressure on the health care system if enacted rapidly enough. However, economic boundary conditions and in particular the large inequality in South Africa – and similarly in many other countries of the Global South – pose additional problems. Thus, in our opinion, in particular complete lockdown seems hardly enforceable and would appear unsustainable especially for a large share of people with lower income, as already weaker containment will likely lead to severe economic consequences in addition to disease related deaths36,37.\n\nHow can the number of critical cases be confined given that the default scenario is already enacted? On April 9th, 2020, the number of cases reported in NMBM was 3038. As the number of reported cases seem to systematically underestimate their actual number by at least a factor of three, as predicted for Austria on April 10, 202010, we estimate that on 9th of April, at least 100 cases existed in NMBM and take that as our initial condition. Starting simulations with the default scenario active, we find that critical cases are likely to vastly exceed the available ICU capacity (Figure 3A) within two months. In contrast, introducing the realistic lockdown scenario immediately, i.e. starting April 13th, might support a successful confinement of the number of critical COVID-19 patients in NMBM to manageable numbers (Figure 3B). In this scenario, the simulations suggest that there is about 80% likelihood that ICU capacity becomes overloaded at some time and that ,if overload occurs, it will be relatively mild (factor of 2-3 of the capacity, in contrast to factor of about 30 when keeping the default measures as they are).\n\nTen stochastic realizations of agent-based simulations (gray lines in (A) and (B)) show increasing typical number of critical cases, quantified by the ensemble mean (dark) and median (light). (A) Stochastic dynamics of the number of critical cases when keeping the currently active default measures in NMBM. The ICU capacity is exceeded in almost all realizations within two months. (B) Switching from default (currently roughly enacted in South Africa) to the realistic lockdown scenario, limiting all activity by 90%, four days after crossing 100 infected people (estimated from 30 actually reported cases) in NMBM (that is on April 13, 2020), yields an exceeded ICU capacity in 80% of realizations, at most five times above the available capacity. (C) Relative exceedance of available ICU capacity in both scenarios. Keeping a default would cause roughly 3000% overload whereas enacting a stricter lockdown on April 13th substantially reduces expected overload to less than 300% and may completely avoid an overload in best cases.\n\nWe remark that the success of such lockdown countermeasures crucially relies on several restrictions and our simulations likely under- rather than overestimate future case numbers. First in our simulations, activities are immediately reduced to the set low values (complete shutdown of public transport, child care and educational facilities, 90% reduction of all other activities) and all inhabitants fully comply with these restrictions. Second, our simulations are based on estimated parameters that thereby come with uncertainties, and stochastic dynamics may create large deviations from the predicted values, in particular also earlier growth and larger total number of critical patients, not last due to an exponential growth of the outbreak in its initial phase without severe lockdown (compare39). Third, our estimates assume that all ICU beds would be available exclusively for COVID-19 patients during the entire time of the outbreak. Those and other constraints call for a more conservative, especially earlier introduction of realistic countermeasures.\n\nWe provide additional measures recorded from simulations on the location of infection events and exact values of the health care system overload in Tables 3 and 4, respectively.\n\nEach event represents an initial infection of one out of 114.346 total agents. While most infections occur at home due to prolonged and close contact, paratransit in terms of minibus taxis is the second largest driver of the spreading process, accounting for approximately 15% of all infection events in the baseline scenario. These numbers decrease under countermeasures and almost disappear under complete lockdown, where only a few people are initially infected before all travel is stopped. A comparison to artificial settings where infections during minibus transit are neglected demonstrate the importance of the minibus taxi service as a driver for the spreading, in particular under partial lockdown.\n\nDuring the first 6 days, the virus spreads uncontrolled. On day seven, countermeasures are initiated that restricts certain activities (see 2). The upper values result from simulations in which minibus taxis are possible locations of infection (presented in the main manuscript). The lower values are drawn from simulations, in which people could not become infected in minibus taxis (compare Table S3).\n\nAcross all scenarios studied, the results thus indicate that it may be hard to enact realistic, socially and economically feasible countermeasures without exceeding ICU capacity and that more drastic measures beyond the current default are rapidly needed. Finally, it seems reasonable to assume that the consequences of countermeasures would be qualitatively the same across South Africa as well as many countries of the Global South.\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 thank Hanne Schlüter for her support during this time.\n\n\nReferences\n\nLi Q, Guan X, Wu P, et al.: Early transmission dynamics in wuhan, china, of novel coronavirus–infected pneumonia. N Engl J Med 2020; 3820 (13): 1199–1207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanche S, Lin YT, Xu C, et al.: The novel coronavirus, 2019-ncov, is highly contagious and more infectious than initially estimated. arXiv preprint arXiv:2002.03268 2020. Publisher Full Text\n\nHufnagel L, Brockmann D, Geisel T: Forecast and control of epidemics in a globalized world. Proc Natl Acad Sci U S A 2004; 101(42): 15124–15129. 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Publisher Full Text\n\nNeumann A, Röder D, Joubert WJ, et al.: Towards a simulation of minibuses in south africa. Journal of Transport and Land Use 2 2015; 8(1): 137–154. Publisher Full Text Reference Source\n\nBischoff J, Maciejewski M, Nagel K: City-wide shared taxis: A simulation study in berlin 2017 IEEE 20th International Conference on Intelligent Transportation Systems (ITSC) 2017; pages 275–280. Publisher Full Text\n\nAlgoa Bus Company: Official Homepage.2020Reference Source\n\nKermack WO, Mc Kendrick AG, Walker GT: A contribution to the mathematical theory of epidemics. Proceedings of the Royal Society of London. Series A, Containing Papers of a Mathematical and Physical Character 1927; 115(772): 700–721. ISSN 0950-1207. Publisher Full Text\n\nKeeling M, Rohani P: Modeling infectious diseases in humans and animals Princeton: Princeton University Press; 2008. ISBN 9780691116174. Reference Source\n\nAnderson RM, May RM: Infectious diseases of humans: dynamics and control Oxford: Oxford Univ. Press; 2010. ISBN 9780198540403.\n\nRoche B, Drake JM, Rohani P: An agent-based model to study the epidemiological and evolutionary dynamics of influenza viruses. BMC Bioinformatics 2011; 12: 87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaidoo K, Singh J, Lalloo U: A critical analysis of icu/hc beds in south africa: 2008-2009. S Afr Med J 2013; 103(10). ISSN 2078-5135. PubMed Abstract \| Publisher Full Text Reference Source\n\nWikipedia: Eastern cape.2020Reference Source\n\nWikipedia: Nelson mandela bay municipality.2020Reference Source\n\nFerguson N, Laydon D, Nedjati Gilani G, et al.: Report 9: Impact of non-pharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand.Reference Source\n\nDepartment: Statistics South Africa - Republic of South Africa: Mid-year population estimates.2019Reference Source\n\nDepartment: Statistics South Africa - Republic of South Africa: Work & Labour Force.2019Reference Source\n\nNomazima Nkosi - Herald LIVE: Nelson mandela bay covid-19 cases continues to rise.2020-04-09Reference Source\n\nMaier BF, Brockmann D: Effective containment explains subexponential growth in recent confirmed covid-19 cases in china. Science 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "88528", "date": "23 Jul 2021", "name": "Cliff C Kerr", "expertise": [ "Reviewer Expertise COVID-19", "epidemiology", "mathematical modeling" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an agent-based model of COVID-19 in Eastern Cape, South Africa, during the very early part of the epidemic (March - April 2020). They \"predict\" that the health system will be overwhelmed even with a strict lockdown. My main comments on the paper are (a) South Africa is towards the end of its third wave, so it is unclear what the significance of this analysis of the first wave is; (b) the predictions made by the model, though they were reasonable as of April last year, do not seem to closely match what actually happened.\nSpecific comments:\n1. The introduction contains a number of statements that are now quite outdated (e.g. \"The transmission of COVID-19 is currently thought to occur through direct inter-person droplet-based infections through coughing and sneezing\").\n2. The introduction discusses SIR models, but COVID is typically modeled as SEIR, and indeed the methods later refer to an incubation time, so it seems an SEIR model is being used.\n3. It's unclear the extent to which long-distance travel contributes to COVID spread. Infection events tend to happen locally (e.g., at restaurants and in multi-generational houses). Thus, the modeling approach used is not obviously capturing the most important features of population contact networks.\n4. Was it really the case in South Africa that private car travel reduced by 80% and public transport by 30%? In other countries, these proportions were roughly reversed.\n5. The use of survey data from 2004 is likely to have some limitations, such as due to the growth Port Elizabeth has seen in that time (according to Wikipedia, by roughly a factor of 4, though that may not be a reliable source). Can you cross-validate with e.g. Google mobility data?\n6. The authors state \"Public minibus taxis are assumed to take an important role in the epidemic simulation\". This is a reasonable assumption, but I do not think subsequent data support this (i.e., whether there is a statistically significant correlation between minibus use and infection rates, after controlling for population density).\n7. Sources should be provided for the disease duration and severity parameters; ideally distributions, rather than point estimates, would be used. Similarly, the values of all parameters in Eq. (1) should be provided.\n8. The epidemic dynamics shown in Fig. 1 seem to have much too short of a timescale, and do not reflect what actually happened. Data on cases, deaths, and (if available) hospitalizations should be plotted on these figures for comparison. For example, the first wave lasted roughly five times as long as that shown here. In addition, ICUs were never overwhelmed during the first wave1.\n\n9. The fact that the model predicted that South Africa would experience a much worse first wave than the UK, when in fact the opposite occurred, would benefit from further discussion.\n10. The authors are encouraged to obtain review and validation of inputs and results by collaborating with an epidemiologist or other public health official who has local expertise in the Eastern Cape region.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "93806", "date": "05 Oct 2021", "name": "Theresa Marié Rossouw", "expertise": [ "Reviewer Expertise Infectious Diseases Immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-conducted simulation of the impact of various COVID-19 containment options in the Nelson Bay Municipality in South Africa. While the article considers containment strategies at the beginning of the COVID-19 pandemic, it could still have important implications for public health measures aimed at containment of subsequent waves of the pandemic.\nThe title should be delimited since data from only one municipality (and not the entire South Africa) were used.\nReference 5 - World Health Organization: Q&A on COVID-19, HIV and antiretrovirals.2020 – does not seem appropriate for this statement: “by April 2020 the World Health Organization (WHO) had confirmed5 about 1.6 million cases worldwide”.\n“Consequently, the COVID-19 epidemic was declared a pandemic as it started to impact economies worldwide.” - This sentence seems to suggest that a pandemic is declared when economies are impacted, but a pandemic is defined as “an epidemic occurring worldwide, or over a very wide area, crossing international boundaries and usually affecting a large number of people” Porta, M. A Dictionary of Epidemiology (Oxford University Press, USA, 2008).\n\n“The significance of timely countermeasures could be seen in the development of infections in the United States, where cases surged from 1,678 to 307,318 between March 16, and April 6, 20206,7.” - This sentence seems to suggest that the United States had timely countermeasures in place, but this is not evident from the number of infections. Do the authors rather mean that the significance of the absence of timely countermeasures is evident in these figures?\n\nThe reference source of the following reference “World Health Organization: Global research on coronavirus disease (COVID-19). 2020” does not seem appropriate for the following statement: “Most countries of the Global South were affected as well, often with reported case numbers just beginning to surge”.\n“current measures implemented in South Africa as of early April 202023. The reference is not freely available and the article is titled: “Fikile Mbalula relaxes Covid-19 regulations for taxi industry”. - Since it is difficult to see how the following facts could have been derived from this article, a different source should be considered (such as, Disaster Management Act: Regulations to address, prevent and combat the spread of Coronavirus COVID-19: Amendment) and an explanation of the percentage reductions should be provided: “These include the shutdown of childcare and educational institutions, the prohibition of leisure activities of any kind and cutting shopping activity options by about 70%. Moreover, work related and “other” activities (for example trips to health care facilities and visits to public institutions) and travelling as passenger in a car are reduced by 80% and formal and informal public transport is reduced by 30%.”\nPlease amend the word “currently” so that it is clear that it refers to the situation during March/ April 2020 rather than at the present moment.\n\n“Third, as a harsher variation of the lockdown that may be achievable in principle, we study the effect of a realistic lockdown scenario”. - I do not think that the term “realistic lockdown” is the best one to use since the authors themselves state that this may not be achievable in practice, thus making it unrealistic. Would another term, such as “idealistic”, “harsher” or “more restrictive”, not be more appropriate?\n“As scaling the capacity of minibus taxis to 1.5 passengers would strongly underestimate the infections during minibus taxi trips, several test scenarios with different fleet sizes and passenger capacities were carried out.” - Since the restrictions operative during the time of the study was a limitation to 70% of capacity, did the authors specifically consider this scenario?\n“Moreover, people with different places of work and residence mix up at this small space” – please change “at” to “in”.\n“Most of the contact intensities were left at their default since it is reasonable to assume that the contact intensities of most activities do not differ significantly from the values determined by21”. - The reference provided is “Müller SA, Balmer M, Neumann A, et al.: Mobility traces and spreading of covid-19. Technical report Berlin: Technische Universität; 2020.” This article is still a preprint despite being submitted on 30 March 2020. Are there no published articles to base the epidemic parameters on?\nIt is not clear where some of the epidemic parameters had been derived from. For instance, “4.5% of all infected agents become seriously sick on day 10 and of these, 25% become critical the following day.” and “Contagious agents recover 16 days after infection, whereas the infection of patients with severe conditions terminates after 23 days.” - Do the authors imply that patients with severe disease remain contagious up to 23 days? Appropriate references are important since many of these estimates have changed with the new variants.\n“Across all scenarios enacted, outbreak dynamics also causes overload of the approximately ICU beds estimated as baseline for NMBM (Figure 1E,F).” - The colours for the “realistic lockdown” and “complete lockdown” scenarios are difficult to distinguish in these 2 figures. Also please explain that the same data are presented in the 2 figures but on a different scale.\n“As a precondition for successful containment, lockdown needs to be implemented sufficiently early on (in particular less than 7 days after the first 100 infected, not the first 100 confirmed infected).” - It is not clear where this precondition comes from. It does not appear that any modelling is shown to support this statement.\n\n“Out of the total 267 ICU beds available in (public and private) hospitals in the whole Eastern Cape Province (data from 2008,32)” – from the reference article it seems that there were only 252 (and not 267) ICU beds in the Eastern Cape province in 2008. Since the data are very old, the authors should consider adding that these data are from the last national audit of ICU resources in South Africa.\n“only about 50 would be available for critical cases from the NMBM (scaled proportional to the relative population counts in 201133,34)”. - It would be useful for readers to see the calculations for this estimate. In addition would a more reputable resources, such as the National Government website, not be a more appropriate source of data than Wikipedia?\n“The integral is taken over the entire time of simulation, 64 days after the 100 people are infected in NMBM.” - Please explain why 64 days were selected since it seems to be 67 from the epidemic parameters: “At the beginning of the simulation, 10 randomly selected agents (corresponding to 100 people) are initially infected. After 7 days of uncontrolled spread, various countermeasures are implemented. We consider the dynamics until the countermeasures are in effect for 60 days.”\n\nFigure 3: It would be useful to name figures 3A and 3B as default scenario and realistic lockdown scenario (or whatever new name is selected) respectively.\nTable 3: “KiGa” – please explain the meaning of this abbreviation.\nReference 10: Could the authors please re-look at the reference source since it lands on a search page.\nPlease be consistent with the use of capitals for the words Global North and Global South.\nPlease amend for clarity: “with many infected responding entirely asymptomatic”.\nPlease be consistent with the use of United States and U.S.\nSome of the same authors have also published an article1 making use of the same methodology and should consider citing this article for the sake of transparency, unless they can argue that the methodology was significantly different?\nI could not find the supplemental material that is referred to in the text.\n\nFinally, with the benefit of hindsight, we now know that the ICU’s were not as overwhelmed as predicted by the scenarios presented in this manuscript. The authors should consider and discuss the possible reasons for this.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-98
https://f1000research.com/articles/9-1211/v1	08 Oct 20	{ "type": "Software Tool Article", "title": "CompoundHetVIP: Compound Heterozygous Variant Identification Pipeline", "authors": [ "Dustin B. Miller", "Stephen R. Piccolo", "Dustin B. Miller" ], "abstract": "A compound heterozygous (CH) variant occurs when a person inherits two alternate alleles, one from each parent, and these alleles occur at different positions within the same gene. Therefore, CH variant identification requires distinguishing maternally from paternally derived nucleotides, a process that requires numerous computational tools. Using such tools can be challenging and often introduce unforeseen challenges such as installation procedures that are operating-system specific, software dependencies, and format requirements for input files. To overcome these challenges, we developed Compound Heterozygous Variant Identification Pipeline (CompoundHetVIP), which uses a single Docker image to encapsulate commonly used software tools for phasing, annotating, and analyzing CH, homozygous alternate, and de novo variants in a series of 13 steps. To begin using our tool, researchers need only install the Docker engine and download the CompoundHetVIP Docker image. The tools provided in CompoundHetVIP can be applied to Illumina whole-genome sequencing data of individual samples or trios (a child and both parents), using VCF or gVCF files as initial input. Each step of the pipeline produces an analysis-ready output file that can be further evaluated. To illustrate its use, we applied CompoundHetVIP to data from a publicly available Ashkenazim trio and identified two genes with candidate CH variants and one gene with a candidate homozygous alternate variant after filtering. While this example uses genomic data from a healthy child, we anticipate that most researchers will use CompoundHetVIP to uncover missing heritability in human diseases and other phenotypes. CompoundHetVIP is open-source software and can be found at https://github.com/dmiller903/CompoundHetVIP; this repository also provides detailed, step-by-step examples.", "keywords": [ "Genetics", "compound heterozygous", "genome analysis", "trio", "phasing", "reproducibility" ], "content": "Introduction\n\nA compound heterozygous (CH) variant occurs when a person inherits two alleles, one from each parent, and these alleles are located at different positions within the same gene1. The compound effects of these alternate alleles may lead to phenotypic effects as seen in some cases of human disease, including ataxia telangiectasia, NGLY1 deficiency, and various types of pediatric cancer2–4. For example, CH variants in the mismatch repair gene, MSH6, have been identified in pediatric patients with colorectal cancer, medulloblastoma, high-grade glioma, glioblastoma, non-Hodgkin’s lymphoma, and acute lymphoblastic leukemia4. To detect CH variants in whole-genome sequencing data, it is necessary to differentiate between paternally and maternally derived nucleotides1. Laboratory-based methods such as fosmid-pool-based or linked-read sequencing can be used; however, if DNA libraries are prepared and sequenced without regard to nucleotide inheritance (as is done in most sequencing projects), computational methods can help determine parental inheritance through haplotype estimation (“phasing”)5–7.\n\nAvailable phasing tools require specific input file types (such as VCF or Plink files) and reference files which are not standardized across different phasing software. In addition, many phasing programs require that input files have been aligned to a specific reference genome, do not contain multiallelic positions, are free of repeat positions, and that each chromosome be phased separately8–10. Figuring out how to prepare files for phasing can be challenging as passing files from program to program may result in unforeseen incompatibilities. Additionally, installing some programs can be challenging because of operating-system specific installation processes and software dependencies.\n\nWe have designed Compound Heterozygous Variant Identification Pipeline (CompoundHetVIP) to help researchers overcome these time-consuming challenges when identifying CH variants. CompoundHetVIP encapsulates specific versions of existing tools, required software dependencies, and custom Python scripts into a cohesive computational environment packaged as a Docker image11. Accordingly, researchers need only install the Docker software and download the CompoundHetVIP Docker image to begin performing CH, homozygous alternate, and de novo analyses at the command line. Furthermore, because the source code for CompoundHetVIP is publicly available, other researchers will be able to reproduce the analyses and investigate the specific methodologies used.\n\n\nMethods\n\nThe functionality of CompoundHetVIP is divided into a series of 13 steps (Figure 1). For each step, a Python script is executed within a Docker container. These scripts provide logic for processing data files and invoking third-party tools. By breaking the pipeline into 13 steps, users have flexibility to perform the steps that are most relevant to their analysis. For example, researchers can use input data for an affected individual only or for a trio (an affected individual and both parents). If parental data are unavailable and the variant positions within the VCF file correspond to genome build GRCh37, users may skip the first three steps. A detailed, step-by-step guide is available on GitHub and as Extended data12.\n\nFor step 1, the inputs can be either Variant Call Format (VCF)13 or gVCF14 files. VCF files contain variant sites only, whereas gVCF files include non-variant sites, too. For each parent of a trio being evaluated, our script retains nucleotide positions that are in common with the child. When gVCF files are used (whether for trios or individuals), our script removes all non-variant sites for the child (but retains these for the parents to support determination of CH status). When applied to trio data, some phasing tools, such as SHAPEIT28, require a single input file for each trio. Therefore, in step 2 (used only when working with trios), we combine the variant files for each member of a trio into a single VCF file using either BCFtools (VCF input files)15 or GATK4 (gVCF input files)14. If GATK4 is used, joint-genotyping is also performed on the trio VCF.\n\nThe remaining steps can be applied either to trios or individuals. Some phasing and annotation programs require that data be aligned to genome build GRCh37; thus, we use this reference genome as our standard. For variant files that have been aligned to genome build GRCh38, step 3 uses Picard Tools16 to convert the data to GRCh37 positions using a lift-over process. During lift-over, some sites may be present in GRCh38, but their exact position in GRCh37 is unknown. To avoid ambiguity, these sites are removed during step 4. This step also removes positions that are multiallelic or duplicated to maintain compatibility with programs such as Plink217,18 and SHAPEIT2 (used in steps 5 and 6, respectively). For trio VCF files, sites that contain missing genotype information (i.e. \"./.\") for both parents are removed to improve phasing accuracy.\n\nCompoundHetVIP can perform phasing using SHAPEIT2, Eagle210, and/or Beagle9. Each of these programs requires that each chromosome be phased independently. Additionally, when using SHAPEIT2, it is recommended that PLINK files (.bed, .bim, .fam) be used as input for phasing. Therefore, step 5 divides a VCF file by chromosome into multiple files and creates the necessary PLINK files for each chromosome (when SHAPEIT2 is used for phasing).\n\nStep 6 phases the variants in each chromosome. All supported phasing programs integrate the 1000 Genomes Project phase 3 haplotype reference panel19 and do not require sequence alignment files (.bam), such as those required by read-based programs20,21. In some scenarios, SHAPEIT2 switches the REF and ALT alleles. Therefore, step 7 ensures that the REF/ALT alleles of the phased VCF files are congruent with those of the reference genome. Also, sites with Mendelian errors are removed.\n\nTo make subsequent analysis of the phased files easier, step 8 concatenates all phased chromosomes into a single file. If a user is analyzing multiple trios (or individuals), this script can also merge the data for these trios (or individuals) into a single VCF file.\n\nStep 9 normalizes VCF files as recommended by GEMINI22 (used in step 11). Normalization involves left-alignment and trimming of variants23. This process helps ensure that variants are represented at their left-most position, with as few nucleotides as possible, and unambiguously. This step uses vt tools23. In step 10, SnpEff24 provides information about the effects of variants on function for known genes. Then, in step 11, GEMINI22 loads the annotated VCF into a relational database. Step 12 uses a custom Python script to extract CH variant data from the database. Our provided script identifies CH variants and filters the data based on user-set thresholds for minor allele frequency (MAF) and Combined Annotation Dependent Depletion (CADD) scores25. Variants with a MAF less than or equal to the user-set threshold and that are classified as loss of function or having “HIGH” putative impact severity are included in the final output. In addition, the final output includes variants with a CADD score greater than or equal to the user-set threshold, a MAF less than or equal to the user-set threshold, and a putative impact severity of “MED”. We consider the variants in the final output as candidates for further evaluation. For step 12, we provide two additional scripts that identify homozygous alternate variants and de novo variants using the same user-set thresholds as those described above.\n\nFinally, in step 13, we add Gene Damage Index (GDI) scores26 and gene-length information to the output files. GDI scores quantify accumulated mutational damage in healthy populations as a way to predict whether genes are likely to have disease-causing variants. Genes of longer length (e.g. TTN, MUC5B) tend to have more total damage but typically less disease-causing damage than shorter genes.\n\nBecause CompoundHetVIP executes all scripts within a Docker container, it can be executed on all major operating systems that are commonly used for scientific computing. Depending on input file sizes, the hardware needed to execute CompoundHetVIP will vary from user to user. Certain tasks, such as phasing (step 6), can be memory intensive. A minimum of 40 GB memory is recommended. When creating a relational database with GEMINI (step 11), there is no minimum processing core recommendation, but multiprocessing can significantly speed up the time it takes to load the database. Users can specify how many processing cores GEMINI can use when executing step 11.\n\n\nResults\n\nWe applied CompoundHetVIP to high-confidence, VCF data that were generated with whole-genome sequencing data from an Ashkenazim trio available through the Genome in a Bottle Consortium27. During step 6, we used SHAPEIT2 to phase the data. In the child of this trio, we identified a CH variant in two genes (FLNB and TTN) using a MAF threshold of 0.01 and a CADD score threshold of 15. Genes with a GDI score less than or equal to 13.84 are classified as being more likely to have disease-causing damage from variants26. FLNB (6.2) was lower than this threshold but TTN (42.9) was not. FLNB has an important role in cytoskeleton development and variations in this gene have been associated with many skeletal disorders28,29.\n\nIn addition, we identified two homozygous alternate variants in TBC1D2 using the same MAF and CADD thresholds that we used for CH variant identification. TBC1D2 had a GDI score of 9.7. TBC1D2 codes for a GTPase-activating protein and is involved in E-cadherin degradation30. The role of this gene and how it may relate to human disease is not yet fully understood.\n\nUsing the same MAF and CADD thresholds described above, we sought to identify de novo variants in this trio. However, none passed these thresholds.\n\n\nConclusion\n\nCompoundHetVIP provides the necessary tools for CH variant identification using VCF or gVCF files as initial input and is executed within a Docker container, which allows for cross-platform compatibility and reproducibility. CompoundHetVIP involves 13 steps (Figure 1) that include a breadth of tasks such as liftover, phasing, annotation, and variant summarization. Our results highlight that potentially damaging CH and homozygous alternate variants are observed in seemingly healthy individuals. However, we anticipate that most researchers will use CompoundHetVIP to identify variants in individuals with a known disease.\n\n\nData availability\n\nVCF data used to generate the results were from an Ashkenazim trio, freely-available through the Genome in a Bottle Consortium at ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/release/AshkenazimTrio/27:\n\n- Child: ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/release/AshkenazimTrio/HG002_NA24385_son/latest/GRCh38/supplementaryFiles/HG002_GRCh38_CHROM1-22_v4.1_highconf.vcf.gz\n\n- Mother: ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/release/AshkenazimTrio/HG004_NA24143_mother/latest/GRCh38/HG004_GRCh38_GIAB_highconf_CG-Illfb-IllsentieonHC-Ion-10XsentieonHC_CHROM1-22_v.3.3.2_highconf.vcf.gz\n\n- Father: ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/release/AshkenazimTrio/HG003_NA24149_father/latest/GRCh38/HG003_GRCh38_GIAB_highconf_CG-Illfb-IllsentieonHC-Ion-10XsentieonHC_CHROM1-22_v.3.3.2_highconf.vcf.gz\n\nZenodo: dmiller903/CompoundHetVIP: CompoundHetVIP Initial Release. https://doi.org/10.5281/zenodo.402116912.\n\nThis project contains the following extended data:\n\n- CompoundHetVIP_example.pdf (detailed step-by-step example)\n\n\nSoftware availability\n\nSoftware available from: https://hub.docker.com/r/dmill903/compound-het-vip\n\nSource code available from: https://github.com/dmiller903/CompoundHetVIP\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.402116912.\n\nLicense: MIT", "appendix": "References\n\nKamphans P, Sabri T, Zhu N, et al.: Filtering for compound heterozygous sequence variants in non-consanguineous pedigrees. PLoS One. 2013; 8(8): e70151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiane M, Molinaro A, Soresina A, et al.: Novel compound heterozygous mutations in a child with Ataxia-Telangiectasia showing unrelated cerebellar disorders. J Neurol Sci. 2016; 371: 48–53. PubMed Abstract \| Publisher Full Text\n\nLi R, Pradhan M, Xu M, et al.: Generation of an induced pluripotent stem cell line (TRNDi002-B) from a patient carrying compound heterozygous p.Q208X and p.G310G mutations in the NGLY1 gene. Stem Cell Res. 2019; 34: 101362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller D, Piccolo S: Compound Heterozygous Variants in Pediatric Cancers: A Systematic Review. Front Genet. 2020; 11: 493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuitama J, McEwen GK, Huebsch T, et al.: Fosmid-based whole genome haplotyping of a HapMap trio child: evaluation of Single Individual Haplotyping techniques. Nucleic Acids Res. 2012; 40(5): 2041–2053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng GXY, Lau BT, Schnall-Levin M, et al.: Haplotyping germline and cancer genomes with high-throughput linked-read sequencing. Nat Biotechnol. 2016; 34(3): 303–311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi Y, Chan AP, Kirkness E, et al.: Comparison of phasing strategies for whole human genomes. PLoS Genet. 2018; 14(4): e1007308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelaneau O, Howie B, Cox AJ, et al.: Haplotype estimation using sequencing reads. Am J Hum Genet. 2013; 93(4): 687–696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrowning SR, Browning BL: Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering. Am J Hum Genet. 2007; 81(5): 1084–1097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoh PR, Danecek P, Palamara PF, et al.: Reference-based phasing using the Haplotype Reference Consortium panel. Nat Genet. 2016; 48(11): 1443–1448. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiccolo SR, Frampton MB: Tools and techniques for computational reproducibility. GigaScience. 2016; 5(1): 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\ndmiller903: dmiller903/CompoundHetVIP: CompoundHetVIP Initial Release (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4021169\n\nDanecek P, Auton A, Abecasis G, et al.: The variant call format and VCFtools. Bioinformatics. 2011; 27(15): 2156–2158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoplin R, Ruano-Rubio V, DePristo MA, et al.: Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv. 2017; 201178. Publisher Full Text\n\nLi H: A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011; 27(21): 2987–2993. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPicard Tools. Reference Source\n\nPurcell S, Chang C: PLINK 2.0.\n\nChang CC, Chow CC, Tellier LC, et al.: Second-generation PLINK: rising to the challenge of larger and richer datasets. GigaScience. 2015; 4: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n1000 Genomes Project Consortium, Auton A, Brooks LD, et al.: A global reference for human genetic variation. Nature. 2015; 526(7571): 68–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdge P, Bafna V, Bansal V: HapCUT2: robust and accurate haplotype assembly for diverse sequencing technologies. Genome Res. 2017; 27(5): 801–812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin M, Patterson M, Garg S, et al.: WhatsHap: fast and accurate read-based phasing. bioRxiv. 2016; 085050. Publisher Full Text\n\nPaila U, Chapman BA, Kirchner R, et al.: GEMINI: integrative exploration of genetic variation and genome annotations. PLoS Comput Biol. 2013; 9(7): e1003153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan A, Abecasis GR, Kang HM: Unified representation of genetic variants. Bioinformatics. 2015; 31(13): 2202–2204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCingolani P, Platts A, Wang LL, et al.: A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012; 6(2): 80–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRentzsch P, Witten D, Cooper GM, et al.: CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 2019; 47(D1): D886–D894. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nItan Y, Shang L, Boisson B, et al.: The human gene damage index as a gene-level approach to prioritizing exome variants. Proc Natl Acad Sci U S A. 2015; 112(44): 13615–13620. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZook JM, McDaniel J, Olson ND, et al.: An open resource for accurately benchmarking small variant and reference calls. Nat Biotechnol. 2019; 37(5): 561–566. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou X, Tian F, Sandzén J, et al.: Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development. Proc Natl Acad Sci U S A. 2007; 104(10): 3919–3924. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang CF, Wang CH, H'ng WS, et al.: Filamin B Loss-of-Function Mutation in Dimerization Domain Causes Autosomal-Recessive Spondylocarpotarsal Synostosis Syndrome with Rib Anomalies. Hum Mutat. 2017; 38(5): 540–547. PubMed Abstract \| Publisher Full Text\n\nFrasa MAM, Maximiano FC, Smolarczyk K, et al.: Armus is a Rac1 effector that inactivates Rab7 and regulates E-cadherin degradation. Curr Biol. 2010; 20(3): 198–208. PubMed Abstract \| Publisher Full Text" }	[ { "id": "76619", "date": "20 Jan 2021", "name": "Soohyun Lee", "expertise": [ "Reviewer Expertise genomics", "transcriptomics", "genomic variant calling and analysis", "software and algorithm development", "cloud infrastructure", "docker", "genomic pipeline" ], "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 describes a software pipeline that identifies compound heterozygous (CH) variants from the VCF or gVCF files of whole genome sequencing data from either a single sample or a trio. The pipeline has 13 steps and in brief, performs VCF file merging, liftover from hg38 to hg37, haplotype phasing, variant annotation and filtering based on minor allele frequency (MAF) and Combined Annotation Dependent Depletion (CADD) scores. The pipeline is dockerized for portable and reproducible analysis.\nThe paper addresses an important problem of identifying CH variants. Many researchers seem to end up writing their own custom CH variant detecting tools and it could be a useful addition if that can be avoided.\nI have a few comments which hopefully would improve the clarity of the article and the usability of the pipeline. Before we get to that, I think I have to disclose that I myself and my team member designed and wrote our own CH variant caller as a part of our own variant calling/analysis pipeline, with the help of several experts in the genetics field (not necessarily for publication but for our own use). Therefore, my review is based on my experience tackling a similar problem but most likely with a different set of requirements/constraints.\nFor better clarity and flow of the article, it may be helpful to describe relatively early on (e.g. in the title or early in the abstract) what the pipeline does, including the data type (e.g. whole genome sequencing) and starting point (e.g. VCF/gVCF). The steps the pipeline offers could also be mentioned earlier. For example, it is not clear until near the end of the article that filtering based on MAF and CADD is performed as a part of the pipeline, and one could wonder simply reading the abstract, why only two genes were identified to have CH variants, which is much fewer than expected from a whole genome sequencing data. It is also not clear in the beginning that variant annotation and an optional GATK4-based variant calling is part of the pipeline.\n\nI think the pipeline usability could be improved if it accommodates previously merged and annotated VCF files. A user may start with a VCF that is annotated with their annotation tool of choice (e.g. VEP which is a more recent tool than SnpEff) that they may want to use for other analysis consistently in addition to CH variant detection. It would be useful to support such a use case. Alternatively, the user may have a pre-merged VCF file generated by GATK4 from joint variant calling with the parameters of their own choice. The pipeline’s VCF merging step performs GATK4-based variant calling which may make more sense to be done along with the other GATK4-based upstream steps rather than as part of the compound het detection step, though that can be a matter of preference. Having this kind of modularity could appeal more to bioinformaticians who are setting up a full variant calling and analysis pipeline and just need a CH detection module. It may also be computationally more efficient to first annotate variants with which genes they overlap with and remove intergenic variants before performing CH detection because CH variants by definition would only occur inside a gene.\n\nThe pipeline performs population-based haplotype phasing based on e.g. SHAPEIT2 to identify CH variants. It is not very convincing though how much this adds compared to simply scanning a VCF file once to collect all pairs of heterozygous variants in a gene whose parents’ genotypes indicate that one comes from the mother and the other from the father. The authors of the latest version of the phasing tool, SHAPEIT4, seems to have used Mendelian logics as the truth set to evaluate the performance of its phasing imputation, suggesting that when the parents’ genotypes are directly available, it maybe be more accurate to use those genotypes than using a population-based imputation.1 It may be helpful for a proband-only case where the parents’ genotypes are not available. However, for a trio case, detecting CH variants from a previously annotated VCF file by simply comparing the genotypes of the parents would take one step of scanning the file (rather than 13 steps). I think having a more convincing rationale about why these additional steps are needed for a trio case would be very helpful.\n\nI think the pipeline can definitely be useful as it is for specific users who do not have their own variant calling or annotation capacity and took an unannotated VCF or gVCF file from an external source and simply want to perform CH variant identification without parent information. Again, going back to point 1), having a full description of what the pipeline offers (including variant annotation, filtering, even running GATK4) in the title and early on in the abstract could make it easier to reach the right type of potential users.\n\nLastly a minor point, it would be useful to clarify if the pipeline also works for whole exome sequencing data.\n\nIs the rationale for developing the new software tool clearly explained? No\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6333", "date": "10 Feb 2021", "name": "Dustin Miller", "role": "Author Response", "response": "Thank you for taking the time to review our manuscript. We appreciate your thorough and helpful comments. We have addressed these comments below in a point-by-point response. These changes have made the manuscript more informative and insightful. The reviewers comments are in regular font. Our responses are in bold. 1. For better clarity and flow of the article, it may be helpful to describe relatively early on (e.g. in the title or early in the abstract) what the pipeline does, including the data type (e.g. whole genome sequencing) and starting point (e.g. VCF/gVCF). The steps the pipeline offers could also be mentioned earlier. For example, it is not clear until near the end of the article that filtering based on MAF and CADD is performed as a part of the pipeline, and one could wonder simply reading the abstract, why only two genes were identified to have CH variants, which is much fewer than expected from a whole genome sequencing data. It is also not clear in the beginning that variant annotation and an optional GATK4-based variant calling is part of the pipeline. Thank you for these recommendations. We have updated the abstract to get to the point more quickly, to mention the tools used for each major step, and to clarify that MAF and CADD are used as part of variant identification. We also updated the abstract to mention that whole-exome and targeted exome data can also be used. This updated abstract should provide a clearer understanding of what tools are included in the pipeline, and what data can be used as initial input. 2. I think the pipeline usability could be improved if it accommodates previously merged and annotated VCF files. A user may start with a VCF that is annotated with their annotation tool of choice (e.g. VEP which is a more recent tool than SnpEff) that they may want to use for other analysis consistently in addition to CH variant detection. It would be useful to support such a use case. Alternatively, the user may have a pre-merged VCF file generated by GATK4 from joint variant calling with the parameters of their own choice. The pipeline’s VCF merging step performs GATK4-based variant calling which may make more sense to be done along with the other GATK4-based upstream steps rather than as part of the compound het detection step, though that can be a matter of preference. Having this kind of modularity could appeal more to bioinformaticians who are setting up a full variant calling and analysis pipeline and just need a CH detection module. It may also be computationally more efficient to first annotate variants with which genes they overlap with and remove intergenic variants before performing CH detection because CH variants by definition would only occur inside a gene. Thank you for your thoughtful comments about the modularity of this pipeline. This is something we have thought about over the course of this project, and we want this pipeline to be flexible for diverse types of usage. We have updated the “gemini_load.py” script with an optional argument to allow users to import a file that was annotated with VEP. Because SnpEff is used as part of the pipeline, the script defaults to SnpEff; but if a user chooses to use VEP outside of the pipeline, they can still apply the remaining steps of the pipeline. Intergenic regions are not considered during the CH detection step. Within the “identify_CH_variants.py” script, the GEMINI database is converted to a TSV file; and during this process, intergenic regions are removed to speed up the CH detection process. Accordingly, it would be fine if researchers uploaded a VCF file from which intergenic regions have already been removed. In addition, we will be actively monitoring GitHub for any feature recommendations or any issues that arise. For users who are more experienced with coding and aren’t afraid of source code, the code is publicly available, and these users can adapt the code to meet their needs. 3. The pipeline performs population-based haplotype phasing based on e.g. SHAPEIT2 to identify CH variants. It is not very convincing though how much this adds compared to simply scanning a VCF file once to collect all pairs of heterozygous variants in a gene whose parents’ genotypes indicate that one comes from the mother and the other from the father. The authors of the latest version of the phasing tool, SHAPEIT4, seems to have used Mendelian logics as the truth set to evaluate the performance of its phasing imputation, suggesting that when the parents’ genotypes are directly available, it maybe be more accurate to use those genotypes than using a population-based imputation.1 It may be helpful for a proband-only case where the parents’ genotypes are not available. However, for a trio case, detecting CH variants from a previously annotated VCF file by simply comparing the genotypes of the parents would take one step of scanning the file (rather than 13 steps). I think having a more convincing rationale about why these additional steps are needed for a trio case would be very helpful. We considered coding an initial step that identifies all CH variants that can be inferred via Mendelian logic when both parents' genotypes are available. However, we wanted to make the pipeline consistent regardless of whether trio or individual data were used. Furthermore, SHAPEIT2 uses Mendelian logic when the parents' genotypes are available. The only time it uses compact hidden Markov model (CHMM) logic with trios is when the phase of the child cannot be determined using Mendelian logic alone (i.e. both parents are heterozygous, and the child is as well). Therefore, SHAPEIT2 provides the benefit of Mendelian logic and CHMM, when necessary. (The SHAPEIT4 paper also notes that it used the 500 trio children as validation only when they could be \"phased using Mendel inheritance logic (i.e., no triple hets and no Mendel inconsistencies).\") In addition, there may be scenarios where data is missing for one or both parents for a certain gene region, and population-based phasing with SHAPEIT2 has the added benefit of imputing the missing information. We have updated the manuscript to clarify these points, as suggested. 4. I think the pipeline can definitely be useful as it is for specific users who do not have their own variant calling or annotation capacity and took an unannotated VCF or gVCF file from an external source and simply want to perform CH variant identification without parent information. Again, going back to point 1), having a full description of what the pipeline offers (including variant annotation, filtering, even running GATK4) in the title and early on in the abstract could make it easier to reach the right type of potential users. Please see response # 1. 5. Lastly a minor point, it would be useful to clarify if the pipeline also works for whole exome sequencing data. Thank you for bringing this up. We designed this pipeline with WGS in mind, and did not think about other types of next-gen sequencing data. To test the pipelines on WES/targeted exome data, we ran the pipeline on a publicly available dataset available here: https://my.pgp-hms.org/profile/huF85C76. The data we used was the “Helix Exome+ WES” VCF file (https://my.pgp-hms.org/user_file/download/3888). After filtering the data for positions that passed quality filters and had a quality > 20, we started on step 3 and went through all remaining steps. The pipeline worked well. We have made this more clear in the manuscript. In addition to the changes mentioned above, we have made a few other changes we would like to mention here: 1. We changed the way we filter the variants to be more simple but also more strict. Originally we allowed some variants to be considered if they were of “HIGH” putative impact but were missing CADD or MAF values. To obtain the variants most likely to play a role in disease, we made the requirement more strict. Now, CADD and MAF scores must be available for all variants, variants must be in exonic regions, and variants can have either “MED” or “HIGH” putative impact. Making these changes did not change the number of CH variants identified in the example dataset, but will likely have an effect in other datasets. This change in how variants are filtered may provide the users with a smaller subset of putative variants, but the CompoundHetVIP will provide more information for the users to use in downstream analysis (i.e. CADD and MAF values for all variants rather than just some of the variants). Our description of the filtering method has been updated in the manuscript. 2. There was an error in our script that identified homozygous alternate variants. Some of our previous code required that a gene have 2 variants in order to be written to the output file. This has been fixed, and it slightly changed the results. Originally we reported only 1 gene with a homozygous alternate variant in the example dataset. After updating the script and running it again, there are 2 genes that have a homozygous alternate variant. This result has been modified in the manuscript. 3. Minor updates to the example PDF (https://github.com/dmiller903/CompoundHetVIP/blob/master/CompoundHetVIP_example.pdf) were made for added clarity and explanation." } ] }, { "id": "76628", "date": "25 Jan 2021", "name": "Sushant Patil", "expertise": [ "Reviewer Expertise Bioinformatics", "Computational Biology", "NGS/HTS", "Cancer/Oncology", "Personalized Medicine" ], "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 the manuscript \"CompoundHetVIP: Compound Heterozygous Variant Identification Pipeline\" by Miller et al.\nI find the published software to be of great utility, even though it may not have scientific novelty as such. The manuscript is neat and well-presented. I recommend this manuscript for publication in the journal. However, I request the authors to address the following points in the final revision:\nIt appears that the program can only be used with whole-genome sequencing VCFs. Is that true? How about VCFs generated from WES or targeted panel?\n\nStep 5 - \"CompoundHetVIP can perform phasing using SHAPEIT2, Eagle210, and/or Beagle9.\". Is there a 'default' mode of execution for the software? If not, I suggest to have one and include one or all of these phasing programs as part of it.\n\nIn Figure 1, I recommend labeling each box with the corresponding Step # to relate it to the text in the manuscript.\n\nIdeally all VCFs should be 1-based. In a hypothetical scenario, if the parents' VCFs are 0-based and the child's 1-based (coming from different workflows), will the program be able to detect and handle such discrepancy?\n\nI recommend saying minor allele frequency (MAF) as Global minor allele frequency (MAF) at first instance. It is easy to be mistaken that for mutant allelic frequency in cancer genomics.\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": "6334", "date": "10 Feb 2021", "name": "Dustin Miller", "role": "Author Response", "response": "Thank you for taking the time to review our manuscript. We appreciate your thorough and helpful comments. We have addressed these comments below in a point-by-point response. These changes have made the manuscript more informative and insightful. The reviewers comments are in regular font. Our responses are in bold. 1. It appears that the program can only be used with whole-genome sequencing VCFs. Is that true? How about VCFs generated from WES or targeted panel? Thank you for pointing this out. We designed this pipeline with WGS in mind and did not think about other types of next-gen data. To test the pipelines on WES/targeted exome data, we ran the pipeline on a publicly available dataset available here: https://my.pgp-hms.org/profile/huF85C76. The data we used was the “Helix Exome+ WES” VCF file (https://my.pgp-hms.org/user_file/download/3888). After filtering the data for positions that passed quality filters and had a quality > 20, we started on step 3 and went through all remaining steps. The pipeline worked well. We have made this more clear in the manuscript. 2. Step 5 - \"CompoundHetVIP can perform phasing using SHAPEIT2, Eagle210, and/or Beagle9.\". Is there a 'default' mode of execution for the software? If not, I suggest to have one and include one or all of these phasing programs as part of it. We have a separate script for all 3 phasing programs. That way the user can choose which program to use. Within each script, the phasing software is executed under default parameters. Our example PDF uses SHAPEIT2 and we recommend this as the default phaser to use, especially when using trio data. However, we feel that the choice of phasing program is fundamental to our pipeline and should require an explicit decision from the user; so we require the user to select one. We have made this more clear in the example documentation (https://github.com/dmiller903/CompoundHetVIP/blob/master/CompoundHetVIP_example.pdf) and in the manuscript. 3. In Figure 1, I recommend labeling each box with the corresponding Step # to relate it to the text in the manuscript. Thank you for the recommendation. We have updated the figure to show step numbers. 4. Ideally all VCFs should be 1-based. In a hypothetical scenario, if the parents' VCFs are 0-based and the child's 1-based (coming from different workflows), will the program be able to detect and handle such discrepancy? This pipeline assumes input VCF files are 1-based. We added this disclaimer to the example documentation referenced in response 2. During step 11, GEMINI 0-bases the positions. However, during step 12, they are reverted back to 1-based positions for consistency. 5. I recommend saying minor allele frequency (MAF) as Global minor allele frequency (MAF) at first instance. It is easy to be mistaken that for mutant allelic frequency in cancer genomics. Thank you for this recommendation. We have updated the manuscript to say “global minor allele frequency” when first referenced. In addition to the changes mentioned above, we have made a few other changes we would like to mention here: 1. We changed the way we filter the variants to be more simple but also more strict. Originally we allowed some variants to be considered if they were of “HIGH” putative impact but were missing CADD or MAF values. To obtain the variants most likely to play a role in disease, we made the requirement more strict. Now, CADD and MAF scores must be available for all variants, variants must be in exonic regions, and variants can have either “MED” or “HIGH” putative impact. Making these changes did not change the number of CH variants identified in the example dataset, but will likely have an effect in other datasets. This change in how variants are filtered may provide the users with a smaller subset of putative variants, but the CompoundHetVIP will provide more information for the users to use in downstream analysis (i.e. CADD and MAF values for all variants rather than just some of the variants). Our description of the filtering method has been updated in the manuscript. 2. There was an error in our script that identified homozygous alternate variants. Some of our previous code required that a gene have 2 variants in order to be written to the output file. This has been fixed, and it slightly changed the results. Originally we reported only 1 gene with a homozygous alternate variant in the example dataset. After updating the script and running it again, there are 2 genes that have a homozygous alternate variant. This result has been modified in the manuscript. 3. Minor updates to the example PDF (https://github.com/dmiller903/CompoundHetVIP/blob/master/CompoundHetVIP_example.pdf) were made for added clarity and explanation." } ] } ]	1	https://f1000research.com/articles/9-1211
https://f1000research.com/articles/10-90/v1	10 Feb 21	{ "type": "Research Article", "title": "Influence of polymorphisms in the vascular endothelial growth factor gene on allograft rejection after kidney transplantation: a meta-analysis", "authors": [ "Thanee Eiamsitrakoon", "Phuntila Tharabenjasin", "Noel Pabalan", "Hamdi Jarjanazi", "Adis Tasanarong", "Thanee Eiamsitrakoon", "Phuntila Tharabenjasin", "Hamdi Jarjanazi", "Adis Tasanarong" ], "abstract": "Background: Reported associations of allograft rejection in kidney transplant patients with VEGF single nucleotide polymorphisms (SNPs) have been inconsistent between studies, which prompted a meta-analysis to obtain more precise estimates. Methods: Using the PICO elements, kidney transplant patients (P) were compared by genotype data between rejectors (I) and non-rejectors (C) in order to determine the risk of allograft rejection (O) attributed to the VEGF SNPs. Literature search of four databases yielded seven articles. To calculate risks for allograft rejection, four SNPs were examined. Using the allele-genotype model we compared the variant (var) with the wild-type (wt) and heterozygous (var-wt) alleles. Meta-analysis treatments included outlier and subgroup analyses, the latter was based on ethnicity (Indians/Caucasians) and rejection type (acute/chronic). Multiple comparisons were corrected with the Bonferroni test. Results: Five highly significant outcomes (Pa < 0.01) survived Bonferroni correction, one of which showed reduced risk for the var allele (OR 0.61, 95% CI 0.45-0.82). The remaining four indicated increased risk for the wt allele where the chronic rejection (OR 2.10, 95% CI 1.36-3.24) and Indian (OR 1.44, 95% CI 1.13-1.84) subgroups were accorded susceptibility status. Conclusions: Risk associations for renal allograft rejection were increased and reduced on account of the wt and var alleles, respectively. These findings could render the VEGF polymorphisms useful in the clinical genetics of kidney transplantation.", "keywords": [ "VEGF polymorphisms", "allograft", "renal", "kidney transplantation", "meta-analysis" ], "content": "Abbreviations\n\nA, adenine; AR, acute rejection; C, cytosine; CA or C/A, cytosine/adenine; CEU, European population; CI, confidence interval; CR, chronic rejection; CRAD, chronic renal allograft dysfunction; C/T, cytosine/thymine; du, duplicate; G, guanine; GIH, Gujarati Indian population; GG or G/G, guanine/guanine; het, heterozygous genotype; HWC, Hardy-Weinberg Compliant HWE, Hardy-Weinberg Equilibrium; I2, measure of variability; ITU, Telugu Indian population; KT, kidney transplantation; LD, linkage disequilibrium; n, number of studies; NRJ, non-rejection; OR, odds ratio; Pa, P-value for association; Phet, P-value for heterogeneity; [R], reference of studies; RJ, rejection; SNP, single nucleotide polymorphism; T, thymine; var, variant allele or genotype; VEGF, vascular endothelial growth factor gene; VEGF, vascular endothelial growth factor protein; wt, wild-type allele or genotype\n\n\nIntroduction\n\nChronic kidney disease is a longstanding global health problem with substantial effects on morbidity and mortality1. Even with medical intervention, the likely endpoints in the progression of this disease are end-stage renal disease and kidney failure. In such cases, kidney transplantation (KT) is the current best available therapeutic option1,2. Success of the transplanted organ or an allograft in the recipient is limited by graft rejection3 which is characterized by inflammatory responses toward the graft tissue resulting in structural and functional impairments leading to allograft dysfunction4. Allograft rejection can be categorized largely into acute rejection (AR) which occurs days/weeks up until three months post-KT, or chronic rejection (CR) which is seen as progressive loss of graft function after three months post-KT5. Key factors that contribute to allograft rejection may involve cytokines that are secreted by immune cells and antibodies against graft antigens6. Cytokines have been recognized as potent immunomodulatory biomolecules that mediate physiological and pathological immune responses. These molecules determine the magnitude of alloimmune responses after transplantation, which influence graft survival7. Differences in genetic background of transplant recipients are, in part, the cause of varying immune responses towards grafts8. Recognizing these genetic differences and their effects on the immune response may help establish individualized immunosuppressive regimens that can improve allograft outcome9. This is accomplished by identifying the alleles that may increase risk or confer protection for immune-mediated complications after KT10. Single nucleotide polymorphisms (SNPs) in the cytokine genes may impact graft survival by altering transcriptional activities and levels of gene expression11 which lead to variations in cytokine production12.\n\nOf the cytokine factors related to immune-mediated renal graft injury, the vascular endothelial growth factor (VEGF) is of potential use as a post-transplantation biomarker13. As mediator of vascular formation, VEGF promotes endothelial cell proliferation, differentiation and survival14. It also mediates endothelium-dependent vasodilation and maintains vascular permeability15. Dysregulations of VEGF expression are evident in many renal abnormalities16,17. This suggests a possible pathologic role of this protein in renal diseases including graft injury. Studies of allograft tissues from rat KT models (in both AR and CR events) and human KT recipients with AR showed increased VEGF expression in renal tubules and interstitium18,19. This suggests involvement of this gene/protein in the pathogenesis of allograft rejection. Various SNPs in the VEGF gene have been identified20,21 and reported to be associated either with low or high VEGF protein production21,22. One of the common VEGF SNPs, a cytosine (C) to adenine (A) polymorphism at position 2578 within the promoter region (-2578 C/A), was found to be associated with VEGF expression and allograft rejection. The CC genotype was associated with high VEGF production but varied in its effects on renal allograft outcomes with reduced23 and increased24 rejection risks across the studies. Given the varied influence of these SNPs on renal allograft function, it is opportune to statistically synthesize these study findings using meta-analysis.\n\nOur study aims to provide better understanding of the genetic role of VEGF SNPs on post-KT allograft outcome in term of risk for allograft rejection among recipients, which might guide potential future directions in transplant genetics. To obtain less ambiguous, clearer estimates of the VEGF role in this investigation, we apply meta-analysis techniques (i.e. outlier treatment) in order to strengthen the evidence.\n\n\nMethods\n\nWe searched for association studies on 13 February 2020, the start date for this meta-analysis. Four strings of search terms were used that included combinations of “vascular endothelial growth factor”, “VEGF”, “polymorphism”, “cytokine”, “renal”, “transplant”, “allograft”, and “kidney transplantation” as medical subject heading and text in MEDLINE using PubMed, Google Scholar, Science Direct and Mednar, unrestricted by language. Details of the search strategies for each of these four databases are shown in Table S1 (Extended data25).\n\nReferences cited in the retrieved articles were also hand-screened to identify additional eligible studies. In case of duplicate articles, we selected the one with a later date of publication.\n\nThe following PICO elements were applied in the meta-analysis: (i) Population: renal allograft patients; (ii) Intervention: VEGF gene polymorphisms; (iii) Comparators: rejectors (RJ) versus non-rejectors (NRJ); and (iv) Outcome: allograft rejection post-KT.\n\nInclusion criteria were: (i) case–control design evaluating the association between VEGF SNPs and risk of allograft rejection; (ii) available VEGF genotype frequencies in the presence and absence of allograft rejection and (iii) sufficient genotype frequency data to enable calculation of the odds ratios (ORs) and 95% confidence intervals (CIs). Exclusion criteria were studies that: (i) did not involve renal allografts; (ii) were review articles; (iii) were functional studies; (iv) did not involve VEGF SNPs and with genotype or allele frequencies that were unusable/absent or, when available, combined with SNPs in other genes, preventing proper data extraction.\n\nWe examined four SNPs (Table 1; Extended data: S2 Table25). Observed phenotypic associations have been attributed to the proximity of SNPs in the VEGF gene26–28, termed linkage disequilibrium (LD). LD is the correlation between alleles located near each other29 and is measured in terms of D′ and r2 with a value of 1 indicating complete LD30,31. LD values were based on the European (CEU), and the Indian populations (Gujarati: GIH and Telugu: ITU) from LDlink. Complete LD between rs699947 (-2578C/A) and rs144854329 (-2549 insertion/deletion) merited combination, labeled VEGF1. -1154G/A (rs1570360), and 938C/T (rs3025039) were not in complete LD, thus analyzed separately, notated as VEGF2 and VEGF3, respectively (Table 2).\n\nVEGF: vascular endothelial growth factor; [R]: Reference; y: years; KT: kidney transplantation; RJ: rejection NRJ: non-rejection; AR: acute rejection; CR: chronic rejection; CRAD: chronic renal allograft dysfunction; n: number of studies; * median (range)\n\nVEGF1: vascular endothelial growth factor polymorphisms; AR: acute rejection; CR: chronic rejection; SNPs: single nucleotide polymorphisms; RJ: rejection; NRJ: non-rejection; HWE: Hardy-Weinberg Equilibrium; wt: wild-type; var: variant; du: duplicate; the 5 and 8 after Prakash indicate the last digit of publication year for these articles; values in bold indicate total sample sizes for each VEGF SNP group and significant departure from the HWE; † aggregate statistical power for the VEGF groups.\n\nTwo investigators (TE and NP) independently extracted data and arrived at a consensus. Authors of the component articles were contacted is cases of missing data. The following information were obtained from each publication: first author’s name, year of the study, country of origin, ethnicity, age of the subjects, comparators, VEGF SNPs (rs number), including transplant outcome in term of type of allograft rejection and values needed to tally the Clark-Baudouin score (Table 1). Sample sizes as well as genotype data in RJ and NRJ were also extracted along with calculated outcomes of the minor allele frequency. HWE was assessed using the application in https://ihg.gsf.de/cgi-bin/hw/hwa1.pl, HWE was reported as P-values of the controls from the Pearson's goodness-of-fit χ2-square test.\n\nUsing the GPower program32, we evaluated statistical power. Assuming an OR of 1.5 at a genotypic risk of α = 0.05, power was considered adequate at ≥80%. Methodological quality of the included studies was assessed with the Clark-Baudouin scale33. In this scale, scores of <5, 5–6 and ≥7 represent low, moderate and high quality, respectively.\n\nGiven the hypothesis of association between VEGF SNPs and risk of allograft rejection following KT, we estimated the ORs with 95% CIs for each study by comparing RJ with NRJ among transplant recipients. Table 2 shows the frequencies of the variant (var) and wild-type alleles, as well as wt-var or heterozygous genotype (het). Non-uniformity of the variant (var) allele in VEGF1 and VEGF2 warranted the use of the allele-genotype model for VEGF1 and VEGF2. On the other hand, the var alleles in VEGF3 (rs3025039) were uniform (all < 0.50), so the standard genetic models were suitable: (i) homozygous: var–var and wt–wt genotypes compared with wt–wt; (ii) recessive: var–var versus het + wt–wt; (iii) dominant: var–var + het versus wt–wt; and (iv) codominant: var versus wt. Using raw data for frequencies, study specific risks (ORs) of allograft rejection were estimated and pooled ORs were calculated by comparing the effects on the same baseline. Multiple comparisons were corrected with the Bonferroni test. Subgrouping was based on ethnicity (Indians/Caucasians) and type of rejection (AR/CR). High significance (Pa < 0.0001) indicated strong evidence for association.\n\nHeterogeneity in meta-analysis34 was addressed with the following: (i) its presence warranted use of the random-effects model35, otherwise fixed-effects model36 was used; (ii) estimated with the χ2-based Q test37; (iii) quantified with the I2statistic38; and (iv) sources were outlier treated. Outlier treatment divided the comparisons into pre-outlier and post-outlier.\n\nSensitivity analysis was used to test for robustness of the summary effects. Publication bias was considered for significant (Pa < 0.05) comparisons with ≥ 10 studies44. Significance was set at a two-sided P-value of < 0.05, except for heterogeneity estimation, which was set at Phet < 0.10)37. Data for the meta-analysis were analyzed using Review Manager 5.3 (Cochrane Collaboration, Oxford, England), SIGMASTAT 2.03, and SIGMAPLOT 11.0 (Systat Software, San Jose, CA).\n\n\nResults\n\nFigure 1 outlines the study selection process in a flowchart following guidelines form the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA; Reporting guidelines). Table S1 (Extended data25) shows the initial search using combinations of four search strings applied to four databases resulted in 1,949 citations, followed by a series of omissions that mostly involved duplications (n = 1,924), The gray literature database (Mednar) yielded no additional papers for inclusion. Thus, the final number of included articles for this meta-analysis was seven23,24,39–43.\n\nOf the seven articles, five23,24,39–41 examined more than one VEGF SNP (Table 1). The number of studies VEGF1 (rs699947 and rs144854329), VEGF2 (rs1570360) and VEGF3 (rs3025039) were 10, five and three, respectively (Table 2). Of the 10 VEGF1 studies, seven and three were in Indian39–41 and Caucasian23,24,43 populations, respectively. Of the five VEGF2 studies, three and two were in Indian39–41 and Caucasian23,24 populations, respectively. One Caucasian42 and two Indian40,41 studies comprised VEGF3. Table 1 shows two publications41,43 that investigated CR, which translated to three studies for VEGF1 (Table 2), otherwise, the rest focused on AR (Table 1 and Table 2).\n\nTable 2 shows an aggregate total sample size (663 RJ/956 NRJ) and a statistical power of 97.7% for VEGF1. In contrast, both VEGF2 (105 RJ/254 NRJ) and VEGF3 (265 RJ/290 NRJ) were underpowered (40.5% and 65.2%). Mean age of the subjects was 39.96±6.6 years (± standard deviation) indicating a near to middle-age demographic profile of the KT subjects. The Clark-Baudouin scores (median 6.0, interquartile range 6.0–6.75) indicated that the methodological quality of the component studies was moderate. Control frequencies deviated from the HWE in three studies (from two articles) for VEGF140,41, two studies39,40 for VEGF2, and one study for VEGF340.\n\nVEGF1 associations with KT. Table S2 (Extended data25) shows 32 comparisons, six of which were significant (Pa = 0.0009–0.04). Of the six, five were post-outlier derived and four survived the Bonferroni correction (Table 3). Of the four, three were in wt indicating increased risk (overall: 1.41, 95% CI 1.14-1.75, Pa = 0.002 [Figure 2], Indian: OR 1.44, 95% CI 1.13-1.84, Pa = 0.004, CR: OR 2.10, 95% CI, Pa = 0.0009) and one in var, indicating reduced risk (Indian: OR 0.61, 95% CI 0.45-0.820, Pa = 0.001). Only the CR outcome had zero heterogeneity (I2 = 0%).\n\nVEGF: vascular endothelial growth factor gene; VEGF1: rs699947+rs144854329; VEGF2: rs1570360; VEGF3: rs3025039; wt: wild-type; var: variant; HW: Hardy-Weinberg; n: number of studies; OR: odds ratio; CI: confidence interval; Pa: P-value for association; Phet: P-value for heterogeneity; I2: measure of variability; values in bold survived the Bonferroni correction\n\nDiamond denotes the pooled odds ratio (OR) indicating increased risk (1.41). Squares indicate the OR in each study. Horizontal lines on either side of each square represent the 95% confidence intervals (CI). The Z test for overall effect shows significance (Pa = 0.002).The χ2-square test outcome has low-level heterogeneity (Phet = 0.17, I2 = 31%).\n\nwt: wild-type; VEGF: vascular endothelial growth factor; I2: a measure of variability expressed in %; RJ: rejection; NRJ: non-rejection, L: long-term\n\nVEGF2 associations with KT. Table S2 (Extended data25) shows 18 comparisons, four of which were significant (Pa = 0.001–0.04), were in the wt model and had moderate heterogeneity (I2 = 30%-51%). Three of the four were products of pre-outlier analysis, where the HWC outcome (OR 1.39, 95% CI 1.01-1.91, Pa = 0.04) confirmed the overall outcome (OR 1.48, 95% CI 1.01-2.15, Pa = 0.04). The other overall outcome was post-outlier derived and survived the Bonferroni correction (OR 1.58, 95% CI 1.19-2.09, Pa = 0.0001). The significant Caucasian outcome (OR 1.55, 95% CI 1.06-2.28, Pa = 0.02) contrasted with the non-significant Indian outcome (OR 1.36, 95% CI 0.72-2.58, Pa = 0.34).\n\nVEGF3 associations with KT. Table S3 (Extended data25) shows eight comparisons, two of which were significant (Pa = 0.008–0.01) but did not withstand Bonferroni correction. These two homogeneous (I2 = 0%) pooled ORs indicated reduced risk in the dominant and codominant models (ORs 0.66–0.69, 95% CIs 0.47-0.92).\n\nSummary of significant VEGF associations with KT. Table 3 summarizes the information on the 12 significant outcomes, five of which survived the Bonferroni correction, four in VEGF1 and one in VEGF2, all deemed robust. These outcomes identified three VEGF polymorphisms (rs699947, rs144854329 and rs1570360) that were associated with allograft rejection post-KT. VEGF1 subgroup outcomes identified CR associations and Indians to be at risk. Depending on the genetic model, the Indian population were both susceptible (wt: OR 1.44, 95% CI 1.13-1.84) and protected (var: OR 0.61, 95% CI 0.45-0.82).\n\n\nDiscussion\n\nThe five Bonferroni-filtered findings (wt and var alleles) were either products of outlier treatment and/or subgrouping. Subgrouping identified the ethnicity and rejection type that was significant, thus specifying associations of the VEGF polymorphisms with allograft rejection post-KT. Subgrouping provided contrasts regarding significant outcomes: (i) In VEGF1, significant in Indians (Pa = 0.001–0.004), non-significant in Caucasians (Pa = 0.78–1.00); (ii) in VEGF2, significant in Caucasians (Pa = 0.02) and non-significant in Indians (Pa = 0.34); (iii) in VEFG1, significant in CR (Pa = 0.0009), non-significant in AR (Pa = 0.12). Subjecting these Pa-values to Bonferroni correction and sensitivity treatment raised the level of evidence that facilitated interpretation with greater confidence. We have shown that meta-analytical tools such as subgrouping, outlier and sensitivity treatments are instrumental in generating evidence for association. By design, such features are not present in the component single-study outcomes. This underpins the value of meta-analysis in systematically synthesizing primary study results and providing insight into associations of VEGF SNPs with allograft rejection post-KT. Conflicting outcomes between primary studies may be due to small sample sizes, hence, lack of power. Underpowered outcomes appear to be common in candidate gene studies45 and are prone to the risk of Type 1 error. In spite of the evidence for associations, the complexity of allograft rejection involves interactions between genetic and non-genetic factors allowing for the likelihood of environmental involvement. Gene-gene and gene-environment interactions have been reported to have roles in associations of other SNPs with post-KT allograft rejection. Two articles39,43 examined polymorphisms in other genes that included interleukin 18 (IL18), transforming growth factor beta 1 (TGFB1) and angiotensin II receptor type 1 (AGTR1). None of the seven articles acknowledged gene-environment interaction. Four23,39–41 of the included articles mentioned haplotype analysis with three presenting haplotype data23,39,40. Additional well-designed studies exploring other parameters would confirm or modify our results in this study and add to the extant knowledge about the association of the VEGF SNPs and renal allograft outcome.\n\nVEGF plays a crucial role in kidney physiology with its involvement in maintaining the integrity and permeability of the glomerular capillary basement membrane17. Adaptive response of VEGF toward renal allograft tissue may be related to its angiogenic property on endothelial cells since VEGF contributes to tissue repair response of damaged capillaries23. After KT, the recipient’s neutrophils and macrophages infiltrate the allograft after reperfusion of the transplanted tissue leading to the production of VEGF24. Shahbazi et al. showed that genetically directed variations in VEGF production with increased frequency of VEGF producing alleles seemed to influence susceptibility to acute allograft rejection24. However, Lemos et al. also suggested that renal allograft recipients with genetic potential for high VEGF production had significantly better graft survival compared to recipients with low VEGF production23. Our results along the timeline of post-KT outcomes indicated increased risks, both for AR and CR in the wt allele, which agreed with Shahbazi et al.24 but contrasted with Lemos et al.23. However, the significance of our increased risk CR finding may require caution in its interpretation given the low number of studies (n = 2) and low statistical power (64.4%). More studies may be needed to clarify our CR outcome. In terms of ethnicity, Indians carriers of the wt CC genotype in rs699947 (-2578C/A), were afforded better graft survival than the CA and AA genotypes41. In contrast, Shahbazi et al. found that the -2578 C allele (rs699947) and the -1154 G allele (rs1570360) were associated with increased risk of acute renal allograft rejection in Caucasians conferring greater risk among wt homozygotes (-2578C/C and -1154 G/G) compared to -2578C/A and -1154G/A heterozygous genotypes24. These inconsistent associations among previous studies may be due to the variations in genetic background influenced by differential ethnicities of the patients.\n\nInterpreting our findings should consider its limitations and strengths. Strengths include: (i) VEGF1 combined sample sizes translated to high aggregate statistical power (97.7%); (ii) significant HWC outcomes validated the overall pooled effects in wt. These validations served to reduce the risk of genotyping errors and minimize methodological weaknesses in our study; (iii) subgroup outcomes in CR and Indians point to potential clinical utility in the genetics of renal transplantation; (iv) efficiency of outlier treatment was the key to generating associative significance and eliminating or reducing heterogeneity and (v) stability of the core overall outcomes are underpinned by surviving the Bonferroni correction (minimizing Type 1 error risk) and robustness (determined with sensitivity treatment). On the other hand, limitations include: (i) all the component studies were underpowered; (ii) most of the moderately significant outcomes (67%) were non-robust.\n\n\nConclusions\n\nTo our knowledge, this is the first meta-analysis to examine associations between VEGF SNPs and risk of allograft rejection post-KT. Risks for renal allograft rejection associated with VEGF polymorphisms were shown to be increased up to 1.6-fold for the wt allele and 39% reduced for the var allele. Subgroups found to be susceptible were the Indian population and CR. These highly significant and robust core effects could render the VEGF polymorphisms useful as a prognostic biomarker in allograft rejection post-KT.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nDryad: Influence of polymorphisms in the vascular endothelial growth factor gene on allograft rejection after kidney transplantation: a meta-analysis, https://doi.org/10.5061/dryad.gqnk98skz25.\n\nThis project contains the following extended data:\n\n- S1 Table Overall, modified and subgroup outcomes for VEGF1.\n\n- S2 Table Overall, modified and subgroup outcomes for VEGF2.\n\n- S3 Table Overall, modified and subgroup outcomes for VEGF3.\n\nDryad: PRISMA checklist for ‘Influence of polymorphisms in the vascular endothelial growth factor gene on allograft rejection after kidney transplantation: a meta-analysis’, https://doi.org/10.5061/dryad.gqnk98skz46.\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'd like to thank A Kunjantarachot for her invaluable contributions at the incipient stages of the draft preparation.\n\n\nReferences\n\nLevey AS, Atkins R, Coresh J, et al.: Chronic kidney disease as a global public health problem: approaches and initiatives - a position statement from Kidney Disease Improving Global Outcomes. 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Dataset, 2021. http://www.doi.org/10.5061/dryad.gqnk98skz" }	[ { "id": "121055", "date": "14 Feb 2022", "name": "Ileana Constantinescu", "expertise": [ "Reviewer Expertise Immunology", "Immunogenetics", "Transplantation", "Molecular Biology", "Virology" ], "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 written by Puntilla and colleagues is very interesting and challenging. The subject is very actual, searching for new biomarkers in attempt to maximize long survival after kidney transplantation. The approach of many research teams is to reveal new biomarkers with impact on kidney allograft rejection. The VEGF gene polymorphisms completes other discussed biomarkers with impact on acute and chronic kidney rejection, like microRNAs, anti-angiotensin II type 2 receptor antibody and urinary cytokines, MIG, IP-10 etc.\nTransplant genetics is complex. We have to have in mind genetic background of chronic renal disease and also the genetic status of transplantation per se.\nThe meta analysis of VEGF SNPs is comprehensive well-documented using updated and accurate statistical methods. The literature search is well represented and well documented. The conclusions are clear. The information is valuable but reading the article could be difficult for a person who is not used with such extensive statistical analyses. I strongly recommend the indexing of this article with only one comment: it is very captivating but difficult to follow in its current format and must be converted to an easier content.\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": "119145", "date": "08 Mar 2022", "name": "Iman Karimzadeh", "expertise": [ "Reviewer Expertise Pharmacogenetics in the setting of kidney transplantation." ], "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 in this meta-analysis focused on the possible association between vascular endothelial growth factor gene polymorphism and allograft rejection after kidney transplantation. They demonstrated that risks for renal allograft rejection associated with VEGF polymorphisms were shown to be increased up to 1.6-fold for the wild type allele and 39% reduced for the variant allele. The topic is novel and interesting. The meta analysis was done properly. The manuscript is well-written. Therefore, the manuscript can be considered for indexing after implementing all comments and revisions.\nThe first reference related to the frequency of acute rejection in kidney transplantation should be added into the first paragraph of the introduction section.\n\nThe second reference related to the role of urine VEGF level in allograft rejection should be added and briefly discussed in the second paragraph of the introduction 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": [] } ]	1	https://f1000research.com/articles/10-90
https://f1000research.com/articles/9-1014/v1	20 Aug 20	{ "type": "Method Article", "title": "Spatial mapping of single cells in the Drosophila embryo from transcriptomic data based on topological consistency", "authors": [ "Maryam Zand", "Jianhua Ruan", "Maryam Zand" ], "abstract": "The advancement in single-cell RNA sequencing technologies allow us to obtain transcriptome at single cell resolution. However, the original spatial context of cells, a crucial knowledge for understanding cellular and tissue-level functions, is often lost during sequencing. To address this issue, the DREAM Single Cell Transcriptomics Challenge launched a community-wide effort to seek computational solutions for spatial mapping of single cells in tissues using single-cell RNAseq (scRNA-seq) data and a reference atlas obtained from in situ hybridization data. As a top-performing team in this competition, we approach this problem in three steps. The first step involves identifying a set of most informative genes based on the consistency between gene expression similarity and cell proximity. For this step, we propose two different approaches, i.e., an unsupervised approach that does not utilize the gold standard location of the cells provided by the challenge organizers, and a supervised approach that relies on the gold standard locations. In the second step, a Particle Swarm Optimization algorithm is used to optimize the weights of different genes in order to maximize matches between the predicted locations and the gold standard locations. Finally, the information embedded in the cell topology is used to improve the predicted cell-location scores by weighted averaging of scores from neighboring locations. Evaluation results based on DREAM scores show that our method accurately predicts the location of single cells, and the predictions lead to successful recovery of the spatial expression patterns for most of landmark genes. In addition, investigating the selected genes demonstrates that most predictive genes are cluster specific, and stable across our supervised and unsupervised gene selection frameworks. Overall, the promising results obtained by our methods in DREAM challenge demonstrated that topological consistency is a useful concept in identifying marker genes and constructing predictive models for spatial mapping of single cells.", "keywords": [ "Single cell RNA-seq", "spatial mapping", "feature selection", "particle swarm intelligence", "nearest neighbor" ], "content": "Introduction\n\nSingle cell RNA sequencing (scRNA-seq) is a cost-efficient, high throughput technology that has dramatically enhanced our understanding of developmental biology such as cell type identification, regulatory network inference, and cell trajectories1–7. Despite many breakthroughs in biological sciences made possible by this technology, it yet suffers from the drawback that native cell location in e.g. embryo or complex tissue is often lost, except for in a few experimental methodologies which are either expensive, require highly specialized tools, or are not as widely applicable as standard scRNA-seq protocols8–11. Given the substantial benefit offered through cell location recovery, such as obtaining a basic understanding of tissue function and disease pathology12,13, the cell spatial reconstruction was specifically addressed in recent Single Cell Transcriptome DREAM challenge as a community-wide effort.\n\nMany promising computational approaches dealing with the spatial reconstitution problem are centered around the main idea that an in situ atlas of a set of landmark gene’s expressions is used as a guideline to be combined with scRNA-seq profiles of individually measured cell14,15. For instance, Seurat14 first imputes the noisy scRNAseq data then predicts the cell locations by comparing the scRNAseq gene expression pattern to its binary expression level measured by in situ data. This step is done through a mixture model. Finally, original cell location is retrieved by evaluating a posterior probability function constructed for cell-bin pairs. DistMap15 was a successful method for spatial reconstruction (of Drosophila embryo) with near single cell resolution, much higher compared to that of Seurat (3039 bins versus 128 bins). It predicts top candidate positions for a given cell by calculating the Mathews Correlation Coefficients (MCC) of binarized landmark gene expressions for every cell-bin combination. While DistMap was to some extent successful in dealing with the cell spatial mapping problem, it was limited to binarized data rather than continuous, utilized simplistic MCC analysis, and more importantly it treats each single cell independently whereas it might be more beneficial to account for collective interrelationships between cells. To more extensively explore the space of better predictive strategies, DREAM challenge aimed to exploit the atlas provided by DistMap with the hope of resolving spatial reconstruction by using incrementally fewer landmark genes (i.e. 60,40,20). Achieving this goal will help with eliminating the need for a priori reference atlas, which is expensive and time-consuming to obtain, in the future transcriptomic studies.\n\nIn this work, we proposed a top-performing method (evaluated based on three distinct scoring criteria defined by DREAM challenge) which allows us to predict the cell location consistently as accurate as DistMap while requiring fewer number of landmark genes. The details of our method and evaluation metrics are provided later in the text.\n\n\nMethods\n\nThe general overview of our method is such that in the first step we investigate both supervised and unsupervised feature selection methods by defining two biologically rational metrics optimizing the consistency between gene expression similarity and cell proximity. In the unsupervised version we do not use the predicted cell locations given in 15 to obtain the set of most informative genes (e.g. 60,40,20), thus avoiding overfitting. On the other hand, the supervised version uses the cell locations given by DistMap as a reference. In the next, to predict the final cell locations, we use a PSO algorithm to assign proper weights to genes based on fitness functions defined by gene expression patterns. This reflects the intuition that different landmark genes are expected to demonstrate different potential in guiding us toward the proper embryo reconstruction. Finally, we use the information embedded in the cell topology to adjust the associated cell-location score with the hope to improve the predictions.\n\nTo reconstruct Drosophila embryo from single cells, we need reference dataset (in situ), spatial coordinates, and scRNA-seq data, the details of which along with the preprosseing steps are given in the following.\n\nReference database The reference database (denoted as W) provides the in situ expression values as a W3039×84 matrix where rows and columns correspond to bin locations and marker genes, respectively. The original data comes from Berkeley Drosophila Transcription Network Project (BDTNP) and in here we used the binarized format as explained in 15.\n\nSpatial coordinates The spatial coordinate information from one half of Drosophila embryo (denoted as L) is an L3039×3 matrix where the columns are x, y, and z coordinates of 3039 rows of bins.\n\nSingle cell RNA sequencing The scRNA-seq data (denoted as Y) gives the gene expression values as a Y1297×8924 matrix where rows and columns are single cells and genes, respectively. In here we followed the normalization process as implemented by 15. Briefly, the raw data was first normalized with respect to the total number of unique molecular identifiers (UMI) for each cell, followed by a pseudo count addition and a log transformation. The binarization process was implemented such that the quantile was varied in order to obtain the minimum mean squared root error between the gene correlation matrix of binarized atlas and binarized scRNA-seq.\n\nIn this study, our first goal is to identify a subset of genes whose expression patterns are predictive of cell locations. We have proposed two different feature selection methods (supervised and unsupervised) to select informative genes. In the supervised method, our metric was defined based on true cell locations (gold standard). To prevent overfitting we applied a 10 fold cross validation. On the other hand, we designed an unsupervised method based on the intuition that the current locations obtained by matching the normalized and binarized scRNA-seq expression patterns with the in situ expression patterns are not necessarily the true locations of these cells. These two methods are discussed in detail in the following sections.\n\nAs we believe the current locations obtained by matching the normalized and binarized scRNA-seq expression patterns with the in situ expression patterns are not necessarily the true locations of these cells, we decided to take an unsupervised feature selection approach, which does not depend on the current locations of the cells to be predicted, and therefore avoid overfitting.\n\nThe key rationale in our unsupervised feature selection method is that if a set of genes can be used as predictors of cell locations, then the cells showing similar expression patterns of these genes must be geometrically close to each other. Therefore, we defined two complementary metrics to quantitatively measure the proximity of cells with similar expression patterns for different gene subsets, and developed a greedy algorithm to search for a gene subset with the optimal (minimal) score combining the two metrics.\n\nMetrics to measure the power of gene signatures as location predictors. The first metric relies solely on the in situ gene expression patterns in the 3039 location bins, and is calculated as follows: given a set of genes G as features, the pairwise Pearson Correlation Coefficient (PCC) is computed between the in situ expression data for every pair of the 3039 location bins; the top-10 locations with the highest PCC is then identified for each location bin; the metric M1G is defined as the average Euclidean distance between each location bin and its top-10 most similar location bins:\n\nThe second metric uses information from both the in situ expression data and the scRNA-seq expression data, and is calculated as follows. Given a set of genes G as features, the pairwise PCC is computed between the scRNA-seq expression pattern of each of the 1297 cells and the in situ expression pattern of each of the 3039 location bins; then for each of the 1297 cells, the top-10 location bins with the highest PCC is identified; the metric M2G is defined as the average Euclidean distance between the geometric coordinates of the location bin most similar to cell c and the geometric coordinates of the top-10 most similar location bins (including the most similar location):\n\nNote that the currently known most possible location of each cell c, lc* which is predicted using all 84 genes with uniform weights, are not used in either M1 and M2; therefore, the gene selection process is not biased towards identifying genes to match the original locations predicted by the 84 genes. Rather, the metric provides an intrinsic measurement of the power of any subset of genes as location predictors, independent of the locations predicted with the 84 genes. In fact, the quality of the 84 genes as predictors can also be measured using these two metrics, and compared to any other gene sets; it is possible that a subset of the 84 genes can receive higher scores in these two metrics than the original 84 genes. In contrast, using a supervised feature selection method, where the “true” location is defined using all 84 genes, any subset of genes will be necessarily inferior to the complete set of 84 genes.\n\nStep-wise backward elimination feature selection algorithm. We used a standard backward elimination algorithm to identify a subset of genes G with the minimal sum of M1GandM2G. Briefly, starting with a set of q genes, we computed M1GandM2G for all possible subsets of q − 1 genes by removing one gene at a time from the set. The subset with the minimal M1G+M2G is then recorded as the best subset of size q − 1. This procedure is then repeated until a desired number of genes is reached. As this algorithm is a greedy approach, it does not guarantee to find the optimal solution. We have also attempted to combine backward elimination with forward selection, which only improved the solution slightly. Due to the excessive running time required, we opted to use the simple algorithm described above while leaving additional improvement as future work.\n\nWhile in the unsupervised approach metrics M1 and M2 were optimized, in the supervised version a single metric N was defined as explained below. This metric, which relies on both the scRNA-seq gene expression patterns in the 1297 cells and the gold standard location of each cell, is calculated as follows: given a set of genes G as features, the pairwise PCC is computed between the scRNA-seq expression data for every pair of the 1297 cells; the top-10 cells with the highest PCC is then identified for each cell; the metric NG is defined as the average Euclidean distance between the gold standard geometric coordinates of each cell and its top-10 most similar cells:\n\nIt is intuitive to assume that the contribution of genes in determining cell locations are not equal. Therefore, we look for a way to learn how to assign proper weight to each selected gene for more accurate prediction of cell locations. To this end, we chose a supervised learning approach, using the cell locations predicted by the highest MCCs with the 84 signature genes as “gold standard” locations. To avoid overfitting, we performed 10-fold cross-validation: gene weights were determined using the scRNA-seq data of 90% of cells; these weights are then used to predict the locations of the remaining 10% of the cells not used in training. The splitting of the data is saved, for reproducibility of the results.\n\nThe basic idea of the PSO algorithm is as follows. We created a set of agents, each of which is initiated with a gene weight vector wi of size \|G\|× 1. Each weight vector is evaluated by how closely the weighted gene expression pattern can be used to predict the cell locations when compared to the “gold standard” locations obtained with the 84 genes:\n\nDuring the search, each agent keeps track of a personal best weight vector P besti, and the global best solution from all agents is denoted Gbest. At each iteration, the weight vector of each agent is updated by the differences between the current weight and the personal best and global best weight vectors:\n\nThe location prediction for each single cell relies on the (weighted) similarity between the expression pattern of selected signature genes in the cell and every location bin. It is important to note that the expression patterns in neighboring cells should be similar in general, and therefore the overall prediction should take the expression of nearby location bins into consideration. Intuitively, if the globally highest scoring location is far away from locations with slightly lower but comparable scores, the confidence score for the highest-scoring location should be reduced; on the other hand, a locally highest-scoring location close to other high-scoring locations should be upweighted. Therefore, to make the final prediction for a given cell, we adjusted the prediction score based on the prediction scores from neighbor locations.\n\nFormally, let C = (cij)n×m be the bin-cell association matrix, where cij is the PCC between the (weighted) scRNA-seq data and the in situ hybridization data for every pair of cells and locations. n = 3039 is the number of candidate location bins, and m is the number of cells in the test set, Let D = (dij)n×n be the Euclidean distance matrix between the geometric coordinates of every pair of location bins. We define an affinity matrix A = (aij)n×n such that aij=e−dijd, where d is a parameter to control how many neighbor locations can impact the final prediction score. A smaller d* value means fewer neighbor locations to be considered. To have a robust measure of how geometrically close two location bins can be, we first measure the distance between each location and its nearest location, and then computed the median of these shortest distances as d*. As a result, most aijs’ are much smaller than e−1, and only a limited number of neighbor locations with very high scores can impact the final prediction score for each cell.\n\nThe final prediction score matrix P = (pij)n×m is calculated by P = A × C. Since aii = 1 and aij ≤ 1 for all j ≠ i, it is easy to see that\n\nFrom the final predicted bin-cell association matrix, we reported the 10 locations with the highest scores for each cell as the most likely positions in embryo.\n\nIn subchallenge 1 we performed both PSO and neighbor weighting. In subchallenge 2, we did not perform PSO; therefore, the 40 genes obtained from gene selection are utilized with uniform weights. In subchallenge 3, genes were weighted with the PSO procedure, but we chose not to perform neighbor weighting.\n\nIn this phase to evaluate the robustness and soundness of the method, a 10 fold CV scenario was performed to obtain 10 different sets of informative genes using a subset of cells. To compare the similarity of the selected genes, Jaccard similarity was defined as follows:\n\nOur method was designated as a top-performing method among 34 participating teams. To evaluate and rank the teams, the challenge organizers had defined three scoring metrics s1,s2, and s3, which were not disclosed to participants at the time of submission. The details of each metric are available at https://github.com/dream-sctc/Scoring. Here, we briefly explain the general intuition and purpose behind each scoring metric. Roughly speaking, s1 stands for how the gene expression of the 10 most probable predicted position for all cells closely approximates that of the in situ expression pattern of the gold standard locations (as predicted by DistMap). On the other hand, s2 only considers how the averaged location prediction of the 10 most probable predictions using 60, 40, and 20 genes is compared to that of the one predicted by using all the 84 genes. As is evident, this metric does not include either of the accuracy of the in situ expression profile prediction and the closeness of in situ and scRNA-seq data. Meanwhile, s3 accounts for how the scRNA-seq expression of 60,40, and 20 genes of the best predicated locations is closely approximating that of the in situ expression patterns.\n\nThe method proposed here is written in Matlab 2018b and the source code is available from GitHub16\n\nIt does not utilize or rely on any specific Matlab toolbox. Therefore by following the clear detailed formulation provided in manuscript this method can readily be implemented in any open-access software.\n\n\nResults and Discussion\n\nIn the two supervised and unsupervised methods discussed so far there exist two important components implemented with the intention to both select a set of most informative genes and locate cells based on the information buried in cell neighborhood network topology. To elucidate the effectiveness of these two components, we designed a set of four experiments in which the first component was replaced either by random gene selection or a simple strategy of high degree gene selection and the second component was inactivated. The subchallenge scores for these total of six methods are given in Table 1. To quantify the effectiveness of the two mentioned components we calculated the average score of the group A, containing supervised and unsupervised, and group B, containing the remaining four methods. Our analysis showed that on average the score of group A were higher by 14%, 20%, 30% in the subchallenges 1, 2, and 3 respectively. The reason for the greater performance in subchallenge 3 was expected given that fewer number of genes were used compare to the other two subchallenges.\n\nThe predictions mentioned above did not involve any additional pre-processing steps, e.g. imputation, on the provided input data. We simply used the binarized and normalized in situ hybridization and scRNA-seq data. However, for the sake of completeness we also examined the possible role of \"imputation\" and using raw data instead of the binarized scRNA-seq data. We tried to impute the dropouts in scRNA-seq data using SAVER17 and netImpute18, but no significant improvement was gained in terms of enhancing our metric scores. On the other hand, although our analysis indicated that using raw data instead of the binarized data can potentially increase the consistency between gene expression pattern similarity and cell proximity in this challenge (according to M1 and M2 metrics), we are limited by the fact that the true locations of the cells to be predicted are unknown, and prediction accuracy is at least partially defined by comparing to the “gold standard” location obtained from binarized data. We speculate that anyone using raw data would probably be disadvantaged. It is noteworthy that our method is applicable if one prefers to use raw data instead of binarized data, and our results (data not shown; available as underlying data) indicate that there is benefit of using raw data instead of binarized data.\n\nIn the post-challenge phase of the competition the data set was divided into train and test subsets using 10-fold cross-validation in order to further investigate to what degree the set of most informative genes are consistent across different subset of cells selected through the 10 fold CV analysis. The results given in Figure 1 show that the Jaccard similarity between different folds are higher than the expected similarity in all three subchallenges indicating that there in fact exists a consistency in the most-informative genes selected across different folds. Moreover, as the number of genes allowed in a subchallenge decreases (from subchallenge 1 to subchallenge 3) the difference between Jaccard similarity of the most-informative genes and its expected value becomes more and more pronounced.\n\nBlue stars represent expected Jaccard similarity.\n\nFigure 2 shows the Venn diagram of 20 most informative genes selected from supervised and unsupervised methods. Out of 20 genes selected by each method, there are 11 common genes identified by both methods, which is more than expected (p-value < 0.0005, Fisher’s exact test).\n\nThe 12 genes denoted by red color are the scRNA cluster-specific genes reported in DistMap.\n\nAnother interesting observation is that cluster-specific genes (denoted by red color) are prevalent in the set of most informative genes obtained from both supervised and unsupervised methods. This finding highlights our method was in fact able to take advantage of those 12 cluster-specific genes which contain cell location information.\n\nTo virtually reconstruct gene expression patterns, the result of our method (i.e. bin-cell association matrix) was processed based on the methodology of vISH - a tool developed in 15 to derive the expression pattern of each of the 84 genes across the location bins, and compared with the expression patterns obtained by DistMap. Figure 3 shows the distribution of the PCC between DistMap and our results from the three subchallenges. Overall, there is a high correlation among reference patterns (DistMap) and patterns generated by our method. The average correlation in the three sub-challenges are 0.81, 0.76, and 0.68, respectively. In sub-challenge 1, almost all genes have been reliably reconstructed, while for sub-challenge 3, a small number of genes have fairly low reconstruction rate.\n\nFigure 4 shows the reconstructed expression patterns for three genes: twi, cad, and ftz, which play key roles in the regulatory network of early Drosophila development. Overall, there is good agreement between our predictions and that of DistMap. In case of twi, our method and DistMap both very precisely predicted the in situ expression pattern. In fact, twi is one the 20 genes selected by both the supervised and unsupervised feature selection methods, due to its distinct expression patterns associated with cell spatial arrangement in the embryo. For cad, DistMap and our method with as few as 20 genes predicted very similar expression patterns, where there is a higher expression in the posterior domain, consistent with the current knowledge of cad in embryo development19. On the other hand, the predicted expression patterns seem to be much more diffused than the in situ expression pattern, potentially because of the binarization of the in situ data, which caused loss of weaker signals. Finally, for ftz, while the predicted expression pattern by our method with 60 genes is in general agreement with DistMap and in situ data, our method with 40 or 20 genes failed to reconstruct the expression pattern of ftz associated with the segmentation of Drosophila embryos20. While it is possible that more refined parameters such as a smaller number of neighbor cells may improve the prediction of our method, we believe the striped pattern of ftz makes it difficult, if not impossible, for any method that aims at a much reduced number of marker genes for spatial mapping.\n\n\nConclusion\n\nIn this work, we proposed a method to identify gene markers for RNAseq-based reconstruction of cell spatial information that were lost during single-cell transcriptomics sequencing of Drosophila embryo. The main hypothesis of this study is that the topology of the marker gene expression based cell-cell similarity graph should be consistent with the topology of the cell-cell geometric location map. To test the hypothesis, several metrics were defined based on this biological rationale to capture the consistency between gene expression similarity and cell proximity. A greedy step-wise backward elimination feature selection algorithm was implemented to find a set of most informative genes to optimize these metrics. Next, a Particle Swarm Optimization algorithm was developed to obtain optimal gene weights to construct the cell-location association matrix. Finally, the prediction score of a cell’s location was further improved by considering the expression similarity between neighboring locations. It was shown that our method can successfully identify markers genes capable of predicting cell locations with high accuracy. In addition, it was also demonstrated that our method can recover the spatial expression patterns of most embryo marker genes. Even though the method proposed here was custom designed for this Drosophila embryo problem, it has the potential to be readily applied to other organisms as well.\n\n\nData availability\n\nThe challenge datasets can be accessed at https://www.synapse.org/#!Synapse:syn16782375\n\nChallenge documentation, including the detailed description of the Challenge design, overall results, scoring scripts, and the clinical trials data dictionary can be found at: https://www.synapse.org/#!Synapse:syn15665609/wiki/582909\n\n\nSoftware availability\n\nSource code is available from: https://github.com/mary77/scSpatialMapping.git\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.387757716\n\nLicense: MIT", "appendix": "References\n\nKolodziejczyk AA, Kim JK, Svensson V, et al.: The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015; 58(4): 610–620. 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Science. 2017; 358(6360): 194–199. PubMed Abstract \| Publisher Full Text\n\nZand M, Ruan J: mary77/scspatialmapping: First release of scspatialmapping. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3877577\n\nHuang M, Wang J, Torre E, et al.: SAVER: gene expression recovery for single-cell RNA sequencing. Nat Methods. 2018; 15(7): 539–542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZand M, Ruan J: Network-based single-cell rna-seq data imputation enhances cell type identification. Genes (Basel). 2020; 11(4): 377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStauber M, Lemke S, Schmidt-Ott U: Expression and regulation of caudal in the lower cyclorrhaphan fly megaselia. Dev Genes Evol. 2008; 218(2): 81–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim B, Fukaya T, Heist T, et al.: Temporal dynamics of pair-rule stripes in living drosophila embryos. Proc Natl Acad Sci U S A. 2018; 115(33): 8376–8381. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "69945", "date": "07 Sep 2020", "name": "Tianci Song", "expertise": [ "Reviewer Expertise Computational Biology", "Machine Learning", "Biological Network Analysis", "Single-cell Genomics", "Spatial 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\nThis manuscript describes the methodology the authors employed to perform well in the DREAM Single cell Transcriptomics Challenge. The theme of the challenge is to select a subset of 84 known marker genes to characterize the spatial gene expression patterns in Drosophila embryo. The team developed methods for unsupervised gene selection, supervised gene selection, gene weighting and neighbor-smoothing based prediction of cell location. Overall, the manuscript provides sufficient detail to reproduce the results and some interpretations. It appears some further improvements can be done as follows:\nWhere it is understood that the team has optimized the hyper-parameter including k, alpha and beta with some fixed values for this dataset. For others to use the same methods on other similar studies, there is no clue how to tune or select the hyper-parameters. Discussion or more experimental results should be provided in this regard.\n\nSimilarly, it is also a mystery why different strategies of combining the methods are necessary for the three sub-challenges. There is no discussion of how the combinations are selected or optimized.\n\nThe results in Table 1 are very poorly explained. How to calculate the 14%, 20%, 30% improvement over the three measures is unexplained. Possibly, it is also helpful to explain the metric used to rank different methods in the competition.\n\nIn Figure 4, why not also plot the original in-situ hybridization and scRNAseq expressions of the three genes for comparison? The use of vISH seems to be unnecessary.\n\nIn the section \"Datasets and pre-processing steps\", a subsection explaining the \"gold standard\" prediction by DistMap should be added. It is confusing in the description of supervised vs unsupervised gene selection, when the DistMap prediction and the nearest locations are used in the measures in equation (2) and (3).\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6331", "date": "09 Feb 2021", "name": "Maryam Zand", "role": "Author Response", "response": "We thank the reviewers for the overall positive comments, as well as the detailed critics that have helped to improve the manuscript. Response to Reviewer1 comments This manuscript describes the methodology the authors employed to perform well in the DREAM Single cell Transcriptomics Challenge. The theme of the challenge is to select a subset of 84 known marker genes to characterize the spatial gene expression patterns in Drosophila embryo. The team developed methods for unsupervised gene selection, supervised gene selection, gene weighting and neighbor-smoothing based prediction of cell location. Overall, the manuscript provides sufficient detail to reproduce the results and some interpretations. It appears some further improvements can be done as follows: (Q1) Where it is understood that the team has optimized the hyper-parameter including k, alpha and beta with some fixed values for this dataset. For others to use the same methods on other similar studies, there is no clue how to tune or select the hyper-parameters. Discussion or more experimental results should be provided in this regard. (A1) We thank the reviewer for bringing up this important point. It should be noted that as the challenge participants were blind to the evaluation metrics at the time of results submission, which made a direct assessment of the performance of the proposed methods inaccessible, our team did not perform extensive parameter tuning. In the revised manuscript we have provided some insights and reasonings behind the selection of hyperparameter values used in our work. This added information may be used as a general guideline for hyperparameter selection for applying the proposed method to other datasets of similar nature. Nonetheless, it should be mentioned that optimum hyperparameters is problem-specific and may be found through ad hoc tests, sensitivity analysis, and detailed investigations. The following has been added in the manuscript. “In this work, k is set to 10 because the evaluation of the prediction results is based on 10 best locations for each single cell. Also, based on the number of location bins (n), we believe 10 is a reasonable choice for the number of nearest neighbors.” “alpha and beta are acceleration coefficients, also referred to as trust parameters. alpha expresses how much confidence a particle has in itself, while beta expresses how much confidence a particle has in its neighboring particles. Particles draw their strength from their cooperative nature and are most effective when alpha and beta coexist in a good balance. Most applications use alpha=beta. Low values for alpha and beta result in smooth particle trajectories, while high values cause more acceleration, with abrupt movement towards or past good regions. In this study we used alpha=beta=0.2, with 200 agents and the maximum number of iterations set to 40. These parameters were manually tuned by observing the values of the fitness function to reach desired search granularity and speed of convergence.” (Q2) Similarly, it is also a mystery why different strategies of combining the methods are necessary for the three sub-challenges. There is no discussion of how the combinations are selected or optimized. (A2) During the challenge period, each team was given a limited number of attempts to test the success of their proposed approach(es) - evaluation results and ranking of all teams were shown in a leaderboard, with no details of the evaluation metrics. It was made clear that different methods could be used for different sub-challenges. Therefore, it was a natural choice for us to try a diverse combination of the main ingredients of the method for different subproblems to get some insights of these ingredients. During the post-challenge stage, further experiments have been designed by the DREAM organizers for additional insights (see ref. [16]). In this manuscript, we have also added a few baseline approaches to test these components (see Table 1). While we could repeat the whole experiments with a common strategy for all three sub-challenges, we thought it would be best for the readers to see the results in a way consistent with our final results submitted to DREAM challenge. Admittedly, we did not use PSO for sub-challenge 2 because of lack of time towards the end of the competition. The rationale of using neighbor weighting in subchallenge 1 and 2 but not in subchallenge 3 is that as subchallenge 3 used substantially fewer genes, the quality of the location prediction may be relatively low and therefore using gene expression information from the predicted neighbors could actually degrade the final prediction. The revised manuscript now provides some details of these rationales. (Q3) The results in Table 1 are very poorly explained. How to calculate the 14%, 20%, 30% improvement over the three measures is unexplained. Possibly, it is also helpful to explain the metric used to rank different methods in the competition. (A3) In the revised manuscript we have further explained the results presented in Table1 to make it clearer. The metrics used to rank different methods is now explained in section “Evaluation metrics”. The details of formulation and additional explanation can be found in the reference article [16]. Additional discussion of the evaluation results has also been included in the revised manuscript (see below for your convenience). “Table 1 shows the results of our supervised and unsupervised methods on the three subchallenges, evaluated by the three metrics (s1, s2, and s3) proposed by the DREAM challenge organizers. The details of these metrics are discussed in Method Section “DREAM consortium evaluation metrics”. A more detailed analysis of the results and comparison with other top-performing algorithms are presented in 16 and is not repeated here. To obtain some additional insights of our algorithms’ performance, we present here the results of some variations of our proposed methods. Both our supervised and unsupervised methods have two important components, (1) gene selection, and (2) neighbor-weighted cell location prediction, integral to selecting a set of most informative genes and locating cells based on the information buried in cell neighborhood network topology. To understand the importance of these two components, we designed a set of baseline studies incorporating four experiments. In these experiments, the gene selection strategy was replaced by either selecting genes randomly, or selecting genes expressed in the most number of cells (high degree genes). The neighbor-based reweighting component was also removed in two of these experiments. The subchallenge scores corresponding to our method (supervised and unsupervised) along with these four baseline studies are listed in Table 1 under the group A and group B, respectively. The method with highest score (s1, s2, s3) in each of the three subchallenges is shown in boldface. It can be seen that the supervised and unsupervised methods (group A) achieved comparable results, and significantly outperformed the baseline approaches (group B) on average, for all three sub-challenges. For subchallenge 3, which is the most difficult task, both of our methods significantly outperformed the baseline approaches in all three metrics. On the other hand, for subchallenge 1, for which the goal is to select 60 genes to best approximate the cell locations determined by 84 genes, random gene selection coupled with neighbor-based reweighting achieved almost the same performance as our unsupervised approach, and is only slightly inferior to the supervised approach. This is understandable because of the extensive overlap between the randomly selected genes and the “optimal” gene set. High degree selection achieved somewhat less accurate results than random selection, indicating that some less frequently expressed genes are important determinants of cell locations. For subchallenge 2, our proposed methods outperformed all four baseline approaches in s2, and three out of the four baseline approaches in s1 and s3. Finally, comparing the four baseline approaches suggest that the neighbor-based reweighting component significantly improved s2, but its impacts on the other two metrics are somewhat mixed. Overall, the significant performance gain in subchallenge 3 compared to random gene selection and high degree gene selection supports that the small set of genes we identified are important for predicting cell locations.” (Q4) In Figure 4, why not also plot the original in-situ hybridization and scRNAseq expressions of the three genes for comparison? The use of vISH seems to be unnecessary. (A4) The original binarized in situ hybridization data is what the first subplot in Figure 4 shows in fact. For other subplots vISH was used because we wanted to compare our results with Distmap which also uses vISH. Moreover, vISH computes the expression pattern for each gene by combining the cell-bin mapping scores with the original normalized gene expression levels. (Q5) In the section \"Datasets and pre-processing steps\", a subsection explaining the \"gold standard\" prediction by DistMap should be added. It is confusing in the description of supervised vs unsupervised gene selection, when the DistMap prediction and the nearest locations are used in the measures in equation (2) and (3). (A5) In the revised version we have added “Gold standard cell locations” subsection. “For each of the 1297 cells, the Mathews Correlation Coefficients (MCC) is calculated at each of the 3039 location bins between the binarized 84 RNAseq expression values for the 84 driver genes and the binarized in situ expression values for the same 84 genes. The location bin with the maximum value of MCC score is defined as the gold standard location for each cell.”" } ] }, { "id": "69942", "date": "18 Sep 2020", "name": "Adi Tarca", "expertise": [ "Reviewer Expertise Machine learning", "genomics", "statistics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe their approach to the DREAM Single cell prediction challenge that was designed to find optimal subsets of 20, 40, and 60 genes that would allow prediction of single cells locations in a dme embryo with minimal accuracy loss relative to locations based on 84 genes. The method could be useful in similar applications, yet I have the following comments:\n\nMajor:\nThe “Unsupervised gene selection” section defines two metrics (M1 and M2) to assess the quality a given gene set G of size 20, 40, or 60 out of all 84 genes with in situ data available. The optimal set of genes is identified by a greedy algorithm. However, metric M2 can not be considered unsupervised because it takes the samples from the set where prediction of locations are expected (cells from the scRNA-seq data) and correlates their expression values with profiles of all in situ bins and finds those genes that ensure a maximum correlation. While the authors do not use the information of the in situ cell assigned by DistMap to a given single cell (gold standard), by expecting that there should be some 10 bins with gene expression patterns that correlate with the profile of each single cell, they are implicitly creating their own gold standard. Therefore, not only M2 should be stated as supervised instead of unsupervised, the authors should add as a limitation in the discussion the fact that they have used in situ data from all 84 genes in selecting the best subsets of 20, 40, 60 which was against the DREAM challenge rules.\n\nThe PSO algorithm to set the gene weights optimization seems to be computationally intensive. Can the authors provide some sense of computation time involved?\nMinor:\n\nThe authors state, “We used a standard backward elimination algorithm to identify a subset of genes G with the minimal sum of M1 and M2”. I believe they meant identifying the subset of genes G with a minimal loss (decrease) in M1+M2, since you want the sum to remain as high as possible. So in the end this is a maximization not minimization problem.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6332", "date": "09 Feb 2021", "name": "Maryam Zand", "role": "Author Response", "response": "We thank the reviewer for the overall positive comments, as well as the detailed critics that have helped to improve the manuscript. Response to Reviewer2 comments The authors describe their approach to the DREAM Single cell prediction challenge that was designed to find optimal subsets of 20, 40, and 60 genes that would allow prediction of single cells locations in a dme embryo with minimal accuracy loss relative to locations based on 84 genes. The method could be useful in similar applications, yet I have the following comments: Major: (Q1) The “Unsupervised gene selection” section defines two metrics (M1 and M2) to assess the quality a given gene set G of size 20, 40, or 60 out of all 84 genes with in situ data available. The optimal set of genes is identified by a greedy algorithm. However, metric M2 can not be considered unsupervised because it takes the samples from the set where prediction of locations are expected (cells from the scRNA-seq data) and correlates their expression values with profiles of all in situ bins and finds those genes that ensure a maximum correlation. While the authors do not use the information of the in situ cell assigned by DistMap to a given single cell (gold standard), by expecting that there should be some 10 bins with gene expression patterns that correlate with the profile of each single cell, they are implicitly creating their own gold standard. Therefore, not only M2 should be stated as supervised instead of unsupervised, the authors should add as a limitation in the discussion the fact that they have used in situ data from all 84 genes in selecting the best subsets of 20, 40, 60 which was against the DREAM challenge rules. (A1) Firstly, we agree that the “unsupervised” version of our method does not strictly follow the DREAM challenge rule of not using in situ data directly. The reason is that we were not aware of the rule until after our submission. (The rule was posted in the discussion forum instead of on the challenge main webpage, and there were a lot of discussions about this issue among other participants as well). This is why we were allowed to submit, “unofficially”, a supervised version after discussing with the DREAM organizers. Despite violating the DREAM challenge rule, we believe the “unsupervised” method we describe here has been evaluated rigorously and the ideas are valuable, based on both post-challenge analysis results, as well as arguments presented below. Secondly, we argue that the M2 metric that we proposed is indeed unsupervised instead of supervised learning. In a supervised method, you optimize parameters in order to achieve the minimal deviation from ground truth (or gold standard in this work). Since the gold standard in this work is defined with all 84 genes, when you reduce the number of genes, you expect to gradually increase the gap between the gold standard and the prediction, and the goal is to minimize the gap with K genes. Our proposed metric M2 does not aim to reduce deviation from ground truth. In fact, the values of M2 achieved with the genes that we selected have better M2 values than the initial 84 genes, which potentially indicate that the initial 84 genes are not “optimal”. Another way to look at this is that we do not use the “gold standard” position in determining how good or bad the selected genes are. The definition of M2 relies on lcG , the location bin whose expression profile of gene set G is the most similar to cell c among all location bins. Although initially when G has all 84 genes, lcG is indeed identical to the gold standard, lc* , this set of locations moves away from the gold standard when the gene set changes – so you could say that we have a “moving” gold standard if you wish, and the fact is that with 20 genes, lcG is quite different from lc*. Furthermore, while we implemented a stepwise backward elimination method to choose G genes from 84 genes, we could have started with any random set of genes or the whole RNAseq dataset (with much longer running time, however). In fact, our analysis results showed that the M2 values on test data can often be better than the M2 values on training data, further supporting that this is not a supervised method. (Q2) The PSO algorithm to set the gene weights optimization seems to be computationally intensive. Can the authors provide some sense of computation time involved? (A2) We have included this information in the revised manuscript: “The running time of PSO algorithm is about 15 hours when running on a system with 16 GB of memory. However, the running time could depend on different optimization settings such as parameter alpha and beta.” Minor: (Q3) The authors state, “We used a standard backward elimination algorithm to identify a subset of genes G with the minimal sum of M1 and M2”. I believe they meant identifying the subset of genes G with a minimal loss (decrease) in M1+M2, since you want the sum to remain as high as possible. So in the end this is a maximization not minimization problem. (A3) M1 and M2 were defined based on Euclidean distances and our objective was indeed to minimize M1+M2, not to maximize it." } ] } ]	1	https://f1000research.com/articles/9-1014
https://f1000research.com/articles/10-85/v1	09 Feb 21	{ "type": "Study Protocol", "title": "Understanding the factors enabling and blocking sustained implementation of cholera interventions in a fragile region of Nigeria: a multi-phase group model building study protocol", "authors": [ "Kelly Elimian", "Carina King", "Karin Diaconu", "John Ansah", "Sebastian Yennan", "Chinwe Ochu", "Emmanuel Pembi", "Gandi Benjamin", "Birger Forsberg", "Chikwe Ihekweazu", "Tobias Alfvén", "Carina King", "Karin Diaconu", "John Ansah", "Sebastian Yennan", "Chinwe Ochu", "Emmanuel Pembi", "Gandi Benjamin", "Birger Forsberg", "Chikwe Ihekweazu", "Tobias Alfvén" ], "abstract": "Introduction: Adamawa and Bauchi are cholera endemic states in the north-east region of Nigeria, each with local government areas classified as cholera hotspots. Ineffective implementation of multi-sectoral cholera interventions in both states could make obtaining the global target for cholera control in Nigeria out of reach. A major contributing factor to this challenge is fragility of the region due to persistent Boko Haram insurgency activities, often characterised by the destruction of health infrastruture and displacement of communities to areas with suboptimal living conditions. Given the complexity of disease control in such a fragile setting, this study aims to systematically examine the barriers and/or facilitators influencing the implementation of existing cholera interventions in these states.\n\nMethods: The study will use a systems dynamic approach. First, we will conduct a health facility survey to determine the current health system capacity to support multi-sectoral cholera interventions, and conduct key informant interviews with purposely selected state and national cholera stakeholders to identify the context-specific facilitators and barriers to the implementation of cholera interventions in these states. We will then conduct nine group model building workshops (four in both the Adamawa and Bauchi states and one in Abuja) among cholera stakeholders similar to those recruited for the interviews. Conclusion: By engaging diverse and relevant cholera stakeholders, including community members, this study has the potential to provide a rich understanding of context-specific factors influencing the implementation of multi-sectoral cholera interventions in a fragile region of Nigeria, with a view to achieve sustainable progress towards cholera control in the country. Moreover, this study could have an impact on the control of other water-borne diarrheagenic diseases in the country.", "keywords": [ "Cholera", "Fragility", "Multi-sectoral", "System dynamic", "Group model building", "Adamawa", "Bauchi", "Nigeria" ], "content": "Introduction\n\nDespite the possible underestimation due to limited surveillance and reluctance to report cases for economic reasons, approximately 2.9 million cholera cases and 95,000 cholera-related deaths are recorded annually worldwide1. These occur predominantly in 47 endemic countries2. Notably, since 2017, the inequitable burden of cholera has continued to increase, with the poorest and most vulnerable populations in fragile settings with inadequate portable water, sanitation and hygiene most at risk3. Cholera elimination as a target, sits across the United Nation’s (UN) Sustainable Development Goals (SDG) agenda4, given its roots in inequity, poverty, environmental threats and conflict. This is because public health measures targeting cholera have an explicit implication for water-related diseases within SDG3 (ensuring healthy lives and promoting wellbeing for all) and an implicit implication for tracking progress towards achieving SDG6 –universal and equitable access to safe and affordable drinking water, and access to adequate and equitable sanitation and hygiene for all. Additionally, cholera transmission is an important motivation for the development of the 1952 World Health Organization’s (WHO) International Sanitary Regulations5, given its capacity to impact global health security and economic growth6.\n\nWhile substantial progress has been made towards cholera control in the SDG era, a combination of climate change, natural and man-made disasters, and rapid unplanned urbanisation continue to support transmission7. The WHO’s Global Task Force on Cholera Control (GTFCC), revitalised in 2014, is a network of more than 50 partners with this global mandate2. In 2017, it launched and adopted the ‘Ending Cholera: A Global Roadmap to 2030’ strategy. Unlike the previous approach to cholera outbreaks, which was more reactive and short-term, this strategy sets out a long-term plan for cholera elimination for 20 of the 47 endemic countries, with the target of 90% reduction in cholera-related deaths by 20302.\n\nThe attainment of these goals hinges upon six interventions: water, sanitation and hygiene (WASH); surveillance and reporting; use of oral cholera vaccine (OCV); healthcare system strengthening; leadership and coordination; and community engagement and empowerment. According to the joint WHO and UN Children’s Fund (UNICEF) updated estimates for WASH in households in 2017, 8 out of 10 people in rural areas in sub-Saharan Africa still lacked basic drinking water; 7 out of 10 lacked basic sanitation services; and 3 billion people lacked basic handwashing facilities at home8. These conditions are drivers of recurrent cholera transmission, especially given that WASH interventions are often reactive during cholera outbreaks in many cholera endemic settings9. An analysis of the global use of OCV stockpile from inception in July 2013 to the end of 2018 noted challenges relating to timeliness of response to cholera outbreaks and contextualisation of strategies for OCV delivery10; a significant improvement in vaccine acceptance and safety was however noted. Additionally, existing government's surveillance systems can become weakened due to limited access to conflict areas, contributing to delayed notification and under-estimation of cholera cases and poor implementation of appropriate public health measures11.\n\nCholera endemic countries are required to contextualise and implement the GTFCC strategic roadmap; however a significant constraint—and a possible source of reversal to current gains—is fragility. This can be the result of violence and prolonged conflict, political and economic instability, marginalisation and inequality, weak and distorted national governance structures and processes, and significant environmental threats and natural disasters12. Fragility can impact public health by limiting a population’s capacity to respond and adapt to stressors and shocks, such as a disease outbreak in a humanitarian setting. A 2020 WHO report indicates a decrease in progress towards cholera control, with nearly double the number of cholera cases during 2019 (923,037) than 2018 (499,447); the number of cholera-related deaths however decreased by 36% (from 2,990 in 2018 to 1,911 in 2019)13. Notably, 93% of cases in 2019 were from Yemen, a country that has become extremely fragile by recurrent armed conflicts with frequent, widespread cuts in water supplies13. A similar trend was also noted in Mozambique, where—amidst successive cyclones with heavy rains and population displacement in 2019—over 7,000 cholera cases was recorded in comparison to 910 cases in the previous year13.\n\nIn Nigeria, the burden of diarrhoeagenic diseases is extremely high, having the second highest total of under-5 child deaths from pneumonia and diarrhoea in the world14. Nigeria has witnessed two major cholera outbreaks in recent years, with over 40,000 cases and 1,716 deaths in 201015 and approximately 50,000 cases and 850 deaths in 201816. The country is currently classified as a cholera high-burden country17, with 83 local government areas (LGAs) in 14 states determined as cholera ‘hotspots’. Notably, over half of these cholera hotspot LGAs are located in the north-east region of the country including Borno, Adamawa and Yobe States, where Boko Haram insurgency activities are predominant18. Boko Haram insurgency activities are often characterised by the disruption of WASH services, displacement of populations to overcrowded camps, and emigration of health workers to safer areas19. It is therefore no surprise that the highest cholera prevalence and case fatality ratio during the outbreak of cholera in Nigeria in 2018 were recorded by north-eastern states16.\n\nWhile reiterating the difficulty of addressing disease outbreaks in the context of fragility, the concentration of cholera burden in the north-east region of Nigeria suggests suboptimal control strategies, particularly with the implementation of existing multi-sectoral cholera interventions. Previous responses to cholera outbreaks in north-east Nigeria have been hampered by inadequate training of healthcare workers, limited supply of emergency response kits and personal protective equipment20, and poor diagnostic capacity and community misconceptions towards WASH and OCV interventions (with consequent reluctance to accept these interventions)21. Furthermore, recurrent transmission of cholera in the region is further compounded by the influx of reactive humanitarian responses which often set up reactive and parallel interventions, without long-term goals in mind22. Thus, given the fragility and cholera endemicity in this region, attaining the global roadmap strategic goals by 2030 in Nigeria will be challenging.\n\nTo improve the likelihood of success, it is important to engage all stakeholders to develop a context-specific understanding, and adopt a whole system perspective of the dynamic interactions between health system capacity to support cholera progammes. Furthermore, it is crucial to identify local facilitators and barriers to the implementation of multi-sectoral cholera interventions, and to explore leverage points for interventions and collaboration across stakeholder groups. The systems modelling methodology of group model building (GMB), is an established methodology for engaging stakeholders to gain mutual understanding of complex problems. GMB works with stakeholders to deeply and actively involve them in the process of model construction through exchange, assimilation, and integration of mental models into a holistic system description23. This methodological approach seeks to understand the non-linear behaviour of complex systems over time, recognising the value of engaging all the relevant stakeholders directly, with a view to generate findings that are locally relevant and implementable24,25. To the best of our knowledge, there is a dearth of evidence on the use of GMB approaches to address cholera both in Nigeria and elsewhere. Therefore, the overarching aim of this study is to collaboratively work with cholera stakeholders to examine the factors enabling and blocking sustained implementation of multi-sectoral cholera interventions in the Adamawa and Bauchi states, with a view to addressing recurrent cholera transmission and inform the development of a locally adapted roadmap for Nigeria.\n\nThe study’s specific objectives are:\n\n1. To describe current health system capacity to support WASH, OCV, case management and surveillance, and current policy, governance and community engagement structures for cholera action.\n\n2. To describe barriers and facilitators for the implementation of multi-sectoral cholera interventions.\n\n3. To identify potential opportunities in the existing multi-sectoral cholera interventions for strengthening multi-sectorial collaboration.\n\n\nMethods\n\nThis study will utilise a mixed-methods design, conducted in two phases. The first phase will use a cross-sectional descriptive design to address objective 1, and a qualitative study design using key informant interviews, underpinned by the social constructionism philosophical worldview, to address objective 2. The second phase (for objectives 2 and 3) will utilise a GMB approach, underpinned by community-based participatory theoretical framework26. This will identify the dynamic interactions between health system capacity to support cholera progammes and local and national barriers and facilitators for implementing multi-sectoral cholera interventions; and identify leverage points (potential opportunities) for interventions and collaboration across stakeholder groups. GMB introduces social dynamics, which can affect the quality of model, buy-in from stakeholders, and ultimately the likelihood that recommendations from the model will be accepted and implemented by stakehoders. It also provides the opportunity for stakeholders to share their mental models on cholera and adjust their mental models as they learn from other stakeholders through moderated participatory engagement.\n\nNigeria is made up of 36 states and the Federal Capital Territory (Abuja), with each state further disaggregated into several LGAs (there are 774 LGAs in Nigeria). The study will be conducted in Adamawa and Bauchi states, in north-eastern Nigeria (Figure 1). These states were selected because they were among the most affected states during the cholera outbreak in Nigeria in 201816. Adamawa has some LGAs that have been directly affected by Boko Haram insurgency while Bauchi has some of its LGAs serving as host communities to persons displaced from Boko Haram affected states of Adamawa, Borno and Yobe. Adamawa state has its capital city in Yola and has an estimated population of 4.7 million people across 24 LGAs27. Bauchi state has its capital in Bauchi and has an estimated population of 7.5 million people across 20 LGAs27. The estimated number of primary, secondary and tertiary health facilities per 100,000 persons in Adamawa is 24.70, 0.69 and 0.03 respectively; 16.40, 0.36 and 0.03 in Bauchi28. In addition to Adamawa and Bauchi, we will also conduct both key informant interviews and GMB in Abuja in order to capture the perspectives of national cholera stakeholders.\n\nStates in north-east Nigeria are coloured yellow, while the FCT (part of north-central Nigeria) is coloured blue. Source: StraightNews Nigeria, with permission.\n\nPhase 1: Quantitative cross-sectional study\n\nPreparations for data collection\n\nA minimum of three research assistants per state will be recruited and trained for data collection, as well as taking informed consent and adhering to ethical practices. Data collection tools will be updated following piloting with healthcare workers in a purposely selected health facility in Abuja to determine completeness, clarity and accuracy. Data collection will be done using Open Data Kit (ODK) Collect installed on password-protected mobile devices.\n\nSampling\n\nBased on pragmatic considerations (e.g. cost and time), we will adopt a purposive stratified sampling approach to recruit 20 rural and 30 urban health facilities in each state (n=100), with a history of confirmed cholera cases during the 2018 outbreak. Only health facilities (government- and private-owned primary, secondary or tertiary) considered functional by the state health authority will be eligible for selection into the study. The selection of contrasting LGAs (urban vs rural) is due to the dependence of cholera attack and case fatality rates on setting type in Nigeria, with higher values in urban than in rural areas16. Convenience sampling of healthcare workers of various professional cadres present on the day of the survey will be done.\n\nData collection\n\nIn determining the health system’s capacity to support multi-sectoral cholera interventions in both the Adamawa and Bauchi states, we will adapt the WASH health care facility core indicators29 and the WHO Service Availability and Readiness Assessment tool30. The trained research assistants will spend 1–2 days at each selected health facility. They will administer a structured questionnaire with the manager or administrative head regarding the current health system’s capacity to support various cholera interventions (e.g. WASH, OCV, surveillance etc.). Additionally, the research assistants will administer a questionnaire on cholera case management and knowledge with a convenience sample of five healthcare workers who meet predefined eligibility criteria—i.e. willingness to participate in the study and to sign an informed consent form—on the day of health facility survey. Prior to the survey, information about the proposed study will be communicated to healthcare workers via a formal letter to the facility manager or administrator. When possible, the research assistants will also perform discrete observations to objectively ascertain the presence of cholera interventions. In terms of duration, data collection will be completed within six weeks of the study start date. The study materials and consent form can be found as extended data31.\n\nData analyses\n\nThe current health system capacity to support various cholera interventions will be determined using descriptive analyses. Continuous variables will be described using mean and standard deviation for normally distributed variables, median (IQR) for non-normal continuous variables, and frequency and percentages (%) for categorical or binary variables. The status of a cholera intervention will be described by calculating its available indicators divided by the total number of indicators. Analyses will be stratified by state, study setting (urban vs rural) and health facility type. The responses on knowledge will be scored as ‘1’ while other responses, such as ‘incorrect’ or ‘don’t know’, will be scored as ‘0’ (zero). The scores will be added to obtain a total score for each study participant and a median score will then be calculated. High knowledge score will be defined as a total score ≥median score and low knowledge score as a score <median score. Factors (e.g. cadre, state, training, recent case management experience etc.) potentially associated with knowledge will be explored using multivariable logistic regression. The findings will be presented as adjusted odds ratios with 95% confidence intervals. A p-value of <0.05 will be considered statistically significant. All analyses will be performed using Stata version 13 (StataCorp, College Texas).\n\nPhase 1: Qualitative study\n\nStudy participants\n\nThree groups of participants (see Table 1 for their distribution by location) will be purposely recruited for the key informant interviews (KIIs). Participants will include:\n\nNA = not applicable\n\nAt the state level:\n\nCommunity members, including previous cholera patients and caregivers, local food retailers, school teachers, local health promoters, community and religious leaders.\n\nHealthcare professionals, including state epidemiologists, disease notification and surveillance officers, health educators/promoters, community health extension workers, nurses, clinicians, traditional healers, academia, the staff of state ministries (health, water resources, environment, primary health care development agency), technical partners and funders (WHO, MSF, UNICEF).\n\nAt the federal or national level:\n\nGovernment and non-government cholera stakeholders (e.g. staff of NCDC, federal ministries of water resources, health, environment and primary health care development agency, as well as technical partners and funders including WHO, World Bank, UNICEF, United States Centers for Disease Control and Prevention (US CDC), Africa Field Epidemiology Network etc.)\n\nAll study participants must be willing to participate in the study and to sign an informed consent form, and considered a cholera stakeholder (community or healthcare professional). All KIIs will be conducted by the lead researcher (KE) either in a health facility or an office at a time convenient for each study participant. Being a qualitative study without a fixed sample size, we will aim for at least 20 participants in each study location, but recruitment will stop once saturation (i.e. when study participants have provided a range of information or perceptions about the study until no additional information is being provided) is reached32. The semi-structured interview guides will be developed separately for each study participant type (i.e. community members, healthcare providers, national publichealth stakeholders) and piloted before data collection. Where possible, the data collection process will be aided using an audio-recorder. The collected will be transcribed verbatim and analysed using a thematic approach, aided by Nvivo software. Thematic analysis will follow the six-phased approaches recommended by Braun and Clarke33, bearing in mind that these phases are not a linear process, but more of a recursive process where one moves back and forth as needed. The six phases include familiarisation with data, generating initial codes, searching for themes, review of initial themes, defining and naming themes, and producing the report.\n\nFindings from this study component, alongside those from the cross-sectional study, will be triangulated to finalise the script development for GMB in phase 2.\n\nPhase 2: Group model building\n\nGMB workshop process\n\nWe will conduct a total of nine all-day GMB workshops, two with community participants and two with health professionals in both states (total of 8), and one with stakeholders in Abuja (i.e. federal government and partners). Table 2 provides an overview.\n\nWe will use locations with minimal distractions to the participants, such as an event centre within a hotel or government ministries. The rationale for participant-specific GMB workshops is to minimise the effect of power dynamics and recognise variation in understanding of cholera by various stakeholders. Between 7–10 participants (12–15 in Abuja given it’s a one-day event) with similar characteristics as those in the qualitative study component will be purposely selected for each GMB session. Invitation of potential participants will be facilitated by a combination of letter/email and phone call. We will ensure that public health preventive measures with regards to coronavirus disease 2019 (COVID-19) (e.g. physical distancing and use of face-masks) are in place during each workshop. The protocol and scripts for each workshop will be modified from those available from Scriptapedia34 and based on preliminary findings from study phase 1. The research team members will be assigned roles as outlined in Table 3. After each GMB workshop, the research team will have a debrief session to reflect on processes and to make necessary adjustments as needed.\n\nGMB tools\n\nThe GMB process will utilise three interactive system mapping tools (see provisional scripts for these tools in Extended Data): graph over time; cognitive mapping; and causal loop diagrams.\n\nGraphs over time allow the participants to share their understanding of cholera as well as perceived drivers of its recurrent transmission in their community over a specific time period. Using an empty graph with time on the x-axis (with a vertical line for the present time) and a variable on the y-axis, the participants will be guided by the researchers to fill in the graph. They will be prompted with questions such as: “What is the trend of cholera in your community since the 2015 presidential elelction in Nigeria?”, “What are the factors influencing the transmission of cholera in your community?”, “What are the interventions available for cholera control in your community?”. As well as drawing on the empty graphs the historical trends of cholera, the participants will be asked to identify two future pathways they predict would occur if current cholera trends continued or if intervention occurred.\n\nCognitive mapping is a visual tool that will introduce participants to systems thinking by exploring their understanding of the facilitators/barriers to the implementation of cholera interventions (e.g. surveillance and oral cholera vaccination) and consequences of implementing these interventions successfually or not25. For this activity, a template will be developed and provided for the participants to complete.\n\nCausal loop diagrams capture the dynamic nature of an issue and the presence of feedback in systems25. This activity focuses on providing the participants with an understanding of why feedback is important in a system, using pictoral examples from relevant studies. Here, simple feedback loops which are a basic operating unit of systems35, will be designed from participants’ previous activities (e.g. barriers to the implementation of cholera interventions and consequences of action and inaction). To further consolidate findings from the causal loop diagrams, a sub-set of of participants will be asked to critique the collective models by adding, deleting and modifying structures in the map. We will also seek to explore potential interventions to identified barriers to implementing cholera interventions in the both Adamawa and Bauchi states. The primary focus here would be to use the following questions to probe for possible actions which could be taken to address the identified barriers: “What variables could you increase or decrease?” “How could you impact connections: strengthen, or weaken a connection, speed it up or slow it down, add or delete connections?” This activity will require the participants to write potential actions on post-it notes and place them on the causal loop diagrams where they consider appropriate. Finally, all the participants will be required to select the top three actions for each intervention that their group would like to see progressed.\n\nPost-GMB workshop\n\nGMB data, alongside field notes and reflections from the research team, will be analysed thematically. Additionally, findings from the GMB workshops will be supported with quotes from thematic analysis of transcripts from the KIIs. Lastly, GMB findings will be digitised using VenSim software, version: 8.2 (an open-source software).\n\nThe protocol for this study has been reviewed and approved by the Nigeria National Health Ethics Research Committee (NHREC Approval Number NHREC/01/01/2007-24/08/2020). Additionally, participants for the qualitative and GMB workshops will be required to provide informed consent after reading or listening to the study information sheet, and will be assured freedom to withdraw from the study at any stage. Autonomy and confidentiality will also be maintained throughout the conduct of this study; For example, we will delete all personal identifiers from the dataset prior to management and analyses, and only the research team members will have access to the dataset on a password-protected laptops. Lastly, while participants will be informed that there are no direct benefits from participating in the study, those in the GMB workshops will however be given stipends to compensate the cost of transportation and potential loss of income for the day—this is appropriate in the context of GMB workshops25. Expected findings will be disseminated through peer-reviewed publications and local and international scientific meetings.\n\n\nDiscussion\n\nThis study will take a systems modelling approach to examine complex factors influencing the implementation of multi-sectoral cholera interventions in a cholera endemic and fragile region of Nigeria. The participatory nature of the study has the potential to improve the quality of findings, acceptance of findings from cholera stakeholders, and ultimately increase the likelihood for implementation of recommendations36.\n\nPrevious attempts aimed at strengthening the healthcare systems in the north-east region of Nigeria have so far failed to address the complex and persistent burden of cholera in the region. For instance, a health facility survey conducted in Adamawa found the state’s capacity to deliver healthcare services to be extremely poor due, in part, to the destruction of health infrastructure by Boko Haram terrorism group37. This survey was however generic without a specific focus on the state’s capacity to respond to or manage communicable disease outbreaks including cholera. Therefore, determining the health capacity of both the Adamawa and Bauchi states to support multi-sectoral cholera interventions could serve two important purposes: to identify needs or gaps for investments and to potentially serve as a benchmark for monitoring progress towards closing the identified gaps.\n\nA study in the state of Yobe, one of the most affected states by Boko haram insurgency, using systems dynamics modelling found a significant decrease in access to health care and human resources for health, largely due to the outward migration of health workers and suspension of public health programmes38. A dearth of evidence on the dynamics of health care access and provision in the context of cholera was evident, limiting robust planning. Thus, findings from our proposed study would be timely in ensuring the holistic identification and implementation of appropriate public health measures for efficient cholera control in this Nigerian region.\n\nThis evidence will be useful to policymakers and funders given the expected impact of the COVID-19 pandemic on SDG progress, with vulnerable populations in fragile settings projected to be the most affected39. In addition to the novelty of using a systems approach to study cholera dynamics in a setting made fragile by armed conflicts, findings from our study could generate new framework for how cholera interventions are constructed or implemented in fragile contexts, while also offering a new perspective to explore potential leverage points with regards to cholera policy and response.\n\nThe qualitative nature of the GMB approach is often considered a limitation, hence there has been a call by researchers to go beyond the qualitative model to create a quantitative simulation model that allows for quantifying the postulated causal relationships established in the qualitative models40. As such, we will seek to conduct a post-GMB quantitative simulation model using historical cholera surveillance data from Nigeria CDC, published literature and other publicly available data (e.g. population census and health metrics) to strengthen the evidence to inform policy decision making. For instance, the quantitative simulation model could provide insights on the relative impact of different cholera interventions (e.g. WASH) on cholera outcomes. Another potential limitation of the GMB approach is limited generalisability of findings, although the aim of a systems modelling approach is to have a highly contextualised understanding of an issue through the prioritisation of stakeholders’ engagement. Nonetheless, we will make deliberate efforts to recruit participants from diverse occupational, socio-economic and gender groups in order to enrich the diversity of the study participants and consequently the expeected findings.\n\n\nConclusion\n\nThe engagement of a diverse range of cholera stakeholders, including community members, in a participatory process could contribute to a renewed desire to bring about positive change to cholera control in an endemic and fragile region of Nigeria. Moreover, this study could have an implicit impact on the control of other water-borne diarheagenic diseases in the country.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: Understanding the factors enabling and blocking sustained implementation of cholera interventions in a fragile region of Nigeria: a multi-phase group model building study protocol. https://doi.org/10.6084/m9.figshare.13686073.v131\n\nThe project contains the following extended data:\n\n- Questionnaire and scripts (all data collection materials)\n\n- Consent form\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Contributors\n\nKE, CK and TA conceptualised the study. KE, CK, KD, JA, BF, CI and TA are implementing the study. KE had primary responsibility for final content. All authors participated in writing, read and approved the final manuscript.\n\n\nAcknowledgements\n\nWe are grateful to the Adamawa State Ministry of Health, Bauchi State Ministry of Health, and the Nigeria Centre for Disease Control for supporting the development of this study protocol. Lastly, we are grateful to the WHO Global Task Force on Cholera Control for its technical support towards the implementation of this study.\n\n\nReferences\n\nAli M, Nelson AR, Lopez AL, et al.: Updated global burden of cholera in endemic countries. PLoS Negl Trop Dis. 2015; 9(6): e0003832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlobal Task Force on Cholera Control: Ending Cholera: A Global Roadmap to 2030. Annecy, 2017; 32. Reference Source\n\nWorld Health Organization: Cholera. WHO. 2019. Reference Source\n\nUnited Nations: Transforming our world: the 2030 Agenda for Sustainable Development. Department of Economic and Social Affairs. 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Chelsea Green Publishing Company, 2008. Reference Source\n\nAnsah JP, Islam AM, Koh V, et al.: Systems modelling as an approach for understanding and building consensus on non-communicable diseases (NCD) management in Cambodia. BMC Health Serv Res. 2019; 19(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealth Sector Nigeria and World Health Organization: Health Resources Availability and Monitoring System (HeRAMS): Adamawa State. 2017. Reference Source\n\nAger A, Lembani M, Mohammed A, et al.: Health service resilience in Yobe state, Nigeria in the context of the Boko Haram insurgency: a systems dynamics analysis using group model building. Confl Health. 2015; 9: 30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnited Nations Department of Economic and Social Affairs: The Sustainable Development Goals Report 2020. 2020. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "79187", "date": "23 Feb 2021", "name": "Kenny Moise", "expertise": [ "Reviewer Expertise Research on operationalization and ecology of infectious 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 a well-thought study protocol elaborated by Kelly Emilian et al. with the objectives of describing the current health system capacity to support multi-sectoral cholera interventions in two fragile states in Nigeria, describing barriers and facilitators for the implementation of such interventions and identifying potential strengthening opportunities for multi-sectoral collaboration. The authors propose a 2-phase study project with quantitative cross-sectional and qualitative methods in the first part and a group model building (GMB) in the second.\nThe aim of the study is clear; the title relevant and informative. Relevant and up-to-date references are used (although a suggestion is made below). The study setting is clearly described and the selection process of individuals is clear. Furthermore, the study is highly relevant for the Nigerian context of fragility and cholera endemicity. It is as relevant for other fragile states around the world battling cholera. As such, the research method is suitable to reach the objectives and enough details are provided in order to replicate it.\nHowever, the research question is not clearly outlined in the introduction. In this section, I wonder if the authors question the factors influencing sustained implementations of cholera interventions or the use of a specific method, namely the GMB or the likelihood of eliminating cholera in the context of fragility (lines 1-8 of the 7th paragraph of the introduction). Therefore, in regards to what is already known about the topic as stated by the authors, and the lack of clarity of the research question, the gap in knowledge they are trying to fill is also not clearly established. The authors stated that “there is a dearth of evidence on the use of GMB approaches to address cholera both in Nigeria and elsewhere.” As stated, they shift the focus from the factors influencing sustained cholera interventions to the GMB methodology.\nTherefore, I would suggest beginning the 7th paragraph with “In order to fully comprehend the factors enabling and blocking sustained implementation…” and switch the knowledge gap statement to “to the best of our knowledge, there is a dearth of evidence on factors enabling or blocking sustained… especially using the GMB method to grasp such factors”.\nTo shorten the introduction (5 paragraphs instead of 7), I would suggest omitting detailed information regarding the SDG and the GTFCC as they are not crucial to establish the background of this study, given the objectives. Furthermore, the last paragraph of the introduction stating the objectives of the study can be shortened with the objectives presented in the text, not numbered. The details describing the group model building should be moved from the introduction to be incorporated in the methods section while avoiding repeats.\nIn the methods section, please include details in the text regarding the variables and indicators that will be used in the descriptive analysis to determine the capacity of the health system to support the cholera interventions (lines 3 and 8 of data analyses sub-heading) and references indicating how they were chosen. Also, provide complete details/definitions about the factors that will be incorporated in the multivariate regression model to assess knowledge (line 16-18 of data analyses sub-heading).\nFurthermore, regarding the discussion, the authors define fragility as “…the result of violence and prolonged conﬂict, political and economic instability, marginalisation and inequality, weak and distorted national governance structures and processes, and significant environmental threats and natural disasters (paragraph 4 in the introduction, lines 4-8)”. This concept seems intertwined with the notion of complex emergencies (CEs) defined in a study by Bompangue et al. as “a humanitarian crisis in a country, region or society where there is a total or considerable breakdown of authority resulting from internal or external conflict, and which requires an international response that goes beyond the mandate or capacity of any single and/or ongoing United Nations (UN) country programme”.\nBompangue et al. concluded that CEs may facilitate the spreading of already existing cholera outbreaks.1 In light of this parallel, I would like to read some discussions regarding how the study proposed by the authors may integrate and deepen the knowledge from Bompangue’s study in the Democratic Republic of the Congo.\n\nOverall, the study protocol is well structured and has the potential to bring relevant and critical knowledge to fighting cholera in a fragile state in order to implement context-specific interventions.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "89013", "date": "19 Jul 2021", "name": "Godfrey Bwire", "expertise": [ "Reviewer Expertise Public Health", "Infectious diseases", "Cholera" ], "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\nReviewers report for published study protocol, “Understanding the factors enabling and blocking sustained implementation of cholera interventions in a fragile region of Nigeria: a multi-phase group model building study protocol [version 1]”\nGeneral comment This is a well written study protocol by Kelly Elimian et al., on an important public health issue that is a major cause of morbidity and mortality in study area, Nigeria and globally. The study protocol aims to document factors responsible for sustained cholera outbreaks in Adamawa and Bauchi States of Nigeria. The authors state clearly the study objectives and propose to use robust methodology to achieve these objectives. Furthermore, the proposed study by Kelly Elimian et al., if implemented, has the potential to contributed positively towards the achievement of the UNSDGs targets, specifically UNSDG No. 3 by 2030. However, there are major flaws with this study protocol which have to be addressed if the overall goal of preventing cholera so as to attain UNSDG targets by 2030 is to be achieved.\nMajor comments Abstract and introduction I like the way the authors summarized the abstract and how they wrote the introduction citing relevant literature in order to build their case.\nKelly Elimian et al., rightly state that the Boko Haran conflict is a major factor that is responsible for sustaining cholera outbreaks in the study areas. This is in line with known facts which links the conflicts and cholera outbreaks1,2,3.\nThe authors state this from the very beginning (abstract) that Boko Haram destructive activities are important in cholera persistence, \"A major contributing factor to this challenge is fragility of the region due to persistent Boko Haram insurgency activities, often characterized by the destruction of health infrastructure and displacement of communities to areas with suboptimal living conditions\" and later (introduction), \"Notably, over half of these cholera hotspot LGAs are located in the north-east region of the country including Borno, Adamawa and Yobe States, where Boko Haram insurgency activities are predominant18. Boko Haram insurgency activities are often characterized by the disruption of WASH services, displacement of populations to overcrowded camps, and emigration of health workers to safer areas19. It is therefore no surprise that the highest cholera prevalence and case fatality ratio during the outbreak of cholera in Nigeria in 2018 were recorded by north-eastern states\".\nHowever, subsequently there is little in the study protocol to show how they (authors) plan to collect data that can result in conflict resolution, a root cause of sustained cholera outbreaks in affected areas. Therefore, for this protocol to be more comprehensive, the authors should revise the introduction and method sections to include an objective that can contribute to conflict resolution and articulated in the method section how they plan to achieve that objective.\nThe title The title of the protocol, “Understanding the factors enabling and blocking sustained implementation of cholera interventions in a fragile region of Nigeria: a multi-phase group model building study protocol”, is open to misinterpretation and may not facilitate replication of the study findings by other interested researchers since a fragile region of Nigeria (a vast country, with 36 states) may be difficult to pinpoint. For example Borno and Yobe states are also Boko Haram affected states that are cholera hotspots but are not study area. Therefore, the authors could revise the title of the study protocol so that it is more specific. Kelly Elimian et al., could consider revision such as “ Understanding the factors enabling and blocking sustained implementation of cholera interventions in Adamawa and Bauchi states of Nigeria”\nWhole study protocol (Abstract, introduction..)\nThe authors in several statements keep on referring to cholera stakeholders which is an assumption that the readers are conversant with cholera and they know who the stakeholders are globally, in Nigeria and in the study area. Given that this study protocol addresses an important public health issue and may have global readership with some audience based in communities where cholera was eliminated several decades ago, it would be more understandable if what is meant by stakeholders is well defined. For instance, a reader or a researcher who is knowledgeable on conflict resolution may think of the Government of Federal Republic of Nigeria, Boko Haram leadership, local communities, UN agencies and other as the key stakeholders. While others may omit Boko Haram leadership yet they are one of the actors who may destroy investment put in place to prevent cholera. Furthermore, this clear operational definition may facilitate replication of the study findings by other researchers.\nIntroduction Kelly Elimian et al., clearly state the overall objective, “Therefore, the overarching aim of this study is to collaboratively work with cholera stakeholders to examine the factors enabling and blocking sustained implementation of multi-sectoral cholera interventions in the Adamawa and Bauchi states, with a view to addressing recurrent cholera transmission and inform the development of a locally adapted roadmap for Nigeria”.\nThis is a good statement. However, my concern is related to proposing to use finding from two states of Adamawa and Bauchi to develop plan for entire Federal Republic of Nigeria which is a vast country with 36 federal states. Therefore, the authors will need to revise this statement to fit the proposed study.\n\nMinor comments These are minor issues but are essential to achieving the objectives of the study.\nMethods The authors include Abuja as a study area yet Abuja is not cholera hotspot area and is not conflict affected state which are major criteria for selection of the study area. It is true, the central team in Abuja will be interviewed, however they will be selected because of the role they play in supporting cholera prevention or their role in conflict resolution in the two states. Therefore, the authors should review study area to remove Abuja state.\nData collection, management and analysis The authors focus on health sector and give little attention to other sectors. According to WHO, GTFCC Cholera prevention and elimination require all actors starting from the political head and this is articulated in the GTFCC framework of 2019 that is a guide for development of the national cholera plans. According to these framework several government sectors (prime minister, finance, water, health and others) are required. However, in this study protocol the participants of the study are mainly health workers and organization supporting health care. These is stated by the authors in the protocol, “Data collection tools will be updated following piloting with healthcare workers in a purposely selected health facility in Abuja to determine completeness, clarity and accuracy. Data collection will be done using Open Data Kit (ODK) Collect installed on password-protected mobile devices” .\n\nThe same (focus on health sector) is also seen in the analysis of data where the data to be analyzed is from the health system and none from other ministries. This is reflected in this statement, “The current health system capacity to support various cholera interventions will be determined using descriptive analyses…”.\nTherefore, Kelly Elimian et al., will need to revise this sections to ensure that the relevant data on cholera prevention is collected from all key ministries and analysed to comprehensively inform the policy on cholera prevention.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/10-85
https://f1000research.com/articles/10-83/v1	08 Feb 21	{ "type": "Research Article", "title": "Exploring engagement with authors of randomised controlled trials to develop recommendations to improve allocation concealment implementation and reporting", "authors": [ "Laura Clark", "Natasha Mitchell", "Catherine Hewitt", "David Torgerson", "Natasha Mitchell", "Catherine Hewitt", "David Torgerson" ], "abstract": "Background: Reviews have consistently shown that allocation concealment is frequently implemented and reported suboptimally in randomised controlled trials (RCTs). This research aims to pilot engaging with authors of RCTs to explore their knowledge and understanding of allocation concealment implementation and reporting to ascertain areas and mechanisms for their improvement.\nMethods: Authors that published RCTs in core clinical journals in one month in 2019 were identified. Authors were invited to complete questionnaires to elicit their views and experiences on the implementation and reporting quality focussing on allocation concealment. Methodological quality of allocation concealment was evaluated in this sample by assessing adherence to the Consolidated Standards of Reporting Trials (CONSORT).\nResults: Reporting was suboptimal, with only 57% of allocation concealment methods reported to be implemented which were judged as adequate, with 18% using sealed envelopes and more than 40% not adequately reporting allocation methods. When exploring allocation concealment, implementation and reporting questionnaires were found to elicit a low response rate amongst authors of RCTs.\nDiscussion: Following analysis of the themes that emerged from the questionnaires, the main recommendations to improve reporting quality are: journals need to endorse, adhere and promote reporting guidelines, a methodologist could review methodological details of publications simultaneously to peer review, envelopes as a form of allocation concealment are poorly implemented and reported, so careful review of these is required, funders need to insist on more robust allocation concealment methods are employed if the RCT setting allows, and authors need to acknowledge their responsibility for transparent reporting of RCTs.", "keywords": [ "reporting quality" ], "content": "Introduction\n\nAllocation concealment is a key criterion of quality for a rigorously conducted randomised controlled trial (RCT). It is the most basic methodological piece of information that should be reported1,2. Allocation concealment has been defined by the Consolidated Standards of Reporting Trials (CONSORT) group as:\n\n‘A technique used to prevent selection bias by concealing the allocation sequence from those assigning participants to intervention groups, until the moment of assignment. Allocation concealment prevents researchers from (unconsciously or otherwise) influencing which participants are assigned to a given intervention group.’ Allocation Concealment, CONSORT.\n\nA common misconception is that allocation concealment is the same as blinding3,4. Unlike allocation concealment, blinding is not always possible or even desirable to be carried out at all in RCTs5. This confusion may inadvertently lead to the use of poor allocation concealment.\n\nHistorically, sequentially numbered, opaque, sealed, envelopes (SNOSE) were used to conceal allocation sequences and are still widely used. Despite descriptions of ‘relatively’ tamper proof methods using envelopes6 they can still be easily subverted, such as by opening them in advance of patient recruitment, therefore findings from RCTs using envelopes ought to be open to greater levels of scrutiny3,7. If envelopes are to be used then methods minimising tampering need to be put into place and reported. A more secure approach for allocation concealment is to use central randomisation– this is a method which is more difficult to subvert as it separates the randomisation sequence generation from the allocation of participants3,7. Research has shown that trials with inadequate or unclear allocation concealment are associated with increased effect sizes of greater than 40%1.\n\nUnfortunately, RCT publications frequently do not report essential methodological factors in full, with many methodological elements, including allocation concealment, missing entirely4,8 In 1996 the CONSORT statement was published, after a growing body of evidence identified a lack of consistent and high-quality reporting of RCTs9, with further revisions for different research designs8,10,11. Many, but not all, journals are now ‘CONSORT endorsing’ journals as they have adopted the CONSORT checklist for RCTs as a basic methodological checklist that authors should adhere to12.\n\nResearch recognises that insecure allocation concealment methods are still being implemented and reporting is still suboptimal despite CONSORT13–16. An area that needs exploring is understanding why personnel involved in the conduct of RCTs are implementing insecure methods and why these methods are still not being properly reported. Engaging with this group will enable targeted recommendations to be made to improve both the implementation and reporting of allocation concealment.\n\n- Pilot the use of a questionnaire to engage with authors of published RCTs to investigate their knowledge and understanding of allocation concealment implementation and reporting.\n\n- Use these findings to ascertain areas and mechanisms which are acceptable to these authors to improve implementation and reporting of allocation concealment.\n\n- Assess whether a questionnaire is an effective way to engage with authors of RCTs regarding methodological information to inform future research.\n\n- Determine the reporting quality of allocation concealment in this sample by assessing the adherence to CONSORT.\n\n\nMethods\n\nWe identified all RCTs published in one month in 2019 in the ‘Abridged Index Medicus’. We do not have ethical approval to specify the month that the articles are published as this would identify the authors invited to participate in the research and not keep their identities secure. We undertook double data extraction of the randomisation method and allocation concealment method where the data was initially extracted and then second checked for any typological errors. We used this data to assess whether papers adhered to CONSORT. Table 1 details the criteria used.\n\nWe contacted corresponding authors via the email address cited in the publication and asked them to anonymously fill in a short questionnaire17 assessing their views on trial reporting. The questionnaire consisted of two questions with closed responses and one open ended question. The purpose of this questionnaire was to establish if authors could define, and identify what were adequate methods of, allocation concealment. Participants were asked to leave their email address if they consented to fill in an in-depth 18 item questionnaire. Both questionnaires were created and distributed using QUALTRICS18. A reminder email was sent two weeks after the initial, requesting participation in the survey.\n\nThe purpose of the second questionnaire was to elicit comprehensive knowledge and views on implementation, reporting quality and mechanisms to improve trial reporting quality in relation to allocation concealment.\n\nAuthors were sent the questionnaire and asked to respond within two weeks, after one week a reminder email was sent. The QUALTRICS survey was closed two months after the end date to ensure if there were late responses could still be submitted. For both questionnaires, where there were open/free text boxes we performed a thematic analysis of the content of the questionnaire responses. The analysis was performed by LC and NM. Text was systematically examined, and themes identified by coding and categorising text according to key themes that emerged Data was initially organised in order to determine what participants knew about allocation concealment methodology, what method they had experience of using, if they knew what essential methodological information to report and what mechanisms could improve the reporting quality of RCTs and the opinions of suggestions to improve reporting quality of allocation concealment methodoloy These categories were further coded and mapped, as detailed participant responses were analysed to identify beliefs, experiences and barriers and facilitators to improving reporting quality, these lead to identifying and developing emergent themes.\n\nAn internal pilot was performed on each questionnaire with research staff in the York Trials Unit who had experience of being authors of RCTs and trial methodology, following piloting we reordered some of the questions but did not change the content\n\nEthical approval was granted by the University of York Health Sciences Research Governance Committee in March 2019. Information regarding study information and the process of consent was provided at the start of the questionnaire, submission of the completed questionnaire by the participant was considered as implicit consent. We provided contact details to enable recipients of the questionnaire to contact the research team with any queries, advised them on the length of time the survey was expected to take, that all responses were anonymous and findings from the survey would be published.\n\n\nResults\n\nIn total, 95 RCTs were identified19, and 87% (n=83) were published in CONSORT endorsing journals. Of these, 94 had a corresponding author email address. The corresponding authors were a combination of clinicians and academics.\n\nOf the identified RCTs, 75.8% (n = 72) reported the allocation sequence generation method and 71.6% (n =68) reported the allocation concealment method (see Table 2 for details), with CONSORT endorsing journals reporting these methodological details more frequently.\n\nTable 3 shows that almost half of RCTs in CONSORT endorsing journals did not report with sufficient clarity that a judgement could be made as to the robustness of allocation concealment. It also indicates that 21.1% (n=20) were double blind placebo blind trials which were judged as adequate, of these only 60% (n=12) reported their allocation sequence generation method.\n\nEnvelopes were used to conceal the allocation sequence in17.9% (n=17) of the included RCTs, with 52.9% (n = 9) describing the security of the envelope, 29.4% (n=5) stating who prepared the envelopes, and 35.3% (n=6) stating who opened the envelope. The reporting quality of necessary methodological information was poor for studies using envelopes; only 17.6% (n=3) were published adequately with all necessary information to assess the methodological rigour reported. Central randomisation was performed in 33 (34.7%) RCTs.\n\nQuestionnaire analysis. In total, 12 (12.7%) anonymous authors returned the shorter questionnaire. The participants had a varied range of experience of how many RCTs they had conducted. Most participants (n=8) had conducted >3 RCTs with one participant stating they had conducted >300 RCTs. Six authors (6.4%) consented to being sent the second larger questionnaire and five authors completed this. From examining the email addresses, one participant was a clinician and five were academics. Figure 1 shows the summary of response rates to the questionnaires.\n\nAll participants would use both central randomisation services and envelopes in future research. Of the participants, 80% (n = 4) believed that central randomisation should be the gold standard if appropriate to the trial design. Most participants supported the use of envelopes but did acknowledge that they were more at risk of subversion than central randomisation.\n\n‘I would say for the envelopes they are adequate if they are SNOSE. While they are adequate they are rather impractical sometimes, and they are not completely immune to subversion of course. Central randomisation is therefore preferred’ (ID 2.5)\n\nParticipants were keen to emphasise that the choice of allocation concealment and randomisation is setting dependent.\n\n‘In my experience, it [selection of allocation concealment method] is dependent on the type of setting; e.g. in a busy clinical ward with 24hr/7 recruitment, envelopes are far more user-friendly, especially when relying on busy clinical staff to take consent and randomise. Alternatively, in a 9–5pm environment, telephone or web-based can be applied easily’ (ID 2.2)\n\n‘Envelopes [are chosen] because it was the next best thing in lieu of central randomisation for sites with poor internet connection etc. Central randomisation [is chosen] because it’s the best way’ (ID 2.5)\n\nThere was a large amount of support for the implementation of envelopes as an allocation concealment method where appropriate. The main reasons stated were trial setting, including the country and budget.\n\n‘[Reasons for envelope use] for cases where you are running trials in LMICs [low and medium income countries] with, e.g. limited internet access - envelopes may be more practical. I also used them in an older trial which did not have funding for a central randomisation service. I would only use them for these niche cases and not as routine.’ (ID 2.5)\n\nOne participant selected that they did not think envelopes were an adequate allocation concealment method but they had used them in the past due to budget constraints.\n\nCentral randomisation was accepted and described as being the ‘best’ with no elaboration as to why. One author considered the problematic reporting of central randomisation.\n\n‘Just saying its 'done centrally' [when reporting allocation concealment methods implemented] I also find problematic. If you can call someone up to randomise and they have an open allocation list, there is still a risk that there is some collusion between the two parties to ensure certain patients are randomised one way or the other’ (ID 2.5)\n\nWe asked authors to list the top three methodological items to consider when conducting and reporting an RCT. Each response included the randomisation method employed with only one response stating that allocation concealment as important. Of the responses gained, 75% included blinding as an important methodological item to report, which could suggest that blinding and allocation concealment are being confused as past research has highlighted3,4.\n\nFrom the short questionnaire 60% of participants stated they could not define allocation concealment. The most general way of defining allocation concealment was to discuss how the randomisation sequence should be hidden, but with no consideration as to why as defined by CONSORT only one participant included selection bias in their response.\n\n‘If done effectively it [allocation concealment] should reduce the risk of selection bias by preventing researchers from manipulating when individual participants are enrolled/randomised based on prior knowledge of the upcoming randomisation sequence’ (ID 1.1)\n\nThis suggests that many of those involved within the conduct of RCTs do not appear to see allocation concealment as an essential element to report.\n\nThere was a misconception that allocation concealment was the same as blinding the patients and outcome assessors by one participant, when asked to state what is important to report in RCTs.\n\n‘Method of concealing the allocated treatments (blinding of patients and outcome assessors) [is important to report in RCTs]’ (ID 2.4)\n\nWe determined whether participants were aware of how to adequately implement and report envelopes in RCTs by asking them to state what essential methodological information is necessary. No author knew all essential quality factors to include within a publication. Only one participant stated that the individual who opened the envelope should be reported, and no authors stated that it was essential to report who prepared the envelopes. All participants knew that envelopes should be sequentially numbered, and that this should also be reported. Participants were mostly aware of additional security measures regarding the envelope that could be taken and reported to decrease the risk of subversion.\n\n‘There are probably extra measures which may help to prevent subversion such as instructing staff to write the patient name etc. on the envelope prior to opening it, and ensuring that the seal is tamper proof - not e.g. a staple which can be carefully unpicked and re-stapled if someone was so motivated. A wax seal comes to mind although that sounds rather impractical too!’ (ID 2.5)\n\nInvestigator integrity emerged as a theme here, with participants claiming that investigators had to implement envelopes correctly but not acknowledging what needed to be reported. This also supports the theme that there is a lot of focus on trial conduct rather than trial reporting, this is discussed further on in this publication\n\n‘Investigators have to use them [envelopes] properly. But investigators have to properly do all aspects of the trial. Basically, we always count on investigator integrity.’ (ID 2.3)\n\n‘If I had to pick something [essential methodological information when reporting envelope use within an RCT] it would be processes to ensure adherence to correct use of the envelopes/prevent violation of the study protocol.’ (ID 2.1)\n\nParticipants recognised that reporting quality needed improving, and different mechanisms were explored:\n\nRole of journal editor. Of those surveyed, 80% (n=4) believed that the journal editor should ensure that key methodological information is reported within an RCT publication. It was also acknowledged that the editor does not necessarily have the expertise to do this.\n\n‘Journal editors should do their best to review methodological information but if they don't have sufficient methodology expertise across all experimental designs that may be included in submissions to their journal, then they are not the most appropriate person for this task’ (ID: 2.1)\n\nWord limits imposed by journals. We asked participants if they thought word limits imposed by journals impacted the amount of methodological information reported and thus the quality of the publication. There was agreement that although it could impact the quality, increasing the word count would most likely not help as the additional word limit may not be used to report the methodology.\n\n‘Increasing journal word counts is merely one mechanism to facilitate (but not ensure) more comprehensive reporting of methods/findings. At authors discretion they could simply use the additional word count to expand their discussion’ (ID 2.1)\n\nPre-submission checks. We found there was minimal support from our surveyed population for journals to perform pre-submission checks on a manuscript prior to full submission. We proposed that key methodological information of an RCT including allocation concealment and randomisation procedures were submitted to a journal for review. Only those manuscripts that include transparent and detailed methodological information would then be invited to submit their full manuscript. This could lead to improved reporting quality of allocation concealment and randomisation methodology.\n\nLinked to this was the criticism of journals and the time it takes for the peer review process to be completed. This influenced participant opinions when considering improvements to reporting quality. Although all participants agreed there was a need to increase reporting quality, they did not want to add any additional barriers to slow down publications.\n\n‘It could add value but the length of the peer review process already substantially delays the release of scientific advances, and adding another rate-limiting step would exacerbate the delay’ (Paper ID 2.1)\n\n‘All these points are important [to improve reporting quality] but I don't want more \"process” [when trying to submit a manuscript to a journal]’ (ID 2.3)\n\nMethodologist. The largest support was given to a methodologist always assessing the methods section of any publication submitted to a journal, similar to the statistical review some journals subject manuscripts to. Support was given to this if it was performed in a streamlined way and done simultaneously whilst the manuscript was going through the peer review process; participants were not supportive of any method of improve=ing trial reporting that they perceived to hold the process up further.\n\n‘There is no reason why specialised methodology review could not be included as part of the standard external peer review process.’ (ID 2.1)\n\nRole of Funders. Support for funders allocating specific funding for a third-party randomisation service if the RCT setting and design suited this emerged.\n\n‘These [allocated funding for a third-party randomisation service] can help ensure robust methods before trial conduct, and thus the quality of the trial and report’ (ID 2.2)\n\nThere was also a suggestion for funding organisations to mandatorily require and fund external independent assessors\n\n‘It would strengthen the robustness of any trial results if funding organizations would demand and financially support external independent assessors’ (ID: 2.4)\n\nBoth of these points however fail to recognise that robust trial design and methodology does not necessarily mean the RCT publication will be written to reflect this; these suggestions will not prevent poor reporting quality they only impact trial conduct. This theme is considered later in this manuscript.\n\nAll participants stated that they used CONSORT guidelines when writing their RCT. Participants emphasised that some authors may not be aware of CONSORT, and that even though some journals do endorse the CONSORT statement there is a difference in how they are adhered to between journals which may impact reporting quality.\n\n‘I think that many clinicians may be unaware of the full consort statement’ (ID 2.4)\n\n‘Journals should adhere strictly to appropriate research reporting guidelines (e.g. CONSORT for RCTs)’. (ID 2.1)\n\nThere was universal support for CONSORT guidelines and strong agreement that there were adequate guidelines and support to aid the robust and transparent publication of research; rather it was adherence to the existing guidelines that is needed, with a focus on researchers ensuring this occurred.\n\n‘Guidelines for acceptable processes are readily available and reporting of that information is commonly required in key research reporting guidelines. If all researchers adhere to those recommendations we should have very little risk of selection bias in published research…..Spend the effort/time/money advocating for better adherence to existing reporting guidelines.’ (ID 2.1)\n\nLinked to this was that there were already mechanisms in place to enable trials to be reported adequately and that these need to be adhered to.\n\n‘Many journals require submission of a supplementary document demonstrating adherence to an appropriate reporting guideline, which commonly directs readers to the appropriate section/page of a manuscript to find relevant information.’ (ID 2.1)\n\nA recurring theme was the idea that if the trial was conducted robustly it would lead to a robust publication automatically. A lot of emphasis was placed on those researchers conducting themselves correctly, with integrity and following the protocol with the implication this would lead to a robust publication.\n\n‘These options [suggestions to aid reporting quality] can help ensure robust methods before trial conduct, and thus the quality of the trial and report’ (ID 2.2)\n\n‘[to improve reporting quality] ensure adherence to correct use of the envelopes/prevent violation of the study protocol’ (ID: 2.1)\n\n\nDiscussion\n\nOur research highlighted again that there is suboptimal implementation and reporting of allocation concealment. Only 57% of RCTs published in CONSORT endorsing journals contained adequate allocation concealment methods.\n\nAnalysis of the questionnaire data indicated that some authors could not accurately define allocation concealment, and it was at times confused with blinding. Only one participant considered it to be within the top three methodological elements of an RCT to report, indicating that it may not be reported adequately as it is not seen as a priority.\n\nTo improve the reporting of allocation concealment there was support for a methodologist to review submitted articles to ensure essential methodological information was reported. There are a number of barriers to operationalise this particular mechanism. One solution could be to nominate a trial team member to be the guarantor for required methodological information on submission of a manuscript, if journals do not have the capacity to support this specialist process.\n\nThe views of this sample of participants suggest that envelopes are going to be employed as a method of allocation concealment in future research for a variety of reasons such as budget, but authors had poor knowledge on which methodological factors should be reported.\n\nCentral randomisation was accepted as the gold standard but cost was a barrier for its implementation. Authors did not consider that choosing a less expensive allocation concealment method could result in inadequate methods being employed, which, could lead to research that is judged as a high risk of bias20,21. We agree with the discussion point raised by a participant stating that randomisation that was ‘performed centrally’ does not give the reader enough information on what was carried out, this is suboptimal reporting.\n\nThis research demonstrated that a survey elicits a low response rate when engaging with authors regarding allocation concealment knowledge, and different mechanisms to engage are needed. The corresponding author of a publication in healthcare is frequently a clinician and not a methodologist, which could explain the low response rate to a questionnaire about methodological information. We intentionally did not limit word counts within the free text boxes to enable full exploration and elicitation of views, this enabled us to capture rich data. Despite the response rate the data was consistent amongst the participants, those that did respond showed high engagement with the topic and this has enabled a targeted list of recommendations to be made on improving the implementation and reporting of allocation concealment for this sample.\n\nThere are many methodological quality factors that could have been addressed when assessing reporting quality. Nevertheless, allocation concealment was our focus due to it being the central part of an RCT. There was a low response rate to the questionnaire with the majority of responses from academics rather than clinicians, which limits the generalisability of the findings amongst the RCT author population. The low survey response rate is a finding that has been observed in other research with a trend of decreasing participant engagement over time22–24, it is postulated that frequent survey requests and work schedules may influence participation, particularly with health professionals25. We did not repeat the survey to increase the sample size as an aim of this research was to assess the effectiveness of this method on author engagement, the response rate demonstrated that this method did not yield a high participation rate for this research area.\n\nFurther work into how best to engage with RCT authors is necessary, interviews may be a better method to engage and unpick what is underpinning suboptimal implementation and reporting of allocation concealment. Identifying who is the methodologist within the author list and engaging with this individual may enable us to determine how the methods section was written and reported and why poor reporting was occurring by examining the internal editing processes that occurs within research teams, including any comments received from reviewers and methodological information that could have been lost during the publication process. Clinicians were under represented in this research, and exploring their views may elicit different mechanisms to improve allocation concealment implementation and reporting.\n\nWork into exploring different mechanisms and reporting platforms where there is an instant publication and a transparent reviewing process is recommended (such as with F1000 Research) which could lead to recommendations in speeding up reviewing time and processes which was highlighted as an issue with this surveyed sample.\n\nThis research highlighted that there needs to be greater knowledge of allocation concealment amongst authors of RCTs, as well as the understanding that securely implementing it does not automatically generate a well written RCT publication.\n\nFrom the findings of this research to improve allocation concealment implementation and reporting we recommend:\n\n- CONSORT is endorsed and adhered to by each journal as it was accepted by all participants in this research\n\n- Trials using envelopes need to be discouraged and if they are to be used then they should be robustly implemented and reported with careful checking that key methodological information is included in publications, as knowledge around this was suboptimal\n\n- Funders assess proposed methodology and insist on the use of robust allocation concealment methods if the RCT setting allows (such as central randomisation)\n\n- If central randomisation is implemented full methodological details are reported within the publication, including the names of external centres involved and the method of randomisation (simple, restricted) and details such as stratification variables and block size.\n\n- Authors need to take responsibility for ensuring key methodological factors are included in their RCT publications and should report all methods transparently even if not methodologically robust\n\n- A methodologist reviews the RCT publication to ensure key methodological information is included simultaneously to the peer review process. Although this is a recommendation based from the findings of this research the difficulties in the practicalities is discussed.\n\n\nData availability\n\nFigshare: Methodological details of included studies\n\nhttps://doi.org/10.6084/m9.figshare.13550348.v119\n\nThis project contains the following underlying data:\n\n• Methodological details of included studies.csv (Methodological details reported in publications of randomised controlled trials included in the study).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).\n\nWe can only currently share the questionnaires sent to participants and the non-identifiable data from the data extraction of the allocation concealment judgement where we identified included studies. We are only able to share the data extracted related to the journal CONSORT endorsement, quality judgements we made on the allocation concealment methods and the breakdown of different methods used (such as central randomisation and envelopes use), as to share the full extracted methods section and title from each included paper would enable the paper and therefore participants to be identified.\n\nIf readers or reviewers would like to apply for access to the data they would have to contact Laura Clark at laura.clark@york.ac.uk. We are required to liaise with the University of York Health Sciences Research Governance Committee (https://www.york.ac.uk/healthsciences/research-information/rsg/) to request permission to share the requested data and adhere to the conditions under which they will grant access.\n\nFigshare: Questionnaire to authors\n\nhttps://doi.org/10.6084/m9.figshare.13550351.v117\n\nThis project contains the following extended data:\n\n• Questionnaires used in study.docx (Questionnaire sent to authors)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "References\n\nJüni P, Altman DG, Egger M: Assessing the quality of controlled clinical trials. BMJ. 2001; 323: 42–46. Publisher Full Text\n\nSchulz KF: Subverting randomization in controlled trials. JAMA. 1995; 274(18): 1456–8. PubMed Abstract \| Publisher Full Text\n\nSchulz KF, Chalmers I, Hayes RJ, et al.: Empirical evidence of bias. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopewell S, Dutton S, Yu LM, et al.: The quality of reports of randomised trials in 2000 and 2006: comparative study of articles indexed in PubMed. BMJ. 2010; 340: c723. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClark L, Mitchell N, Hewitt C, et al.: Questionnaire to authors. figshare. Figure. 2021. http://www.doi.org/10.6084/m9.figshare.13550351.v1\n\nQUALTRICS: Qualtrics. 2019.\n\nClark L, Mitchell N, Hewitt C, et al.: Methodological details of included studies.csv. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.13550348.v1\n\nHiggins JPT, Altman DG, Sterne JAC: Chapter 8: Assessing risk of bias in included studies. In: JPT H and Green S, (eds.). The Cochrane Collaboration, 2011 Cochrane Handbook for Systematic Reviews of Interventions Version 510 (updated March 2011),2011. Reference Source\n\nHiggins JPT, Savović J, Page MJ, et al.: Assessing risk of bias in a randomized trial. Cochrane Handbook for Systematic Reviews of Interventions. 2019; 205–228. Publisher Full Text\n\nBlanco D, Hren D, Kirkham JJ, et al.: A survey exploring biomedical editors’ perceptions of editorial interventions to improve adherence to reporting guidelines. [version 3; peer review: 3 approved]. F1000Res. 2019; 8: 1682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMulligan A, Hall L, Raphael E: Peer review in a changing world: An international study measuring the attitudes of researchers. J Am Soc Inf Sci Technol. 2013; 64(1): 132–161. Publisher Full Text\n\nPrice A, Schroter S, Clarke M, et al.: Role of supplementary material in biomedical journal articles: surveys of authors, reviewers and readers. BMJ Open. 2018; 8(9): e021753. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlanigan TS, McFarlane E, Cook S: Conducting survey research among physicians and other medical professionals - a review of current literature. In: Proceedings of the Survey Research Methods Section, American Statistical Association. 2008; 4136–4147. Reference Source" }	[ { "id": "84202", "date": "17 May 2021", "name": "Fang Hua", "expertise": [ "Reviewer Expertise Clinical epidemiology", "evidence-based 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 is an interesting and important study regarding allocation concealment. The authors assessed the reporting and methodological quality of allocation concealment among RCTs published in “core clinical journals” in one month of 2019. They also carried out online questionnaires to investigate the knowledge of the RCT authors about allocation concealment. The study was generally well-conducted, but the article could be improved in the following aspects.\nA main limitation of this study is its sample size, with only 95 RCTs and 5 participants included. These need to be properly stated in the limitation paragraph. The authors explained that they did not repeat the survey to increase sample size as it was an aim to assess the effectiveness of questionnaire survey on author engagement. If this is the case I suggest abridging the part of introducing findings of this survey, as the findings were based on a very small sample and might not be very representative / reliable anyway. In addition, please provide the rationale for sample size determination, i.e. why did you decide to include RCTs published in one month of 2019, and was the sample size calculated a priority.\n\nThe authors mentioned that they used CONSORT to assess methodological quality, but CONSORT is a reporting guideline. Maybe it should be stated that methodological quality was assessed using a checklist developed based on CONSORT.\n\nThe authors used the term “implementation and reporting of allocation concealment” for several times. “Conduct” or “execution” might be a better word than “implementation”, as the latter has a different meaning in “implementation science”.\n\nAbstract – the total number of included RCTs should be included in the Results section; the sentence in Discussion section is quite long and difficult to follow.\n\nKeywords – please add more keywords to facilitate indexing and identification of this paper.\n\nMethods – “We identified all RCTs published in …”—how were these RCTs identified (e.g. electronic search or hand search the journals’ issues), and when was the last search conducted? If an electronic search was performed, the search strategy needs to be provided.\n\nTable 1 – “Double-blind placebo-controlled trials were considered adequate as they are considered a robust trial design and difficult to subvert”. This criterion seems inappropriate, as according to Cochrane Handbook allocation concealment is to avoid selection bias, while blinding is related to performance & detection biases. Please provide rationale for this criterion, or add this to the limitations paragraph.\n\nResults – It will be better if the characteristics of included RCTs were described, so that readers can better understand the context of these findings.\n\nReferences #7 and #20 are the same.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "96107", "date": "27 Oct 2021", "name": "Jelena Savovic", "expertise": [ "Reviewer Expertise Evidence synthesis", "RCTs", "non-randomised studies", "bias", "epidemiology", "meta-epidemiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInformative study, but based on a very small sample of authors and very poor response rate. Methods used are appropriate.\nI answered 'Partly' to the Q, 'Are all the source data underlying the results available to ensure full reproducibility?' because the month of the search for RCTs could not be revealed due to ethical reasons. This would make the study more difficult to reproduce. However, not disclosing the participants is more important than being able to reproduce the exact study the authors did. Interested parties would be able to reproduce the study for a different publication period, which would be a more useful endeavour.\nAlthough the recommendations made are sensible, is it really possible to make recommendations based on responses from 6 authors? Perhaps a stronger caveat to these recommendations would help? This was the reason I answered 'Partly to the last reviewer question. I feel that the main finding is that engaging RCT authors via surveys does not work.\nIn the text when the % of participants are given, can the actual numbers be given as well please? E.g. 'From the short Q-re 60% of participants stated...' to 'From the short Q-re 7 (60%) of participants stated...' or to state 7 (out of 12). I find this especially important with such small numbers.\nThe findings of the first part of the study, based on the analysis of reporting in 95 RCTs published in one month in 2019, are perhaps more interesting. There are some additional items I would be interested to read about, that the authors may be able to extract from their sample of 95 RCTs:\nComparative adequacy of reporting by type of corresponding author (clinician vs academic). Perhaps this could identify some training needs in these groups.\n\nUnder Allocation concealment methods: envelopes - Can you extract data on how many of the RCTs that used the envelope method described any sort of process for ensuring that the next envelope is not opened until the next participant is irreversibly enrolled? I find that this part of the concealment process for envelopes is often ignored and almost never reported. It would be interesting to see if any of the RCTs in your sample reported it.\nMinor issues:\nThere are several spelling errors/typos and missing punctuation in the manuscript. - e.g:\nIn Methods, para 4: \"...could improve the reporting quality of RCTs and the opinions of suggestions to improve reporting quality of allocation concealment methodoloy These categories were further coded and mapped, as detailed...\" - \"methodoloy\" is misspelt and there is a missing period after it.\n\nIn the Methods para 5: \"An internal pilot was performed on each questionnaire with research staff in the York Trials Unit who had experience of being authors of RCTs and trial methodology, following piloting we reordered some of the questions but did not change the content \" - The sentence should break after 'methodology' and the new sentence to start with 'Following'. Also the period is missing at the end of the para.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-83
https://f1000research.com/articles/10-82/v1	08 Feb 21	{ "type": "Research Article", "title": "Differential activation of c-Fos and Egr1 during development of the mouse visual cortex", "authors": [ "Anna Ivanova", "Pavel Rogozin", "Grigori Enikolopov", "Konstantin Anokhin", "Alexander Lazutkin", "Anna Ivanova", "Pavel Rogozin", "Grigori Enikolopov", "Konstantin Anokhin" ], "abstract": "Background: Critical periods (CP) in brain development are characterized by heightened neural plasticity in the relevant brain regions. They are associated with changes in gene expression cascades, in particular with altered expression of genes involved in plasticity regulation, such as immediate early genes. Here we examine the expression of the immediate early genes c-Fos and Egr1 at different stages of mouse visual cortex (VC) development. Methods: Mice 11, 25, and 50 days of age were maintained under standard light-dark conditions, deprived of light for 5 days, or deprived of light for 5 days and then exposed to light for 90 min. Their brains were analyzed at PND16 (before the onset of the CP), PND30 (during the CP) and PND55 (after the CP) to determine the changes in the number of cells expressing c-Fos and Egr1 in the binocular primary visual and primary somatosensory cortices. Results: We found highly specific induction of c-Fos expression in the primary VC in response to light. We also observed transient cross-modal activation of c-Fos in the barrel field of the primary somatosensory cortex in response to light before and during the CP; such activation disappeared after the CP. Expression of Egr1 was not induced by light in the VC before the CP, but was evident during and after the CP, although the induction was much less pronounced than that of c-Fos. Conclusions: Dynamic changes in c-Fos and Egr1 expression may reflect their contribution to the VC plasticity during the CPs of postnatal brain development.", "keywords": [ "Critical period", "IEG", "Somatosensory cortex", "Ccular dominance", "Cortical development", "Plasticity" ], "content": "Introduction\n\nPostnatal development of the neocortex includes crucial stages when proper experience defines the subsequent maturation of sensory, motor, cognitive and other functions. There is a defined time window, described as a critical period (CP), when a particular developing brain structure is especially sensitive to environmental stimuli and when a relevant experience can produce large-scale permanent changes to neural circuits1–7. CPs are characterized by exceptionally high levels of plasticity, and have been best documented for sensory systems, particularly for the development of vision1,4,7–9. Experience-dependent modification of the mammalian visual cortex (VC) occurs during a narrow temporal window and is indispensable for the subsequent proper connectivity and vision development. Perturbations during the CP of the visual system development lead to deficiencies in the processing of the visual signals, and in humans may result in disorders of sight such as amblyopia4,10,11.\n\nThe development of the primary visual cortex (V1) in mice occurs in several stages7. The first stage includes formation of precise topographic maps; it takes place before the eyes opening and before the axonal projections from the dorsal lateral geniculate nucleus organize high-resolution point-to-point connections with neurons in layer 4 of V1 (L4). During the second stage of V1 development, around the time of eye opening and within several days of the first visual responses in the retina, V1 neurons acquire orientation selectivity. In the third stage of V1 development, the selective properties of neurons are refined and become similar through both eyes. For the mouse VC, the CP corresponds to PND21–35. After that period, the circuitry and responses of V1 appear mature and normally remain stable throughout life7.\n\nAlthough a large part of the VC corresponds to a monocular zone that receives inputs predominantly from the contralateral eye, a smaller binocular zone of V1 receives inputs from both eyes. Neuronal responses in the binocular region are driven more strongly by visual inputs received from the contralateral eye than the ipsilateral eye, in a phenomenon characterized as ocular dominance (OD). OD is highly influenced by visual experience during CP and changes in OD (OD plasticity) are possible only during early postnatal development through monocular deprivation of visual stimuli3,4,7.\n\nCPs in brain development are associated with pronounced changes in gene expression, driven by the changes in expression of key transcriptional regulatory factors5,12. For instance, the highest levels of pCREB, the phosphorylated (active) form of the transcriptional factor CREB, are found during and after the CP of VC development13,14. Activation of CREB induces expression of its downstream targets, such as immediate early genes (IEGs) c-Fos, Egr1, or Arc, which are themselves important regulators of gene expression cascades; moreover, induction of IEG expression (e.g., of c-Fos) is often considered a proxy of neuronal activation. Changes in IEG expression have been thoroughly investigated for the developing visual cortex of mice, rats, cats, and primates8,15–35, with the results providing strong evidence for the regulatory input of IEGs into the development of the visual system. Such studies have been particularly informative for the mouse model, with the conclusions being extrapolated to human brain development and human vision disorders3,4,10,11. Here we asked whether c-Fos and Egr1, two IEGs broadly used as markers of neuronal activity, show distinct patterns of activation in the mouse brain, as related to the CP of the visual system development. Towards this goal, we explored the dynamics of basal and induced expression of c-Fos and Egr1 in the binocular zone of V1 at different stages of mouse VC development – before, during, and after the CP, when they may be required for the proper development of vision.\n\n\nMethods\n\nIn this study we used C57Bl/6J mice of both sexes (RRID:IMSR_JAX:000664, The Jackson Laboratory, USA). All experiments were performed in accordance with the requirements of the Ministry of Health of the Russian Federation (Decree no.267 of 19/6/2003) and the “Regulations for studies using experimental animals” (Research Institute of Normal Physiology, Russian Academy of Medical Sciences, Protocol no.1 of 3/9/2005).\n\nBefore the experiment, animals were kept under normal conditions at room temperature in 12/12h light-dark cycle and maintained on ad libitum food and water. For breeding, adult animals (one male and 2–3 females) were kept together in the colony room in standard cages 36×21×13.5 cm. Pregnant dams were transferred to individual cages a few days before labor, and appearance of pups was taken for the first day of postnatal development (PND1). Litters of 6–8 pups were considered normal and selected for the experiments. Pups from each litter were kept with mothers until PND21. In further experiments each pup was considered to be an experimental unit.\n\nUntil the end of the experiment, the animals were not subjected to pain or stressors. Pups were kept with dams (without other mice in the cage) until the end of weaning period to prevent infanticide and to ensure comfortable conditions for the animals. The deprivation camera was well ventilated and quiet. All manipulations were performed with the utmost speed and care. PND16 mice were euthanized by transposition of cervical vertebrae to minimize animal distress as recommended for the juvenile mice by the American Veterinary Medical Association (AVMA) Guidelines for the euthanasia and the European Commission. We applied the same method to the mice of other ages for experimental consistency.\n\nA total of 51 mice were divided into three groups: home cage group (HOME), a total of 15 animals: n=4 for PND16, n=5 for PND30 and n=6 for PND55; dark control group (DARK), a total of 17 animals: n=6 for PND16, n=6 for PND30 and n=5 for PND55; and experimental group (EXP), a total of 19 animals: n=5 for PND16, n=7 for PND30 and n=7 for PND55. The number of animals per experimental group was based on the information on the variation of the response as measured by the number of c-Fos-expressing cells and mean and standard deviation of the response, as obtained from our previous experience and pilot studies. This information was used for statistical power analysis to estimate the group size that is necessary to test our hypotheses, with significance level alpha of 0.05 (95%).\n\nThe average weight of an animal of the corresponding age was 4–8g for PND16, 10–20g for PND30 and 20–25g for PND55. Atypically low weight of the animal at the time of euthanasia was considered as an a priori exclusion criterion. All animals in this series met this inclusion criterion and were therefore used for the experiments.\n\nOn PND9, PND23, or PND48 (i.e., before, during and after CP), the cages containing mice from the EXP and DARK groups were placed into a light deprivation camera (a ventilated plastic box, 55×39×46.5сm, fully isolated from external light) and kept there for 2 days in a standard light-dark cycle, with two cages in the camera at a time. For the next 5 days, the EXP and DARK groups were deprived of light. Deprived mice from each cage were randomly allocated into DARK or EXP group by the experimenter so that each group was sex-balanced. On PND16, 30 or 55, respectively, EXP mice were exposed to bright light for 90 min before euthanasia; DARK mice were euthanized immediately after opening of the deprivation camera. HOME mice were maintained under a standard light-dark cycle for the entire time before euthanasia.\n\nAfter euthanasia, brains were quickly removed and frozen under liquid nitrogen vapor. Investigators could not be blinded before collecting brain samples due to different location of the HOME group and the time of euthanasia of the DARK and EXP groups. Collected brain samples were double-blind coded and decoded only after all images were obtained and cell counting was completed.\n\nSerial 20 µm thick coronal sections through the VC and SC were cut with a freezing microtome (Leica CM 1950, Germany). Sections were fixed in 4%PFA for 10 min at 4°C, rinsed in 1x PBS three times for 5 min each at RT and placed in an immunostaining chamber (Sequenza Immunostaining Center, Shandon, UK). Sections were incubated with 2.5% normal horse serum (Vector Laboratories, USA) in 1x PBS for 40 min at 4°C and then with primary polyclonal antibodies against c-Fos (anti-rabbit, 1:500, sc-52, RRID:AB_2106783, Santa Cruz Biotechnology, USA) or Egr1 (anti-rabbit, 1:1000, sc-189, RRID:AB_2231020, Santa Cruz Biotechnology, USA) in 1x PBS, 2.5% normal horse serum and 0.01% NaN3 overnight at 4°C. After 3 rinses in 1x PBS, the sections were incubated with secondary biotinylated anti-rabbit IgG polyclonal antibody (ImmPress PK-7401, RRID:AB_2336529, Vector Laboratories, USA) for 50 min at RT. After being rinsed three times in 1x PBS, the sections were incubated in DAB solution (0.06% DAB and 0.003%H2O2 in 1x PBS) for 5–7 min at RT, rinsed again with dH2O, dehydrated with a graded concentrations of ethanol series (70%EtOH, 96%EtOH, and 100%EtOH) and xylene for 5 min each step and then coverslipped with Mount-Quick (DAIDO SANGYO Co, Japan).\n\nThe brain sections (three coronal sections per brain) were captured with a computer assisted microscope system Olympus VS110 (10x, NA 0.40, Olympus, Japan). Images were converted into gray scale in Photoshop CS5 v.12.0.1 (RRID:SCR_014199, Adobe Systems Inc., USA), and the number of c-Fos+ or of Egr1+ cells was counted in the VC and SC with ImagePro Plus v.3.0 software (RRID:SCR_016879, Media Cybernetics, USA). Cell density in the region structure (cell number/area of the structure, mm2) was calculated and statistically analyzed with a set of density values for different sections of the same region averaged over the number of slices.\n\nAt this stage, a priori criteria for excluding animals from the analysis were the absence (mainly due to cutting or staining errors) of three slices from the selected region (mainly due to cutting or staining errors); immunohistochemical staining of improper quality (too pale or too bright); or poor morphology of slices that did not allow counting the number of cells in all three slices from the region. As a result of applying these criteria, 1–2 animals from some Egr1 groups were excluded. The final number of 51 mice remain unchanged for the statistical analysis of c-Fos. Egr1 expression was analyzed in total of 43 animals: a total of 15 mice in the EXP group: n=5 for PND16, n=5 for PND30, and n=5 for PND55; a total of 15 mice in the DARK group: n=5 for PND16, n=5 for PND30, and n=5 for PND55; and a total of 13 mice in the HOME group: n=4 for PND16, n=4 for PND30 and n=5 for PND55.\n\nStatistical analysis was performed in Statistica v.7 (RRID:SCR_014213, StatSoft) and GraphPad Prism 6.01 (RRID:SCR_002798, GraphPad Software). Kruskal-Wallis test with multiple comparisons of mean ranks for all groups (KWmc test) used for intergroup comparisons. For the comparison of VC and SC, Wilcoxon matched pairs test (Wmp test) was used. Differences were considered statistically significant at p < 0.05. All data are presented as a mean ± standard error of the mean.\n\n\nResults\n\nTo investigate the CP-related dynamics of the cortical cell populations carrying markers of neuronal activation, we first defined the basal level of c-Fos-expression in the brains of the home caged mice (control HOME group mice). The brains were analyzed for c-Fos-expressing cells in the primary VC before (PND16), during (PND30), and after (PND55) the CP. In parallel, we determined the changes in c-Fos+ cells in the barrel field of the primary SC of the same brain preparations. We found very few c-Fos expressing cells (1–10 cells per mm2) in both the VC and SC at each of the three analyzed time points: 0.84±0.42 in the VC and 0.39±0.15 in the SC at PND16; 1.45±0.45 in the VC and 1.27±0.14 in the SC at PND30; and 10.17±4.96 in the VC and 2.83±1.14 in the SC at PND55 (Figure 1A and B).\n\nFor clarity and comparable axes values, the data are presented as grouped by the cortical areas: VC (A) and SC (B) and as grouped by days: PND16 (C), PND30 (D), and PND55 (E). Data for the HOME, DARK and EXP groups at the same age are compared using KWmc test for all groups, with * - p<0.05; - p<0.01; * - p<0.005. Data for the same group at different ages are compared using KWmc test, +++ - p<0.005. Data for VC and SC are compared using Wmp test, # - p<0.05.\n\nNext, we determined c-Fos expression in mice deprived of light for five consecutive days (the DARK group). In these dark-reared animals, similarly to the HOME group, the number of c-Fos-expressing cells was very low in both the VC and SC at each of the three time points: 1.12±0.44 in the VC and 1.26±0.62 in the SC at PND16; 3.96±1.64 in the VC and 3.17±1.34 in the SC at PND30; and 1.05±0.35 in the VC and 0.80±0.24 in the SC at PND55 (Figure 1A and B).\n\nWe then determined whether brief exposure to light (the EXP group) altered c-Fos expression. Indeed, at each analyzed time point the number of c-Fos-positive cells was significantly higher in both the VC and SC in the EXP group than in the HOME or DARK groups. On PND16, before the visual input CP, c-Fos expression in EXP mice was 49.32±2.36 in the VC and 17.53±2.78 in the SC (Figure 1A and B), i.e., 47- and 35-fold higher (VC) and 36- and 11-fold higher (SC) than that in the HOME and DARK groups, respectively (p=0.0048, KWmc test). Note that c-Fos induction was 2.8-fold higher in the VC than in the SC at this time point (p=0.04, Wmp test, Figure 1C).\n\nDuring the CP, at PND30, c-Fos expression in EXP mice was also significantly higher than that in the HOME and DARK groups in both cortical areas: 502.98±35.76 cells in the VC and 9.24±0.91 cells in the SC (Figure 1A and B). For the VC this corresponded to a level 350- and 127-fold greater than those in the HOME and the DARK group, respectively and for the SC this corresponded to levels 7.7- and 2.9-fold greater than those in the HOME and the DARK groups (respectively, p=0.004 and 0.018 for VC and p=0.016 and 0.035 for SC in KWmc test). As it was observed for PND16, induction of c-Fos after light exposure was significantly higher in the VC than in the SC (54-fold, p=0.018, Wmp test, Figure 1D).\n\nFinally, after the CP, on PND55, exposure to light also resulted in a significant increase in c-Fos expression in the VC (166.73±29.59 cells, Figure 1A and E), which corresponded to a level 16.4- and 154-fold greater than that in the HOME and DARK groups (p=0.038 and 0.002, respectively, KWmc test for all groups). In the SC, in contrast to the PND16 and PND30 results, light exposure did not increase the number of c-Fos-expressing cells (3.41±0.76, Figure 1B and E) and there was no statistically significant difference relative to the HOME and DARK groups.\n\nIn parallel to the analysis of c-Fos-expressing cells, we analyzed the number of Egr1-expressing cells in the same preparations. On PND16, the number of Egr1-expressing cells was comparable in all groups in both cortical areas (Figure 2A-C): 632.23±49.70, 605.93±100.22, and 599.24±45.65 cells/mm2 in the VC in the HOME, DARK, and EXP groups, respectively, and 564.93±53.47, 532.04±88.36, and 720.70±72.60 cells/mm2 in the SC in the HOME, DARK, and EXP groups.\n\nFor clarity and comparable axes values, the data are presented as grouped by the cortical areas: VC (A) and SC (B) and as grouped by days: PND16 (C), PND30 (D), and PND55 (E). Data for the HOME, DARK and EXP groups at the same age are compared using KWmc test for all groups, with * - p<0.05; - p<0.01; * - p<0.005. Data for the same group at different ages are compared using KWmc test, +++ - p<0.005. Data for VC and SC are compared using Wmp test, # - p<0.05.\n\nOn PND30 (Figure 2A, B and D), i.e., during the CP, the basal level of Egr1 expression (HOME group) in the VC was significantly lower than that on PND16 (331.36±52.20, p=0.04, KWmc test). However, light exposure produced significant induction, with 793.85±122.71 Egr1-expressing cells/mm2, a level 2.5-fold greater than that in the DARK group (p=0.038, KWmc test). This induction was specific to the VC and was not observed in the SC at that time point. In the VC, as compared with the SC, the number of Egr1-cells in the EXP group was 1.7-fold higher (p=0.043, Wmp test).\n\nOn PND55 (Figure 2A, B and E), light exposure produced a significant induction of Egr1-expressing cells in the VC (3.3-fold greater levels in the EXP group (931.67±84.13 cells/mm2) than in the corresponding DARK group (310.51±49.89 cells/mm2), p=0.007, KWmc test), but not in the VC or SC in the other groups. When compared across the time-points, the number of Egr1-expressing cells increased at PND55 in the VC (1.55-fold increase over the PND16 values) but not the SC.\n\n\nDiscussion\n\nSeveral attempts have been made to identify genes differentially expressed in the VC before and after the CP, and c-Fos and Egr1 have been suggested to act as important modulators of the CP4,5,8,9,36,37. Our results support this possibility by showing time- and region-specific response of the VC to visual stimuli, as revealed by the induction of c-Fos-positive cells and, to a lesser degree, of Egr1-positive cells.\n\nWe investigated the dynamics and specificity of the IEG induction at the key stages of the mouse cortical development – before, during, and after the visual CP. Our results show that exposure to light at the CP (PND30) results in a markedly high (130–350-fold) increase in c-Fos-expressing cells in the VC relative to the levels in the DARK and HOME groups, and to the levels before and after the onset of CP, and relative to an adjacent unrelated cortical area (SC).\n\nc-Fos induction in response to light was also observed before the CP, but to a lesser degree (11–36-fold). Strong increase in c-Fos+ cells in the VC was also observed after the CP (153-fold at PND55). Induction of c-Fos-expressing cells in response to light was also observed in the SC during the CP, albeit at a significantly smaller scale. This increase in the SC disappeared after the CP, by PND55.\n\nEgr1, another potential marker and effector of CP, responds to light during the CP by increased induction in the VC, although at a much smaller scale than c-Fos (a 2.5-fold increase). Induction of Egr1-positive cells was also preserved after the CP. The overall number of Egr1-expressing cells in the VC in response to light was similar before, during, and after the CP (with a small 1.55-fold increase from PND16 to PND55). Thus, although Egr1 responds to light during the CP, its specificity with respect to the CP appears to be less than that of c-Fos. At the same time, a lack of Egr1 response before the CP may reflect a shift in the role of Egr1 during development and its involvement in the plastic changes occurring during the CP, such as changes in the excitatory/inhibitory balance.\n\nThe validity of c-Fos and Egr1 as markers of the visual response, was corroborated by a much less pronounced induction of c-Fos (and no significant induction of Egr1) in the somatosensory, as compared to the visual, cortex. At the same time, the SC still responded to light with a robust increase in c-Fos+ cells, which was particularly pronounced before the CP, when the c-Fos induction was close to that in the VC (35–47-fold in the VC vs. 11–35-fold in the SC). This cross-modal activation was transient: it was less pronounced during the CP and disappeared after the CP. Cross-modal activation was not observed for Egr1, which may indicate greater specificity of Egr1 in the plastic rearrangements accompanying cortical maturation.\n\nIn sum, our results demonstrate dynamic changes in the expression of IEGs and suggest that IEG activation cascades underlie high cortical plasticity observed during the critical periods of visual cortex development.\n\n\nData availability\n\nDryad: Differential activation of c-Fos and Egr1 during development of the mouse visual cortex. https://doi.org/10.5061/dryad.k0p2ngf7938.\n\nThis project contains the following underlying data:\n\n- Data file 1: Egr 1 cell density in PND 16 mice\n\n- Data file 2: Egr 1 cell density in PND 30 mice\n\n- Data file 3: Egr 1 cell density in PND 55 mice\n\n- Data file 4: Fos cell density in PND 16 mice\n\n- Data file 5: Fos cell density in PND 30 mice\n\n- Data file 6: Fos cell density in PND 55 mice\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 thank Drs. Irina Zaraiskaya (P.K. Anokhin Institute of Normal Physiology) and Evgeny Amelchenko (Stony Brook University) for critical reading of the manuscript.\n\n\nReferences\n\nHensch TK: Critical period regulation. Annu Rev Neurosci. 2004; 27(1): 549–579. PubMed Abstract \| Publisher Full Text\n\nHensch TK: Critical period plasticity in local cortical circuits. Nat Rev Neurosci. 2005; 6(11): 877–888. PubMed Abstract \| Publisher Full Text\n\nHooks BM, Chen C: Circuitry Underlying Experience-Dependent Plasticity in the Mouse Visual System. Neuron. 2020; 106(1): 21–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHensch TK, Quinlan EM: Critical periods in amblyopia. Vis Neurosci. 2018; 35: E014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBegum MR, Sng JCG: Molecular mechanisms of experience-dependent maturation in cortical GABAergic inhibition. J Neurochem. 2017; 142(5): 649–661. 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PubMed Abstract \| Publisher Full Text\n\nWorley PF, Cole AJ, Murphy TH, et al.: Synaptic regulation of immediate-early genes in brain. Cold Spring Harb Symp Quant Biol. 1990; 55: 213–23. PubMed Abstract \| Publisher Full Text\n\nBeaver CJ, Mitchell DE, Robertson HA: Immunohistochemical study of the pattern of rapid expression of C-Fos protein in the visual cortex of dark-reared kittens following initial exposure to light. J Comp Neurol. 1993; 333(4): 469–84. PubMed Abstract \| Publisher Full Text\n\nLennartsson A, Arner E, Fagiolini M, et al.: Remodeling of retrotransposon elements during epigenetic induction of adult visual cortical plasticity by HDAC inhibitors. Epigenetics Chromatin. 2015; 8: 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMontero VM: Amblyopia decreases activation of the corticogeniculate pathway and visual thalamic reticularis in attentive rats: a 'focal attention' hypothesis. Neuroscience. 1999; 91(3): 805–17. PubMed Abstract \| Publisher Full Text\n\nPizzo R, Gurgone A, Castroflorio E, et al.: Lack of Cdkl5 Disrupts the Organization of Excitatory and Inhibitory Synapses and Parvalbumin Interneurons in the Primary Visual Cortex. Front Cell Neurosci. 2016; 10: 261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSato MT, Tokunaga A, Kawai Y, et al.: The effects of binocular suture and dark rearing on the induction of c-fos protein in the rat visual cortex during and after the critical period. Neurosci Res. 2000; 36(3): 227–33. PubMed Abstract \| Publisher Full Text\n\nChoi SY: Synaptic and circuit development of the primary sensory cortex. Exp Mol Med. 2018; 50(4): 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTropea D, van Wart A, Sur M: Molecular mechanisms of experience-dependent plasticity in visual cortex. Philos Trans R Soc Lond B Biol Sci. 2009; 364(1515): 341–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazutkin A, Ivanova A, Rogozin P, et al.: Data for: Differential activation of c-Fos and Egr1 during development of the visual cortex, Dryad. Data Collection. http://www.doi.org/10.5061/dryad.k0p2ngf79" }	[ { "id": "81585", "date": "14 Apr 2021", "name": "Alison Barth", "expertise": [ "Reviewer Expertise Neuroplasticity and neocortical circuits" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor points of concern:\nThe finding that somatosensory cortex showed an increase in fos after light exposure is interesting, but these experiments do not appear to be well controlled and is thus not clearly attributable to the single variable of light exposure. Fos is highly sensitive to handling and change in environment. For example, if the animals were handled to move them to a new cage for light exposure, that could drive broad fos expression. The methods should be clarified on this point: “On PND16, 30 or 55, respectively, EXP mice were exposed to bright light for 90 min before euthanasia” is incomplete but critical to interpretation of the data. If the animals were handled to enable light exposure, this must be indicated. If this was the procedure used, it suggests that cross-modal activation of somatosensory cortex by visual input is less likely than bona fide somatosensory stimulation.\n\nThe figures are sparse and the analysis is unsophisticated. Current analysis of critical period effects on immediate early gene expression relates activity to specific cortical circuits, at least by layer if not by cell type. The current analysis is not attentive to these important variables. At the very least, the authors should show images of the c-fos and egr histology.\n\nThe layers in which fos and egr were observed is important; however, analysis was of bulk labeling. The coarseness of this quantitation will make this manuscript significantly less impactful in the field, and does not represent the state of the art in the field. It would be simple to redo the analysis to show which layers the IEGs are activated in. Also, quantitation of “cells per mm2” is difficult to interpret. Can the authors represent the immunopositive cells as a percent of the total? The data sheets in the repository are difficult to interpret.\n\nWas there any difference in the intensity of staining for individual cells, across either different cells or under different conditions?\n\nThe data presentation is redundant and somewhat confusing. Figure 1 and 2 A and B are replotted in a different configuration in Figure 1 and 2 C-E. A scatter plot of values from different animals should be shown, so that the reader can appreciate variability in this measurement. Also, there are a bewildering number of statistical comparisons – were post-hoc corrections done for all? This kind of broad statistical analysis is usually accomplished with an ANOVA, but given the large number of comparisons with corrections it is possible that nothing will appear significant. That by itself is not a sticking point for the differences observed, but the statistical analyses do not appear to be done properly.\n\nIn the discussion, the authors attempt to compare induction of egr1 and cfos, but the resolution of the data presented is too coarse to enable any specific insight and regulatory processes. Did the same cells induce both transcription factors, or was expression restricted to different layers/subsets of neurons?\n\nMinor:\n“DARK mice were euthanized immediately after opening of the deprivation camera.” Please define the precise time interval between removal and tissue harvesting. IEGs can be induced within as little as 15-30 minutes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "88399", "date": "27 Jul 2021", "name": "Marina Yu Khodanovich", "expertise": [ "Reviewer Expertise Neuroscience", "immmunohistochemistry", "animal models" ], "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 of the dynamics the expression of the immediate early genes c-Fos and Egr1 in response to visual stimulation depending on the stage of development is new and interesting. The work was performed on a representative sample, uses adequate design and methods. However, there are a number of questions for authors that would help improve the presentation of their research and strengthen the interpretation of the results:\nImage processing. Were the sizes of regions of interest the same for all groups and animals that were taken? What is the size of the regions of interest for cell counting in the visual and somatosensory cortex? Which layers of the cortex were taken for counting?\n\nStatistical analysis. Why were the non-parametric Kruskal-Wallis and Wilcoxon tests used? If the distribution was normal, then it would be more correct to use Repeated Measures ANOVA followed by post-hoc tests, while taking into account complex design and multiple intergroup comparisons. However, even if the distribution differs from normal, the rank-based Newman-Keuls test for multiple comparisons can be used.\n\nPresentation of the results. In Figures 1 and 2, diagrams c-e actually duplicate a and b. At the same time, histological images are not shown in the figures. They are necessary to assess the quality of the staining and the presence of the background.\n\nThe authors used animals of both sexes to form groups. Were there gender differences in the expression of c-Fos and Egr1?\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": [] } ]	1	https://f1000research.com/articles/10-82
https://f1000research.com/articles/8-394/v1	08 Apr 19	{ "type": "Research Article", "title": "Patient-centered benefit-risk analysis of transcatheter aortic valve replacement", "authors": [ "Kevin Marsh", "Natalia Hawken", "Ella Brookes", "Carrie Kuehn", "Barry Liden", "Natalia Hawken", "Ella Brookes", "Carrie Kuehn", "Barry Liden" ], "abstract": "Background: Aortic stenosis (AS) treatments include surgical aortic valve replacement (SAVR) and transcatheter aortic valve replacement (TAVR). Choosing between SAVR and TAVR requires patients to trade-off several benefits and risks. The objective of this research was to determine which outcomes associated with TAVR and SAVR patients consider most important, collect quantitative data about how patients weigh these benefits and risks, and evaluate patients’ preferences for SAVR or TAVR. Methods: Patients with aortic stenosis were recruited from advocacy organization databases. An online adapted swing weighting (ASW) method was used to elicit attribute tradeoffs from 93 patients. The ASW exercise consisted of a series of pairwise comparisons of attributes. Survey data were used to estimate the weight that patients put on the AS treatment attributes, which were incorporated into a quantitative benefit-risk analysis (BRA) to evaluate patients’ preferences for TAVR and SAVR. Results: On average, patients put greater value on attributes that favored TAVR than SAVR. The value patients placed on the lower short-term mortality rate, reduced procedural invasiveness, and quicker time to return to normal quality of life associated with TAVR, offset the value they placed on the time over which SAVR has been proven to work. There was substantial heterogeneity in patients’ preferences. This was partly explained by age, with differences in preference observed between patients <60 years to those ≥60 years. A Monte Carlo Simulation found that 75.1% of patients prefer TAVR. Conclusions: Most AS patients are willing to tolerate sizable increases in clinical risk in exchange for the benefits of TAVR, resulting in a large proportion of patients preferring TAVR to SAVR. Further work should be undertaken to characterize the heterogeneity in preferences for AS treatment attributes. Shared decision-making tools based on attributes important to patients can support patients’ selection of the procedure that best meets their needs.", "keywords": [ "TAVR", "aortic valve", "transcatheter", "patient preference", "benefit-risk analysis" ], "content": "Introduction\n\nAortic stenosis (AS) is a progressive cardiovascular condition resulting from narrowing (or stenosis) of the aortic valve. This narrowing prevents the valve from fully opening, decreasing blood flow out of the heart and forcing it to work harder to maintain sufficient blood flow. If left untreated, AS can lead to severe cardiovascular complications and death1. As of 2015, 12.4% of the United States population over age 75 (nearly 2.5 million people) were reportedly diagnosed with AS2,3. More than one in eight people (13.3%) over the age of 75 in the US have moderate-to-severe AS4. Patients with AS may be asymptomatic for many years until the valve is narrowed severely enough to cause symptoms. Symptoms of AS include chest pain and angina, syncope, dyspnea, fatigue, and palpitations, all of which are exacerbated by physical activity5. Undertreatment of AS patients is common, with more than half of patients referred to cardiologists failing to receive surgical treatment6. Once symptoms appear, often between the ages of 70 and 80 years old, the prognosis of untreated patients is poor7. Among untreated patients, average survival is 50% at two years and 20% at five years after the onset of AS symptoms.\n\nThe traditionally recommended treatment for AS is surgical aortic valve replacement (SAVR). Such invasive surgery, involving a large incision in the chest, may not be suitable for all patients, especially those with comorbidities. The alternative, transcatheter aortic valve replacement (TAVR), is a minimally invasive, catheter-based procedure to replace the aortic valve in patients with AS. TAVR is the first-line therapy for inoperable patients with severe AS and an alternative to SAVR in operable high-risk patients. Among patients who are at intermediate surgical risk, both TAVR and SAVR are associated with improvements in disease-specific and generic health status8. However, TAVR is associated with a reduced rate of complications and a quicker recovery time, with patients returning to a normal quality of life more quickly9. When first available, the benefits of TAVR were offset by reportedly increased risks of stroke and the need for a pacemaker10–12. Recent clinical data reveal similar, if not improved rates of stroke and need for pacemaker among TAVR patients13. Furthermore, at a median two-year follow-up, all-cause mortality for patients undergoing TAVR was 20.2% compared with 21.9% for patients undergoing SAVR14.\n\nThe choice of TAVR or SAVR involves patients making trade-offs between multiple treatment attributes, including invasiveness, speed of recovery, mortality rates, and risks of complications. Given the challenging nature of this decision, tools have been developed to support patient decision-making15. However, little is known about the weights that patients assign to which attributes, how they make trade-offs between these attributes, and whether and how these preferences vary between patients. The objective of this research was to determine which outcomes associated with TAVR and SAVR patients consider most important, collect quantitative data about how patients weigh the benefits and risks associated with TAVR and SAVR, and to use this data to evaluate patients’ preferences for SAVR or TAVR.\n\n\nMethods\n\nPatients’ preferences for TAVR or SAVR were assessed using a quantitative benefit-risk assessment (BRA). This involved identifying attributes that distinguish TAVR and SAVR, measuring TAVR and SAVR performance on these attributes, eliciting patients’ preferences for these attributes, and combining performance and preference in a BRA to determine what proportion of patients prefer TAVR or SAVR. The patient-preference survey upon which the BRA is based was fielded in July–August 2018.\n\nA long list of potential attributes was identified by reviewing the attributes highlighted in previous patient preference studies for heart value surgical interventions, published meta-analyses and clinical studies, and regulators’ assessments of related products. Additional attributes were identified based on consultation with TAVR and SAVR clinical experts and from patient input. The final attributes used in the study were selected based on clinical and regulatory relevance, whether or not the attribute distinguishes between TAVR and SAVR, and to comply with the attribute set properties required of an additive BRA16. For example, to avoid overlap with ‘mortality’, the ‘stroke’ attribute was were defined as ‘disabling, non-fatal strokes.’\n\nDescriptions of the final attributes included in the BRA are summarized in Table 1.\n\nTAVR and SAVR performance against the final attributes were identified from the published literature and from clinical data (Table 2), focusing on data that had a high degree of reference and use within the clinical community. Available data for stroke risk, defined as “all stroke” (both fatal and non-fatal) in the literature, and independence, defined by Kansas City Cardiomyopathy Questionnaire (KCCQ)17 score, required transformation to estimate performance as defined by the final study attributes. To estimate the risk of non-fatal stroke only, available stroke risk data was adjusted using the mortality rate among patients with severe aortic stenosis enrolled in the PARTNER trial who suffered a stroke compared to the mortality rate among those in the trial who did not suffer a stroke18. ‘Independence’ was defined as the probability of achieving relief from AS symptoms within a month of a procedure. Given available data, this was estimated as the probability of achieving a total score of 75 on KCCQ17. The KCCQ is a standard patient reported outcome measure used in clinical trials of surgical and transcatheter heart valves19. Estimated mean KCCQ score and variation in this measure were transformed into the proportion of patients achieving a KCCQ total score of 75 at 1 month using procedures previously described in Marchini20.\n\nCI, confidence interval; AS, aortic stenosis.\n\nAn adapted swing weighting (ASW) exercise was administered online to elicit patients’ preferences for treatment attributes21. The objective of the ASW exercise was to identify the level of change in an attribute that patients would be willing to accept in exchange for improving their procedure from ‘invasive’ to ‘minimally invasive’ (see Table 1 for definitions). The ASW exercise consisted of a series of pairwise comparisons of attributes—the ‘invasiveness’ attribute and one other attribute. Participants were shown ‘current’ and ‘improved’ levels on each attribute and asked which improvement they would prefer to make (an example choice question is shown in Figure 1). The ‘current’ levels were chosen to reflect the attribute performance levels of TAVR and SAVR (Table 2), to ensure they had credibility with patients, adjusted to ensure that patients had sufficient range to indicate the change in the attribute that would have the equivalent value as reducing invasiveness. Therefore, the exercises were not designed to directly elicit patients’ willingness to tolerate the actual change observed with TAVR.\n\n‘Type of Procedure’ was used in every pairwise comparison. Only the comparison attribute was varied across different attributes and performance levels.\n\nEach set of pairwise comparisons included five iterations of the choice question. Depending on the answer to the choice question, the level of improvement offered on the non-procedure attribute was updated in the direction that made the value of improvements on the two attributes more similar than in the previous question. The levels used in each of the five iterations, and how these changed depend on previous responses are shown in extended data, Appendix 124.\n\nA pilot of the survey among five AS patients, carried out over a 4-week period in June 2018, ensured acceptable cognitive burden, clarity of the instructions, and ease of use of the elicitation software. Before completing the ASW exercises, participants were introduced to the attributes and their definitions. Participants also completed two ASW practice questions; interpretation of their response to these practice questions was tested to ensure participants understood how to complete each of the pairwise comparisons. Only six participants incorrectly interpreted the meaning of their response to the first practice question, and no participants incorrectly interpreted their response to the second practice question. Each participant completed a proportion of the possible pairwise question—either 3 or 4 sets of pairwise comparison questions. Participants also completed clinical and demographic questions, and health literacy and numeracy questionnaires, available as extended data24.\n\nPotentially eligible participants were recruited from the membership of Heart Value Voice and Mended Hearts patient organizations through e-mails and advertisements on social media platforms. Patients from the American Heart Association membership who had given prior permission to receive mailings were also invited to participate. Potential participants were directed to an online screening tool, where their eligibility for the survey was determined. Participants had to meet specific inclusion/exclusion criteria to participate (Box 1). There were no predefined enrollment targets or stratification by other demographic characteristics. Following completion of the online screening tool, eligible participants were directed to the web survey. They were also sent a link to the web survey, available as extended data24, via e-mail so that they were able to complete the study at a time that was convenient for them.\n\nInclusion criteria\n\nBe at least 19 years of age\n\nHave a self-reported diagnosis of aortic valve disease\n\nAble to read and understand English\n\nWilling and able to complete an online survey\n\nWilling and able to provide (electronic) consent to participate in study\n\nBe a resident of the United States\n\nHave been treated procedurally (TAVR or SAVR) for AS in the past 10 years\n\nor\n\nIf untreated, must have experienced at least one of the following symptoms of AS:\n\n■ Breathlessness/Shortness of breath\n\n■ Tiredness/Lack of energy/Fatigue\n\n■ Chest pain\n\n■ Dizziness or blackouts/Syncope\n\nBe willing and able to participate in a telephone interview, and to be audio-recorded (qualitative pilot only)\n\nExclusion criteria\n\nHave a cognitive impairment, hearing difficulty, visual impairment, acute psychopathology\n\nHave insufficient knowledge of English, which could interfere with the patient’s ability to provide written consent and complete the web survey\n\nAre not experiencing at least one of the symptoms of AS, as described in the inclusion criteria\n\nParticipant responses to the ASW questions identified the level of change in an attribute that patients would be willing to accept in exchange for improving their procedure from ‘invasive’ to ‘minimally invasive.’ For instance, a participant might assign the same value to a 2% reduction in mortality risk as reducing the invasiveness of the procedure. Inverting this relationship, responses we’re used to estimate the maximum acceptable increase in risks (MIR) or the maximum acceptable reduction in benefits (MRB) that participants would be willing to tolerate in exchange for moving from an invasive procedure to a minimally invasive procedure. In the above example, the participant would be willing to tolerate a MIR of 2% in mortality risk to reduce the invasiveness of the procedure.\n\nIn cases of incomplete or missing data, no data imputation was performed, and unanswered questions were coded as missing. Data from participants who completed only a portion of the survey were included in the analyses if they had completed all the questions in at least one ASW exercise.\n\nParticipant preferences were incorporated into the following benefit-risk model constructed in Microsoft Excel.\n\n\n\nWhere\n\nU(x) is the overall value generated by procedure x.\n\nwi is the weight associated with attribute i,\n\nvi is the partial value function for attribute i, which was assumed to be linear\n\nxi is the performance of performance x on attribute i\n\nParticipants’ MIR/MRB was converted into an attribute weight (wi) by setting the weight for ‘type of procedure’ to 1, and then dividing the range in performance between TAVR and SAVR on that attribute (Table 2) by the MIR/MRB. For instance, if patients were willing to tolerate a 2% increase in mortality risk in exchange for a reduction in invasiveness, and reducing invasiveness is given a weight of 1, then the 4.8% change in mortality risk covered by the benefit-risk model (0.5%–5.3% range identified in Table 2) would be given a weight of 1.4 (4.8%/2%). Weights across all attributes in the model were then normalized to sum to 100.\n\nFour outputs were generated from the benefit-risk model to evaluate patients’ preferences for TAVR and SAVR. First, the incremental overall value generated by TAVR:\n\n\n\nSecond, the incremental partial value on each attribute generated by TAVR:\n\n\n\nWhere\n\nxTAVR is the performance of TAVR on attribute i\n\nxSAVR is the performance of SAVR on attribute i\n\nThird, a threshold analysis was undertaken, estimating the level of performance (minimum acceptable benefit or maximum acceptable risk) on each attribute that would leave patients indifferent between TAVR and SAVR. More specifically:\n\n\n\nWhere\n\nMARa or MABa is the maximum acceptable risk or minimum acceptable benefit of attribute a\n\nTAVRa is the performance of TAVR on attribute a (see Table 2).\n\nx1 unit a is a single unit of performance on attribute a\n\nTable 3 outlines the definitions of MIR, MRB, MAR, and MAB.\n\nTAVR, transcatheter aortic valve replacement; SAVR, surgical aortic valve replacement.\n\nFourth, a Monte Carlo Simulation (MCS) was run to explore uncertainty in model inputs. That is, the benefit-risk model was run 10,000 times for both TAVR and SAVR. In each instance, the model drew from the distribution around both performance and weight inputs. Specifically, performance inputs were drawn from the distribution around TAVR and SAVR performance data (Table 2). Weight inputs were drawn in a manner that reflected the probability that participants identified different MIR/MRBs in their responses to the survey. For each iteration of the MCS, TAVR and SAVR, we’ve ranked based on U (Equation 2) and the proportion of instances that TAVR ranked first was estimated.\n\nIn accordance with ethical practice, Institutional Review Board (IRB) approval was obtained through Advarra (approval MOD00300398) to comply with human participants research requirements prior to initiation of participant recruitment or administration of measures in the pilot or main studies. Informed consent was recorded electronically via the online survey platform. Any change to the protocol and/or informed consent form was resubmitted to the IRB for review and approval prior to implementation. The study was available for monitoring, auditing, IRB review, and regulatory inspection as applicable.\n\n\nResults\n\nA total of 93 patients completed the survey (Table 4). Raw data are available from Open Science Framework24. The majority of patients were 60 years old or older (n=69, 74.3%). More than half of the respondents were female (n=53, 57.0%) and almost all participants were white (n= 87, 93.5%). Over half of the sample was retired (n=57, 61.3%) and completed a college degree or higher (n=61, 65.6%). Most of the participants lived with a partner/spouse, family, or friends (n =74, 79.6%). No participants reported experiencing severe limitations to their physical activity. Very few patients demonstrated low health literacy (n=3, 3.2%) or numeracy (n=6, 6.5%), and no patients were low on both scales.\n\n1 Responses were scored between 0 (always) and 4 (never). Each participants’ scored responses were averaged for a composite score ranging from 0–4. A low score if ≤2.\n\n2 Participants were given one point for each correctly answered question (maximum numeracy score = 5). A low score if given is ≤2 answered incorrectly. ³Overall low: individuals who scored low on both educational level and objective health literacy.\n\nNYHA, New York Heart Association.\n\nWhen responding to the ASW questions, only a small proportion (10%) of participants ‘straight-lined’ on all questions—consistently choosing to improve either ‘procedure’ or the comparison attribute across all five iterations of the choice question. These responses may be a valid reflection of participants’ preference—suggesting a strong preference either for avoiding an invasive procedure, or a strong preference to prioritize improving other procedure attributes. Thus, these responses were included in the analysis. The impact of excluding these data were tested, and it was determined that results of the BRA were not sensitive to whether these data were included or excluded.\n\nTable 5 shows the difference in performance of TAVR compared with SAVR on each attribute, and patients’ willingness to accept this difference in exchange for the lesser invasiveness of TAVR. The increase in risks or the reduction in benefits that patients are, on average, willing to accept in exchange for reducing procedure invasiveness is reported in the middle three columns. For instance, patients would be willing to tolerate a 6.55% increase in the probability of experiencing disabling, non-fatal stroke in exchange for the reduction in invasiveness associated with receiving TAVR instead of SAVR. In each case, patents were on average willing to accept TAVR’s performance on any attribute in exchange for its lower invasiveness. In the case of four attributes (mortality, disabling non-fatal stroke, independence, and dialysis), TAVR performs better than SAVR. Where SAVR performs better than TAVR (the need for new permanent pacemaker and time over which the procedure has been proven to work), patients would, on average, be willing to accept TAVR’s performance given its lower invasiveness. For example, participants are willing to tolerate a 7.6% increase in the risk of a new permanent pacemaker, while the probability of needing a new permanent pacemaker only increases by 3.3% with TAVR.\n\n†See Table 2 for source of data\n\n* p<0.1\n\nThe standard errors in patients’ MIR/MRB suggests a substantial heterogeneity in patients’ responses to the ASW exercise (see extended data, Appendix S224 for more detail). Some of this heterogeneity was associated with participants’ age. MIR/MRB for three attributes—probability of having a new permanent pacemaker, probability of requiring dialysis, and period over which a procedure had been proven to work—were associated with whether patients are over or under 60 years old. Older patients were more willing to tolerate increases in risks/reductions in benefit to avoid having to undergo an invasive procedure. Younger patients (<60 years old) would, on average, still be willing to accept the increased risk of needing a new permanent pacemaker associated with TAVR, but had a tolerance for a reduction in the period over which a procedure had been proven to work of less than the 10 years. That is, everything else being equal, younger patients (<60 years old) may prefer to undergo a more invasive procedure to ensure the procedure had had been proven to work for 10 years or longer.\n\nThere were a large proportion of participants whose individual MIR/MRB was greater than the change in attribute performance achieved with TAVR (Table 5). For attributes where performance is better with TAVR compared to SAVR (mortality, disabling non-fatal stroke, independence, and dialysis), 100% of patients would prefer the improved performance and reduced invasiveness of TAVR. For the two attributes on which attribute performance is better with SAVR compared to TAVR, 61% of patients would be willing to accept the increased risk of needing a new permanent pacemaker, and 38% of participants would be willing to accept the shorter period for which TAVR had been proven to work in order to experience TAVR’s reduced invasiveness.\n\nThe above analysis compares the performance of TAVR on each attribute separately. A complete comparison of TAVR and SAVR should do so across all attributes simultaneously and take into account observed heterogeneity (in this case across age groups). This objective is accomplished by means of the benefit-risk model (see Equation 2 and Equation 3). Figure 2 and Figure 3 show the incremental value of TAVR (overall and by attribute) observed among patients 60 years old or older (Figure 2) and among patients less than 60 years old (Figure 3). These figures reveal that, overall, TAVR is of greater value to patients than SAVR. Specifically, patients placed greater value on TAVR based on a lower short-term mortality rate, reduced procedural invasiveness, and a quicker time to return to normal quality of life (independence) offsetting the value patients placed on longer period over which the procedure has been proven to work and reduced risk of needing a pacemaker generated by SAVR. Similar patterns were observed among younger and older patients, though younger patients place a lower value on reducing the invasiveness of their procedure, the speed with which they return to a normal quality of life, and a higher value on the period over which the procedure has been proven to work compared to older patients.\n\nTable 6 reports a threshold analysis, which shows the minimum amount of benefit that patients would accept before preferring TAVR, or the maximum amount of risk that patients would tolerate before preferring TAVR. For example, given the incremental value that patients attach to TAVR (as reflected in Figure 2 and Figure 3) they would be willing to tolerate a mortality risk of 12.4% following TAVR before they would be indifferent between TAVR and SAVR.\n\nThe MCS shows that 75.1% of patients would prefer TAVR over SAVR. When the analysis is run separately for patients less than and greater than 60 years old, the proportions of patients preferring TAVR are 78.3% and 73.9% respectively. Removing patients who ‘one-lined’ in response to ASW exercises does not impact the results of the MCS, with 75.3% of patients still preferring TAVR.\n\n\nDiscussion\n\nThe choice between TAVR and SAVR for the treatment of AS involves making trade-offs between procedure invasiveness; the period over which the procedure has been proven to be effective; mortality, stroke and independence outcomes; and risks such as the need for a new pacemaker or dialysis. This study elicited patients’ preferences for AS procedure attributes to determine how they make these trade-offs, and thus whether they prefer TAVR or SAVR. Results suggest that, given the potential benefits and risks of TAVR and SAVR, on average, patients attach more value to TAVR, and the majority of patients would prefer TAVR. Patients placed a greater value on the lower invasiveness, quicker speed of recovery, and reduced risk of mortality, stroke and need for dialysis associated with TAVR than they did on longer period over which the procedure has been proven to work and reduced risk of needing a pace maker associated with SAVR.\n\nCurrent guidelines from the American Heart Association for the procedural treatment of AS do not take into account recent clinical data supporting the use of TAVR25. Based on the recent clinical results of TAVR and the findings of this study, regulators may reach different conclusions about the need to protect patients from risks historically associated with TAVR. For instance, TAVR may not result in the increased risk of stroke that regulators might expect it to, and patients may be willing to tolerate the greater need for a permanent pacemaker in order to experience the benefits of TAVR.\n\nThe BRA revealed substantial heterogeneity in patient preferences for AS treatment. Some preference heterogeneity is explained by patient age, with older patients being less willing to tolerate the invasiveness of SAVR, instead preferring to accept greater potential risks associated with other procedure attributes in order to reduce the invasiveness. However, preference heterogeneity raises concerns about the attendance of participants to the preference elicitation tasks. A small proportion (10%) of participants straight-lined on all questions. While this might indicate a lack of attendance, it may also be capture strong preferences for/against the invasiveness of SAVR. Furthermore, all participants interpreted their response to the practice questions correctly, and a very small proportion of respondents demonstrated low health literacy or numeracy. This provides some reassurance that the preference heterogeneity observed in this study reflects a genuine difference in preference, rather than being the result of patients failing to complete the survey in a meaningful way.\n\nTwo other studies that used ASW to elicit patient preferences have been published26,27. Both studies also observed substantial preference heterogeneity. One of these studies27 provided evidence supporting the validity and reliability of the preference outputs, both by replicating the results of the ASW with a thresholding exercise, and by comparing participants’ responses with their qualitative statements on the basis for their answers. This provides some reassurance about the validity of responses to the ASW exercise used for the current study. This may suggest that methods such as ASW, which elicit individual-level patient preferences, capture more preference heterogeneity than population-level methods, such as discrete choice experiments. Further work could usefully continue to validate the results of ASW exercises and test the hypothesis that individual-level preferences method captures greater heterogeneity in patient preferences.\n\nOnly one other study of AS patients’ treatment preferences has been published to date28. The study design was sufficiently different to the current study—focusing on patients’ willingness to accept the mortality risk associated with interventions—that it is not possible to directly compare the results. However, the study by Hussain et al.28 did reveal a higher risk tolerance among patients with greater disease burden (defined as weekly incidence of restricting symptoms, perceived change in health compared with 1 year earlier, EQ-VAS scores, and the New York Heart Association (NYHA) classification). Our study failed to identify an association between patient preferences and NYHA classification, though this might be due to the limited sample size and the small proportion of the sample in the more severe stages of the NYHA classification. Further research could usefully gather data from a larger sample of AS patients to determine if NYHA classification is associated with preferences.\n\nWhile a majority of patients in the current study preferred TAVR, a number of patients (around 25%) preferred SAVR. This, and the underlying heterogeneity in patient preferences, support the need for a shared decision-making (SDM) tool that will help patients and surgeons choose procedures based on both clinical indications and patient risk tolerance. The Patient-Centered Outcomes Research Institute (PCORI) has developed a SDM tool to support patients choose between SAVR and TAVR15. However, this tool includes a narrower range of treatment attributes—stroke risk, mortality risk and discharge home—than those included in the analysis reported in this study. Furthermore, the tool does not include a component to elicit a patient’s preferences.\n\nFinally, the current study is based on a relatively small sample of patients, and the sample is healthier and younger than the population currently eligible for TAVR and SAVR29. Further AS patient preference research should replicate this study in a larger sample of patients, including more patients with more severe disease burden.\n\n\nConclusions\n\nMost AS patients are willing to tolerate sizable increases in clinical risk in exchange for the benefits associated with TAVR. A BRA incorporating data from patients’ preferences for the attributes of AS treatments revealed a strong preference for TAVR compared to SAVR. The analysis also revealed substantial heterogeneity in individual patient preferences, partly associated with patient age. Further work is required to understand this heterogeneity, and whether additional patient characteristics such as NYHA class are associated with different preferences. In the meantime, SDM tools should incorporate the factors identified in this model to assist patients and clinicians in achieving a more patient-centered treatment decision.\n\n\nData availability\n\nOpen Science Framework: AS patient preference data. https://doi.org/10.17605/OSF.IO/RTDS624.\n\nThis project contains the following underlying data:\n\nTAVR Manuscript_Full Dataset.csv (full dataset of patient responses).\n\nTAVR Manuscript Datamap.xlsx (codebook for the dataset).\n\nOpen Science Framework: AS patient preference data. https://doi.org/10.17605/OSF.IO/RTDS624.\n\nThis project contains the following extended data:\n\nTAVR Manuscript Appendices S1_S2.docx (contains S1 Appendix, including S1 Table 1 and legends for S1 Figures 1-6; and S2 Appendix, including legends for S2 Figures 1-6).\n\nTAVR Manuscript Figures.docx (Figure 1–Figure 3, S1 Figures 1-6 and S2 Figures 1-6).\n\nTAVR Survey Contents.docx (a copy of the questionnaire given to each participant).\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\nFunding for this project was provided by Edwards Lifesciences, Washington D.C., US.\n\n\nAcknowledgements\n\nThe authors would like to thank Heart Value Voice and Mended Hearts for their support with the design of the survey, and their support recruiting patients.\n\n\nReferences\n\nPerera S, Wijesinghe N, Ly E, et al.: Outcomes of patients with untreated severe aortic stenosis in real-world practice. N Z Med J. 2011; 124(1345): 40–8. PubMed Abstract\n\nBach DS, Radeva JI, Birnbaum HG, et al.: Prevalence, referral patterns, testing, and surgery in aortic valve disease: leaving women and elderly patients behind? J Heart Valve Dis. 2007; 16(4): 362–9. PubMed Abstract\n\nUS Census Bureau: Updated statistic from 2015- (Population Division, June 2015). 2015.\n\nNkomo VT, Gardin JM, Skelton TN, et al.: Burden of valvular heart diseases: a population-based study. Lancet. 2006; 368(9540): 1005–11. PubMed Abstract \| Publisher Full Text\n\nThaden JJ, Nkomo VT, Enriquez-Sarano M: The global burden of aortic stenosis. Prog Cardiovasc Dis. 2014; 56(6): 565–71. PubMed Abstract \| Publisher Full Text\n\nBach DS: Prevalence and characteristics of unoperated patients with severe aortic stenosis. J Heart Valve Dis. 2011; 20(3): 284–91. PubMed Abstract\n\nRoss J Jr, Braunwald E: Aortic stenosis. Circulation. 1968; 38(1 Suppl): 61–7. PubMed Abstract \| Publisher Full Text\n\nBaron SJ: Valve Disease. 2017. Reference Source\n\nLeon MB, Smith CR, Mack M, et al.: Transcatheter aortic-valve implantation for aortic stenosis in patients who cannot undergo surgery. N Engl J Med. 2010; 363(17): 1597–607. PubMed Abstract \| Publisher Full Text\n\nBaron SJ, Arnold SV, Wang K, et al.: Health Status Benefits of Transcatheter vs Surgical Aortic Valve Replacement in Patients With Severe Aortic Stenosis at Intermediate Surgical Risk: Results From the PARTNER 2 Randomized Clinical Trial. JAMA Cardiol. 2017; 2(8): 837–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGargiulo G, Sannino A, Capodanno D, et al.: Transcatheter Aortic Valve Implantation Versus Surgical Aortic Valve Replacement: A Systematic Review and Meta-analysis. Ann Intern Med. 2016; 165(5): 334–44. PubMed Abstract \| Publisher Full Text\n\nSiontis GC, Praz F, Pilgrim T, et al.: Transcatheter aortic valve implantation vs. surgical aortic valve replacement for treatment of severe aortic stenosis: a meta-analysis of randomized trials. Eur Heart J. 2016; 37(47): 3503–12. PubMed Abstract \| Publisher Full Text\n\nYu PJ, Mattia A, Cassiere HA, et al.: Should high risk patients with concomitant severe aortic stenosis and mitral valve disease undergo double valve surgery in the TAVR era? J Cardiothorac Surg. 2017; 12(1): 123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiemieniuk RA, Agoritsas T, Manja V, et al.: Transcatheter versus surgical aortic valve replacement in patients with severe aortic stenosis at low and intermediate risk: systematic review and meta-analysis. BMJ. 2016; 354: i5130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatient-Centered Outcomes Research Institute (PCORI): Optimizing Health Outcomes in Patients with Symptomatic Aortic Valve Disease. Reference Source\n\nMarsh K, IJzerman M, Thokala P, et al.: Multiple Criteria Decision Analysis for Health Care Decision Making--Emerging Good Practices: Report 2 of the ISPOR MCDA Emerging Good Practices Task Force. Value Health. 2016; 19(2): 125–37. PubMed Abstract \| Publisher Full Text\n\nMishra RK, Yang W, Roy J, et al.: Kansas City Cardiomyopathy Questionnaire Score Is Associated With Incident Heart Failure Hospitalization in Patients With Chronic Kidney Disease Without Previously Diagnosed Heart Failure: Chronic Renal Insufficiency Cohort Study. Circ Heart Fail. 2015; 8(4): 702–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKapadia S, Agarwal S, Miller DC, et al.: Insights Into Timing, Risk Factors, and Outcomes of Stroke and Transient Ischemic Attack After Transcatheter Aortic Valve Replacement in the PARTNER Trial (Placement of Aortic Transcatheter Valves). Circ Cardiovasc Interv. 2016; 9(9): pii: e002981. PubMed Abstract \| Publisher Full Text\n\nGreen CP, Porter CB, Bresnahan DR, et al.: Development and evaluation of the Kansas City Cardiomyopathy Questionnaire: a new health status measure for heart failure. J Am Coll Cardiol. 2000; 35(5): 1245–55. PubMed Abstract \| Publisher Full Text\n\nMarchini J: Lecture 6: The Normal Distribution: Department of Statistics. University of Oxford. Reference Source\n\nPostmus D, Mavris M, Hillege HL, et al.: Incorporating patient preferences into drug development and regulatory decision making: Results from a quantitative pilot study with cancer patients, carers, and regulators. Clin Pharmacol Ther. 2016; 99(5): 548–54. PubMed Abstract \| Publisher Full Text\n\nThourani VH, Kodali S, Makkar RR, et al.: Transcatheter aortic valve replacement versus surgical valve replacement in intermediate-risk patients: a propensity score analysis. Lancet. 2016; 387(10034): 2218–25. PubMed Abstract \| Publisher Full Text\n\nLeon MB, Smith CR, Mack MJ, et al.: Transcatheter or Surgical Aortic-Valve Replacement in Intermediate-Risk Patients. N Engl J Med. 2016; 374(17): 1609–20. PubMed Abstract \| Publisher Full Text\n\nMarsh K: AS patient preference data. 2019. http://www.doi.org/10.17605/OSF.IO/RTDS6\n\nNishimura RA, Otto CM, Bonow RO, et al.: 2017 AHA/ACC Focused Update of the 2014 AHA/ACC Guideline for the Management of Patients With Valvular Heart Disease: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation. 2017; 135(25): e1159–e95. PubMed Abstract \| Publisher Full Text\n\nPostmus D, Richard S, Bere N, et al.: Individual Trade-Offs Between Possible Benefits and Risks of Cancer Treatments: Results from a Stated Preference Study with Patients with Multiple Myeloma. Oncologist. 2018; 23(1): 44–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSri Bhashyam S, Quartel A, Gershman A, et al.: editors. PSY202 - The occupational hazards of measuring risk tolerance: Convergent validity in preference elicitation. ISPOR European Conference; Barcelona, Spain. Value Health. 2018; 21(Supplement 3): S470. Publisher Full Text\n\nHussain AI, Garratt AM, Beitnes JO, et al.: Validity of standard gamble utilities in patients referred for aortic valve replacement. Qual Life Res. 2016; 25(7): 1703–12. PubMed Abstract \| Publisher Full Text\n\nOtto CM, Kumbhani DJ, Alexander KP, et al.: 2017 ACC Expert Consensus Decision Pathway for Transcatheter Aortic Valve Replacement in the Management of Adults With Aortic Stenosis: A Report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents. J Am Coll Cardiol. 2017; 69(10): 1313–46. PubMed Abstract \| Publisher Full Text" }	[ { "id": "46833", "date": "25 Apr 2019", "name": "Ross Jaffe", "expertise": [ "Reviewer Expertise Internal medicine physician with prior experience in outcomes research and shared decision making. Not actively involved in research in my present role as a venture capitalist", "but I led the MDIC Patient Centered Benefit-Risk Project that developed a framework for incorporating patient preferences into FDA medical device regulation and developed the first catalog of patient preference methodologies included as an appendix to that report." ], "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 by Marsh et al assesses patient preferences regarding the important medical decision about whether to undergo traditional surgical aortic valve replacement (SAVR) versus the newer transcatheter aortic valve replacement (TAVR) for aortic stenosis. This is an important effort to understand what attributes of benefit and risk from aortic valve replacement are most important to patients and understand how patients tradeoff those attributes in deciding which therapy to choose. With shared decision-making becoming increasingly used as our US healthcare system evolves to be more patient-centric, studies such as this one are important to expanding our understanding of how best to provide the information needed by patients to make informed decisions about their care. This study confirms a belief that many clinicians have that most patients prefer TAVR to SAVR because it is less invasive, providing evidence that about 75% patients prefer the less invasive approach. These patients are willing to tolerate a somewhat higher risk of stroke, pacemaker placement, and dialysis, and less evidence of long-term duration for the benefits of less invasiveness. It also identifies that younger patients (<60 yo) perspectives may differ from those of older patients (>60 yo) somewhat, although both groups generally prefer the less invasive procedure.\n\nI support the publication of this study. While this study is not perfect from my point of view, it is an important contribution to the literature, both about how to treat aortic stenosis and about patient preference assessment more generally. Understanding what treatment attributes are most important to patients and how patients trade off benefits and risks is important to understanding how best to inform patients about their treatment options and help them make decisions that best reflect their individual preferences. As the paper notes on p. 11, there has only been one other, more limited study of patient preferences in aortic stenosis, so this study is an important expansion of our understanding of patient preferences that clinical area.\n\nAdditionally, from a methodological perspective and as also noted in the paper, there are only a few other studies that use adapted swing weights (ASW) to assess patient preferences. Swing weighting is one of only fourteen methods identified in the Medical Device Innovation Consortium (MDIC) review of preference assessment methodologies. (See: MDIC Patient Centered Benefit Risk Project Report, Appendix A: Catalog of Methods for Assessing Patient Preferences for Benefits and Harms of Medical Technologies, May 2015, available at: https://mdic.org/resource/patient-centered-benefit-risk-pcbr-framework/). This study is an important contribution to the literature about swing weighting methods, and should allow comparison to studies of patient preference using other methods to help researchers and clinicians better understand how best to assess patient preferences.\n\nThis study does have a few issues that should be highlighted to help put the results in context. I also note an area for further assessment of the data as well as areas for future research focus. Please note that I come at this study as someone with an interest in patient preferences from clinical and regulatory policy perspective, and do not have the expertise to comment on the specific methodology or statistical analysis, which I will leave to experts in those areas.\n\nMajor concerns: 1. The mixing of treated and untreated patients in the participant population: In Box 1 on p. 6, the inclusion criteria describe that patients in the study could have already had a procedure within the last 10 years or could be untreated. From a shared decision making point of view, patients express their preferences prior to treatment – so it is most important to understand the benefit-risk attributes that are most important to patients not yet treated and how such patients trade off such risks. The mixing of both untreated and treated patients may make it hard to understand how the pre-procedure patients view these issues.\n\nPrior treatment could significantly influence a patient’s preference for one treatment option or another, but it his hard to know a priori how treatment would influence a patients preferences. Prior treatment may introduce a confirmation bias that patients tend to prefer the procedure that they chose to have, and therefore require much greater benefit or much less risk from the alternative procedure compared to treatment naïve patients. Alternatively, if patients had a negative experience with their prior procedure, they may find the benefit/risk profile of the alternative procedure much more attractive than that of the procedure that they had. Additionally, their experience of specific benefits or risks from their procedure may skew their weighting of those specific attributes compared to other patients.\n\nFor this study, it would be important in Table 4 to add a breakdown of the patient treatment history, specifically the number and percentage of patients that have had SAVR, TAVR, or were untreated.\n\nAdditionally, it would be helpful to add a comparison of the preferences of patients in each of these categories to show how prior treatment affects the MIR/MRB for each attribute, perhaps in a table similar to Table 5 except substituting treatment category for age. One concern is that with a sample size of 93 patients, sub-categorization by treatment status may result in too few patients in any one category to have confidence in the results. If this is a problem, it should be acknowledged that future studies may be needed to better understand the effect of prior treatment on preferences in aortic valve replacement.\n\n2. Representativeness of patients involved in member organizations: The study recruited participants from two member organizations (Heart Value Voice and Mended Hearts). While it is understandable why the membership of these organizations facilitated identifying patients with aortic valve disease. However, patients who are motivated enough to join such organizations may have different preferences than the broader population of patients with the disease eligible for treatment of their aortic valve disease. There is no way to assess this potential difference in this study, but the authors could acknowledge this potential source of bias in the sample population in their discussion of the results and encourage future study in one or more different aortic valve disease populations.\n\n3. Lack of clarity in the attribute of “Time over which the procedure has been proven to work”: From the definition of this attribute in the paper, it is difficult to know whether patients interpreted this attribute as a measure of how long they could expect benefit, i.e, duration of effect, or whether patients also viewed this as how much clinical experience there was with a treatment, i.e., uncertainty in the knowledge about the effect. (See section 2 of the MDIC Patient Centered Benefit Risk Report for a nice discussion of uncertainty and how it relates to patient preferences.). This ambiguity raises the question about whether this attribute as described elicited preferences about expected duration, or elicited preferences about patient tolerance of uncertainty about the effect of TAVR, or some combination of the two. Showing an example of how this “proven to work” attribute was shown to patients akin to Figure 1 might clarify this ambiguity. Future studies might try to separate these attributes, particularly comparing an established therapy like SAVR with a newer treatment like TAVR.\n\nMinor issues and typos:\nIntroduction, last paragraph of left column, p. 3: the beginning of the second sentence is awkward: “However, little is known about the weights that patients assign to which attributes, . . . . “ I would suggest “However, little is known about the weights that patients assign to each attribute. . . .” Methods, Attribute selection, last sentence (p.3): note “was were”. I would remove the were. Table 1, p. 4: Disabling non-fatal stroke: in the description, first sentence, note the “one 1” – should be “one month”. Table 2, p. 4, description of “independence” attribute: the description of the measure of this attribute does not mention that this is measured at one month, whereas all the other measures note the time frame of the measure.\n\nAnalysis, p. 6, top right column, first paragraph: there looks to be a calculation error in the next to the last sentence. 4.8%/2% = 2.4, not 1.4 as written. Comparisons of TAVR and SAVR, p. 7: right column, top paragraph – “In each case, patents. . . . ” should be “In each case, patients. . . .” Figures 2 and 3, p. 9: Each x-axis should be labeled with what the unit of measurement is. This will help the figures be more self-explanatory.\n\nI hope that these comments are helpful. Given my interest in the use of patient preferences in the FDA regulatory process and increasing shared decision making in medicine broadly, I am pleased that the authors undertook this study to better understand patient preferences in aortic stenosis treatment, and I support its publication. It should be a nice addition to both the aortic stenosis treatment literature and the patient preference 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?\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": "4632", "date": "14 May 2019", "name": "Kevin Marsh", "role": "Author Response", "response": "Review 1: Ross JaffeThis article by Marsh et al assesses patient preferences regarding the important medical decision about whether to undergo traditional surgical aortic valve replacement (SAVR) versus the newer transcatheter aortic valve replacement (TAVR) for aortic stenosis. This is an important effort to understand what attributes of benefit and risk from aortic valve replacement are most important to patients and understand how patients tradeoff those attributes in deciding which therapy to choose. With shared decision-making becoming increasingly used as our US healthcare system evolves to be more patient-centric, studies such as this one are important to expanding our understanding of how best to provide the information needed by patients to make informed decisions about their care. This study confirms a belief that many clinicians have that most patients prefer TAVR to SAVR because it is less invasive, providing evidence that about 75% patients prefer the less invasive approach. These patients are willing to tolerate a somewhat higher risk of stroke, pacemaker placement, and dialysis, and less evidence of long-term duration for the benefits of less invasiveness. It also identifies that younger patients (<60 yo) perspectives may differ from those of older patients (>60 yo) somewhat, although both groups generally prefer the less invasive procedure. I support the publication of this study. While this study is not perfect from my point of view, it is an important contribution to the literature, both about how to treat aortic stenosis and about patient preference assessment more generally. Understanding what treatment attributes are most important to patients and how patients trade off benefits and risks is important to understanding how best to inform patients about their treatment options and help them make decisions that best reflect their individual preferences. As the paper notes on p. 11, there has only been one other, more limited study of patient preferences in aortic stenosis, so this study is an important expansion of our understanding of patient preferences that clinical area. Additionally, from a methodological perspective and as also noted in the paper, there are only a few other studies that use adapted swing weights (ASW) to assess patient preferences. Swing weighting is one of only fourteen methods identified in the Medical Device Innovation Consortium (MDIC) review of preference assessment methodologies. (See: MDIC Patient Centered Benefit Risk Project Report, Appendix A: Catalog of Methods for Assessing Patient Preferences for Benefits and Harms of Medical Technologies, May 2015, available at: https://mdic.org/resource/patient-centered-benefit-risk-pcbr-framework/). This study is an important contribution to the literature about swing weighting methods, and should allow comparison to studies of patient preference using other methods to help researchers and clinicians better understand how best to assess patient preferences. Response: We thank the reviewer for their interest in our studyThis study does have a few issues that should be highlighted to help put the results in context. I also note an area for further assessment of the data as well as areas for future research focus. Please note that I come at this study as someone with an interest in patient preferences from clinical and regulatory policy perspective, and do not have the expertise to comment on the specific methodology or statistical analysis, which I will leave to experts in those areas. Major concerns:1. The mixing of treated and untreated patients in the participant population: In Box 1 on p. 6, the inclusion criteria describe that patients in the study could have already had a procedure within the last 10 years or could be untreated. From a shared decision making point of view, patients express their preferences prior to treatment – so it is most important to understand the benefit-risk attributes that are most important to patients not yet treated and how such patients trade off such risks. The mixing of both untreated and treated patients may make it hard to understand how the pre-procedure patients view these issues. Prior treatment could significantly influence a patient’s preference for one treatment option or another, but it his hard to know a priori how treatment would influence a patients preferences. Prior treatment may introduce a confirmation bias that patients tend to prefer the procedure that they chose to have, and therefore require much greater benefit or much less risk from the alternative procedure compared to treatment naïve patients. Alternatively, if patients had a negative experience with their prior procedure, they may find the benefit/risk profile of the alternative procedure much more attractive than that of the procedure that they had. Additionally, their experience of specific benefits or risks from their procedure may skew their weighting of those specific attributes compared to other patients. For this study, it would be important in Table 4 to add a breakdown of the patient treatment history, specifically the number and percentage of patients that have had SAVR, TAVR, or were untreated. Additionally, it would be helpful to add a comparison of the preferences of patients in each of these categories to show how prior treatment affects the MIR/MRB for each attribute, perhaps in a table similar to Table 5 except substituting treatment category for age. One concern is that with a sample size of 93 patients, sub-categorization by treatment status may result in too few patients in any one category to have confidence in the results. If this is a problem, it should be acknowledged that future studies may be needed to better understand the effect of prior treatment on preferences in aortic valve replacement. Response: This is an important observation. We have updated Table 4 as suggested. We have not replicated Table 5, but have instead added the following note that no difference in preferences were observed between participants who had / hadn’t been treated previously for their AS.“No other correlation was found between participant characteristics and MIR/MRB. This includes whether a participant reported having previously undergone treatment for their AS. While this might be expected to influence preferences, the ability of the analysis to identify this influence is limited by the small sample size and the relatively small proportion of participants who reported not having previously received AS treatment (11.8%).”The discussion section has also been updated to include the following statement: “Further research could usefully gather data from a larger sample of AS patients to determine the association of preferences and patient characteristics, such as NYHA classification or whether patients have previously undergone treatment for AS.” 2. Representativeness of patients involved in member organizations: The study recruited participants from two member organizations (Heart Valve Voice and Mended Hearts). While it is understandable why the membership of these organizations facilitated identifying patients with aortic valve disease. However, patients who are motivated enough to join such organizations may have different preferences than the broader population of patients with the disease eligible for treatment of their aortic valve disease. There is no way to assess this potential difference in this study, but the authors could acknowledge this potential source of bias in the sample population in their discussion of the results and encourage future study in one or more different aortic valve disease populations. Response: We agree that the recruitment strategy may be a source of bias in the preference data. We have acknowledged this in the discussion, which now includes the statement: “Finally, the current study is based on a relatively small sample of patients, and the sample is healthier and younger than the population currently eligible for TAVR and SAVR. AS patients were recruited from the membership of Heart Valve Voice and Mended Hearts, and it is possible that patients who are motivated to join these organizations may have different preferences than the broader population. Further AS patient preference research should replicate this study in a larger sample of patients, including more patients with more severe disease burden”.3. Lack of clarity in the attribute of “Time over which the procedure has been proven to work”: From the definition of this attribute in the paper, it is difficult to know whether patients interpreted this attribute as a measure of how long they could expect benefit, i.e, duration of effect, or whether patients also viewed this as how much clinical experience there was with a treatment, i.e., uncertainty in the knowledge about the effect. (See section 2 of the MDIC Patient Centered Benefit Risk Report for a nice discussion of uncertainty and how it relates to patient preferences.). This ambiguity raises the question about whether this attribute as described elicited preferences about expected duration, or elicited preferences about patient tolerance of uncertainty about the effect of TAVR, or some combination of the two. Showing an example of how this “proven to work” attribute was shown to patients akin to Figure 1 might clarify this ambiguity. Future studies might try to separate these attributes, particularly comparing an established therapy like SAVR with a newer treatment like TAVR. Response: We have updated Figure 1 to use an example of a survey question that includes the ‘time proven to work’ attribute.Minor issues and typos: Introduction, last paragraph of left column, p. 3: the beginning of the second sentence is awkward: “However, little is known about the weights that patients assign to which attributes, . . . . “ I would suggest “However, little is known about the weights that patients assign to each attribute. . . .” Response: Updated as suggested Methods, Attribute selection, last sentence (p.3): note “was were”. I would remove the were. Updated as suggested Table 1, p. 4: Disabling non-fatal stroke: in the description, first sentence, note the “one 1” – should be “one month”. Response: Updated as suggested Table 2, p. 4, description of “independence” attribute: the description of the measure of this attribute does not mention that this is measured at one month, whereas all the other measures note the time frame of the measure. Response: The current description includes a timeframe. No update made Analysis, p. 6, top right column, first paragraph: there looks to be a calculation error in the next to the last sentence. 4.8%/2% = 2.4, not 1.4 as written. Response: Tank you for noticing this. Updated. Comparisons of TAVR and SAVR, p. 7: right column, top paragraph – “In each case, patents. . . . ” should be “In each case, patients. . . .” Response: Updated Figures 2 and 3, p. 9: Each x-axis should be labeled with what the unit of measurement is. This will help the figures be more self-explanatory. Response: Labels have been added to the Figures." } ] }, { "id": "46834", "date": "07 May 2019", "name": "Megan Coylewright", "expertise": [ "Reviewer Expertise shared decision making" ], "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 co-authors have an ambitious agenda to identify patient preferences regarding the differing attributes for the treatment of aortic stenosis. They are to be commended for tackling an important topic, and employing known methodologic standards for addressing the question.\n\nThey deploy a methodology of adapted swing weighting (ASW), identifying which treatment attributes patients would tolerate to move from an invasive (surgical aortic valve replacement, or SAVR) option to a minimally invasive (transcatheter aortic valve replacement, or TAVR) option. A set of pairwise comparisons were made between 'less invasive\" and other features of AVR, with 5 different iterations presented.\n\nThe study is written by employees of one of the TAVR companies (Edwards LifeSciences), as well as employees of a company that specializes in patient preference work, paid for by Edwards. It is not apparent that there was engagement of other stakeholders in the study design, such as patients, families, clinicians, administrators, policy makers, etc. There were 5 patients with the disease condition that trialed the exercises before the start of the study to ensure understandability. There is no information how these patients were recruited, or what changes were made in response to their feedback.\n\nThe authors’ conclusions are that patients are willing to tolerate big increases in risk and exchange for benefits of a minimally invasive procedure. Importance of the research question: Given the recent reconsideration of the TAVR national coverage determination, data as needed to inform interventions designed to increase the degree to which patient preferences are included in final decision making. This study suggests that there is heterogeneity in patient preferences and that patients may tolerate larger differences in risk than physicians and regulators traditionally have deemed acceptable when faced with a minimally invasive procedure. Originality of the work: There is not a large amount of data on the preferences of patients for TAVR.\n\nStrengths: Identifying patient preferences and how they influence decision-making is an important topic. Pair-wise comparisons can be helpful to delineate tolerance of risk in one domain versus another. In the decision aid that was presented to the patient's demonstrating differences between the 2 therapies, the same picture was used for both surgery and transcatheter procedure which did potentially reduce bias.\nWeaknesses: The largest weakness is the fact that the attributes were not selected by patients known to be affected by the disease condition; a survey was asked of participants in advocacy groups online. The eligible patients were created from the membership of heart valve voice and mended hearts. They were directed to an online screening tool where patient reported disease conditions were recorded. The study sample was largely white and highly educated. No patients had severe limitations to physical activity, which has been shown to significantly impact decision-making. This has been a consistent weakness in study design for clinical trials as well. The patients selected for the survey, in addition, were not necessarily patients that would be considering this decision.\nData analysis: Responses from the survey were used to compute the maximum acceptable increase in risk or maximum acceptable reduction in benefits that participants would be willing to tolerate in exchange for being involved in a less invasive procedure.\nInterpretation of data: 93 patients completed the survey. Patients were not representative of the TAVR population and described above in the weaknesses. The authors importantly describe substantial heterogeneity and patient's responses to the exercise. Importantly, this correlated with patient age, which was lower in the sample than the TAVR population. Patients were more likely to prefer TAVR when considering benefits of independence or risks of mortality or disabling stroke. They were more likely to favor surgical AVR when considering risks of pacemaker and uncertainty regarding durability.\nPresentation of data and story:\n\nAbstract: The abstract describes that the purpose of the research is to determine which outcomes associated with TAVR and S AVR patients consider most important. Patients were not included in the selection of potential outcomes associated with the therapies. The abstract is stand-alone, describing the findings of trade-offs for risks and benefits. However, there should be a further statement about whether or not the patients had aortic stenosis and whether they were symptomatic as this is known to impact decision-making.\n\nIntroduction: The introduction would need to be revised regarding the literature around pacemaker and stroke. Otherwise, the introduction lays out the rationale and explains the goals for the study.\n\nMethods: The methods provide sufficient detail that experiments could be repeated however, much of the methods could be relegated to a supplement online.\n\nResults: The results are hypothesis generating but are limited in the reliability given uncertainty regarding patient diagnosis, severity of AS and symptoms status. In addition, the attributes that the patients are commenting on are defined by the research team, with leadership from the company, and therefore have potential bias.\n\nThe findings are clinically relevant as they describe differences and patient preferences around risk and benefit. They highlight the need for shared decision making approach. They also raise the issue of which outcomes are most important to patients. Additional research is needed to identify that from patients with symptoms themselves.\n\nDiscussion: The discussion around the relationship between the guidelines and regulators needs editing. It is not clear on what the authors mean when they say that the current guidelines \"do not take into account recent clinical data supporting the use of TAVR\". There is an important point about how patients willingness to tolerate certain risks may differ from policy makers and physicians and an argument can be made to include patient preferences into final decision making. Limitations described include how well the participants responding to the survey paid attention to the questions. This is described as a reason for some of the heterogeneity. Limitation would be the small size of the patient's sample and those patients not having the disease that is being studied nor being symptomatic. The limitations focus primarily on the selection of ASW exercises as a way to assess patient preferences rather than the patient population.\n\nThe tables and figures do stand-alone. The references appear appropriate. I appreciate the opportunity to review this interesting 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?\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": "4631", "date": "14 May 2019", "name": "Kevin Marsh", "role": "Author Response", "response": "Review 2: Meghan Coylewright The co-authors have an ambitious agenda to identify patient preferences regarding the differing attributes for the treatment of aortic stenosis. They are to be commended for tackling an important topic, and employing known methodologic standards for addressing the question. They deploy a methodology of adapted swing weighting (ASW), identifying which treatment attributes patients would tolerate to move from an invasive (surgical aortic valve replacement, or SAVR) option to a minimally invasive (transcatheter aortic valve replacement, or TAVR) option. A set of pairwise comparisons were made between 'less invasive\" and other features of AVR, with 5 different iterations presented. The study is written by employees of one of the TAVR companies (Edwards LifeSciences), as well as employees of a company that specializes in patient preference work, paid for by Edwards. It is not apparent that there was engagement of other stakeholders in the study design, such as patients, families, clinicians, administrators, policy makers, etc. There were 5 patients with the disease condition that trialed the exercises before the start of the study to ensure understandability. There is no information how these patients were recruited, or what changes were made in response to their feedback. The authors’ conclusions are that patients are willing to tolerate big increases in risk and exchange for benefits of a minimally invasive procedure. Importance of the research question: Given the recent reconsideration of the TAVR national coverage determination, data as needed to inform interventions designed to increase the degree to which patient preferences are included in final decision making. This study suggests that there is heterogeneity in patient preferences and that patients may tolerate larger differences in risk than physicians and regulators traditionally have deemed acceptable when faced with a minimally invasive procedure. Originality of the work: There is not a large amount of data on the preferences of patients for TAVR. Strengths: Identifying patient preferences and how they influence decision-making is an important topic. Pair-wise comparisons can be helpful to delineate tolerance of risk in one domain versus another. In the decision aid that was presented to the patient's demonstrating differences between the 2 therapies, the same picture was used for both surgery and transcatheter procedure which did potentially reduce bias. Weaknesses: The largest weakness is the fact that the attributes were not selected by patients known to be affected by the disease condition; a survey was asked of participants in advocacy groups online. The eligible patients were created from the membership of heart valve voice and mended hearts. They were directed to an online screening tool where patient reported disease conditions were recorded. The study sample was largely white and highly educated. No patients had severe limitations to physical activity, which has been shown to significantly impact decision-making. This has been a consistent weakness in study design for clinical trials as well. The patients selected for the survey, in addition, were not necessarily patients that would be considering this decision. Response: We thank the reviewer for their interest in the study and their comments. We agree with concerns about the representative of the sample. The observation of the limitation of the study has been extended in the discussion section to read: “Finally, the current study is based on a relatively small sample of patients, and the sample is healthier and younger than the population currently eligible for TAVR and SAVR. AS patients were recruited from the membership of Heart Value Voice and Mended Hearts, and it is possible that patients who are motivated to join these organizations may have different preferences than the broader population. Further AS patient preference research should replicate this study in a larger sample of patients, including more patients with more severe disease burden”. Data analysis: Responses from the survey were used to compute the maximum acceptable increase in risk or maximum acceptable reduction in benefits that participants would be willing to tolerate in exchange for being involved in a less invasive procedure. Interpretation of data: 93 patients completed the survey. Patients were not representative of the TAVR population and described above in the weaknesses. The authors importantly describe substantial heterogeneity and patient's responses to the exercise. Importantly, this correlated with patient age, which was lower in the sample than the TAVR population. Patients were more likely to prefer TAVR when considering benefits of independence or risks of mortality or disabling stroke. They were more likely to favor surgical AVR when considering risks of pacemaker and uncertainty regarding durability. Presentation of data and story: Abstract: The abstract describes that the purpose of the research is to determine which outcomes associated with TAVR and S AVR patients consider most important. Patients were not included in the selection of potential outcomes associated with the therapies. The abstract is stand-alone, describing the findings of trade-offs for risks and benefits. However, there should be a further statement about whether or not the patients had aortic stenosis and whether they were symptomatic as this is known to impact decision-making. Response: The following statement has been added to the abstract: All patients self-reported as being diagnosed with AS, and as either having received treatment for their AS in the previous 10 years or as experiencing limitations in their physical activity as a result of their AS. Introduction: The introduction would need to be revised regarding the literature around pacemaker and stroke. Otherwise, the introduction lays out the rationale and explains the goals for the study. Response: We have not updated the introduction. Perhaps the reviewer could provide more detail on the literature that should be summarized in the introduction. Methods: The methods provide sufficient detail that experiments could be repeated however, much of the methods could be relegated to a supplement online. Response: We have not moved any content to a supplementary appendix. We would be happy to consider doing so if the editors thought that was more appropriate given the audience for the journal. Results: The results are hypothesis generating but are limited in the reliability given uncertainty regarding patient diagnosis, severity of AS and symptoms status. In addition, the attributes that the patients are commenting on are defined by the research team, with leadership from the company, and therefore have potential bias. Response: We agree that it would have been preferable to have been able to engage patients more in the design of the study. The following statement has been added to the discussion section: “it was not possible to engage patients in the design of the elicitation exercise. Instead, the patient voice was reflected in the design through a review of the limited preference research undertaken with AS patients to date and through the engagement that the experts who were consulted had themselves had with patients. Further work would usefully confirm with patients that no attributes were excluded from the study”. We have also added to the paragraph in the discussion on limitations the fact that we relied on patient self report of their diagnoses and AS severity. The findings are clinically relevant as they describe differences and patient preferences around risk and benefit. They highlight the need for shared decision making approach. They also raise the issue of which outcomes are most important to patients. Additional research is needed to identify that from patients with symptoms themselves. Response: We agree this would improve the research. As above, a note to this effect has been added to the discussion. Discussion: The discussion around the relationship between the guidelines and regulators needs editing. It is not clear on what the authors mean when they say that the current guidelines \"do not take into account recent clinical data supporting the use of TAVR\". There is an important point about how patients willingness to tolerate certain risks may differ from policy makers and physicians and an argument can be made to include patient preferences into final decision making. Limitations described include how well the participants responding to the survey paid attention to the questions. This is described as a reason for some of the heterogeneity. Limitation would be the small size of the patient's sample and those patients not having the disease that is being studied nor being symptomatic. The limitations focus primarily on the selection of ASW exercises as a way to assess patient preferences rather than the patient population. Response: See earlier responses. The discussion of the limitations of the study has been extended to cover these concerns." } ] } ]	1	https://f1000research.com/articles/8-394
https://f1000research.com/articles/10-81/v1	08 Feb 21	{ "type": "Review", "title": "Digital screen time during the COVID-19 pandemic: a public health concern", "authors": [ "Abida Sultana", "Samia Tasnim", "Md Mahbub Hossain", "Sudip Bhattacharya", "Neetu Purohit", "Abida Sultana", "Md Mahbub Hossain", "Sudip Bhattacharya", "Neetu Purohit" ], "abstract": "Due to the restrictions imposed to contain the coronavirus disease 2019 (COVID-19) pandemic, different population groups have adapted to varying screen time levels, which may have profound implications on their physical and mental wellbeing. Several empirical studies included in this review reported a sudden upward change in screen time across different population groups. A higher number of people with increased screen time compared to their pre-pandemic state and prolonged duration of total screen time substantiates such assertions. The available evidence suggests that screen time is associated with obesity, hypertension, type 2 diabetes, myopia, depression, sleep disorders, and several non-communicable diseases. This elevated burden of diseases is more prevalent among individuals who have sedentary lifestyles and other unhealthy behaviors that are likely to increase during quarantine or isolation due to COVID-19. Hence, it is critical to assess the adverse health outcomes that may appear as long-term consequences of such behavior. Researchers and practitioners need to revisit the available guidelines and incorporate evidence-based interventions for preventing unhealthy screen time among the affected individuals. Such interventions may address harmful behaviors associated with screen time and promote active lifestyles that may improve health across populations during and after this pandemic.", "keywords": [ "Screen time", "Screen use", "COVID-19", "Coronavirus", "Health promotion." ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) pandemic has critically impacted physical and mental health globally1,2. Many countries have adopted varying measures to minimize the transmission of the disease, including adopting shelter in place policies, staying at home, limiting access to nursing homes, and prohibiting gatherings at places where people can potentially come into closer contact3. Populations with stricter preventive strategies, higher contact tracing, and faster clinical care may result in better public health outcomes during this pandemic4–6. However, the closure or remote operations of schools, offices, and other organizations have resulted in higher use of digital media such as desktops, laptops, tablet computers, and mobile devices for interpersonal communications and other organizational activities7,8. People staying at home or closed places spend higher hours watching television or using digital media for entertainment purposes9. Previous research shows that screen time is associated with a wide range of non-communicable diseases among vulnerable individuals10–13. Recent empirical studies suggest a rapid increase in digital screen time in different populations during the COVID-19 pandemic14–17. This necessitates a comprehensive understanding of the potential public health impacts and how preventive strategies should be adopted to address these.\n\nIn this narrative literature review, we aimed to describe the epidemiological burden of prolonged screen time and associated problems in COVID-19 from available articles retrieved from Medline and Google Scholar using a non-systematic approach. It was not possible to conduct a systematic literature review due to the low number of empirical research articles. The epidemiological burden is primarily described as the prevalence and the average duration of screen time of the individuals or population studied. Secondarily, key factors associated with these problems are summarized to inform the risk and protective factors. Lastly, we have discussed how the current evidence may inform future research, policy development and practice, to improve screen-based behaviors amid the COVID-19 pandemic and future public health emergencies.\n\n\nDiscussion\n\nA growing body of literature concerns the rising trends of screen time and its associated health outcomes during the COVID-19 pandemic. A study of 254 Canadian families with young children reported increased screen time in mothers, fathers, and children during COVID-19 at 74%, 61%, and 87%, respectively17. Moreover, a study conducted in China found that about 70% of 1033 participants spent more time looking at screens after the COVID-19 outbreak16. Another study used a longitudinal design to evaluate health behavior changes during COVID-19 and found a perceived increase in screen time among the participants15. Furthermore, a study conducted in Poland reported that 49% of the participants experienced an elevated screen time during the COVID-19 pandemic14. Additionally, a study conducted in Turkey during the final days of the long-standing lockdown found that nearly 72% of the children studied had a higher screen time compared to the previous years18. In that group, the average duration of screen time was 6.4 hours per day, which is much higher than the 2 hours per day recommendations of the American Academy of Child and Adolescent Psychiatry (AACAP)18. Another study recruited 4108 participants from nine European countries and found a 65% increased screen time among the participants during this pandemic19. According to the survey conducted by Majumder et al., among 203 Indian day job holders and university students, the use of electronic devices such as televisions, computers and cell phones has increased significantly during the lockdown phase20. These studies provide early evidence on rising trends of screen time in diverse populations, which necessitate an investigation into how such trends may impact public health in the global scenario.\n\nA study from the UK found that the participants had an average of 7.2 hours of screen time, which was higher in younger adults aged below 34 years compared to those aged 65 years or above21. This study reported a positive association between screen time and poor mental health among the participants (OR=1.07, 95% CI=1.02–1.13), which was more significant in women (OR=1.07, 95% CI=1.01–1.14) and adults aged 35–64 years (OR=1.13, 95% CI=1.05–1.22). Another large-scale study from China conducted among more than 12,000 participants from all central provinces reported that the average screen time was more than 4 hours per day while they were staying at home. They also found that individuals with more strenuous physical activities had a better emotional state and lesser screen time than those with lighter physical activity22. A study from Canada reported that men and women had better general and mental health status if they had an active lifestyle with lesser screen time compared to those who had a sedentary lifestyle with higher screen time23. Xiao and colleagues surveyed 1680 Chinese adolescents and reported that screen time was negatively associated with mood24. Additionally, children with lesser screen time and increased physical activity had a reduced number of conflicts with their parents24. The available evidence confirms that the early impacts of screen time on different age groups can range from physical to psychosocial conditions with varying risks, which may require longitudinal studies focusing on the relationships between screen time and the multiple variables that may explain the causality, as well as the long-term consequences of screen time.\n\nThe current evidence on adverse health outcomes associated with increased screen time may require an ecological evaluation by expanding the focus on correlated factors such as dietary practices and physical activities among the affected population. For example, Pišot and colleagues reported an increased body mass that could be explained by meal sizes, unhealthy food consumption, sports time, and screen time19. Similarly, Górnicka found that 43% of respondents had experienced a reduction in physical activity, and 34% had increased food consumption in a sample where 49% of individuals reported an increased screen time14. A study from China assessed the physical and psychosocial health impacts of the nationwide lockdown and found that more than half of Chinese adults had a sedentary lifestyle with inadequate physical activity, increased screen time, and poor emotional state16. These studies suggest that a synergistic effect may exist between these co-occurring health behaviors, which can be prevalent among individuals affected by this pandemic. Screen time alone or in combination with other sedentary behaviors may have detrimental effects on populations affected by COVID-19, which should be examined in the context of global evidence from previous research on screen time and associated health outcomes.\n\nGlobal evidence suggests that screen time is associated with multiple health outcomes in different population groups. Syntheses of primary studies summarized in numerous systematic reviews and meta-analyses may provide a more substantial evidence base on a health problem of interest25–27. Multiple studies have shown adverse ophthalmological impacts associated with screen time. A meta-analytic review of 15 studies found a pooled odds ratio (OR) for myopia of 1.02 (95% confidence interval [CI]: 0.96 – 1.08) in a sample of 49789 children13. A significant proportion of evidence-based reviews report higher risks of non-communicable diseases associated with screen time. For example, a meta-analysis of 16 studies found that the odds of overweight or obesity was 1.67 (95% CI: 1.48 – 1.88, P < .0001) in children who had screen time ≥2 hours per day28. A dose-response meta-analysis found linear associations between television viewing and type 2 diabetes and hypertension and a non-linear association with overweight or obesity among adult participants29. This study also reported that each added hour of television viewing increased the risks of hypertension and type 2 diabetes by 6% and 8%, respectively29.\n\nMoreover, the risks of non-communicable diseases associated with sedentary activities that increase screen time may provide critical insights into how screen time can be prevalent alongside other health behavior and yield poor health outcomes across population groups. A large hospital-based cohort study confirmed a direct deleterious relationship between screen time and cardiovascular disease (CVD) events and all-cause mortality30. It indicated individuals who engaged in screen-based entertainment for more than 4 hours per day were at 1.5 times higher risk of all-cause mortality and 2.3 times increased risk of clinically confirmed CVD events compared to those with less than 2 hours of screen time per day30. A meta-analysis of 20 studies analyzed 32 effect sizes on the relationship between non-active video gaming and body mass31. This study found a positive relationship between these two constructs, whereas moderator analyses revealed that the relationship was pronounced among adults compared to children or adolescents.\n\nSeveral evidence-based reviews have reported the mental health impacts of elevated screen time. For example, another meta-analytic review of 12 cross-sectional and seven longitudinal studies found that individuals with higher screen time had significantly elevated risks of depression (OR: 1.28, 95% CI: 1.17 – 1.39, p < .01)10. Another review of 31 studies reported that screen time was associated with poorer sleep outcomes in infants, toddlers, and preschoolers11. Such psychosocial health outcomes associated with screen time may not depend on the quantity of time using the screen, only the quality and contents of screen use may critically impact individuals. A meta-analytic review of 43 studies with a sample of 31,162 participants found that mass trauma television coverage was associated with acute stress reactions and posttraumatic stress outcomes, highlighting the psychosocial implications of television content for the users of this media32. These evidence-based reviews may enable reflection on how the pattern of screen time in the current pandemic may be associated with multiple health outcomes globally.\n\nContemporary evidence suggests a rapid increase in digital screen time during the COVID-19 pandemic. In contrast, pre-pandemic research syntheses indicate that such incremental use of screen-based media may result in adverse physical and mental health consequences in the affected populations. Such challenges may impose an added burden of non-communicable diseases globally in the post-pandemic world. It is critical to acknowledge this upcoming public health crisis and adopt mitigation strategies to prevent the health hazards associated with increased screen time.\n\nIt is necessary to strengthen the knowledge base to make informed policies, guidelines, and treatments for reducing the adverse health impacts of screen time. Although digital screen time is a global issue, there is a lack of research on this topic from low- and middle-income countries. Therefore, strengthening collaborative research which engages global nations to combat common public health challenges associated with COVID-19 can improve international evidence and future practices33. Such collaborative research on the effects of screen time on diverse population groups would need multi-specialty research teams to better understand the epidemiological variances across populations. Moreover, leveraging digital technologies to assess behavioral dimensions and psychosocial correlates can be useful for generating user-level insights that may guide the development of future interventions34–36. Particularly, assessing dose-response relationships between screen time and health outcomes in different COVID-affected population groups may facilitate the development of informed practice guidelines.\n\nMany studies during COVID-19, as well as in the pre-pandemic era, assessed screen time in children and younger populations11,37, which can be a research trend attributable to the fact that these populations are more vulnerable to increased screen time with lesser self-control and this is likely to have long-term consequences. Although screen-based activities in moderation pose no harm, children and teenagers often lack the discipline and insight to limit screen time on their own. It is essential to further specify the safe levels of screen time for age-specific groups through assessing available evidence and ensuring consensus among pediatricians, general practitioners, parents, teachers, social workers, and other key stakeholders. There is a growing body of evidence suggesting that the home environment also plays a vital role in screen time. The presence of a TV in the bedroom, lack of house rules for TV watching, and infrequent family meals are associated with increased duration of screen time14,38–41. Many studies have identified that setting house rules can be effective in limiting screen time for children. For example, a randomized control trial by Barr-Anderson et al., reported that a change in a parent's total screen time and parental rules limiting TV watching were associated with reduced screen time among the children in the study42. Similarly, Birken et al., found that eating lunch in front of the TV and an increase in parental screen time by one hour significantly increased children's screen time. However, a fixed time for screen time reduced hours spent in front of the TV by 30 minutes per day among children43. Additionally, Hu et al., reported that Chinese preschoolers belonging to households with regulations to limit screen time had improved social and cognitive skills. They also found that mothers hold a pivotal role in modeling children's screen behavior44.\n\nParents can be instrumental in reducing screen time for children by ameliorating the overall environment of the house, improving family bonding, and regulating screen time. Although parents' role has become more critical during this COVID-19 pandemic, many are struggling to achieve a work-life balance as they must navigate working from home45–47. This struggle is more pronounced in lower-income families as they were hit disproportionately by the current catastrophe48. The government can mobilize resources to train and support parents to guide their children. Teachers and pediatricians can increase awareness among parents regarding screen time through social media campaigns, webinars, and other online forums. Training modules can be developed for parents to improve household regulation regarding screen time and better guide their children. Lastly, social workers and community health workers can also be employed to train parents and provide psychosocial support as necessary.\n\nSimilar approaches can be adopted for special population groups such as working professionals who may have similar patterns of screen use. Identifying such trends and the underlying psychosocial reasons associated with screen use may enable the development and adoption of common strategies addressing elevated screen time and associated health outcomes. In this regard, guidelines provided by the World Health Organization (WHO) and other institutions may offer some strategic directions regarding how existing guidelines should be revisited and used to develop future guidelines and recommendations. However, such efforts must consider COVID-related psychosocial factors and age-specific behavioral constructs for achieving optimal appropriateness.\n\nThe prevention of unhealthy screen use and associated health outcomes may require specific interventions that acknowledge the unique variances of the quantity and quality of different screen types. For instance, individuals watching television may have different screen use levels than those using social media in smart devices. Targeted interventions should emphasize delivering mass media and online-based health communications, focusing on diverse populations with varying screen time. Such segments of individuals with specific digital behavior may require personalized interventions for preventing unhealthy screen use. Additionally, place and population-specific limitations and opportunities for digital health technologies should be explored for optimal implementation of such intervention49,50. Strategies such as setting limits for screen-based educational or institutional activities complemented by planned exercises that involve offline communications and activities can be useful for people staying at home and attending online sessions51.\n\nBoth digitally and traditionally delivered interventions should aim not only to make people aware of the adverse consequences of screen time but also enable them to engage in active lifestyles, improved dietary practices, and healthier behaviors that promote individual health and wellbeing35,52. It would be necessary to create enabling environments at home or communities that may allow off-screen in-person physical and psychosocial activities that do not compromise safety measures related to COVID-19 while protecting individuals from an unhealthy lifestyle. Nonetheless, psychosocial interventions during COVID-19 should aim for improving social capital and community-level determinants of health that facilitate sustainable health and wellbeing53. Such interventions should be evidence-based and culturally appropriate, incorporating the perspectives of primary users, healthcare providers, and communities. Local, national, and global healthcare organizations and scientific societies may play critical roles by providing updated evidence and recommendations that should be widely communicated for developing multilevel strategies that promote healthier screen use choices for individuals and populations51,54.\n\n\nConclusions\n\nCOVID-19 has affected many aspects of human lives, including the patterns of digital screen use. Previous literature describes the varying levels of health impacts associated with screen time, whereas a growing number of recent studies have shown a rising trend of screen time in different populations with possible health impacts. Prospective research may provide further insights into how different types and amounts of screen time may influence health outcomes across populations. As different populations may have varying screen use behavior, and associated health outcomes, healthcare providers and decision-makers should emphasize on empowering those populations to adopt healthier lifestyles and behaviors. It is critical to use the available evidence and adopt multilevel measures for preventing unhealthy screen time and other behaviors that may impact health and wellbeing among individuals at risk.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nActer T, Uddin N, Das J, et al.: Evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as coronavirus disease 2019 (COVID-19) pandemic: A global health emergency. Sci Total Environ J. 2020; 730: 138996. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHossain MM, Tasnim S, Sultana A, et al.: Epidemiology of mental health problems in COVID-19: a review [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 636. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltmann DM, Douek DC, Boyton RJ: What policy makers need to know about COVID-19 protective immunity. Lancet. 2020; 395(10236): 1527–1529. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuguyo O, Kengne AP, Dandara C: Singapore COVID-19 Pandemic Response as a Successful Model Framework for Low-Resource Health Care Settings in Africa? OMICS. 2020; 24(8): 470–478. PubMed Abstract \| Publisher Full Text\n\nMcKee M: Learning from success: how has Hungary responded to the COVID pandemic? GeroScience. 2020; 42(5): 1217–1219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEsposito S, Principi N, Leung CC, et al.: Universal use of face masks for success against COVID-19: evidence and implications for prevention policies. Eur Respir J. 2020; 55(6): 2001260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobbins T, Hudson S, Ray P, et al.: COVID-19: A new digital dawn? Digit Health. 2020; 6: 2055207620920083. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTing DSW, Carin L, Dzau V, et al.: Digital technology and COVID-19. Nat Med. 2020; 26(4): 459–461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKirály O, Potenza MN, Stein DJ, et al.: Preventing problematic internet use during the COVID-19 pandemic: Consensus guidance. Compr Psychiatry. 2020; 100: 152180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Li Y, Fan H: The associations between screen time-based sedentary behavior and depression: a systematic review and meta-analysis. BMC Public Health. 2019; 19(1): 1524. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJanssen X, Martin A, Hughes AR, et al.: Associations of screen time, sedentary time and physical activity with sleep in under 5s: A systematic review and meta-analysis. Sleep Med Rev. 2020; 49: 101226. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Z, Lin X, Kaminga AC, et al.: Impact of the COVID-19 Epidemic on Lifestyle Behaviors and Their Association With Subjective Well-Being Among the General Population in Mainland China: Cross-Sectional Study. J Med Internet Res. 2020; 22(8): e21176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarroll N, Sadowski A, Laila A, et al.: The Impact of COVID-19 on Health Behavior, Stress, Financial and Food Security among Middle to High Income Canadian Families with Young Children. Nutrients. 2020; 12(8): 2352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOzturk Eyimaya A, Yalçin Irmak A: Relationship Between Parenting Practices and Children's Screen Time During the COVID-19 Pandemic in Turkey. J Pediatr Nurs. 2021; 56: 24–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPišot S, Milovanović I, Šimunič B, et al.: Maintaining everyday life praxis in the time of COVID-19 pandemic measures (ELP-COVID-19 survey). Eur J Public Health. 2020; 30(6): 1181–1186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMajumdar P, Biswas A, Sahu S: COVID-19 pandemic and lockdown: cause of sleep disruption, depression, somatic pain, and increased screen exposure of office workers and students of India. Chronobiol Int. 2020; 37(8): 1191–1200. PubMed Abstract \| Publisher Full Text\n\nSmith L, Jacob L, Trott M, et al.: The association between screen time and mental health during COVID-19: A cross sectional study. Psychiatry Res. 2020; 292: 113333. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQin F, Song Y, Nassis GP, et al.: Physical Activity, Screen Time, and Emotional Well-Being during the 2019 Novel Coronavirus Outbreak in China. Int J Environ Res Public Health. 2020; 17(14): 5170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColley RC, Bushnik T, Langlois K: Exercise and screen time during the COVID-19 pandemic. Health Rep. 2020; 31(6): 3–11. PubMed Abstract \| Publisher Full Text\n\nXiao S, Yan Z, Zhao L: Physical Activity, Screen Time, and Mood Disturbance Among Chinese Adolescents During COVID-19. J Psychosoc Nurs Ment Health Serv. 2020; 1–7. PubMed Abstract \| Publisher Full Text\n\nHossain MM, Khan N, Sultana A, et al.: Prevalence of comorbid psychiatric disorders among people with autism spectrum disorder: An umbrella review of systematic reviews and meta-analyses. Psychiatry Res. 2020; 287: 112922. PubMed Abstract \| Publisher Full Text\n\nHossain MM, Sultana A, Tasnim S, et al.: Prevalence of mental disorders among people who are homeless: An umbrella review. Int J Soc Psychiatry. 2020; 66(6): 528–541. PubMed Abstract \| Publisher Full Text\n\nHossain MM: Umbrella Review as an Emerging Approach of Evidence Synthesis in Health Sciences: A Bibliometric Analysis. SSRN Electron J. 2020. Publisher Full Text\n\nFang K, Mu M, Liu K, et al.: Screen time and childhood overweight/obesity: A systematic review and meta-analysis. Child Care Health Dev. 2019; 45(5): 744–753. PubMed Abstract \| Publisher Full Text\n\nGuo C, Zhou Q, Zhang D, et al.: Association of total sedentary behaviour and television viewing with risk of overweight/obesity, type 2 diabetes and hypertension: A dose-response meta-analysis. Diabetes Obes Metab. 2020; 22(1): 79–90. PubMed Abstract \| Publisher Full Text\n\nStamatakis E, Hamer M, Dunstan DW: Screen-based entertainment time, all-cause mortality, and cardiovascular events: population-based study with ongoing mortality and hospital events follow-up. J Am Coll Cardiol. 2011; 57(3): 292–299. PubMed Abstract \| Publisher Full Text\n\nMarker C, Gnambs T, Appel M: Exploring the myth of the chubby gamer: A meta-analysis on sedentary video gaming and body mass. Soc Sci Med. 2019; 112325. PubMed Abstract \| Publisher Full Text\n\nPfefferbaum B, Nitiéma P, Newman E: Is Viewing Mass Trauma Television Coverage Associated With Trauma Reactions in Adults and Youth? A Meta-Analytic Review. J Trauma Stress. 2019; 32(2): 175–185. PubMed Abstract \| Publisher Full Text\n\nHossain MM: Current status of global research on novel coronavirus disease (COVID-19): a bibliometric analysis and knowledge mapping [version 1; peer review: 2 approved with reservations]. F1000Res. 2020; 9: 374. Publisher Full Text\n\nAhasan R, Hossain MM: Leveraging GIS and spatial analysis for informed decision-making in COVID-19 pandemic. Health Policy Technol. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarbadhikari S, Sarbadhikari SN: The global experience of digital health interventions in COVID-19 management. Indian J Public Health. 2020; 64(Supplement): S117–S124. PubMed Abstract \| Publisher Full Text\n\nHossain MM, McKyer ELJ, Maa P: Applications of artificial intelligence technologies on mental health research during COVID-19. SocArXiv. 2020. Reference Source\n\nStiglic N, Viner RM: Effects of screentime on the health and well-being of children and adolescents: A systematic review of reviews. BMJ Open. 2019; 9(1): e023191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGingold JA, Simon AE, Schoendorf KC: Excess screen time in US children: Association with family rules and alternative activities. Clin Pediatr (Phila). 2014; 53(1): 41–50. PubMed Abstract \| Publisher Full Text\n\nBauer KW, Neumark-Sztainer D, Fulkerson JA, et al.: Familial correlates of adolescent girls’ physical activity, television use dietary intake, weight, and body composition. Int J Behav Nutr Phys Act. 2011; 8: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarlson SA, Fulton JE, Lee SM, et al.: Influence of limit-setting and participation in physical activity on youth screen time. Pediatrics. 2010; 126(1): e89–96. PubMed Abstract \| Publisher Full Text\n\nSisson SB, Broyles ST, Newton RL, et al.: TVs in the bedrooms of children: Does it impact health and behavior? Prev Med. 2011; 52(2): 104–8. PubMed Abstract \| Publisher Full Text\n\nBarr-Anderson DJ, Fulkerson JA, Smyth M, et al.: Associations of American Indian children’s screen-time behavior with parental television behavior, parental perceptions of children’s screen time, andmedia-related resources in the home. Prev Chronic Dis. 2011; 8(5): A105. PubMed Abstract \| Free Full Text\n\nBirken CS, Maguire J, Mekky M, et al.: Parental factors associated with screen time in pre-school children in primary-care practice: A TARGet Kids! study. Public Health Nutr. 2011; 14(12): 2134–2138. PubMed Abstract \| Publisher Full Text\n\nHu BY, Johnson GK, Wu H: Screen time relationship of Chinese parents and their children. Child Youth Serv Rev. 2018; 94: 659–669. Publisher Full Text\n\nDavis CR, Grooms J, Ortega A, et al.: Distance Learning and Parental Mental Health During COVID-19. Educ Res. 2020; 50(1): 61–64. Publisher Full Text\n\nHjálmsdóttir A, Bjarnadóttir VS: \"I have turned into a foreman here at home.\" Families and work-life balance in times of Covid-19 in a gender equality paradise. Gender Work Organ. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarbe A, ogurlu U, Logan N, et al.: Parents’ Experiences with Remote Education during COVID-19 School Closures. Am J Qual Res. 2020; 4(3): 45–65. Publisher Full Text\n\nDooley DG, Bandealy A, Tschudy MM: Low-Income Children and Coronavirus Disease 2019 (COVID-19) in the US. JAMA Pediatr. 2020; 174(10): 922–923. PubMed Abstract \| Publisher Full Text\n\nHossain MM, Tasnim S, Sharma R, et al.: Digital interventions for people living with non-communicable diseases in India: A systematic review of intervention studies and recommendations for future research and development. Digit Health. 2019; 5: 2055207619896153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhasan R, Alam MS, Chakraborty T, et al.: Applications of GIS and geospatial analyses in COVID-19 research: A systematic review [version 1; peer review: awaiting peer review]. F1000Res. 2020; 9: 1379. Publisher Full Text\n\nWiederhold BK: Children's Screen Time During the COVID-19 Pandemic: Boundaries and Etiquette. Cyberpsychol Behav Soc Netw. 2020; 23(6): 359–360. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "136401", "date": "09 May 2022", "name": "Michael Johnson Jr.", "expertise": [ "Reviewer Expertise Television", "Political Economics", "Media Industries", "LGBTQ", "Race & Ethnicity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author offers an interesting analysis about the impact of screen time exposure on the physical and mental condition of people pre-pandemic. It ties the residential captivity of populations due to the COVID-19 epidemic, with the pre-existing co-morbidities which often accompany sedentary media consumption. \"...the closure or remote operations of schools, offices, and other organizations have resulted in higher use of digital media such as desktops, laptops, tablet computers, and mobile devices for interpersonal communications\" and it links previous research which demonstrated increased screen time with \"a wide range of non-communicable diseases among vulnerable individuals\".\n\nTo accomplish this task the authors conducted a meta-analysis of the extant literature, despite a very limited number of studies on the subject. Importantly the authors conclude that remedies to screen time consumption \"may require specific interventions that acknowledge the unique variances of the quantity and quality of different screen types...It would be necessary to create enabling environments at home or communities that may allow off-screen in-person physical and psychosocial activities that do not compromise safety measures related to COVID-19 while protecting individuals from an unhealthy lifestyle\". The authors make a number of cogent observations which are informed by the research examples they utilize to good effect. Its conclusions are sound and limited to specifics that recognize different levels of media consumption across a variety of viewing demographics and amidst wide disparities in global healthcare.\nThe only issue with the authors' narrative is the numerical inconsistency between what they describe as limited research on the subject with 195 (by my count) studies reviewed in their research. They should make a disclaimer to clarify how these two seemingly disparate facts can exist simultaneously. Despite this minor reservation, I believe the article provides a useful starting point that expands upon the literature of media consumption and therefore I can recommend indexing. As I note in my recently published 2021 article on the subject1, “There’s no question that an important percentage of domestic American consumers are interested in the a la carte consumption habits, ease of accessibility (especially on mobile devices), and demand for “original” content, streaming will continue to have an appeal” and this observation has significance for the author’s contention that screen time correlates with increased comorbidities that endanger the population’s public health during the pandemic. If streaming does increase in popularity, as I fully anticipate that it will, then the screen time consumption dynamic the authors have identified will become even more concerning as time under COVID 19 continues.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "126292", "date": "09 May 2022", "name": "Sukhpreet Tamana", "expertise": [ "Reviewer Expertise Mental health", "screen time", "sleep", "behavioral health", "physical health", "sedentary behavior", "epidemiology", "interventions." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important and timely synthesis of data reporting on a topic of growing concern around screen time use and associated risk of health problems in the context of the COVID-19 pandemic. The authors summarize in this unsystematic narrative review, the prevalence and average duration of screen time exposure across different populations during the COVID-19 pandemic. The article also describes evidence assessing associations between screen time and various health outcomes and proposes potential mitigation strategies. The article is clearly written and generally well organized.\n\nA few considerations that the authors may consider:\nMajor points to consider\nIt is important for the authors to explain and describe available screen time recommendation guidelines developed for adults and children (for example the American Academy of Pediatrics, Canadian 24-Hour Movement Guidelines). This would be helpful to the reader when interpreting the studies of screen time exposure the authors summarize in this paper.\n\nThe authors interpretation or synthesis of the data does not consider any differences between studies such as populations sampled, sample size, adjustment for covariates, and differences in study design and how screen time was assessed (e.g., total number of hours, range). This could be addressed as a section explicitly stating any limitations.\n\nMinor points to consider\n1. Title\nWe suggest the authors include “narrative review” or at a minimum “review” in the title.\n2. Abstract\nConsider replacing the term “a higher number” with “a higher percentage.”\n3. Introduction\nIn the second paragraph, the authors explain how the articles were retrieved but do not indicate the time frame. Please clarify the text as follows “..from available articles retrieved between XX and XX from Medline..”.\n\nThe scope of the literature search appears to be limited. Other databases beyond Google Scholar and Medline should have been included. Please clarify.\n\nIn the second paragraph, line 3, consider rephrasing “problems in COVID-19” to “the associated problems with the COVID-19 pandemic”.\n4. Discussion\nSection ‘COVID-19 and rising trends of screen time: public health concerns\": Paragraph 1, reference number 18 appears to be the incorrect citation. Please amend.\n\nSection ‘Global evidence on the association between screen time and health outcomes’: Here the authors summarize data from studies conducted pre-pandemic. We suggest removing the term ‘global’ from the title and/or replace it with the term “pre-COVID-19 or pre-pandemic”. The authors should indicate any limitations. Were all the studies identified of high or moderate quality?\n5.Conclusions:\n\nIn the text “…those populations to adopt healthier lifestyles and behaviors.” Consider replacing “healthier lifestyles and behaviors” with “healthier screen time use.”\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": [] } ]	1	https://f1000research.com/articles/10-81
https://f1000research.com/articles/9-103/v1	11 Feb 20	{ "type": "Research Article", "title": "A qualitative investigation of surrogacy as a panacea for infertility in Nigeria", "authors": [ "Oluwatobi Joseph Alabi" ], "abstract": "Background: Up until recently, adoption is the most common alternative to recommended to couples struggling to procreate. However, with the advancement in medical technology, it is now possible to procreate through assisted reproductive technology (ART). Debates continue to ensue on the contentious issues emanating from various ART procedures, for instance regarding surrogacy there are concerns that this fragments womanhood, motherhood and parenthood, and there is a dereliction of the sacredness and cultural sanctity of the family system, most especially in an African context. However, as infertility becomes more prevalent among couples trying to have children in Nigeria, it has become important that alternate mediums of reproduction be examined within the socio-cultural milieu of the country. This study set out to examine surrogacy as a panacea to infertility in Nigeria through a qualitative lens. Methods: 15 stakeholders (traditional birth attendants, medical gynaecologists and legal professionals within the social, medico-legal framework of reproductive health) in Nigeria were engaged in an in-depth interview to unravel the challenges surrogacy might or is encountering as an ART in Nigeria. Results: There are various social, traditional, cultural and religious beliefs that police the reproductive sphere of Nigeria, which has grave implications on fertility treatment. These socio-cultural and religious factors do not provide a fertile ground for surrogacy to thrive in Nigeria. Hence, it is important that the socio-cultural framing of reproducing in Nigeria become receptive to modern medical reproductive alternatives and innovations. Conclusions: For surrogacy to permeate the reproductive terrain of the country there is a need to jettison several socio-cultural and religious sentimental beliefs policing reproduction in Nigeria.", "keywords": [ "Surrogacy", "reproduction", "infertility", "culture", "religion." ], "content": "Introduction\n\nInfertility in most parts of Africa is usually not well researched as a vital part of sexual reproductive health, yet its impact can be highly consequential1. Having biological children is highly desirable and inability to conceive is often demonized, socially stigmatized, often leads to divorce or results in adverse psychological and health effects on the partners involved2. It is also important to understand the gender narratives around infertility in most of sub-Saharan Africa; while men and women can both be potentially infertile, women are often blamed and punished for childlessness3,4. Within this very complex socio-cultural milieu that prioritizes fertility and links procreation to the completeness of a man or woman, couples are put under pressure to bear children and failing to do so is regarded as an existential failure. Studies show that the pressure to have children has increased risky sexual behaviour in sub-Saharan Africa among couples4. However, it is important to note that infertility treatment has not been holistic as a result of the limited access to various ART alternatives and stigmatizing attitudes towards medical reproductive alternatives2,5.\n\nInfertility is described as a global public health challenge affecting between 13–17% of couples trying to conceive in sub-Saharan Africa, with a high rate of about 32% in most of the countries sampled in the survey6; this is according to the infertility survey conducted in 27 sub-Saharan countries including Nigeria. As the percentage of infertility remains high in most countries in sub-Saharan Africa (32%), the pressure on couples to procreate and find solutions to their stigmatized status becomes very complex. The search for infertility treatment is a complex terrain to navigate because infertility possesses socio-cultural attributes and challenges at both foundational and experimental levels7. Explanations of the aetiology of infertility differ to an extent between health care providers, patients and the society in Nigeria7. Western medicine will diagnose infertility biologically and administer clinical recommendations; however, indigenous medicine believes factors causing infertility could be biological and even supernatural7. This dichotomy in the aetiology of sickness in general and infertility, in particular across Africa, is foundational for the various socio-cultural narratives surrounding reproduction in Nigeria. There are various socio-cultural beliefs policing fertility in Nigeria and these beliefs are of grave concern for the perception of and attitude towards infertility2. The cultural environment is paternalistic and as such, children are highly desired, and parenthood is culturally mandatory2. Any standard below this paternalistic cultural expectation is regarded as cultural deviation and often stigmatized2.\n\nThe challenge of infertility in sub-Saharan Africa is debated among researchers, especially because of the complex socio-cultural narratives surrounding reproduction in the region. As such, various treatment alternatives have been debated and discussed in the literature, showing that adoption has been the most popular route for definite cases of infertility among couples in Nigeria8. Notwithstanding the popularity of this alternative, it is a very challenging and complex process to embrace in Nigeria9. A previous study among the Yoruba people of Western Nigeria revealed that the acceptability of child adoption has several socio-cultural factors that influence the success of adoption, including lack of adequate information about the adoption process10. These limitations have also been discussed as eminent in Eastern Nigeria11. These socio-cultural challenges are not particular to the South-West or Eastern parts of Nigeria as it is important to note that despite the heterogeneous nature of the Nigerian society, a common narrative across these cultures is the premium placed on children, emphasis on parenthood and the norm that every woman should conceive and carry children to term2. Fertility is highly valued across genders and it often seen as a validation of womanhood or manhood.\n\nThe medical technological advancement that led to the birth of baby Louise Brown in 1978 (the first baby born by in vitro fertilization in England) rekindled the hope of many childless couples across the world on the possibility of having children that are genetically connected to them and many have used this platform since then12. However, in Africa, there are limited reproductive centers because they are driven mostly by private sectors and quite expensive to patronize13. In addition, the cost of access, low success rate, and financial commitment are barriers to utilizing assisted reproductive technologies in Nigeria10. Even though there are many options for infertility treatment across the globe, the success of such processes depends on etiological factors, availability of medically advanced diagnostic tools, skills of the attending physician and financial status of the couples involved14,15. This study recognises and expands on the various challenges already narrated in the literature influencing the success and use of ARTs in Nigeria, and furthers the conversations around other fertility treatments in Nigeria, particularly surrogacy. Surrogacy is a process where a third party (woman) of childbearing age carries a pregnancy for a commissioning parent with the intention of relinquishing the baby after birth16. This study discusses the socio-cultural factors influencing the practice/acceptance of surrogacy in Nigeria. By engaging informed stakeholders within healthcare and legal systems, it is able to assess the possibilities of having surrogacy as a panacea to infertility in Nigeria.\n\n\nMethods\n\nThis research is qualitative, and its strategy of inquiry is explorative. In utilizing this exploratory approach, the researcher harvested the perception of society by presenting informed opinions of gynaecologists, traditional birth attendants (TBA) and legal professionals on the practice of surrogacy in Nigeria. Exploratory qualitative research allows the researcher to collect data from persons who are experts about a phenomenon and helps develop a composite description of the essence of their experience for all individuals17.\n\nThis study was approved by the University of KwaZulu-Natal ethical clearance Committee (Protocol number: HSS/0705/017M) after due consultation and gatekeepers' consent was provided by Ekiti State Teaching Hospital, Ado-Ekiti, Ekiti- State and Oyedeji Ayodele & Co, Abuja Nigeria.\n\nThe study population consisted of three categories of participants above the penal code age (18 years) in Nigeria. These included ten gynaecologists, five TBAs and five legal professionals. This study was conducted in two cities in Nigeria: gynaecologists and TBAs were recruited and interviewed at Ekiti State Teaching Hospital, Ado-Ekiti, Ekiti- State Nigeria; legal professionals were recruited and interviewed at Oyedeji Ayodele & Co, Abuja, Nigeria.\n\nConsidering that surrogacy is not a popular practice in Nigeria, purposive sampling was used to select participants who have expertise regarding surrogacy in Nigeria. Both institutions served as host to the researcher and provided access and to participants and premises. The Ekiti State Teaching Hospital in Ado-Ekiti is one of the foremost fertility clinics in the South-western part of the country and thus a suitable location for the study; while, Oyedeji Ayodele legal chamber is one that has handled some legal disputes about children paternity and reproductive contract over the years. Both locations had the criteria to provide the required information this study needed.\n\nInformed consent detailing the purpose of the study and the strategies to ensure participants’ anonymity and confidentiality were explained to the participants before the study began, after which they signed the consent voluntarily participated. The informed consent included that the participants could withdraw from the study at any point if they no longer feel comfortable.\n\nPurposive sampling was adopted to recruit participants in the study. The institutions used as a host for the research provided access to legal professionals, gynaecologist and TBA’s. Upon receiving ethical clearance from the University of KwaZulu-Natal, the gynaecological centre of Ado Ekiti Teaching Hospital was visited and appointments were made to interview gynaecologists at their convenience, the department also served as a link to the association of TBAs in Ekiti State. Oyedeji Ayodele & Co. was also visited in Abuja where interested legal professionals within the chamber were approached and interviewed. In total, 10 gynaecologists, 5 TBAs and 5 legal professionals were interviewed in the study.\n\nFace-to-face in-depth interviews were conducted with each participant using a semi-structured interview schedule that contained three sets of questions. An audio recorder and field notes were used to collect the data. The participants consented to tape the interview with the audio recorder expressly on the informed consent. The first set of questions examined surrogacy within a cultural perspective, the second questioned medical opinions on surrogacy arrangements, and the third examined the legal trepidations in the practice of surrogacy. The interview schedule was translated into Yoruba, the local language of this Western region of Nigeria for the benefit of the TBAs who might not be English speakers. Interviews lasted on average of 30 minutes.\n\nNot all participants approached participated in the study. Considering the nature of the medical and legal profession, the timing was a major constraint; several of the legal practitioners and gynaecologists cancelled appointments more than twice and some were finally not interviewed. However, data saturation was a major guide during the process of the fieldwork. The narratives from participants were constantly accessed to see that new information was derived and when there were very frequent repetitions data saturation had been reached and the fieldwork came to an end.\n\nAudiotapes were transcribed by the researcher and interview transcripts and summaries were checked with participants to ensure that their narratives were well captured and not distorted in any way. The interview lasted an average of 30 minutes.\n\nThematic content analysis was employed to analyse the field notes and transcripts. Thematic content analysis is an approach to the analysis of documents and texts (which may be printed or visual) that seeks to quantify content in terms of predetermined categories (in this research emerging categories from participants’ narratives) in a systematic and replicable manner18. Transcripts from the interviews were organized into a logically readable format, after which recurrent patterns and conceptual issues were identified and developed into themes that formed the basis for the analysis. Participant numbers and characteristics (age, gender, and job; Table 1) were used in reporting the findings of this study to ensure the anonymity of participants as noted in the informed consent.\n\n\nResults\n\nThe dynamic nature of human societies coupled with varying regulatory normative codes across cultural boundaries creates the need to examine the perception of surrogacy within various cultural settings to appreciate the differences and similarities that are obtainable in the growth of the practice as a core ART process. The imperative questions that steered this study included a need to understand how surrogacy is conceptualized in Nigeria; and a concern to understand the socio-cultural imperatives influencing the practice in Nigeria. The themes discussed include the social perception of surrogacy; the socio-cultural intricacies of surrogacy and its implication for the normative understanding of a ‘woman’; the gendered nature of infertility; and, the effect of surrogacy on the structure of parenthood.\n\nThe participants provided varying thoughts on the popularity of surrogacy as an Assisted Reproductive Technology (ART) in Nigeria. Some of these seemed to be influenced by access to global information and medical practices. A careful examination of the responses revealed complex narratives that were probably influenced by access to information relative to the individual’s job. Gynaecologists and legal professionals expressed more informed opinions on the practice of surrogacy while TBAs discussed surrogacy essentially from cultural and religious points of view:\n\nSurrogacy in Nigeria is not a popular practice, it is not that popular because when viewed from a socio-cultural lens, there are lots of beliefs, controversies that surround surrogacy. In most quarters in Nigeria, surrogacy is not popular at all and I will say the popularity and acceptance of the practice are hugely influenced by socio-cultural, legal and spiritual factors. The legal space has not created platforms that support or regulate the process and the socio-cultural and religious aspects of our society are not in favour of such acts that distort natural order. (Gynaecologist, M, 48 years.)\n\nAll the gynaecologists interviewed believed surrogacy will gain prominence with time and as the country continues to transform; however, they also believe that it is still in its infancy, backward and grossly rudimentary. From these narratives these gynaecologists believe surrogacy is not yet very popular in Nigeria from their expert point of view. They also emphasized that the popularity of the process is hampered by socio-cultural beliefs and values regulating reproduction. However, surrogacy is becoming a thriving venture in major urban settlements like Abuja, Lagos, and Port-Harcourt.\n\nWhile the majority of legal professionals interviewed (n=3) believed surrogacy to be a controversial issue in Nigeria with very little patronage, the others believed the practice is practically non-existent within the country since patronage is very low.\n\nThe opinions shared by TBAs were grounded in religious and cultural beliefs, leading to opposition to its practice:\n\nOur prayer is that this act won’t gain prevalence and acceptance in Nigeria. Because it is anti-cultural and anti-religion. God has established a process for that and no one or advancement should change it. It is a devilish act championed by the western world and it won’t succeed in our land. How do you expect a woman that did not gestate a child to know the real worth and value of that child? Or how does the child become a true member of the kin when another woman gestated him/her. God forbid, it won’t work here. I have heard people are doing it already but God will destroy their plans. (TBA, F, 58 years.)\n\nAll the TBAs shared the same opinion that surrogacy is becoming a social discussion in Nigeria; however, they hoped it would not thrive within the country because it goes against cultural and religious normativity for procreation. The enterprise of surrogacy is indeed a very debatable area with very little social knowledge in the general Nigerian population, and currently does not constitute an important aspect of public discussion19,20.\n\nWhat surrogacy means and the social reception it enjoys differ across cultural and social boundaries. Surrogacy may create numerous ethical concerns that are derivatives of social, cultural and religious fundamentals of the community21. Hence, it is important to understand how this practice is perceived and engaged within Nigeria to properly examine its possibility as an alternative to infertility.\n\nSurrogacy and its practice are borne out of a desire to have a child22, and contemporary practice is highly hinged on a desire to have a child with a biological connection23,24. Perceptions of surrogacy may also be grounded in descriptions that suggest commercialization25,26. A surrogate could be referred to a woman who rents her womb. TBAs in this research described surrogacy as an act that distorts the cultural normative process for procreation. Surrogacy in Yoruba language is translated as “agbabi odi omo eni”, meaning ‘contracted pregnancy does not become yours’. The meaning and definition of the process in this local language suggests a difference between the surrogate and child, as well as between the commissioning parent and the child, and may lead to the creation of stigmas. The TBAs stated that surrogacy is perceived as a way of expressing how one woman was unsuccessful in conceiving a child; this role, however, has been taken up by another woman who has the physiological capability to conceive and carry a child to term. Moreover, socio-cultural normativity about procreation views surrogates as deviants and even in cases of altruism, her altruism is regarded as ranging beyond normative boundaries27. In conclusion, the perception of surrogacy in Nigeria is influenced by cultural, social, traditional and religious values.\n\nThe significance of women in African society cannot be overstated. They play a significant role in the continuity of the community and the operation of social life, and are considered the fabricators of life and the mothers of humankind. As a major player in the personal rituals associated with birth, puberty, and death, the symbolism of these rituals shows the important cultural meanings of womanhood28. There is a link between fertility, culture, and religion in most African cultures29. While the ability to conceive and carry a child to term is regarded as a gift and part of the doings of a supernatural being, the cultural definition of a woman is her ability to perform the gestational role of carrying a child to term. In Africa, “high fertility was not only a divine reward but evidence of the right behaviour. Among the Chaga of Tanzania, the wife in complying with the divine order has been described in these words: she corporates with her husband, the ancestors, even God, in creating the child”30. Hence, it can be inferred that fertility is a product of complete obedience to God, the ancestors, and the maintenance of good societally approved behaviour. Children are highly desirable and women are regarded on their ability to conceive, carry to term and nurture the child. The birth of a child is celebrated and seen as a sign of divine approval by both living and dead/ancestors. However, infertility is regarded most times as a woman’s problem and evidence of sin and disapproval by both God and ancestors30. These are very strong gendered assumptions that continue to domesticate women, sexualise their bodies, and reproduces female subjects and oppression within the study location.\n\nThe perceptions shared by the participants are diverse but entrenches the notion that fertility and children are important parts of the African community. A woman’s ability to conceive and carry a child to term defines her and serves as a rite of passage to womanhood. In addition, religion is fundamental to the conception of fertility, while infertility is an aberration that results from sin. When most of the participants were asked about how they think the practice of surrogacy will influence the conceptualization of womanhood, various issues emerged, including:\n\nPersonally, it is against the definition of womanhood and even the surrogate is not thoughtful enough, what will the surrogate tell her own husband after she must have done something like this. Will she say she has not given birth before or what? What if she cannot put to bed again? Even if she marries and now have a child through the cultural medium with her husband, would she now refer to this present child as her first seed or the surrogate child? This is disturbing and against our culture. (TBA, F, 53.)\n\nThe idea is that surrogacy encroaches on what defines a woman and it is not a practice any of the parties involved will be proud to identify with. In addition, surrogacy was seen as a negation of religious procedure for reproduction:\n\nGod created the man and woman. There is a plan and process designed by the creator for procreation. God endowed women for reproduction and he did not sanction in vitro processes or surrogate practice. The Bible says there shall be non-barren in the land. That there will be children in your loins, so it does affect the definition of womanhood. (TBA, F, 40 years.)\n\nThe responses captured above further reiterate the intersection between religion and culture in the perception of fertility and procreation within this context and across most African communities. It is an intersection that continues to hinder the possibilities of ARTs in Nigeria and further produces assumptions that seek to police women’s sexuality and reproductive abilities.\n\nThe perception that infertility is regarded as evidence of sin or recklessness was discussed by participants. For example, it was noted that infertility may be the result of the reckless lifestyle the woman must have lived as a youth:\n\nWe cannot change the gender of the woman because she can’t carry a child but it must be that the woman that requires the service of a surrogate must have lived a reckless life as a youth, maybe she must have aborted 3 to 4 times before marriage. So, she will later have problems with giving birth but the husband won’t know. So, she’s still a woman that has no experience or knowledge of what it means to be pregnant and carry the baby to full term. Most times these couples struggling to give birth especially the women have been promiscuous and reckless, so she’s reaping the fruit of her labour. Instead of going through this ungodly medium, she must cry unto God and ask for forgiveness. It is only God that can give children. (TBA, F, 65 years.)\n\nThe perception that infertility is punishment assumed that women are solely responsible for infertility; therefore, reiterating that infertility is highly gendered and usually regarded as a woman’s problem:\n\nChildlessness is a taboo, a curse and seen as the woman’s fault mostly. Even our doctors stereotypical and superstitious perception of the causes infertility is absolutely amazing, I was surprised when a doctor said, childlessness is 30% the woman’s fault, 30% the man’s fault and 40% unknown causes. But up until now the way it is treated is as if when you are childless it is the woman’s fault. (Legal professional, F, adult.)\n\nIt is deducible from the response above that the perception of infertility because of a supernatural/unknown force is shared by educated individuals, such as medical professionals. The connection between a woman’s ability to birth a child and her attainment of womanhood in the society was expanded upon by other participants:\n\nWomanhood in this environment is seen in terms of a woman’s ability to have delivery. So, a woman that adopt the option of surrogacy will not have an opportunity of giving birth by herself and that will question her womanhood, a lot of people will see her as not being woman enough because she has not been seen to be pregnant or heard of her delivery of a child. People attach being able to deliver as a mark of womanhood, so surrogacy will affect womanhood negatively especially where people place a lot of emphasis on a woman’s ability to get pregnant, gestate effectively and carry the child to term. (Gynaecologist, M, 45 years.)\n\nFrom this it can be suggested that without gestating and carrying a foetus to term, it is culturally believed that the woman is incomplete and has failed in her role, and as such transferred this responsibility to another. ‘Renting’ a womb for surrogacy, as discussed in the literature31,32, is seen in this context as a cultural anomaly and misnomer:\n\nIt really doesn’t influence it except to prove that one woman failed and another woman is doing her work for her. In the sense that we have linked womanhood especially in Africa to motherhood, the ability to conceive and birth a child. That’s what we call a complete woman, the average woman to wants to be a mother so when there’s a woman that can’t give birth we might want to re-evaluate what womanhood means. It also means that society is evolving and when things are not the way they should be 100% we have found ways of filling the lacunae and bridging the gap. (Legal professional, M, adult.)\n\nThe failure of a woman to fulfil her cultural role as a woman has essentially created a gap that other women will fill, either for altruistic reasons, e.g. helping women fulfil their social expectations of becoming mothers, or for financial gain inherent in the practice of commercial surrogacy. One participant stated that while the practice negates the normative ways of procreation, it also fulfils a cultural goal of helping other infertile couples build a family:\n\nIn my own view, how it affects the definition of womanhood is; it depends on how you look at it, especially in an Africa setting where it is believed that if a woman does not bear a child then the woman is not fulfilled or a marriage that does not have a child. However, now we have women coming out to help women in the actualization of their desire to have a baby, so I think this has really gone a long way and as put smile on the faces of lots of females around the world and it as really make the womanhood more appreciated than what you can ever think of. (Gynaecologist, M, 39 years.)\n\nEmbracing this positive aspect of surrogacy to help individuals fulfil the normative social expectation of raising a family where children are regarded as a premium. In addition, individuals should have the agency to take whatever reproductive routes they desire within the acceptable legal frames of their society. These need to be identified as a fundamental part of human rights within the Nigerian society.\n\nAnother insightful perspective shared by the participants was that even though surrogacy negates cultural norms, it has redefined the essence of the family institution and parenthood. It brings the fundamental reasons why parenthood should go beyond genetics to the fulfilment of being actively responsible in the care of children:\n\nSurrogacy redefines the whole essence of the family and parenthood. It makes us think of a woman beyond just bearing children. It reminds us there is more to a woman than just bearing children because at the end of the day even if there is a problem with the man, everybody stigmatizes the woman in a childless union. It means a woman can be a woman without necessarily bearing a child. (Legal professional, M, 34 years.)\n\nHmmm…. Well the definition of womanhood generally around here is complex because in the real sense being a mother is not about giving birth like we also say being a father is not about fathering a child but the roles and responsibilities assumed. However, we find out that being a woman has been defined with being able to conceive in the African sense, so surrogacy thus affects this definition of womanhood. Not being able to conceive is more unless like you are less than a woman, in fact, I have seen where people have written that if you give birth through caesarean operation (CS) then you are not a true woman. So, I can imagine that in that circle giving birth through a surrogate tamper with the traditional definition of womanhood, however, the world is evolving and people are defining motherhood and fatherhood by roles and responsibilities assumed in the life of a child. (Legal professional, F, adult.)\n\n\nDiscussion\n\nThis study has showcased various perceptions regarding surrogacy in Nigeria. The medical possibility of the process is not denied or questioned, but the views captured in this study are fundamental socio-cultural and religious musings questioning the morality of the process and thus affecting its popularity and social acceptance in Nigeria. The study revealed that even though surrogacy appears to be practiced in Nigeria, it is hidden and unpopular. While surrogacy might be glorious news for couples struggling to have children where natural conception is impossible, opinions of the current participants were very divergent on the ethical, religious and socio-cultural morality of the process. Another study supports that surrogacy in Nigeria is very complex and controversial because of the legal, ethical, socio-cultural, psychological and religious concerns surrounding the process overshadows whatever benefits it provides20.\n\nSome of the socio-cultural factors influencing the growth of surrogacy in Nigeria are the gendered nature of infertility; women are often seen as problematic partners when couples are struggling to conceive and this perception is accompanied by various stigmas that labels, diminishes and reduces woman to reproductive entities. For example, in the presents study, the ability of a woman to gestate is attached to her completeness or her attainment of womanhood, when she fails to fulfil this social expectation, she becomes stigmatized as a failure and incomplete as a woman. This attitude will discourage women from seeking fertility treatment and may lead to couples with fertility challenges becoming secretive about their medical concern. It was also noted by the present study that women having challenges conceiving are stigmatized as promiscuous and reckless, and it was noticeable that when issues of infertility are discussed, women often occupy the centre of the discussion and very little is discussed regarding male infertility.\n\nHence, the practice of surrogacy is seen to be distorting the original socio-cultural definition of a woman. ‘Renting a womb’ as it is popularly described is perceived to be anti-religion and anti-culture. Aside from these various limiting debates around surrogacy, the practice is one of the most progressive ways of addressing infertility and it has stirred up conversations around the social-reorganization of parenthood. While it is a desirable fertility alternative, it is meted with several socio-cultural and religious complications in Nigeria.\n\nThe findings of this study were limited by time and resources. The focus was on one of the fertility clinics at the Ekiti-State Teaching Hospital, and as such do not provide sufficient data for generalising findings. However, it provides a detailed empirical framework for examining surrogacy within the Nigerian context.\n\n\nConclusions\n\nThis study has unpacked the various socio-cultural and religious trepidations in the practice of surrogacy in Nigeria. Through engagement with medical professionals, legal practitioners and traditional birth attendants, the study unravelled that there are several socio-cultural and religious factors policing the reproductive sphere in Nigeria. The overarching influence of cultural beliefs on medical concerns such as infertility may have limited the extent to which people seek help. In the study location, it was revealed that infertility is highly gendered and women often occupy the centre of discussions around infertility. It became evident from the findings that there are many cultural rights that define a woman within the context of this study. It ranges from the importance attached to the gestational process as sacrosanct for defining a woman and her ability to value a child; the connection between kinship and the process of birth. The prevalence of these cultural beliefs in the perception of fertility, infertility, and treatments are daunting challenges to the growth of surrogacy in Nigeria. The social space does not create a fertile environment for alternatives processes for assisted reproduction to thrive and as such, there is a need to properly educate the population about the medical advantages of ARTs. It also became evident that the socio-cultural factors discussed above are limiting the growth of surrogacy as an ART in Nigeria.\n\n\nData availability\n\nFigshare: Surrogacy Transcript. figshare. Dataset. https://doi.org/10.6084/m9.figshare.11120891.v133.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Interview schedule, https://doi.org/10.6084/m9.figshare.10264904.v134.\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\nI want to appreciate Dr Mariam Seedat-Khan who supervised my master’s research, she gave me the right impetus and guidance to investigate an area of research that is seemingly grey and unexplored. I am also grateful to Dr Babatunde Olofinbiyi and Mr Sunday of Ekiti-State Teaching Hospital Ado-Ekiti for their support in the data collection process. Miss Damilola Oginni and Miss Gbemisola Oginni were also very crucial in the success of my fieldwork.\n\n\nReferences\n\nPolis CB, Cox CM, Tunçalp Ö, et al.: Estimating infertility prevalence in low-to-middle-income countries: an application of a current duration approach to Demographic and Health Survey data. Hum Reprod. 2017; 32(5): 1064–1074. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlabi OJ: Socioeconomic Dynamism and the Growth of Baby Factories in Nigeria. SAGE Open. 2018; 8(2): 2158244018779115. Publisher Full Text\n\nCui W: Mother or nothing: the agony of infertility. Bull World Health Organ. 2010; 88(12): 881–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhont N, van de Wijgert J, Coene G, et al.: 'Mama and papa nothing': living with infertility among an urban population in Kigali, Rwanda. Hum Reprod. 2011; 26(3): 623–629. PubMed Abstract \| Publisher Full Text\n\nAsemota OA, Klatsky P: Access to infertility care in the developing world: the family promotion gap. Semin Reprod Med. Thieme Medical Publishers. 2015; 33(1): 17–22. PubMed Abstract \| Publisher Full Text\n\nOkonofua F, Menakaya U, Onemu SO, et al.: A case-control study of risk factors for male infertility in Nigeria Asian J Androl. 2005; 7(4): 351–361. PubMed Abstract \| Publisher Full Text\n\nFehintola AO, Fehintola FO, Ogunlaja OA, et al.: Social Meaning and Consequences of Infertility in Ogbomoso, Nigeria. Sudan Journal of Medical Sciences. 2017; 12(2): 63–77. Publisher Full Text\n\nOmosun AO, Kofoworola O: Knowledge, attitude and practice towards child adoption amongst women attending infertility clinics in Lagos State, Nigeria. Afr J Prim Health Care Fam Med. 2011; 3(1): 259. Publisher Full Text \| Free Full Text\n\nNwaoga CTN: Socio-religious implications of child adoption in Igboland South Eastern Nigeria. Mediterr J Soc Sci. 2013; 2(11): 705. Publisher Full Text\n\nOladokun A, Arulogun O, Oladokun R, et al.: Acceptability of child adoption as management option for infertility in Nigeria: evidence from focus group discussions. Afr J Reprod Health. 2009; 13(1): 79–91. PubMed Abstract\n\nOmeire CO, Iheriohanma EBJ, Osita-Njoku A, et al.: The challenges of child adoption and the emergence of baby factory in South Eastern Nigeria. Int J Educ Res. 2015; 3(8): 63–74. Reference Source\n\nSteptoe PC, Edwards RG: Birth after the reimplantation of a human embryo. Lancet. 1978; 2(8085): 366. PubMed Abstract \| Publisher Full Text\n\nGiwa-Osagie OF: The need for infertility services in the developing world: the WHO point of view. Gynecol Obstet Invest. 2004; 57(1): 58. PubMed Abstract\n\nOkonofua FE: Infertility in sub-Saharan Africa. Contemporary Obstetrics and Gynaecology for Developing Countries. 2003; 8: 128–56.\n\nOkunlola MA, Adebayo OJ, Odukogbe AA, et al.: Assessment of tubal factor contribution to female infertility in a low resource setting (southwest Nigeria): hysterosalpingography vs laparoscopy. J Obstet Gynaecol. 2005; 25(8): 803–804. PubMed Abstract \| Publisher Full Text\n\nBanerjee S, Basu S: Rent a womb: Surrogate selection, investment incentives and contracting. J Econ Behav Organ. 2009; 69(3): 260–273. Publisher Full Text\n\nCreswell JW: Standards of validation and evaluation. Qualitative inquiry and research design: choosing among five approaches. 2013; 2: 201–21. Reference Source\n\nBryman A: Social research methods. Oxford university press. 2016. Reference Source\n\nBello FA, Akinajo OR, Olayemi O: In-vitro fertilization, gamete donation and surrogacy: Perceptions of women attending an infertility clinic in Ibadan, Nigeria. Afr J Reprod Health. 2014; 18(2): 127–133. PubMed Abstract\n\nUmeora OUJ, Umeora MC, Emma-Echiegu NB, et al.: Surrogacy in Nigeria: Legal, ethical, socio cultural, psychological and religious musings. Afr J Med Health Sci. 2014; 13(2): 105–109. Publisher Full Text\n\nArmour KL: An overview of surrogacy around the world: trends, questions and ethical issues. Nurs Womens Health. 2012; 16(3): 231–236. PubMed Abstract \| Publisher Full Text\n\nDanna D: Contract Children: Questioning Surrogacy.. ibidem-Verlag/ibidem Press. 2015. Reference Source\n\nDeomampo D: Defining parents, making citizens: nationality and citizenship in transnational surrogacy. Med Anthropol. 2015; 34(3): 210–225. PubMed Abstract \| Publisher Full Text\n\nMelhuus M, Syse A: Gestational Surrogacy in Norway. Handbook of Gestational Surrogacy: International Clinical Practice and Policy Issues. 2016; 217. Publisher Full Text\n\nChatterjee P: Human Trafficking and Commercialization of Surrogacy in India. European researcher. Series A. 2014; 85(10–2): 1835–1842. Publisher Full Text\n\nWhite PM: Commercialization, Altruism, Clinical Practice: Seeking Explanation for Similarities and Differences in Californian and Canadian Gestational Surrogacy Outcomes. Womens Health Issues. 2018; 28(3): 239–250. PubMed Abstract \| Publisher Full Text\n\nTeman E: The social construction of surrogacy research: an anthropological critique of the psychosocial scholarship on surrogate motherhood. Soc Sci Med. 2008; 67(7): 1104–1112. PubMed Abstract \| Publisher Full Text\n\nNhlekisana RO: From Childhood to Womanhood: Puberty Rites of! Xoo Girls of Zutshwa. Marang: Journal of Language and Literature. 2017; 29: 31–41. Reference Source\n\nFernandez R, Alessandra F: Culture: An empirical investigation of beliefs, work, and fertility. Am Econ J-macroecon. 2009; 1(1): 146–77. Publisher Full Text\n\nCaldwell JC, Caldwell P: The cultural context of high fertility in sub-Saharan Africa. Population and development review. 1987; 13(3): 409–437. Publisher Full Text\n\nChang M: Womb for rent: India's commercial surrogacy. Harvard International Review. 2009; 31(1): 11. Reference Source\n\nTwine FW: Outsourcing the womb: Race, class and gestational surrogacy in a global market. Routledge. 2015; 118. Publisher Full Text\n\nAlabi O: Surrogacy Transcript. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.11120891.v1\n\nAlabi O: Interview schedule. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10264904.v1" }	[ { "id": "59860", "date": "20 Feb 2020", "name": "Daniela Danna", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe proposed article is based on personal interviews with professionals in health and law on the subject of surrogacy. The author takes a clear stand for “modernization”, where he considers surrogacy an ART. He presents in a favorable light the positive attitudes towards surrogacy by legal experts and gynecologists (mostly male) and in a negative light the objections expressed by traditional birth attendants (all female).\nThe objective of the article is to identify cultural obstacles to the normalization of this practice, as the author considers it a panacea for infertility, therefore positive, progressive, good. There are multiple problems with his view, the principal one is the blurring of what happens in surrogacy and the imprecision in its description, that is missing its concrete aspects. (This blurring is surprising, considering that my own contribution Contract children, a book exactly describing at the global level the phenomenon and its legal and ethical requisites, is listed in the bibliography. I have since published another article that might be of help to the author: “The subrogation of motherhood: a juridical institution putting kinship on the market”, in Salute e società, n. 2, 2019, pp. 44-591). Instead of providing a description, the author defines surrogacy as an ART or an “alternative to infertility” (p. 5), and the whole paper rests on this faulty premise.\nSurrogacy is not an ART nor a “fertility treatment” (p. 3), as the insemination in some countries is also achieved with coitus (there are documented cases in Mexico and China). It is instead the motherhood of a woman that will not be considered the legal mother – by deception of the authorities or by changes in the filiation laws. It is essentially a third way to become legal parents besides birthing (with or without recognition by the natural father, a recognition that happens automatically when the mother is married) and adoption. Therefore juridical language should be employed, as it is a juridical institution: the subrogation of motherhood. But out of simplicity I will also call this practice “surrogacy”.\nThe mother, that is – in objective language – the woman who has given birth, renounces by contract to her right to the baby (called in law the principle of mater semper certa) in exchange for money, often called “reimbursement”. Where are those “cases of altruism” that the author claims to stand on a par with the commercial version (p. 5)?\nIllegal surrogacy happens, and it is just a practice where the delivery of the baby to third persons is achieved by payment or by illegal force, but when it is regulated by law – despite usually taking place in clinics – is not at all a medical practice, but a pregnancy not essentially different from other pregnancies. The IVF can or cannot take place, and the insemination can happen outside clinics. The fact that surrogacy is clinically managed does not pertain to the core of the practice, which is the legal possibility to sell filiation by the mother to the prospective, and paying, parent(s). It is therefore wrong to state in the abstract that, before surrogacy, adoption was “the most common alternative” to legally become a mother, as adoption was the only alternative to birthing a child. The major problem with the aseptic definition of surrogacy as an ART – that is, as if it were technology – is that this definition takes for granted that the woman who gives birth will renounce her baby, will renounce her right to legally recognize her baby. But this is not inherent in the practice, which is merely based on a promise. The certainty will only come with legally binding documents: a contract that is currently invalid in most countries. She will surely give up the baby only if she is bound by a contract that is deemed legally valid – for this reason is surrogacy a juridical institution. This use of a woman’s body to obtain a son or daughter – I repeat – is in nearly all cases paid for, and the Rapporteur for the U.N. on child trafficking has already declared commercial surrogacy to be trafficking in babies, and – in her last contribution on the subject that I have read – awaited for supporters of “altruistic” surrogacy to demonstrate that such a thing really exist and is not merely a label for the same selling of commissioned newborns. The author apparently ignores the Rapporteur’s conclusion that commercial surrogacy is a violation of the human rights of the newborn, it is an indignity suffered by a mother, and becomes a violation of her human rights, too, when she changes her mind having developed a motherly relationship with her baby but cannot keep him or her because she signed a contract more than nine months before.\nBut how can the author ask others about “how surrogacy is conceptualized in Nigeria” while he has a superficial concept of surrogacy as (just) an ART? Why does he cancel out the legal debate, and does not give the reader the fundamental information of how surrogacy is legally defined in Nigeria? And if it does not appear in law (as far as I know this is the case), what are the consequences for people who may have done informal agreements? Have there ever been real cases? There are sources on forcing pregnancy on women already trafficked for the purpose of prostitution – should this not be discussed?\nHow can he claim that gynecologists have a “more informed opinion” on surrogacy (p. 5) when his own description of it is blurry and essentially wrong? Other questions that come to my mind pertain to the framing of the interviews that he conducted: Do not gynecologist talk from their own cultural point of view, like the TBAs? What is “scientific” about their opinions on surrogacy? It seems to me that the TBAs are closer to target (see end of the review).\nIn general, the interview material could be analysed in more depth and with more quotations. The author’s denomination of “modern” for surrogacy is hardly accurate, as it is well know that there are descriptions in the Bible. The author is worried about stigma creation, but what if surrogacy in itself is violence? Violence against the newborn, who is not a tabula rasa but needs his or her mother, and actively looks for her for the nourishment from her breasts? Violence against the numerous women who do not want to give up their babies after birth but are forced to do it by monetary penalties or even by the force of the State (see the cases of Baby M and Melissa Cook, among many others)? Should acts of violence be free from “stigma”? Should not surrogacy be correctly nominated for what it is: an unnecessary separation of the dyad mother-newborn because intermediaries (and doctors and legal experts, as we see in other countries) profit from the plight of infertile couples, suggesting and organizing this trafficking for their own profit? It seems to me that the explanation for the favorable opinions of doctors and legal experts that the author has found, could be grounded in their own prospect of becoming part of these agreements, organize them and profit from them.\nThere is a class dimension and an inequality basis in surrogacy that are totally foregone in the proposed article: there is no altruistic surrogacy but women in need that accept to become pregnant and give up their babies to richer couples/singles because they are paid. (Of course there is the question of consciousness, of subjectivity: many women who get paid say that this is not their primary goal, but without money they most certainly would not undergo a pregnancy for others.) Therefore there is no “purely altruistic” commitment (very few exceptions apart) as ALL the legislations canceling mater semper certa allow for “reimbursements” that can be conspicuous. Without money, there would be no surrogacy of relevance, just a few cases now and then. But the aim of the author – whether he realizes it or not – is to set up a market for newborns by obscuring what is really taking place in the countries that have introduced surrogacy.\nAnother problem with the general framework of the paper is that, even though the author laments the identification of “woman” with “mother”, he basically suggests a method for a woman to become (by means of paying another woman) a mother even if the couple is infertile. Then we are back to square one with the identification woman=mother. A true alternative would be a change in culture that, instead of working for the social acceptance and normalization of rich couples paying poor women for their babies, would work for the social acceptance of women who cannot, or do not want, to become mothers.\nI am no native English speaker, so I cannot judge the overall quality of the language, but I noted that there is sometimes a lack of grammatical concordances between verb and subject, and there might be other errors (as in my own text). I do not understand for example “data saturation”. Other minor points: at p. 3 what is “holistic” in ART procedures, that is the artificialization of reproduction, that result in babies being born with more health problems than naturally conceived babies? What is a “foundational and an experimental level” (same page)? Why is infertility considered a particular case of “sickness” (same page), when the organism that is infertile is in fact generally healthy? What is a “visual text” (p. 4)? Should “corporate” at p. 6 not be “cooperate” (interesting slip of the tongue, as the article advocates for a new market in newborns)? Finally: at p. 8: surrogacy is not “one of the most progressive ways of addressing infertility”, nor “a desirable fertility alternative”, but buying and selling newborns exploiting the poor women that are forced by need to take up pregnancy as a job (even in the US they sign surrogacy contracts for such goals as paying for the tuitions of their other children). The TBAs might express this and the refusal of such a market in the language of religion, but they are closer to reality than the various experts who stand to gain from the introduction of this juridical institution.\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": [ { "c_id": "5951", "date": "21 Sep 2020", "name": "Oluwatobi Alabi", "role": "Author Response", "response": "Hi Daniela, Thank you for your very insightful comments on this article. I find them very useful for the review and the recommendations in most of the case helped in production of the new version. However, I will like to state emphatically that I stand to benefit nothing from the commercialization of surrogacy in Nigeria like you implied in some instance. In fact, the article does not intend or aim to make a case for/against the practice of surrogacy in Nigeria. Rather, it seeks to investigate how various socio-cultural and religious factors influencing reproduction in Nigeria and south-west Nigeria in particular shape the perception of surrogacy within the studied population. Thank you again for your insightful comments." } ] }, { "id": "60862", "date": "07 Apr 2020", "name": "George Mbara", "expertise": [ "Reviewer Expertise Political Science", "International relations", "cyber security", "security studies", "African philosophy and ethics", "public governance and climate change." ], "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 article presents surrogacy as an aspect of assisted reproductive technology (ART) and explores the challenges inhibiting the acceptance of the practice among Nigerians. The study collected its data from a particular region of the country (South West) and thus, makes generalisations on the findings. Most of the respondents were collected from one ethnic group (the Yorubas) and mostly professional gynaecologists. The findings of the study were erroneously generalised as representing the entire country.\n\nNigeria is not a homogeneous country as presented in the title of this investigation. The country has over two hundred and fifty (250) ethnic nationalities domiciled within its borders1. This diversity transcends religion, culture and socio-political inclinations. Years before the 15th century, most of the ethnic groups that make up present-day Nigeria can all trace their origins to different states. Among the early states were the Benin Kingdom, the Yoruba Kingdoms, Hausa States, Nupe, and the Igbo-ukwu. Countless small states existed around the Lake Chad region which was later swallowed or dispersed during the expansion of Kanem to the North East of Lake Chad (Mbara, 2018:12). Historically, these groups were differentiated based on their socio-political organization. The Yorubas, Hausas and the Binis had a centralized administrative system while the Igbos of the East have variously been described as a “stateless” society because unlike the Hausa, Yoruba and Benin Kingdoms, they did not have a central political institution. Further, the indigenous people that inhabited the country today were profoundly divided by their historical experiences, culture, political development, and religion.\nThus, a topic like this should look at surrogacy based on the teachings and practices of a particular culture or religion. Investigating surrogacy in ‘Nigeria’ as a whole makes the study problematic given the fact that the three major tribes (Igbo, Yoruba and Hausa), for instance, have very diversified cultural and even religious beliefs/inclinations which deeply impact their social existence. The author alludes to this when he declared, “What surrogacy means and the social reception it enjoys differ across cultural and social boundaries” (p.5). For this reason, this study will best suit a particular ethnic nationality or religion in Nigeria. Just as their ethnicity and religion differ, so to their belief systems are very divergent. The influence of Western education on these ethnic nationalities intensified this division among the people leaving some very conservative (the North) and others more progressive (the South) at varying degrees. This makes surrogacy a very contentious and subjective issue.\nConsequently, the best title for this work should be “A qualitative investigation of surrogacy as a panacea for infertility among the Yoruba people of Nigeria” given the setting of the interviews coupled with the fact that “The interview schedule was translated into Yoruba, the local language of this Western region of Nigeria for the benefit of the TBAs who might not be English speakers” (p.4). With the vast size of Nigeria and the heterogeneous nature of the country, getting most of your sample population from Ekiti State will surely not give you enough grounds to caption the findings as Nigeria. Buttressing this view, Alabi (2020:5) in his results submits, “The dynamic nature of human societies coupled with varying regulatory normative codes across cultural boundaries creates the need to examine the perception of surrogacy within various cultural settings to appreciate the differences and similarities that are obtainable in the growth of the practice as a core ART process”. Thus, he cannot lump over 250 ethnic nationalities in Nigeria and discuss them as a single culture. A little search will reveal that surrogacy has been in practice in many Nigerian societies and in different forms.\nFurthermore, for a more holistic view of the subject matter of this investigation, the stakeholders for the interview should have included a Christian, a Muslim and an adherent of the African Traditional Religion (ATR). This is because Nigerians are a deeply religious people and their religion impacts their culture, lifestyles and value systems. Likewise, some custodians of culture like traditional rulers from the major ethnic groups should also constitute the stakeholders. However, the problem of ethnic minorities and the fear of domination will make it more difficult to select the cultural groups that will participate in the investigation. Thereby justifying the need to narrow down the investigation to an ethnic/religious group in the country. If the investigation is done based on the above recommendation, the author will find very diversified views on the subject matter in Nigeria.\nSurrogacy, as presented in this text (ART or fertility treatment), is quite alien to most Nigerian cultures and so, the scholar presents it as a taboo in the country as evidenced in his findings. In the first place, ART is the medical procedures used in treating the problem of infertility. Such procedures include, in vitro fertilization, cryopreservation of gametes or embryos, intracytoplasmic sperm injection, among others. I align my thoughts with the first reviewer who declared, “Surrogacy is not an ART nor a ‘fertility treatment’ (p. 3), as the insemination in some countries is also achieved with coitus (there are documented cases in Mexico and China)” (Danna, 2020:10). The relationship between surrogacy as an ART as presented in the text is shrouded in ambiguities thus leaving room for multiple interpretations. Surrogacy (renting a womb, see pages 7 & 8) has been in practice in different forms in many Nigerian cultures. In some cases, the problem of infertility is solved by the man consenting to a family member impregnating his wife on his behalf. The “sperm donor” lays no claim to the child. The impotent man plays the father figure to the child who bears his name. In other cases, it was the barren wife in a marriage that took a younger wife for herself. Correspondingly, when a man dies childless, a family member can sleep with the woman (his wife) to produce an heir for the deceased (See the Igbos of Eastern Nigeria, the Ibibios of South-south Nigeria). In these cultures, these practices are often altruistic in nature.\nIn particular, among the Igbos of Eastern Nigeria, surrogacy has been in practice in various forms like the woman-woman marriage. Commenting on this practice, Aliyu (2018: para 3) maintains: In these various societies, the wives other women married were ladies-in-waiting, surrogate mothers, and daughters-in-law… Woman-to-woman marriage allowed for greater freedom of sexuality for the wives, they could have boyfriends, anonymous men whose only duty was to supply sperm, Achebe calls them “male sperm donors”, and this was socially accepted. Any child the wives had was taken care of by their female husband and carried her name, and this was legitimate in the eyes of society.\nWoman-to-woman marriage practice did not include any sexual contact between the female husband and her wife. This was not \"lesbianism,\" since none of the women who married other women was drawn to other women either romantically or sexually. Children were the prime attraction, every woman who turned into a female husband only wanted a child considered legitimate in the eyes of society. In the same vein, Nwoko (2012) gives other examples of surrogacy among the Mbaise Igbo where a family's female children jointly paid the pride price of a younger lady after the death of their father in the name of their eldest sister, so that the new bride could procreate and raise male children to maintain the lineage of the family. Essentially, a well-behaved young man was chosen by the young bride from the kindred, but in most cases, the bedmate was chosen from the female husband's blood relationship. This had significant implications as it was aimed at maintaining the blood tie of that particular family and to prevent the young bride from polluting the family by raising children fathered by persons with strange ailments, thieves or miscreants.\nTo actualize the essence of the marriage, Nwoko (2012:76) adds: The female husband remained the sociological father of any resulting offspring. The children belonged to the lineage of her father, not to their biological father. Consequently, she played the role of the father, provider, protector and indeed all the functions and responsibilities enshrined in the patriarchal concept which included physical protection of the family and its territory, the male economic sphere, the spiritual sphere, the social sphere, etc. Surrogacy in the context of ‘renting’ a womb is still a cultural anomaly in most Nigerian cultures, but as an altruistic act, it has been around over the years. Moreover, how can surrogacy be a panacea for infertility in Nigeria? The simple meaning of panacea is “cure-all”. Surrogacy can be an option, not a panacea since there are multiple factors responsible for infertility, some are more complex than others. Various traditional treatments are available to infertile couples in Nigeria. Thus, surrogacy cannot be a panacea as the author implies. Additionally, in the abstract, 15 stakeholders were stated by the author as respondents, but in the methodology, 10 gynaecologists, 5 TBAs and 5 Legal professionals making a total of 20 respondents. Please reconcile the numbers and justify the sample size especially the 10 gynaecologists. This investigation is more of a social phenomenon than medical and should have covered custodians of some traditions/cultures. Gynaecologists can provide us with some scientific aspects of fertility treatment but not surrogacy as a cultural practice. Why were social workers not interviewed? Besides, in the data collection under methodology, the scholar declares, “The first set of questions examined surrogacy within a cultural perspective” (p.4). Who provided this information? In my opinion, the traditional rulers or elders of various cultural groups should be in the best position to provide the necessary information here. They are the custodians of the various cultures, not the TBAs. If due diligence was carried out here, the findings would have been remarkably different from what we have here.\n\n‘Interviews lasted on average of 30 minutes.’ Unnecessary repetitions.\n\nMoreover, in p.5, the author states, ‘The opinions shared by TBAs were grounded in religious and cultural beliefs, leading to opposition to its practice”. This response was perhaps predicated on the false assumption that surrogacy is an “alternative to infertility”. Besides, one can ask, what religion and what culture in Nigeria? This gives room for ambiguities and subjective interpretations. Even when it comes to religions like Christianity in Nigeria, some sects/denominations hold more radical or conservative approach to issues surrounding modern-day surrogacy (Renting a womb with pecuniary incentives). For example, the Catholics and Jehovah Witnesses will surely diverge in their views. The same applies to the Deeper lifers (radicals) and say a Baptist Christian (conservatives). Even in ATRs, the various cultures will have varying degrees of acceptance or abhorrence for surrogacy. There are also individuals in Nigeria who, as a result of Westernisation, have accepted this practice. So many young couples in Nigeria now see surrogacy as a viable option for procreation.\nIn p.6, the scholar declares, “However, infertility is regarded most times as a woman’s problem and evidence of sin and disapproval by both God and ancestors”. The influence of Christianity (Catholicism in particular) is fast changing this view among some Nigerian Catholics as Canon 1084.3 clearly states that sterility does not forbid nor invalidate a marriage (when there has been no form of deceit). A childless marriage is valid, and companionship is equally very important in marriages. In preparatory classes, intending couples are exposed to this reality. While a childless couple keeps seeking a remedy to their condition, they are made to understand that it does not invalidate/nullify their union. For this reason, a marriage contracted under the church cannot be dissolved for the reason of childlessness. This explains why you find pockets of couples who have been in the union for decades despite been childless. This does not, however, negate the fact that fertility is still a very integral part of the people/lay faithful. This investigation should look at surrogacy as a “viable” option in the case of infertility instead of a ‘panacea’ since it is an exploratory study which discovered that the practice is still very alien to the people and unacceptable. The title as it is gives the impression that surrogacy was discovered to be the ‘solution’ instead of an ‘alternative’. If I am right, the author seeks to sell the idea of surrogacy as a viable option in the case of infertility, not a total solution. The word ‘panacea’ denies other viable options available like IVF, adoption, polygamy or cases where a relative impregnating the wife of an impotent brother to preserve the bloodline, or where a woman marries another woman with the sole purpose of procreation.\nThe study pointed out the factors militating against the adoption of surrogacy as a panacea to infertility in Nigeria but failed to establish the need for surrogacy in the country thereby making the title a misnomer. To declare surrogacy a panacea requires that other alternatives were investigated and found wanting, thus necessitating the adoption of surrogacy as a “cure-all” remedy.\nConsider women who give birth through a caesarean section? Some Africans (Nigerians) still do not see them as complete women since they have not felt the pangs of birth.\nLastly, some typographical/grammatical errors were observed in the work – articles were either omitted or wrongly used. For example, subheading (p. 7): “Surrogacy reorganizing the essence of parenthood in Nigeria” (Add colon or dash after ‘Surrogacy’), “While surrogacy might be glorious news…” (p.7. rework), “and it often seen” (p.3. Rework).\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": [ { "c_id": "5952", "date": "24 Sep 2020", "name": "Oluwatobi Alabi", "role": "Author Response", "response": "Hi George, Thank you for your very insightful comments on this article. I find them very useful for the review and the recommendations in most of the case helped in the production of the new version. However, unlike you implied in some instances, the article never assumed to generalize its findings to the entire Nigeria and in no instance was Nigeria as a country presented as the study location. While the topic might have Nigeria named which was a mix up during the editorial process with the journal, the methodology gives the reader a detailed description of the specific study location in Nigeria. Likewise, as a qualitative study, it aim was not to generalize findings but provide a more detailed understanding of surrogacy within the study location. Also, the article does not intend or aim to make a case for/against the practice of surrogacy in Nigeria. Rather, it seeks to investigate how various socio-cultural and religious factors influencing reproduction in Nigeria and south-west Nigeria in particular shape the perception of surrogacy within the studied population. Thank you again for your insightful comments." } ] } ]	1	https://f1000research.com/articles/9-103
https://f1000research.com/articles/9-1496/v1	21 Dec 20	{ "type": "Research Article", "title": "Patient knowledge of fecal calprotectin in inflammatory bowel disease (IBD): An observational study in Mexico", "authors": [ "Brenda Maldonado-Arriaga", "Sergio Sandoval-Jiménez", "Juan Rodríguez-Silverio", "Sofía Lizeth Alcaráz- Estrada", "Tomás Cortés-Espinosa", "Rebeca Pérez-Cabeza de Vaca", "Jonathan Shaw", "Paul Mondragón-Terán", "Cecilia Hernández-Cortez", "Juan Antonio Suárez-Cuenca", "Graciela Castro-Escarpulli", "Brenda Maldonado-Arriaga", "Sergio Sandoval-Jiménez", "Juan Rodríguez-Silverio", "Sofía Lizeth Alcaráz- Estrada", "Tomás Cortés-Espinosa", "Rebeca Pérez-Cabeza de Vaca", "Jonathan Shaw", "Paul Mondragón-Terán", "Cecilia Hernández-Cortez" ], "abstract": "Background: Fecal calprotectin (FC) can be a valuable tool to optimize health care for patients with inflammatory bowel disease (IBD). The objective of this observational study was to determine the level of knowledge of the FC test in Mexican patients with IBD. Methods: A self-report questionnaire was distributed via Facebook to patients with IBD. The survey consisted of 15 questions in two categories: the first category assessed knowledge of IBD diagnosis, and the second category assessed knowledge of the FC test. Results: In total, 460 patients with IBD participated, of which 83.9% (386) had ulcerative colitis (UC) and 16.0% (74) had Crohn’s disease (CD). Regarding IBD diagnosis, 41.9% of participants stated that they did not know of a non-invasive test for fecal matter to identify inflammation of the colon. Regarding the FC test, 57.5% (UC) and 58.1% (CD) stated that they did not know about the test. Additionally, 65.8% (UC) and 51.3% (CD) of participants stated that they had never received the FC test and 82.6% (UC) and 77.0% (CD) recognized that the FC test was difficult to access in their medical practice. Furthermore, 66% (UC) and 52.7% (CD) of participants noted that their specialist doctor had never suggested the FC test to them, yet 89.1% (UC) and 87.8% (CD) stated that they would prefer FC analysis for their IBD follow-up assessments. Conclusions: There is little knowledge of the FC biomarker among Mexican patients with IBD. This suggests the need for greater dissemination of its use and scope as a biomarker in IBD.", "keywords": [ "Inflammatory bowel disease", "Fecal calprotectin", "Level of knowledge", "Ulcerative colitis", "Crohn’s disease." ], "content": "Abbreviations\n\nIBD, Inflammatory bowel diseases; FC, Fecal Calprotectin; UC, Ulcerative Colitis; CD, Crohn’s Disease.\n\n\nIntroduction\n\nInflammatory bowel disease (IBD) is defined as a group of chronic inflammatory disorders of unknown cause that affect the gastrointestinal tract and includes two diseases: Crohn's disease (CD) and Ulcerative Colitis (UC). These diseases are defined according to clinical, radiological, endoscopic, and histological criteria, and are characterized by chronic relapses, which present in outbreaks (active phases) and periods of remission (inactive phases)1.\n\nCurrently, gastrointestinal endoscopy, histological examination of biopsies, and contrast imaging are mandatory techniques for the diagnosis and evaluation of IBD activity. Due to the complexity of the disease and the need for a multidisciplinary approach, diagnosing and treating the condition is a challenge for medical specialists. Patients generally take five to ten years to be diagnosed, which implies that treatments are applied late and therefore, the quality of life of patients with IBD is directly affected2. Patients with IBD also worry about a timely diagnosis, as well as complications of the disease and secondary conditions3,4.\n\nUnfortunately, endoscopy is not easily accessible in many rural areas of the Mexican Republic and when performed are sometimes unnecessary, a problem compounded by the cost of the procedure. Recently, in different international institutions the use of biomarkers or biological markers has been routinely applied. The most prominent biomarker used for IBD is fecal calprotectin (FC), which is a protein derived from neutrophils, released in the feces in response to inflammation of the intestinal mucosa. FC levels have been found to be associated with endoscopic severity, prediction of mucosal healing, and prediction of relapse; therefore, it is a useful biomarker for monitoring patient response to treatment5,6. Additionally, a primary objective of the treatment of IBD is to improve the patient's health-related quality of life regardless of the type of therapy used. Patient knowledge of alternative methods for diagnosis and follow-up of IBD leads to increased well-being of the patients and to a reduction in overall healthcare costs7.\n\nDespite the clinical utility of FC in IBD, the lack of dissemination and knowledge among patients could limit its acceptance and use as a clinical test. Currently in Mexico, there are no official statistics on the use of FC as a diagnostic marker and follow-up test for IBD. Consequently, the present study aims to assess knowledge regarding the use of FC as a diagnostic test among patients with IBD.\n\n\nMethods\n\nThis study used an observational cross-sectional design. Through the “Fundación Vivir con Crohn y CUCI A.C” (a non-profit foundation that offers information and support to people with IBD in Mexico), an electronic questionnaire was disseminated with questions aimed at assessing participant knowledge of FC and its use in IBD treatment. The questionnaire was developed by “Fundación Vivir con Crohn y CUCI A.C”, gastroenterologists and health advocates of Hospital C.M.N. \"20 de Noviembre\", ISSSTE, in 2020. Two gastroenterologists in the group validated the 15 questions. A pilot test of the questionnaire was carried out with 5% of the population (23 people with IBD who attended the hospital), to ensure the validity of the questionnaire. The questionnaire was anonymous and voluntary, and consisted of 15 basic questions about the patient's social and demographic data, as well as questions related to knowledge of the FC test8. A link to the questionnaire was posted on the “Fundación Vivir con Crohn y CUCI A.C”’s Facebook site on the 3rd July 2020. The survey was hosted on Google Forms.\n\nInclusion criteria were Mexican adults, aged 18 to 45 years old and diagnosed with inflammatory bowel disease. The sample size was calculated using the central limit theorem9 with a margin of error of 5% and a confidence interval of 95%.\n\nA database was produced with Excel v19.0. The period of data collection was August to September 2020, where the following data were collected from each patient: 1) Type of IBD (UC and CD), 2) Sex of the patient, 3) Health System where they were treated, 4) Are you familiar with the non-invasive stool test to see if the intestine is inflamed? 5) Do you know of any test to determine if the disease is in the active phase? 6) Do you know of any test to determine if the disease is in the remission phase? 7) Do you consider that colonoscopy is necessary for the follow-up of IBD? 8) Do you know the FC test? 9) Have you ever had the FC test? 10) Does your medical unit have the FC test? 11) How accessible is it to perform the FC test in your medical unit? 12) How often does the specialist doctor suggest that you take the FC test? 13) Do you know approximately the cost of the FC test? 14) Do you think the price of the FC test is lower than the endoscopy study?, 15) For the follow-up of the disease would you prefer to perform Colonoscopy or the FC test?\n\nInitially, a univariate analysis was carried out, where absolute and relative frequencies were used for qualitative variables and mean and standard deviation were used for quantitative variables. All tests were performed with Excel v19.0.\n\nThis study was based on the guidelines for clinical research established in the Declaration of Helsinki, in the Ministry of Health, and in the “Centro Médico Nacional, 20 de Noviembre, ISSSTE”. The Institutional Biosafety, Ethics and Research Committee, approved this study (number 033.2017). Written informed consent was obtained from each patient. As well as in the Biosafety, Ethics and Institutional Research Committees, patient information was deidentified and data were stored in a confidential registry of Hospital C.M.N. \"20 de Noviembre\", ISSSTE.\n\n\nResults\n\nIn total, 460 patients with a diagnosis of IBD were obtained in 29 states of the Mexican Republic10. The following states and cities of the Mexican Republic were the ones that provided the information: Mexico City, Mexico City (131); State of Mexico, Toluca de Lerdo (68); Monterrey, Nuevo Leon (36); Guadalajara, Jalisco (32); Coahuila, Saltillo (20); Leon, Guanajuato (16); Sinaloa, Culiacán (16); Puebla, Puebla (15); Morelos, Cuernavaca (12); Veracruz, Xalapa (12); Baja California, Mexicali (11); Chihuahua, Chihuahua (11); Pachuca, Hidalgo (9); Querétaro, Querétaro (8); Ciudad Victoria, Tamaulipas (8); Hermosillo, Sonora (7); Durango, Durango (6); Chilpancingo, Guerrero (5); Nayarit, Tepic (5); Oaxaca, Oaxaca (5); Quintana Roo, Chetumal (5); San Luis Potosí, San Luis Potosí (5); Tlaxcala, Tlaxcala (5); Yucatán, Mérida (3); Aguascalientes, Aguascalientes (2); Campeche, Campeche (2); Michoacán, Morelia (2); Zacatecas, Zacatecas (2); Tabasco, Villahermosa (1) (Figure 1).\n\nThe colours represent the number of participants recruited in each state. Mexico City and the State of Mexico (dark red) presented the greatest number of participants (over 40) with IBD. Monterrey and Guadalajara (red) presented 21 to 40 participants with IBD. The states of Coahuila, León and Sinaloa (orange) presented 21 to 40 participants with IBD. Puebla, Morelos, Veracruz, Baja Calicfornia and Chihuahua (yellow) presented 11 to 15 participants with IBD. Pachuca, Querétaro, Ciudad Victoria, Hermosillo and Durango (dark green) presented 6 to 10 participants with IBD. Chilpancingo, Nayarit, Oaxaca, Quintana Roo, San Luis Potosí, Tlaxacala, Yucatán, Aguascalientes, Campeche, Michoacán, Zacatecas and Tabasco (light green) presented 1 to 5 participants with IBD.\n\nTable 1 shows the general characteristics of the study participants. Of the 460 patients who participated in the questionnaire, there was a greater number of patients with a diagnosis of ulcerative colitis (386; 83.9%) than Crohn’s disease (74, 16.0%), and a predominance of women (327, 71%) over men (133, 28.9%), as well as greater medical attention in the public health sector.\n\nData are presented as number (%), and median/±\n\nIt was observed that more than 50% of participants were not aware of any non-invasive stool test (i.e. not endoscopy) to test whether the intestine is inflamed. Additionally, more than 50% stated that they did not know of any test to differentiate whether the disease is in the active or remission phase. Moreover, 90% of participants stated that colonoscopy was necessary for the follow-up of IBD (Table 2).\n\nData are presented as number (%)\n\nIt was observed that more than 50% of participants had no knowledge of the FC test and have never had it. Furthermore, 64% of participants did not know if their medical unit has the FC test and 83% consider the test hard to access in their medical unit. Additionally, more than 65% of the participants stated that they had never been offered the FC test. Despite this, almost 90% of patients said they would prefer to use the FC test to monitor their condition (Table 3).\n\nData are presented as number (%)\n\n\nDiscussion\n\nTo ensure high quality care for patients with a chronic disease, it is important for the patient to have adequate information on their diagnosis, treatment and follow-up, and for the doctor to inform and discuss different options with the patient. Determining patient knowledge of their disease can help reduce costs in the health sector and at the same time improve the quality of life of patients. Assessing knowledge of fecal calprotectin (CF) in patients with IBD is, therefore, important to improve patient care for this chronic disease. This study is the first to our knowledge to evaluate this in Mexico.\n\nParticipants were recruited through the “Fundación Vivir con CU y Crohn SA”, and 460 patients with a diagnosis of IBD were surveyed, of which, 83.9% were diagnosed with UC and 16.0% with CD. The predominance of UC over CD is similar to that reported in other countries in Asia and Latin American countries, such as Colombia7,11.\n\nIn the first category of questioning about the participants’ knowledge of IBD tests, participants mostly reported to not know of any test for the state of the disease. This shows that the majority of participants were uninformed, which can lead to a deterioration in the quality of life12. When living with a chronic disease, it is essential to know about advances in medicine, to know the signs and symptoms, and how to better diagnose and monitor the disease to improve quality of life. The benefits of staying properly informed include awareness of useful tools and tests to diagnose and prevent the advancement of the disease11,13,14.\n\nMore than 90% of the participants stated that colonoscopy is necessary for the follow-up of IBD. Colonoscopy can be used in the initial differential diagnosis, in the surveillance of carcinoma, and to evaluate abnormalities on imaging tests. However, undergoing a colonoscopy is invasive, expensive, and intolerable for some patients. In other studies, patients have claimed to have anxiety before and during the colonoscopy, given potential risks such as intestinal perforation. Colonoscopy has also been associated with several unfavorable outcomes, including missed diagnoses and avoidable repeat procedures15,16. In addition, it is difficult to perform an endoscopic evaluation when there is injury to the intestinal mucosa, a frequent problem in IBD17–19.\n\nIn the second part of the questionnaire to identify the degree of knowledge of the CF test, more than 50% of the population in both groups answered that they had no knowledge of the FC test. They also said that the FC test had never been performed on them, and that their specialist doctor had never suggested performing the FC test. This highlights the scarcity of knowledge regarding this test in treating patients with IBD, and that the doctor-patient relationship seems deficient. The doctor must inform the patient of their pathology, the procedures to follow, the treatment possibilities, eventual healing and, in general, must adhere to correct clinical practice to improve the information perceived by patients20,21.\n\nIn addition, it must be clear that the only effective information is that provided by the health professional before the intervention or treatment in question. Adequate information should be provided sufficiently in advance and be honest and easy to understand, so that the patient can make an informed decision22. That is why the dissemination of information to the patient, including the benefits and disadvantages of any potential procedure should be made known23.\n\nThe study participants were questioned about the accessibility of the FC test in their medical unit. More than 70% stated that it was difficult to access this test, and in contrast, more than 50% of the population reported not knowing whether this test is available in their medical unit. The Mexican Consensus for the diagnosis and treatment of IBD suggests that the FC test is useful to evaluate the activity of IBD. The test’s response to medical treatment correlates with mucosal scarring or endoscopic remission, and is a good predictor of relapse, so access should be easily available in any medical unit24. Furthermore, expert guidelines suggest that disease activity should be reassessed every 3 to 6 months25, therefore, it would be important for public and private medical units to have the FC test for regular cost-effective testing17,26.\n\nIn questions about the cost of FC, more than 60% of participants stated that they did not know the cost of the test, and more than 60% believed that the price of FC is less than a colonoscopy. On the contrary, a study carried out by Motaganahalli et al., in 2019, suggests that the introduction of the FC test in the routine health care of IBD not only helps the patient, but is potentially cheaper than a colonoscopy27. The use of colonoscopies could be reduced by more than 80% if the FC test is used as the first approach28. In addition, the use of the FC test over a colonoscopy would be more profitable, indirectly, for the patient, since colonoscopies cause loss of productivity and absence from work, require sedation and attendance at a medical care center29,30.\n\nFinally, the participants were questioned about which test they would prefer to carry out for an IBD follow-up appointment. More than 80% stated that they would prefer the FC test. FC could not only be useful in the differentiation of patients with active IBD and those in remission, but it also correlates well with the degree of inflammatory activity evaluated by clinical indices such as endoscopy and histology, and predicts clinical relapse, as well as the mucosal healing and postoperative recurrence31,32. Other authors have shown that FC levels are associated with the presence of histological alterations in endoscopic biopsies, being lower in the absence of anatomopathological inflammation33–35. In different cohorts it has also been reported that the FC test is able to distinguish IBD from Irritable Bowel Syndrome36,37. Therefore, the use of an alternative non-invasive biomarker such as FC that can accurately differentiate functional or organic diseases, detect inflammation of the intestinal mucosa, and monitor disease activity to avoid invasive and costly testing could be highly useful38,39.\n\nAssessing patient knowledge of non-invasive tests such as FC in patients with IBD may help to improve patient care as well as a reduction in medical costs during follow-up of the disease. This study suggests that knowledge of such tests can be improved with better communication with the medical personnel. The limitations of the study were that there was a much greater number of participants with ulcerative colitis than Crohn’s disease, and the patients were only evaluated cross-sectionally.\n\nIn general, patients with chronic diseases should know about the tests that help to diagnose and monitor their disease. For IBD, assessing a biological biomarker, such as FC, is more specific than clinical indices, less expensive, more comfortable than endoscopic monitoring, and reduces the need for endoscopic examinations. Therefore, patients with IBD should be given the correct information on the use of FC which could be a valid alternative to evaluate the response to treatment and reduce the number of colonoscopies performed. To improve knowledge transfer to patients, we suggest improving accessibility to services, creating comprehensive care units, having medical specialists inform their patients about management of the disease, promoting medical research on the disease, and likewise, raising more awareness in society about IBD.\n\n\nConclusion\n\nUsing the application of a questionnaire, we evaluated the knowledge of patients with IBD about the diagnostic tests that are required for the diagnosis and follow-up of the disease. There was a lack of awareness of the effective and low-cost Fecal Calprotectin test in the participants. In order for patients to make informed decisions regarding the management of their disease, doctors should provide adequate and timely information about the different options available.\n\n\nData availability\n\nHarvard Dataverse: Patient knowledge of fecal calprotectin in inflammatory bowel disease (IBD): An observational study in Mexico. https://doi.org/10.7910/DVN/AHPJOF10\n\nThis project contains the following underlying data:\n\n- Database Maldonado et al.tab. (Excel spreadsheet of questionnaire responses)\n\nHarvard Dataverse: Patient knowledge of fecal calprotectin in inflammatory bowel disease (IBD): An observational study in Mexico. https://doi.org/10.7910/DVN/NOOUQF8\n\nThis project contains the following extended data:\n\n- Questionnaire in English\n\n- Questionnaire in Spanish\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 for publication of the participants’ details was obtained from participants.", "appendix": "Acknowledgments\n\nWe would like to thank the “Fundación Vivir con Crohn y CUCI A.C”, especially Beatriz Capdevielle Gómez, Cristina Guadalupe Rivera Guzmán and Fátima Rangel Vargas. Finally, thank you to Sofia Mulia for kindly correcting the style of the manuscript.\n\n\nReferences\n\nGuan Q: A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Disease. J Immunol Res. 2019; 2019: 7247238. 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Rev Med Chil. 2013; 141(12): 1578–83. Publisher Full Text\n\nElnawsra O, Fok I, Sparrow M, et al.: Faecal calprotectin: current usage and perceived beneficial effects of third-party funding on rates of colonoscopy by Australian gastroenterologists. Intern Med J. 2016; 46(5): 590–5. PubMed Abstract \| Publisher Full Text\n\nTremaine WJ: Nine medico-legal pitfalls in inflammatory bowel disease in the United States. Curr Drug Targets. 2014; 15(11): 1039–41. PubMed Abstract \| Publisher Full Text\n\nPerrenoud B, Velonaki VS, Bodenmann P, et al.: The effectiveness of health literacy interventions on the informed consent process of health care users: a systematic review protocol. JBI Database System Rev Implement Rep. 2015; 13(10): 82–94. PubMed Abstract \| Publisher Full Text\n\nYamamoto-Furusho JK, Gutiérrez-Grobe Y, López-Gómez JG, et al.: Grupo del Consenso Mexicano de Colitis Ulcerosa Crónica Idiopática. The Mexican consensus on the diagnosis and treatment of ulcerative colitis. Consenso mexicano para el diagnóstico y tratamiento de la colitis ulcerosa crónica idiopática. Rev Gastroenterol Mex. 2018; 83(2): 144–167. PubMed Abstract \| Publisher Full Text\n\nZhang W, Wong CH, Chavannes M, et al.: Cost-effectiveness of faecal calprotectin used in primary care in the diagnosis of inflammatory bowel disease. BMJ Open. 2019; 9(4): e027043. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoschetti G, Laidet M, Moussata D, et al.: Levels of Fecal Calprotectin Are Associated With the Severity of Postoperative Endoscopic Recurrence in Asymptomatic Patients With Crohn's Disease. Am J Gastroenterol. 2015; 110(6): 865–72. PubMed Abstract \| Publisher Full Text\n\nWright EK, Kamm MA, De Cruz P, et al.: Measurement of fecal calprotectin improves monitoring and detection of recurrence of Crohn's disease after surgery. Gastroenterology. 2015; 148(5): 938–947. 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PubMed Abstract \| Free Full Text" }	[ { "id": "76438", "date": "06 Jan 2021", "name": "Javier P Gisbert", "expertise": [ "Reviewer Expertise Inflammatory bowel 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\nTITLE\nOK.\n\nABSTRACT\nOK.\n\nINTRODUCTION\nOK.\n\nMETHODS\nIt seems that the questionnaire had been previously used in another study (as a reference is provided for it). Was it previously validated? Please clarify this.\nAs the questionnaire was posted on Facebook site, there may be a bias for including only patients with internet access (which could be associated, for example, with younger age, higher educational status, etc.). This potential limitation should be discussed in the Discussion section.\nIn this respect, inclusion criteria were Mexican adults, aged 18 to 45 years old. Please justify this. This could induce another bias, as older patients could have lower internet access. Therefore, the results of the present study could have been biased to more favorable findings. This potential limitation should also be discussed in the Discussion section.\nIt is stated that the sample size was calculated using the central limit theorem with a margin of error of 5% and a confidence interval of 95%. However, more details should be provided, and the calculated sample size should be given.\nStatistical analysis: It is stated that “Initially, a univariate analysis was carried out”. This seems to imply that a multivariate analysis was also performed, but I cannot see any information regarding it.\n\nRESULTS\nOf the 460 patients who participated in the questionnaire, there was a much greater number of patients with a diagnosis of ulcerative colitis (386; 83.9%) than Crohn’s disease (74, 16.0%). Even though the authors point out that “The predominance of UC over CD is similar to that reported in other countries in Asia and Latin American countries, such as Colombia”, and they comment in the Discussion section that “The limitations of the study were that there was a much greater number of participants with ulcerative colitis than Crohn’s disease”, these explanations seem to be insufficient. Please try to explain better this possible bias.\nThe same is valid for the predominance of women (327, 71%) over men (133, 28.9%), please try to explain this misbalance.\n\nDISCUSSION\nIt is repeatedly stated that: “The majority of participants were uninformed, which can lead to a deterioration in the quality of life”. And that “They also said that the FC test had never been performed on them and that their specialist doctor had never suggested performing the FC test. This highlights the scarcity of knowledge regarding this test in treating patients with IBD, and that the doctor-patient relationship seems deficient”. However, it should also be clarified that the lack of knowledge of the patient does not necessarily imply that the doctor has not informed the patient correctly, since it is possible that the determination of FC is simply not indicated in them. In other words, it is clear that FC is not always indicated in all patients, and for those patients, this information could be correctly omitted. Please clarify this in the Discussion section.\n\nREFERENCES\nOK.\n\nTABLES\nOK.\n\nFIGURES\nOK.\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": [ { "c_id": "6291", "date": "05 Feb 2021", "name": "Brenda Maldonado", "role": "Author Response", "response": "Dear Reviewer 1: METHODS Reviewer 1: It seems that the questionnaire had been previously used in another study (as a reference is provided for it). Was it previously validated? Please clarify this. Answer: Thanks for the comment. The questionnaire was developed by “Fundación Vivir con Crohn y CUCI A.C”, gastroenterologists and health advocates of Hospital C.M.N. \"20 de Noviembre\", ISSSTE, in 2020. It is an original questionnaire, without prior use. Two gastroenterologists in the group validated the 15 questions. A pilot test of the questionnaire was carried out with 5% of the population (23 people with IBD who attended the hospital), to ensure the validity of the questionnaire. Reviewer 1: As the questionnaire was posted on Facebook site, there may be a bias for including only patients with internet access (which could be associated, for example, with younger age, higher educational status, etc.). This potential limitation should be discussed in the Discussion section. Answer: Thank you for the suggestion. It will be added as a limitation in the discussion. ……Although there is growing evidence to suggest that Facebook is a useful recruiting tool and therefore its use should be considered when implementing future health research. However, one of the important limitations of this study is Internet access…. Whitaker, C., Stevelink, S., & Fear, N. (2017). The Use of Facebook in Recruiting Participants for Health Research Purposes: A Systematic Review. Journal of medical Internet research, 19(8), e290. https://doi.org/10.2196/jmir.7071 Reviewer 1: In this respect, inclusion criteria were Mexican adults, aged 18 to 45 years old. Please justify this. This could induce another bias, as older patients could have lower internet access. Therefore, the results of the present study could have been biased to more favorable findings. This potential limitation should also be discussed in the Discussion section. Answer: Thanks for the suggestion. The inclusion criterion based on the age of 18 to 45 years of age, was chosen because in previous studies it is presented primarily to people of productive age (Sairenji, T., Collins, K. L., & Evans, D. V. (2017). An Update on Inflammatory Bowel Disease. Primary care, 44(4), 673–692. https://doi.org/10.1016/j.pop.2017.07.010. Kamp, K., Dudley-Brown, S., Heitkemper, M., Wyatt, G., & Given, B. (2020). Symptoms among emerging adults with inflammatory bowel disease: a descriptive study. Research in nursing & health, 43(1), 48–55. https://doi.org/10.1002/nur.21985) Reviewer 1: It is stated that the sample size was calculated using the central limit theorem with a margin of error of 5% and a confidence interval of 95%. However, more details should be provided, and the calculated sample size should be given. Answer: According to a previous study by Yamamoto et al., 2015, the prevalence of IBD in Mexico is 150,000, and considering an expected prevalence difference and an acceptable error of 5%, the following calculations were made: N=Zα22p1-pd2 N = Sample size calculation; Zα / 2 = Z value of the alpha error with 95% confidence, assigning alpha = 0.05; P = expected population prevalence for the event under study (Yamamoto et al., 2015); d = Difference between the expected population prevalence value and the acceptable error. Values: Zα / 2 = 1.96; p = 0.38; d = 0.30. N= 1.9620.381-0.380.302= 384 This means that if you survey 384 people, the actual data you are looking for will be 95% of the time in the ± 5% range relative to the data you see in the survey. Reviewer 1: Statistical analysis: It is stated that “Initially, a univariate analysis was carried out”. This seems to imply that a multivariate analysis was also performed, but I cannot see any information regarding it. Answer: Thank you for your comment. Only univariate analysis was performed RESULTS Of the 460 patients who participated in the questionnaire, there was a much greater number of patients with a diagnosis of ulcerative colitis (386; 83.9%) than Crohn’s disease (74, 16.0%). Even though the authors point out that “The predominance of UC over CD is similar to that reported in other countries in Asia and Latin American countries, such as Colombia”, and they comment in the Discussion section that “The limitations of the study were that there was a much greater number of participants with ulcerative colitis than Crohn’s disease”, these explanations seem to be insufficient. Please try to explain better this possible bias. Answer: Thanks for the suggestion. We add the explanation “The limitations of the study were that there was a much greater number of participants with ulcerative colitis than Crohn’s disease”. Although the trend of IBD is similar to other countries, the lack of epidemiological data in Mexico is still a challenge to understand the true number of IBD cases. Yamamoto-Furusho, J. K., Bosques-Padilla, F. J., Charúa-Guindic, L., Cortés-Espinosa, T., Miranda-Cordero, R. M., Saez, A., & Ledesma-Osorio, Y. (2020). Inflammatory bowel disease in Mexico: Epidemiology, burden of disease, and treatment trends. Epidemiología, carga de la enfermedad y tendencias de tratamiento de la enfermedad inflamatoria intestinal en México. Revista de gastroenterologia de Mexico, 85(3), 246–256. https://doi.org/10.1016/j.rgmx.2019.07.008 The same is valid for the predominance of women (327, 71%) over men (133, 28.9%), please try to explain this misbalance. Answer: Thanks for the suggestion. We add the explanation The predominance of women (327, 71%) over men (133, 28.9%), this imbalance may be due to environmental factors such as the use of oral contraceptives, psychosocial stress, dietary factors, among others. On the other hand, it has been described that the use of social networks is more frequent in women than in men, so it can also be attributed that there is a greater number of women. Grosberg, D., Grinvald, H., Reuveni, H., & Magnezi, R. (2016). Frequent Surfing on Social Health Networks is Associated With Increased Knowledge and Patient Health Activation. Journal of medical Internet research, 18(8), e212. https://doi.org/10.2196/jmir.5832 Algethmi, W., Baumann, C., Alnajjar, W., Sroji, A., Alsahafi, M., Jawa, H., Qari, Y., Peyrin-Biroulet, L., Saadah, O. I., & Mosli, M. (2020). Environmental exposures and the risk of inflammatory bowel disease: a case-control study from Saudi Arabia. European journal of gastroenterology & hepatology, 32(3), 358–364. https://doi.org/10.1097/MEG.0000000000001619 Wang, X., Fan, X., Deng, H., Zhang, X., Zhang, K., Xu, J., Li, N., Han, Q., & Liu, Z. (2019). Use of oral contraceptives and risk of ulcerative colitis - A systematic review and meta-analysis. Pharmacological research, 139, 367–374. https://doi.org/10.1016/j.phrs.2018.11.036 Ho, S. M., Lewis, J. D., Mayer, E. A., Plevy, S. E., Chuang, E., Rappaport, S. M., Croitoru, K., Korzenik, J. R., Krischer, J., Hyams, J. S., Judson, R., Kellis, M., Jerrett, M., Miller, G. W., Grant, M. L., Shtraizent, N., Honig, G., Hurtado-Lorenzo, A., & Wu, G. D. (2019). Challenges in IBD Research: Environmental Triggers. Inflammatory bowel diseases, 25(Suppl 2), S13–S23. https://doi.org/10.1093/ibd/izz076 DISCUSSION It is repeatedly stated that: “The majority of participants were uninformed, which can lead to a deterioration in the quality of life”. And that “They also said that the FC test had never been performed on them and that their specialist doctor had never suggested performing the FC test. This highlights the scarcity of knowledge regarding this test in treating patients with IBD, and that the doctor-patient relationship seems deficient”. However, it should also be clarified that the lack of knowledge of the patient does not necessarily imply that the doctor has not informed the patient correctly, since it is possible that the determination of FC is simply not indicated in them. In other words, it is clear that FC is not always indicated in all patients, and for those patients, this information could be correctly omitted. Please clarify this in the Discussion section. Answer: Thanks for the comment. We add the explanation. This information cannot necessarily be omitted, since clinical guidelines such as that of European Crohn’s and Colitis Organization [ECCO] the European Society of Gastrointestinal, Abdominal Radiology [ESGAR], British Society of Gastroenterology consensus guidelines and Mexican guidelines for the Diagnosis and Treatment of IBD in the Adult Population, recommend the use of the fecal calprotectin test in various clinical settings, including initial diagnosis, relapse diagnosis and response to treatment. Maaser, C., Sturm, A., Vavricka, S. R., Kucharzik, T., Fiorino, G., Annese, V., Calabrese, E., Baumgart, D. C., Bettenworth, D., Borralho Nunes, P., Burisch, J., Castiglione, F., Eliakim, R., Ellul, P., González-Lama, Y., Gordon, H., Halligan, S., Katsanos, K., Kopylov, U., Kotze, P. G., … European Crohn’s and Colitis Organisation [ECCO] and the European Society of Gastrointestinal and Abdominal Radiology [ESGAR] (2019). ECCO-ESGAR Guideline for Diagnostic Assessment in IBD Part 1: Initial diagnosis, monitoring of known IBD, detection of complications. Journal of Crohn's & colitis, 13(2), 144–164. https://doi.org/10.1093/ecco-jcc/jjy113 Cannatelli, R., Bazarova, A., Zardo, D., Nardone, O. M., Shivaji, U., Smith, S., Gkoutos, G., Ricci, C., Gui, X. S., Ghosh, S., & Iacucci, M. (2020). Fecal Calprotectin Thresholds to Predict Endoscopic Remission Using Advanced Optical Enhancement Techniques and Histological Remission in IBD Patients. Inflammatory bowel diseases, izaa163. Advance online publication. https://doi.org/10.1093/ibd/izaa163 Krzystek-Korpacka, M., Kempiński, R., Bromke, M., & Neubauer, K. (2020). Biochemical Biomarkers of Mucosal Healing for Inflammatory Bowel Disease in Adults. Diagnostics (Basel, Switzerland), 10(6), 367. https://doi.org/10.3390/diagnostics10060367 Ricciuto, A., & Griffiths, A. M. (2019). Clinical value of fecal calprotectin. Critical reviews in clinical laboratory sciences, 56(5), 307–320. https://doi.org/10.1080/10408363.2019.1619159 We greatly appreciate every suggestion." } ] } ]	1	https://f1000research.com/articles/9-1496
https://f1000research.com/articles/10-78/v1	05 Feb 21	{ "type": "Research Article", "title": "Effect of dietary protein level on growth, food utilization, food conversion and survival rate of giant trevally (Caranx ignobilis)", "authors": [ "Abdullah A. Muhammadar", "Firdus Firdus", "Zainal A. Muchlisin", "Samadi Samadi", "Muhammad A. Sarong", "Boihaqi Boihaqi", "Satria Sartira", "Ibnu Sahidir", "Agung Setia Batubara", "Firdus Firdus", "Zainal A. Muchlisin", "Samadi Samadi", "Muhammad A. Sarong", "Boihaqi Boihaqi", "Satria Sartira", "Ibnu Sahidir", "Agung Setia Batubara" ], "abstract": "Background: Proper feed formulation is required for successful fish farming activities. Therefore, it is necessary for fish feed to provide optimal growth so that the cultivation business generates profits. Currently, there is very limited information about the appropriate feed for Caranx ignobilis, causing problems with its development. This study aims to provide feed with different protein levels to C. ignobilis. Methods: We will examine the protein levels’ effects on the daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE), and survival rate (SR). This research was conducted for 35 days, from June to October 2017, at the Center Brackiswater Aquaculture Development (BPBAP) Ujung Batee, Ministry of Marine Affairs and Fisheries, Aceh Besar, Indonesia. This study used a completely randomized design method, with five treatment levels (30%, 40%, 50%, 60%, and 70% protein feed) and four replications. Results: The results showed that feeding with different proteins on C. ignobilis had a significant effect on the mean values of DGR, SGR, AGR, FCR, FE, and SR. The 50% protein feed gave the best results for C. ignobilis, with a mean DGR value of 0.267 ± 0.005 g / day, a mean SGR of 1.722 ± 0.030% / day, a mean AGR of 0.081 ± 0.003 cm/day, a mean FCR of 1.290, a mean FE 77.755% and a mean SR was 86.667%. Conclusions: Furthermore, feed treatment with increased protein content between 30%–50% has a positive correlation with the growth of C. ignobilis. However, the ability to grow fish will decrease if the feed protein content is >50%.", "keywords": [ "Caranx ignobilis", "protein", "growth", "ratio", "efficiency" ], "content": "Introduction\n\nGiant trevally (Caranx ignobilis) is a reef fish species with a high economic value1 because it is in very high demand in domestic and international markets, with a price range of IDR 42,000–80,000 / kg2,3. Furthermore, C. ignobilis does not have muscles between muscles, so it is easy to consume4. Based on these advantages, C. ignobilis is exploited for consumption, economy, and recreation in various ways, including the use of destructive fishing gear (include e.g. bottom trawling, cyanide fishing, dynamite fishing, and ghost fishing)5. Massive coral damage due to environmental damage and the effects of global warming also puts pressure on the species population6. Therefore, it is necessary to conserve C. ignobilis through the domestication process so that numbers caught in nature can be reduced by diverting fishermen to C. ignobilis cultivation activities.\n\nFish that are eligible to become cultivated commodities must meet criteria such as economic value, desirability, fast growth, resistance to disease, and ease of domestication7–10. C. ignobilis has several advantages, such as being euryhaline so that it can live in a salinity range of 15–32 ppt11, it can live in conditions of high density(up to 150 individuals / m2 12), fast growth13, economic value and no history of disease9. Based on these advantages, C. ignobilis can be cultivated in ponds and floating net cages andseveral fishermen in Aceh, Indonesia, have started cultivating this species in with this method14. However, there are several obstacles in developing cultivation, such as a lack of availability of commercial feed with the appropriate protein content. Therefore, we need a study on feed protein that can provide optimal growth in this fish species.\n\nIn cultivation activities, growth is a determining factor for success. The problem that is often faced by fish farming is the low value of protein, so cultivating fish is not optimal. According to Subandiyono and Hastuti15, protein has various roles and functions, including brave protein as a body structure, such as collagen, which is a fibrous connective tissue and has a solid structure to form fish muscles. Fish can grow optimally if protein needs are met. Protein has a role in determining the fish growth process because most of the fish’s body (45–75% dry weight) consists of protein16. Also, protein is an efficient source of energy for aquatic animals, especially carnivorous fish17.\n\n\nMethods\n\nThis research was conducted for 35 days, from June to October 2017, at the Center Brackiswater Aquaculture Development (BPBAP) Ujung Batee, Ministry of Marine Affairs and Fisheries, Aceh Besar, Indonesia.\n\nThis study used a completely randomized design method, with five treatments and four replications. Fish were given experimental feed containing 30–70% protein as follows:\n\nA = Treatment of feed containing protein 30%\n\nB = Treatment of feed containing protein 40%\n\nC = Treatment of feed containing protein 50%\n\nD = Treatment of feed containing protein 60%\n\nE = Treatment of feed containing protein 70%\n\nThis article is reported according to the ARRIVE guidelines. All efforts were made to lessen harm to the animals by complying to the guidelines of ethical animal use in research of Syiah Kuala University (Ethic Code No. 958/2015) and this study was approved by the Animal Research Ethics Committee of the Veterinary Ethics Committee, Faculty of Veterinary Medicine, Syiah Kuala University (Ethic Code No. 63/KEPH/X/2020).\n\nWe obtained 300 C. ignobilis juveniles from Lancang Barat Village, Dewantara District, Aceh Utara Regency. The fish were acclimatized for two weeks at the BBAP Ujung Batee, Aceh Besar Indonesia. They were placed in 20 rearing nets of 1 × 1 meter in size, which were placed in a concrete pond containing 40 tons of water (range of salinity value 29.19-29.44‰), each containing 15 C. ignobilis juveniles.\n\nThe experimental feed was made from fish meal, rebon shrimp flour, rice flour, soybean flour, cornflour, blood meal, coconut oil, CaCO3, Isolesin, L-Tryptophan, DL-Methionine, and premix with a feed protein content of 30–70%. All ingredients were mixed and analyzed for protein content (Table 1).\n\nJuvenile C. ignobilis were selected randomly. Their average weight was 11.41 ± 0.40 g and their average initial total length was 7.70 ± 0.76 cm. Furthermore, the fish are distributed into 20 nets measuring 1 × 1 meter. The net is placed in a concrete pond with a water volume of 40 tons (15 fish/net). The fish were given experimental feed twice a day at 7 am and 5 pm for 35 days, as much as 3% per body weight.\n\nThe parameters measured in this study are DGR, specific growth rate (SGR), AGR, feed conversion ratio (FCR), FE, and survival (SR). DGR and SGR were analyzed based on the formula by Muchlisin et al.18,19. AGR was analyzed based on the Jones20 formula, FCR, and FE were analyzed based on the formula of Shapawi et al.21; SR was analyzed based on the formula of Hseu et al.22.\n\nDGR (g day−1) = (Wt – Wo)/t,\n\nSGR (% day−1) = [(Ln Wt – Ln Wo)]/ t × 100,\n\nAGR (cm day−1) = L2−L1Δt\n\nFCR = F/ (Wt – Wo)\n\nFE = 1FCR× 100%\n\nSR= (Nt)No× 100\n\nNote: Wo is the initial weight of fish when researched (g); Wt is the weight of the fish at the end of the study (g); t is the study duration (days). L1 is the length of the fish at the start ofraising (cm), L2 is the length of the fish at the end of raising (cm), Δt is the length of rearing (days). Nt is the number of test fish at the end of maintenance, and No is the number of test fish at the beginning of rearing.\n\nData of DGR, SGR, AGR, FCR, FE, and SR were analyzed statistically by one way ANOVA (Analysis of Variance) with a 95% degree of confidence (P < 0.05) using SPSS version 20 software. Significant data were tested further with Duncan’s multiple ranges test.\n\n\nResults\n\nThe results showed that diets with different protein content significantly affected (P < 0.05) the values of DGR, SGR, and AGR of C. ignobilis. The best growth of C. ignobilis was found in the feed treatment with 50% protein content, obtaining a mean DGR value of 0.27 ± 0.01 g / day, a mean SGR of 1.72 ± 0.03% / day and a mean AGR of 0.013 ± 0.01. cm / day, followed by 40% protein feed (mean DGR value 0.23 ± 0.01 g / day, SGR 1.50 ± 0.04% / day, and AGR 0.11 ± 0.01 cm / day ), 30% protein feed (mean DGR value of 0.21 ± 0.01 g / day, SGR 1.40 ± 0.07% / day, and AGR 0.08 ± 0.00 cm / day), protein feed 60% (mean DGR value 0.13 ± 0.01 g / day, SGR 0.98 ± 0.07% / day and AGR 0.07 ± 0.01 cm / day), and 70% protein feed (mean DGR value 0.12 ± 0.01 g / day, SGR 0.91 ± 0.08% / day and an AGR value of 0.07 ± 0.02 cm / day) (Table 2).\n\nNote: Numbers followed by different superscripts of letters indicate significant differences (P < 0.05). Numbers followed by a superscript of the same letter show no significant difference (P > 0.05). [A] 30% protein feed, [B] 40% protein feed, [C] 50% protein feed, [D] 60% protein feed, [E] 70% protein feed.\n\nFurthermore, the results showed that the feed formulations with different protein content also significantly affected (P < 0.05) the value of FCR, FE, and SR of C. ignobilis. The best mean FCR, FE and SR values were shown in the 50% protein feed treatment with mean values of 1.29 ± 0.07, 77.76 ± 4.46% and 86.67 ± 5.44% respectively, then followed by treatment of 40% protein feed (mean FCR 1.49 ± 0.17, FE 67.73 ± 7.61%, and SR 80 ± 5.45%), 30% protein feed (mean FCR 1.66 ± 0.09, FE 60.33 ± 3.38%, and SR 78.33 ± 3.34%), 60% protein feed (mean FCR 1.79 ± 0.06, FE 56.03 ± 1.84 %, and SR 68.34 ± 3.33%), and protein feed 70% (mean FCR 1.96 ± 0.15, FE 51.19 ± 3.71%, and SR 65.00 ± 3.34%) (Table 3).\n\nNote: Numbers followed by different superscripts of letters indicate significant differences (P < 0.05). Numbers followed by superscript of the same letter show no significant difference (P > 0.05). [A] 30% protein feed, [B] 40% protein feed, [C] 50% protein feed, [D] 60% protein feed, [E] 70% protein feed.\n\n\nDiscussion\n\nThe analysis showed that C. ignobilis growth occurred optimally with 50% protein feed based on the mean values of DGR, SGR, and AGR. Similar results were also found in its sister, C. melampygus; feed with about the same protein content (45%) provided optimum growth in these fish23. Furthermore, this study revealed that the increased feed protein content had a positive correlation to fish growth, but this only occurred up to 50% protein feed, while fish growth would decrease if the feed protein content was >50%. This is probably because C. ignobilis is only able to absorb feed protein optimally by ≤ 50%. Similar results occurred in Oreochromis niloticus, which was given feed treatment with 17%, 30%, and 35% protein, which showed that the optimum growth results occurred in 30% protein feed treatment, but the 35% protein feed in O. niloticus had no significant effect on fish growth24. Furthermore, another study by Yang et al.25 revealed that Bidyanus bidyanus, which was given feed treatment with protein levels of 13%, 19%, 25%, 31%, 37%, 43%, 49%, and 55%, showed that fish growth continued to increase in size from 13%–37% protein feed. However, fish growth was no longer significant in the range of 43%–55% protein feed. Other research results, also by Yang et al.26 on Spinibarbus hollandi, which received protein feed of 13%, 19%, 25%, 31%, 37%, 43%, 49%, and 55%, showed that fish growth continued to increase from 13%–31% protein feed, but fish were no longer grew significantly in the 37%–55% protein feed. This review shows that the fish’s ability to absorb feed protein differs by species and that fish have a limited ability to absorb protein in feed at certain periods and conditions.\n\nProtein is the main macromolecular component fish need27. The function of protein is prioritized for the synthesis of new proteins according to the fish’s nutritional needs. At the same time, carbohydrates and lipids supply energy availability for fish28. Feed protein will be hydrolyzed in the digestive tract, and only free amino acids can be absorbed by the intestine for tissue and organ purposes as body protein29. However, feed with high protein content may not be used optimally by fish as in this study. Feed with 60% and 70% protein given to C. ignobilis actually decreased the ability to grow, the feed efficiency (FE), and fish survival. In addition, the feed treatment with 60% and 70% protein given to C. ignobilis also decreased the average FE and SR values but increased the FCR value. High FCR will increase feed consumption so that fish farming production costs tend to increase as well. Furthermore, the SR values in protein feed treatment of 60% and 70% only reached 68.34% and 65.00%, probably because the ability to absorb feed (FE) decreases, so that the quantity of fish feces increases and causes the fish to die due to poisoning.\n\nThe mean optimum FE value of C. ignobilis reached 77.755%, which resulted in the 50% protein feed treatment. This result is better than the best average FE value of C. hippos, which reached 19.01% in the combination treatment of 50% fish waste and 50% artificial feed30. Furthermore, another study by Rostika et al.31 also produced the best average FE value for Caranx sp. lower than this study with a value of 30.60% in the treatment of 100% trash fish feed. The different results may be due to different test fish species and feed treatments, so that the fish’s ability to absorb feed nutrients is also different. Another factor that plays a role in FE is the biological value contained in feed protein. Protein in feed with high biological value will stimulate the accumulation of body protein greater than that of low biological value32–34. Protein is a nutrient required in large quantities in fish feed formulations because it needs to be given continuously with sufficient quality and quantity. Buwono35 states that, biologically, the quality of artificial feed shows the nutritional value of the protein contained in the feed. Thus, the feed quality is assumed to be protein quality. The feed protein quality depends mainly on its essential amino acid content; the lower the essential amino acid content, the lower the protein quality36–39.\n\nThe best SR value in this study was shown in the 50% protein feed treatment with a mean value of 86.67%. This result is lower than the studies conducted by Rostika et al.31 and Rostika et al.30 regarding the combination of pelleted feed and trash fish to Caranx sp. and C. hippos, which obtained an SR value of 100% in each treatment. Furthermore, the mean SR value of C. melampygus in the study of Suprayudi et al.23 is also higher than this study, with a value reaching 100% with the best treatment of 45% protein feed. Furthermore, another study by Rombenso et al.40 showed that C. latus, C. crysos, and C. hippos reared in floating net cages near the coast of Brazil by feeding sardines produced an average SR value of 90%. Therefore, further research is needed to increase the SR value of C. ignobilis in the future so that it can increase fish production.\n\nThe best FCR value produced in this study was in the 50% protein feed treatment with a mean value of 1.29. The lowest value was shown was in the treatment of 70% protein feed (mean FCR 1.96). This result is better than the research conducted by Rombenso et al.40, which showed that C. latus, C. crysos, and C. hippos rearing in floating net cages near the coast of Brazil by feeding them sardine produced a mean FCR value of 4.2. This study’s FCR value is also better than the FCR value of other fish species, such as largemouth bass Micropterus salmoides (best FCR value of 1.3941), Asian stinging catfish Heteropneustes fossilis (best FCR value of 1.4242), Thai pangas Pangasius hypophthalmus (best FCR value of 1.6543), and relatively equivalent to the FCR of the Siberian sturgeon Acipenser baeri (maximum of 1.2544) and catfish Ictalurus punctatus (maximum of 1.3045).\n\n\nConclusion\n\nThe results showed that feed with different protein levels had a significant effect on the mean values of DGR, SGR, AGR, FCR, FE, and SR in C. ignobilis. The 50% protein feed gave the best results on C. ignobilis with a mean DGR value of 0.27 ± 0.01 g / day, a mean SGR of 1.72 ± 0.03% / day, a mean AGR of 0.13 ± 0.01 cm/day, a mean FCR of 1.29 ± 0.07, FE was 77.76 ± 4.46%, and SR was 86.67 ± 5.44%. The treatment with the lowest value was 70% protein feed with a mean DGR value of 0.12 ± 0.01 g / day, mean SGR 0.91 ± 0.08% / day, mean AGR 0.07 ± 0.02 cm/day, mean FCR 1.96 ± 0.15, FE 51, 19 ± 3.71%, and the average SR was 65.00 ± 3.34%. Furthermore, feed treatment with increased protein content between 30%–50% has a positive correlation with the growth of C. ignobilis. The ability to grow fish will decrease, however, if the protein content of the feed is >50%.\n\n\nData availability\n\nFigshare: Effect of dietary protein level on growth, food utilization, food conversion and survival rate of giant trevally (Caranx ignobilis). https://doi.org/10.6084/m9.figshare.12936563.v146.\n\nThis project contains the following underlying data:\n\nData of daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE) and survival rate (SR) of Caranx ignobilis. (Raw data for the daily growth rate (DGR), specific growth rate (SGR), absolute growth rate (AGR), feed conversion ratio (FCR), feed efficiency (FE) and survival rate (SR) of all fish examined in this study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThanks to all of the staff at the Ujung Batee Brackish Aquaculture Fisheries Center who assisted with the research. Special thanks go to Ms. Melina, S.Si for her participation during the research.\n\n\nReferences\n\nSun Y, Xu T: Complete mitochondrial genome of Caranx equula (Perciformes, Carangidae): genome characterization and phylogenetic analysis. Mitochondrial DNA B Resour. 2018; 3(2): 786–787. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith GC, Parrish JD: Estuaries as nurseries for the jacks Caranx ignobilis and Caranx melampygus (Carangidae) in Hawaii. Estuar Coast Shelf Sci. 2002; 55(3): 347–359. 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Publisher Full Text\n\nMuchlisin ZA, Afrido F, Murda T, et al.: The Effectiveness of experimental diet with varying levels of papain on the growth performance, survival rate and feed utilization of keureling fish (Tor tambra). Biosaintifika. 2016; 8(2): 172–177. Publisher Full Text\n\nJones CM: Age and growth. Fishery Science: The unique contributions of early life stages. Blackwell Publishing. USA. 2002.\n\nShapawi R, Mustafa S, Ng WK: A comparision of the growth performance and body composition of the humpback grouper Cromileptis altevalis fed on farm-made feed, commercial feeds or trash. J Fish Aquat Sci. 2011; 6(5): 523–534. Publisher Full Text\n\nHseu JR, Chang HF, Ting YY: Morphometric prediction of cannibalism in larviculture of orange-spotted grouper, Epinephelus coioides. Aquaculture. 2003; 218(1–4): 203–207. Publisher Full Text\n\nSuprayudi MA, Ihu MZ, Utomo NP, et al.: Protein and energy: protein ratio in diets for juvenile bluefin trevally Caranx melampygus. Journal of Applied Aquaculture. 2014; 26(2): 187–196. Publisher Full Text\n\nBahnasawy MH: Effect of dietary protein levels on growth performance and body composition of monosex Nile tilapia, Oreochromis niloticus L. reared in fertilized tanks. Pak J Nutr. 2009; 8(5): 674–678. Publisher Full Text\n\nYang SD, Liou CH, Liu FG: Effects of dietary protein level on growth performance, carcass composition and ammonia excretion in juvenile silver perch (Bidyanus bidyanus). Aquaculture. 2002; 213(1-4): 363–372. Publisher Full Text\n\nYang SD, Lin TS, Liou CH, et al.: Influence of dietary protein levels on growth performance, carcass composition and liver lipid classes of juvenile Spinibarbus hollandi (Oshima). Aquac Res. 2003; 34(8): 661–666. Publisher Full Text\n\nCarter CG, Houlihan DF: Protein synthesis. Fish physiology. 2001; 20: 31–75. Publisher Full Text\n\nHiggs DA, Sutton JN, Kim H, et al.: Influence of dietary concentrations of protein, lipid and carbohydrate on growth, protein and energy utilization, body composition, and plasma titres of growth hormone and insulin-like growth factor-1 in non-transgenic and growth hormone transgenic coho salmon, Oncorhynchus kisutch (Walbaum). Aquaculture. 2009; 286(1–2): 127–137. Publisher Full Text\n\nPacheco MTB, Costa-Antunes AE, Sgarbieri VC: New technological and physiological fungsional properties of milk proteins. In Bascoe AB, Listow CR, edidors. Protein Research Progress. New York: Nova Science Publishers Inc. 2008.\n\nRostika R, Rahmanto F, Haetami K, et al.: The use of various proportions of rough fish and pellets on the growth of giant trevally fish (Caranx hippos) in the east coast floating net cages (KJA Pantai timur), pangandaran. Int J Fish Aquat Stud. 2020; 8(1): 197–200. Reference Source\n\nRostika R, Fauzan MI, Lili W: The use of various proportions of rough fish on giant trevally (Caranx sp.) culture in the floating net cages, Pangandaran, West Java. Asian Journal of Fisheries and Aquatic Research. 2020; 5(3): 1–7. Publisher Full Text\n\nWilson RP, Halver JE: Protein and amino acid requirements of fishes. Annu Rev Nutr. 1986; 6(1): 225–244. PubMed Abstract \| Publisher Full Text\n\nLeng RA, Stambolie JH, Bell R: Duckweed-a potential high-protein feed resource for domestic animals and fish. Livestock Research for Rural Development. 1995; 7(1): 36. Reference Source\n\nKaushik SJ, Seiliez I: Protein and amino acid nutrition and metabolism in fish: current knowledge and future needs. Aquac Res. 2010; 41(3): 322–332. Publisher Full Text\n\nBuwono ID: Kebutuhan asam amino esensial dalam ransum ikan. Yogyakarta: Kanisius. 2000. [In Indonesian]. Reference Source\n\nShiau SY, Lan CW: Optimum dietary protein level and protein to energy ratio for growth of grouper (Epinephelus malabaricus). Aquaculture. 1996; 145(1–4): 259–266. Publisher Full Text\n\nLazo JP, Davis DA, Arnold CR: The effects of dietary protein level on growth, feed efficiency and survival of juvenile Florida pompano (Trachinotus carolinus). Aquaculture. 1998; 169(3–4): 225–232. Publisher Full Text\n\nLee SM, Cho SH, Kim KD: Effects of dietary protein and energy levels on growth and body composition of juvenile flounder Paralichthys olivaceus. J World Aquac Soc. 2000; 31(3): 306–315. Publisher Full Text\n\nYassir E, Abdel-Tawwab M, Ahmad MH, et al.: Effect of dietary protein level, initial body weight, and their interaction on the growth, feed utilization, and physiological alterations of Nile tilapia, Oreochromis niloticus (L.). Aquaculture. 2010; 298(3–4): 267–274. Publisher Full Text\n\nRombenso AN, Bowzer JC, Moreira CB, et al.: Culture of Caranx species [Horse-eye Jack Caranx latus (Agassiz), Blue Runner Caranx crysos (Mitchill), and Crevalle Jack Caranx hippos (Linnaeus)] in near-shore cages off the Brazilian coast during colder months. Aquac Res. 2014; 47(5): 1687–1690. Publisher Full Text\n\nCochran NJ, Coyle SD, Tidwell JH: Evaluation of reduced fish meal diets for second year growout of the Largemouth Bass, Micropterus salmoides. J World Aquac Soc. 2009; 40(6): 735–743. Publisher Full Text\n\nSiddiqui TQ, Khan MA: Effects of dietary protein levels on growth, feed utilization, protein retention efficiency and body composition of young Heteropneustes fossilis (Bloch). Fish Physiol Biochem. 2009; 35(3): 479–488. PubMed Abstract \| Publisher Full Text\n\nAmin AR, Bapary MAJ, Islam MS, et al.: The impacts of compensatory growth on food intake, growth rate and efficiency of feed utilization in Thai Pangas (Pangasius hypophthalmus). Pak J Biol Sci. 2005; 8(5): 766–770. Publisher Full Text\n\nRad F, Köksal G, Kindir M: Growth performance and food conversion ratio of Siberian sturgeon (Acipenser baeri Brandt) at different daily feeding rates. Turk J Vet Anim Sci. 2003; 27(5): 1085–1090. Reference Source\n\nPeterson BC, Booth NJ, Manning BB: Replacement of fish meal in juvenile channel catfish, Ictalurus punctatus, diets using a yeast-derived protein source: the effects on weight gain, food conversion ratio, body composition and survival of catfish challenged with Edwardsiella ictaluri. Aquac Nutr. 2012; 18(2): 132–137. Publisher Full Text\n\nBatubara AS: Effect of dietary protein level on growth, food utilization, food conversion and survival rate of giant trevally (Caranx ignobilis). Figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12936563.v1" }	[ { "id": "87825", "date": "05 Jul 2021", "name": "Wing Yin Mo", "expertise": [ "Reviewer Expertise Aquaculture", "environmental 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\n1. In the abstract, please revise the Methods from future tense to past tense, as the authors have already finished the experiment.\n\n2. Professional editing is needed to improve the overall quality of the manuscript.\n\n3. In the introduction, I think it will be more meaningful if the following information is provided:\nProduction volume of the species, exported vs. domestic consumption.\n\nNatural diet composition: Is the fish carnivorous fish? What are the natural preys?\n\n4. One of the major issues of the manuscript is the feed formulation (Methods). More explanation should be provided to polish the manuscript.\nHow did the authors produce the feed? Home-made or produced by manufacturers?\n\nWhere did the authors get the ingredients? Home-made or purchased?\n\nDo the fish feed pellet float or sink? Does the diet fit the preference of fish?\n\nThe design seems like the authors were testing the suitability of blood meal (I prefer “meal” instead of “flour”) rather than testing the effects of protein levels. I did a quick check. There are many studies to replace fishmeal with blood meal. For omnivorous fish, 50% replacement is suggested (Kirimi et al. (20161)). For carnivorous species, such as Murray cod, partial replacement is possible (Abery et al. (20022)). Would it be true that high levels of blood meal affect growth of fish?\n\nThere are too many variables in the diets. To my best knowledge, high levels of carbohydrate is harmful to carnivorous fish. The author might check the paper published by Stone et. al. (20033), for more information about the effects of carbohydrates on different fish species. Thus, it would be possible that fed with Diet A and B resulted in inferior growth is related to the high levels of carbohydrate.\n\nThe diets were tested using juveniles and that should be reflected on the title.\n\n5. The authors suggested fish mortality of groups D and E were related to feces accumulation and poisoning. However, the authors didn’t mention the depth of the experimental pond. In the pond used in the experiment, the authors suggested nets were used. Could the fish feed accumulate inside the cage and kill the fish because of that? Did the fish show any sign of intoxication? Also, is that pond equipped with any aerators? Is there any water treatment facility?\n6. Amino acids and proximate compositions of the diets: The authors calculated the protein content of fish feeds. Did the authors measure the exact protein concentration? Would it be possible that the high level of blood meal resulted in inferior growth, because of insufficient amino acid(s)? In addition to protein, other proximate compositions i.e. lipid, ash, moisture and carbohydrate contents should also be measured and presented.\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": [] }, { "id": "87824", "date": "19 Jul 2021", "name": "Mohammad Bodrul Munir", "expertise": [ "Reviewer Expertise Fish Nutrition" ], "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 MS \"Effect of dietary protein level on growth, food utilization, food conversion and survival rate of giant trevally (Caranx ignobilis)\" is not written well. For example, writing style of the background in the abstract was found to have grammatical errors. The methods in this section are expressed as future tense.\n\nThere was no feed proximate analysis found; however it is necessary to validate the desired protein percentage.\nHow many biological and technical replicates did you take?\nSuperscripts in Table 2 indicated the significance differences among the rows; but the researchers did not express this, therefore it is quiet difficult to understand. Please revise it.\nIn the Table 2, the results of DGR in A and B were not significantly different. It should be same. Please check it. Standard errors of the results are confusing. (how is it possible 0.01, 0.02, etc?)\nExpression of superscripts in Table 3 have the same problem as Table 2, please re-write. I felt confused the FCR data. Was there any relation of 3% body weight feed provided to fish? How did you calculate this 3% body weight?\nOverall not at the standard for indexing using the present format.\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?\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/10-78
https://f1000research.com/articles/10-76/v1	05 Feb 21	{ "type": "Research Article", "title": "Analysis of rs1048943 in the CYP1A1 gene in laryngeal cancer - a case control study", "authors": [ "Marwa Abdalwahab", "Mona Ellaithi", "Marwa Abdalwahab" ], "abstract": "Background: The CYP1A1 gene is essential in the metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs). Numerous studies have investigated the links between the polymorphism Ile462Val rs1048943 A>G and its association with different cancers but the results have been variable. In this study, we studied rs1048943 in Sudanese patients diagnosed with laryngeal cancer. Methods: This is a case-control study of 49 laryngeal cancer cases and 50 healthy controls. Restriction fragment length polymorphisms (RFLP) were used to analyse the fragment region of the CYP1A1 gene that contains rs1048943. Results: The average age of the patients was 51.2 years old and the male to female ratio was 1:1. Well differentiated squamous cell carcinoma constituted most of the histopathological diagnoses. Ile462Val rs1048943 A>G was not found in any of the cases but was found in heterozygous form in four controls (p = 0.1331, OR 0.1044, 95% CI, 0.0054-1.9924). Conclusions: Our study did not show a significant association of CYP1A1 Ile462Val rs1048943 A>G with laryngeal cancer. Future studies may need to test additional single nucleotide polymorphisms (SNPs) in laryngeal cancer tissue samples to identify the effect of those SNPs on laryngeal cancer.", "keywords": [ "CYP1A1 gene", "laryngeal cancer", "rs1048943", "squamous cell carcinoma. Upper Aero-digestive tract cancers." ], "content": "Introduction\n\nSeveral epidemiological studies have suggested that tobacco smoking and alcohol consumption increase the risk of developing laryngeal cancer1–6. Certain genetic polymorphisms have been identified as genetic risk factors specifically for laryngeal squamous cell carcinoma (SCC)7. Cytochrome P450 (CYP) enzymes are the key enzymes in phase I of the metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs)8. PAHs are carcinogens associated with laryngeal cancer. Different studies have pointed to the involvement of CYP in procarcinogen activation9–12. The CYP1A1 polymorphisms rs1048943 A>G and rs4646903 T>C have been the most frequently studied13. CYP1A1 rs1048943 A>G is an amino acid change at codon 462. CYP1A1 rs4646903 T>C is characterized by the T-to-C mutation at nucleotide 3801 in the 3'- flanking region of the gene14. Numerous studies have investigated the links between the polymorphisms rs1048943 and rs4646903 and the risk of laryngeal cancer, but the results appear to be inconsistent. The Ile462Val rs1048943 A>G polymorphism in the CYP1A1 gene is located in exon 7 in the heme binding region and leads to a doubling of enzyme activity15. Heme plays a vital regulatory role in the cell16.\n\nThe objective of this study was to find out if there was an association between the rs1048943 polymorphism and laryngeal cancer in Sudanese patients. The hypothesis was that the polymorphism might play a biological role in laryngeal cancer progression specifically in Sudanese patients.\n\n\nMethods\n\nThis was a case-control study carried out during the years 2019–2020. It involved 49 patients diagnosed with different grades and types of laryngeal cancers and 50 healthy controls. DNA samples for all participants were recruited from the upper aero digestive tract cancer (UADT) biobank. This biobank contains biological materials and data from cancer patients as well as control healthy individuals. These samples were collected from newly diagnosed cases to the biobank during the years 2014–2016 from the Radiation and Isotope Centre Khartoum (RICK), Sudan. The controls group was collected from healthy individuals recruited from the UADT biobank during the same period. The biobank includes a large number of DNA samples from both cancer patients and healthy controls; however, our selection was restricted to the patients diagnosed with cancer of the larynx only and controls were matched to patients by age and sex. The sample size was based on the availability of laryngeal samples in the biobank and matching controls. The biobank had a total number of 49 laryngeal cancer cases, so all were included.\n\nDNA samples from blood were obtained from the UADT biobank. The DNA had been previously extracted using the innuPREP mini Kit from Analytik Jena. DNA quantification (ng/μL) was determined with a 5 µl DNA sample using the NanoDrop 2000 (Cat. No. ND-2000, Thermo Fisher Scientific). The DNA samples were stored at -80°C.\n\nThe region that contains the changes of the nucleotides A>G which identifies rs1048943 was amplified by PCR using universal eubacterial primers to produce an amplicon of 226 bp. The primers were designed using Primer 3. Accordingly, the forward sequence was (CTCACCCCCCTGATAGGATAGTGCTAT) and the reverse sequence was (TTTGGAAGTGCTCAGCAGCAGCAGCAG). The reaction was performed in a total volume of 20 μl using the Maxime PCR premix kit i- startaq (Cat. No 25165): 0.3 μl of each primer, 14.7μl DW and 5 μl of the DNA template. The PCR was performed in a Thermo cycler (Techne TC-412, UK) with an initial denaturation at 95°C for 2 minutes, followed by 35 denaturation cycles at 95°C for 30 seconds, primer annealing at 58°C for 30 seconds extension at 72°C for 30 seconds and a final extension at 72°C for 5 minutes. After that, gel electrophoresis was performed to detect the presence of the amplified DNA fragments in the PCR product. The gel contained agarose gel dissolved in a buffer to 1.5 % to which an ethidium bromide (0.5 μg/ml) was added. The size of the wild type band was 226 bp. The electrophoresis result was photographed with the Syngene InGenius LHR2 gel imaging system (Syngene, Woonsocket, RI, USA).\n\nWe used the NEBcutter V2.0 web tool provided by New England Bio labs to select the restriction enzyme specific to the position of the polymorphism. Accordingly, the 2-base pair product of the CYP1A1 gene was digested with the BSrD1 restriction enzyme. The enzyme recognized a short specific sequence of the nucleotide base, then catalysed hydrolysis (cleavage of the chemical bond that binds the nucleotide) by adding water molecules. The digestions were performed in a total volume of 31 μl. The reaction mixture consisted of 10 μl of the PCR product, 1μl of the restriction enzyme and 2 ml of l0x buffer, with the volume adjusted by 18 μl sterile DW. Electrophoresis was performed on the digested product with 1.5 (w/v) agarose and 0.5 μg/ml ethidium bromide, and photographs were taken with the Sygene InGenius LHR2 gel documentation system (Syngene, Woonsocket, RI, USA). The sizes of the bands after digestion were 150 bp and 76 bp.\n\nStatistical analysis was performed using Stata v11 software (Statacorp, College Station, Texas). Results were presented in frequencies for the grades and types of laryngeal cancer. Genotyping analysis was performed using the Chi-square test in order to estimate the association of the different alleles to laryngeal carcinomas. The statistical level of significance was set at p value 0.05.\n\n\nResults\n\nThere were a total of 23 male and 26 female cases with a male to female ratio of 1:117. Their average age was 51.2 years old (range 13–80 years). The control group contained 23 males and 27 females with a male to female ratio of 1:1.7. Their average age was 51.6 (range 19–80) (Table 1).\n\nHistopathological classification showed that 27 patients (55.1%) were diagnosed with well differentiated SCC, 12 (24.7%) with moderate SCC, 5 (10.2%) with poorly differentiated SCC and 1 (2 %) with SCC without indication of the degree (Table 2). Other histopathological types (adenoid cystic carcinoma, diffuse large cell lymphoma, mucoepidermoid carcinoma, and laryngeal papillomatosis) had a sample of 1 (2%) participant each.\n\nThe genotypic distribution of rs1048943 A>G is shown in Table 3. The wild type homozygous genotype A/A was present in all the cases (100%) yet not the heterozygous (AG) or homozygous mutant (GG) genotype (0%). Of the controls, four had the heterozygous allele AG, 46 had the homozygous wild type AA genotype, and none had the GG genotype (p = 0.1331, OR 0.1044 and 95% CI 0.0054 to 1.9924) (Table 3).\n\n\nDiscussion\n\nLaryngeal cancer is one of the most predominant cancers of the upper respiratory tract18. It is rare in individuals under 60 years of age with three quarters of all diagnoses occurring in people over 6019. In the current study, the mean age of the participants was 51.2 years which is a rare age of onset for laryngeal cancer; however, young patients are expected to have a better prognosis20.\n\nWorldwide, laryngeal cancer occurs more commonly in men than in women21,22. In the Sudan cancer registry for the years 2009–2013, the ratio of men to women diagnosed with laryngeal was about 2.8:1. This contradicts the almost equal ratio we found in this study. This could be because all cases in this study were collected from Radiation and Isotope Centre of Khartoum (RICK). On the other hand, cancer registry cases were collected from numerous hospitals and pathology labs from all over Sudan.\n\nIn this study squamous cell carcinoma was the most common type of laryngeal cancer, which reflect the type of cells in the larynx. Well differentiated squamous cell carcinoma was the dominating grade, suggesting that the cancer was detected early.\n\nThe CYP enzymes, especially CYP1A1, play an important role in laryngeal cancers15 as well as in lung cancer23, prostate cancer24, colorectal cancer14 and renal cell carcinoma25. Here, rs1048943 showed no association with laryngeal cancer (p = 0.1331). In view of the small sample size but with our high statistical power we deem these results to be reliable and accurate. We can conclude that rs1048943 might be a polymorphism found in different ethnicities but not directedly linked to laryngeal cancer in Sudanese patients. Hence, it is necessary to study CYP1A1 using more advanced technology like next generation sequencing (NGS) to identify SNPs associated with laryngeal cancer.\n\n\nConclusion\n\nTo sum up, the present study did not find an association between rs1048943 and laryngeal cancer in Sudanese patients.\n\n\nData availability\n\nDRYAD: rs1048943 in CYP1A1 gene in laryngeal cancer- Sudanese study. https://doi.org/10.5061/dryad.j3tx95xcm17.\n\nThis project contains the following underlying data:\n\n● Sudan-larynex-SNP_analysis.xlsx (demographics and clinical information of participants).\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 for publication\n\nWritten informed consent for publication of the participants’ details was obtained from the participants.\n\n\nEthical consideration\n\nThe project was approved by the National Research Ethical Review Committee Health Research Council, Republic of Sudan National Ministry of Health in 2014 (NO:UADT/6.2014). All samples are part of the UADT cancer biobank. A signed consent form was submitted by all study participants.", "appendix": "Authors contributions\n\nMarwa Abdalwahab conducted the laboratory work and contributed in Manuscript writing. Dr. Mona Ellaithi was the principle investigator and writer of the manuscript.\n\n\nAcknowledgements\n\nThe Establishment of the Upper Aerodigestive Tract (UADT) Biobank reported in this article was undertaken with the support of a Return Grant obtained within the framework of the International Agency for Research on Cancer (IARC) Research Training and Fellowship Programme (CRA No. FEL/13/03).\n\n\nReferences\n\nAltieri A, Garavello W, Bosetti C, et al.: Alcohol consumption and risk of laryngeal cancer. Oral Oncol. 2005; 41(10): 956–65. PubMed Abstract \| Publisher Full Text\n\nPelucchi C, Gallus S, Garavello W, et al.: Cancer risk associated with alcohol and tobacco use: focus on upper aero-digestive tract and liver. 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PubMed Abstract\n\nHayashi S, Watanabe J, Nakachi K, et al.: Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P450IA1 gene. J Biochem. 1991; 110(3): 407–11. PubMed Abstract \| Publisher Full Text\n\nBurton MJ, Kapetanaki SM, Chernova T, et al.: A heme-binding domain controls regulation of ATP-dependent potassium channels. Proc Natl Acad Sci U S A. 2016; 113(14): 3785–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMona E, Marwa A: rs1048943 in CYP1A1 gene in laryngeal cancer- Sudanese study, Dryad. 2021. http://www.doi.org/10.5061/dryad.j3tx95xcm\n\nZhao W, Niu F, Xie Z, et al.: Assessment of the association between ACYP2 and laryngeal squamous cell carcinoma risk in Chinese males. Mol Genet Genomic Med. 2019; 7(7): e00731. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed SAO, Abderhman S, Taha M, et al.: Clinical presentation of laryngeal cancer in Sudanese patients. Int J Curr Res. 2017; 9: 53095–53098. Reference Source\n\nLacy PD, Piccirillo JF, Merritt MG, et al.: Head and neck squamous cell carcinoma: better to be young. Otolaryngol Head Neck Surg. 2000; 122(2): 253–258. PubMed Abstract \| Publisher Full Text\n\nBaselga J: Why the epidermal growth factor receptor? The rationale for cancer therapy. Oncologist. 2002; 7(Suppl 4): 2–8. PubMed Abstract \| Publisher Full Text\n\nSadri M, McMahon J, Parker A: Laryngeal dysplasia: Aetiology and molecular biology. J Laryngol Otol. 2006; 120(3): 170–7. PubMed Abstract \| Publisher Full Text\n\nSan Jose C, Cabanillas A, Benitez J, et al.: CYP1A1 gene polymorphisms increase lung cancer risk in a high-incidence region of Spain: a case control study. BMC Cancer. 2010; 10: 463. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang BL, Zheng SL, Isaacs SD, et al.: Polymorphisms in the CYP1A1 gene are associated with prostate cancer risk. Int J Cancer. 2003; 106(3): 375–8. PubMed Abstract \| Publisher Full Text\n\nMeng FD, Ma P, Sui CG, et al.: Association between cytochrome P450 1A1 (CYP1A1) gene polymorphisms and the risk of renal cell carcinoma: a meta-analysis. Sci Rep. 2015; 5: 8108. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "127855", "date": "06 Apr 2022", "name": "Vijaylakshmi Jain", "expertise": [ "Reviewer Expertise Molecular oncology", "microbiology", "virology", "proteomic", "bioinformatics", "mycology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study entitled ‘Analysis of rs1048943 in the CYP1A1 gene in laryngeal cancer - a case-control study’ focuses on the association of rs1048943 gene polymorphism with laryngeal cancer in the Sudanese population. One of the most common tumors of the upper respiratory system is laryngeal cancer. The CYP1A1 gene is required for carcinogenic polycyclic aromatic hydrocarbon metabolism (PAHs). Numerous studies have looked into the relationship between the polymorphism Ile462Val rs1048943 A>G and various malignancies, however, the results have been mixed. Men were more likely than women to get laryngeal cancer. From the year 2009 to 2013, the ratio of men to women was around 2.8:1, which contradicts the current study. The average age of participants was 51.2 yrs. The most common type of laryngeal cancer in this study was squamous cell carcinoma, which reflects the type of cells in the larynx. The most common grade was well-differentiated squamous cell carcinoma, indicating that the malignancy was discovered early. There was no link between rs1048943 and laryngeal carcinoma in this study (p = 0.1331). The study deduces that rs1048943 is a polymorphism that is common in people of many ethnicities but is not connected to laryngeal cancer in Sudanese patients.\n1) Is the work clearly and accurately presented and does it cite the current literature? A- Partly, as some major papers were not mentioned here. Like, Ahmed S, Abdelrhman S, Taha M, Malik M, El Naseh W. Etiology and Clinical Presentation of Laryngeal Cancer in Sudanese Patients. Inter J Otorhinolaryngology. 2018;5(1):3.1\n2) Is the study design appropriate and is the work technically sound? A- Partly, Smoking is the predominant factor of laryngeal cancer but it has not been associated or linked to the study anyhow. Laryngeal cancer is a multifactorial disease but no such correlations were made in the study.\n5) Are all the source data underlying the results available to ensure full reproducibility? A- Partly, there is mention of the figure but no figures were attached in the manuscript.\n6) Are the conclusions drawn adequately supported by the results? A- No, as already mentioned laryngeal cancer is a multifactorial disease where smoking predominates. But, the study only talks about the age, division of cancer, and genotyping which is insufficient to draw any major conclusion when talking about the Sudanese population. Some biochemical correlations and other factors can be correlated to make such bold decisions on a population. Last but not the least, an increased sample size would be appropriate to conclude its association.\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? No", "responses": [] }, { "id": "127856", "date": "19 Apr 2022", "name": "Tracie Cheng", "expertise": [ "Reviewer Expertise CYP1A1 SNPs in colorectal cancer and molecular 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 to the authors for submitting this work.\n\nThe introduction requires more input regarding the current state of knowledge of polycyclic aromatic hydrocarbons (PAHs) and their carcinogenicity.\n\nHow do PAHs get metabolised from a procarcinogenic state to a carcinogenic molecule via CYP1A1?\n\nWhat role does rs1048943 play if there is an SNP in the heme binding region?\n\nThe authors state numerous studies have investigated CYP1A1 SNPs and laryngeal cancer, could you please cite these studies?\n\nOn what basis in the literature do the authors make their hypothesis?\n\nUnfortunately, the sample size is low in this study, though the authors did use every laryngeal cancer case available in the biobank. The authors hypothesise that rs1048943 contributes to the progression of laryngeal cancer in Sudanese patients, however, their results nullify this.\n\nCan the authors suggest why this SNP is not involved in laryngeal cancer?\n\nInterestingly, 4 healthy controls express heterozygous GA alleles, can this be explained?\n\nThank you again to the authors.\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/10-76
https://f1000research.com/articles/10-75/v1	05 Feb 21	{ "type": "Systematic Review", "title": "Occupational therapy productions in times of pandemic: a systematic review of occupational therapeutic actions against the new coronavirus (COVID-19) epidemic", "authors": [ "Carlos Eduardo Ramos Ataide", "Alberto Gonçalves Matos Junior", "Lorena Henriete Araújo Dias", "Luciane Ferreira Farias", "Carlos Roberto Monteiro de Vasconcelos Filho", "Aristela de Freitas Zanona", "Carlos Eduardo Ramos Ataide", "Alberto Gonçalves Matos Junior", "Lorena Henriete Araújo Dias", "Luciane Ferreira Farias", "Carlos Roberto Monteiro de Vasconcelos Filho" ], "abstract": "Background: In March 2020, the World Health Organization classified infection and contagion of coronavirus disease 2019 (COVID-19) as a worldwide pandemic. Since then, health professionals have been tirelessly researching the symptoms and consequences of this disease in people's daily lives. Occupational therapists have also been mobilized to implement effective actions in order to guarantee the functionality and quality of life of individuals and groups. The aim of this article was to investigate what available resources there are regarding occupational therapy during the pandemic, and thus support and enrich professional practice during this time. Methods: A search for was carried out for materials such as technical notes, guidelines for working with COVID-19 patients, clinical trials, and reflections and debates on occupational therapeutic practice during the COVID-19 pandemic. Articles in English, Portuguese and Spanish were included. The search was carried out in the Virtual Health Library (VHL), Google Scholar, Latin American & Caribbean Health Sciences Literature (LILACS), Scientific Electronic Library Online (SCIELO) databases, Medical Literature Analysis and Retrieval System Online (MEDLINE), Pubmed, and the websites of occupational therapy entities and organizations. Results: A total of 44 materials were found and categorised into performance guides, technical notes and scientific articles. The materials dealt with different themes, with a predominance of guidelines for hospital practices. However, there was also a significant amount of notes on the positioning of representative bodies in the preparation of intervention guides in wards, intensive care units, and outpatient and patient homes to support these professionals. Conclusions: The results demonstrate the quick response of authors in the field of occupational therapy, to combat the challenges and barriers of the COVID-19 pandemic. The texts promote scientifically-based strategies to provide the best assistance to patients during the pandemic period.", "keywords": [ "Occupational Therapy", "Coronavirus Infections", "Coronavirus", "Pandemics" ], "content": "Introduction\n\nThe coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, impacts the daily lives of people, families and communities, as well as having an emergency impact on public health worldwide. In view of this, challenges arise for the multidisciplinary teams working on the front line to fight the virus, to adopt patient-centered approaches and care during the acute phase of the disease and after-care1.\n\nCOVID-19 spreads through contact with droplets on contaminated surfaces followed by direct contact with eyes, nose or mouth. The main symptoms of the disease are high temperature, continuous cough, and loss or change to sense of smell or taste2. Emergency measures have been adopted to slow down the contagion curve and prevent health systems from being overloaded. Social distancing has been the main measure used to reduce the spread of the virus; however, social distance and isolation has had a negative impact on people's daily lives2.\n\nAccording to the World Federation of Occupational Therapists3, there are several changes to and consequences of the way people perform their occupations in times of isolation, including access to resources in terms of activities of daily living (ADLs), mobility, communication, social interaction and well-being. The COVID-19 pandemic has had a devastating impact on the occupations of millions of people around the world. In this sense, occupational therapy, as part of the multidisciplinary team on the front line to face the crisis, presents itself as a fundamental specialty in reducing the repercussions of symptoms, hospitalization and social isolation4.\n\nThroughout history, occupational therapy has been important when dealing with social emergency scenarios, starting after the first world war, when thousands of people had physical and / or mental disabilities. In the current situation, all knowledge accumulated over the years must be accessed, mainly because in the beginning of the pandemic, health professionals did not know which strategies would be better for treating the symptoms and consequences of COVID-19. It is worth noting that occupational therapists use evidence-based practices to ensure effective treatment and improve the quality of life of their patients5.\n\nFollowing this, the aim of this article was to perform a systematic review of scientific publications that make reference to occupational therapy during the COVID-19 pandemic. This was in order to provide guidance for the clinical practice of occupational therapy professionals and students.\n\n\nMethods\n\nThis systematic review was carried out according to the PRISMA recommendations6 using the following electronic databases: Virtual Health Library (VHL), Google Scholar, Latin American & Caribbean Health Sciences Literature (LILACS), Scientific Electronic Library Online (SCIELO) databases, Medical Literature Analysis and Retrieval System Online (MEDLINE), Pubmed, and the websites of occupational therapy entities and organizations.\n\nThe databases were searched between May and September 2020. The terms and combinations used for the search were derived from the Health Sciences / DeCS Descriptors, which were: Occupational Therapy, Coronavirus, Pandemic and COVID-19, and their equivalents in Spanish and Portugese. The complete strategy consisted of searching the entry terms in the MeSH, referring to the research question based on PICO (population, intervention, comparison and outcomes): what are the actions taken by occupational therapists during the pandemic period caused by the new coronavirus? The terms were searched using the Boolean operators AND and OR.\n\nThe search strategy was as follows: Occupational Therapy [Mesh] OR Occupational Therapy [tiab] OR Therapy Occupational [tiab] OR Occupational Therapies [tiab] OR Therapies Occupational [tiab] AND Coronavirus [Mesh] OR Coronavirus Infections [Mesh] OR Coronavirus Infection [tiab] OR Infection, Coronavirus [tiab] OR Infections, Coronavirus [tiab] OR Middle East Respiratory Syndrome MERS (Middle East Respiratory Syndrome) [tiab] AND Pandemics [Mesh] OR Pandemic [tiab] AND COVID-19 [Mesh] OR COVID 19[tiab] OR COVID-19 Virus Disease [tiab] OR COVID-19 Virus Diseases[tiab] OR Disease, COVID-19 Virus [tiab] OR Virus Disease, COVID-19 [tiab] OR COVID-19 Virus Infection [tiab] OR COVID 19 Virus Infection [tiab] OR COVID-19 Virus Infections [tiab] OR Infection, COVID-19 Virus [tiab] OR Virus Infection, COVID-19 [tiab] OR 2019-nCoV Infection [tiab] OR Coronavirus Disease-19 [tiab] OR 2019 Novel Coronavirus Disease [tiab] OR 2019 Novel Coronavirus Infection [tiab] OR Coronavirus Disease 2019 [tiab] OR Disease 2019, Coronavirus [tiab] OR SARS Coronavirus 2 Infection [tiab] OR SARS-CoV-2 Infection [tiab] OR Infection, SARS-CoV-2 [tiab] OR SARS CoV 2 Infection [tiab] OR SARS-CoV-2 Infections [tiab] OR COVID-19 Pandemic [tiab] OR COVID 19 Pandemic [tiab] OR COVID-19 Pandemics [tiab] OR Pandemic, COVID-19 [tiab].\n\nNo filters were used (neither time nor language) so that the results were as comprehensive as possible.\n\nArticles were included if they were written in English, Spanish and Portuguese, written by occupational therapists and / or representative entities, and published in late 2019 until September 2020, consistent with the first documentation of the virus in December 2019. For this review, randomized clinical trials, reflective articles, performance guides and normative articles from professional associations were included. Experimental studies in animals, systematic reviews and letters to the editor were excluded.\n\nThe data collection was carried out by two independent researchers. The articles from the search were first judged on: the title of the study, the type of study, and the summary. Articles that did not meet the eligibility criteria were excluded. After the first filter, a complete reading of the studies was performed to judge and list the information relevant to the research.\n\nFor the analysis, a synthesis matrix was prepared for the presentation and qualitative organization of information. For each identified category, the matrix contained the title, authors, study objective, results and the type of study. We simplified the main guidelines and results of the studies in order to provide a quick overview of the included articles.\n\n\nResults and discussion\n\nOf an initial 733 articles found, 44 were included in the analysis (Figure 1), of which 21 were in Portuguese, 19 in English and two in Spanish. These were categorized into three categories, being scientific articles in journals (19), performance guides (18) and technical notes published by entities representing occupational therapy (7) (Figure 1). The 19 scientific articles were divided into experience reports and original articles.\n\nThe 18 performance guides found were produced by associations and official entities of occupational therapy around the world. They address guidelines for the performance and operation of occupational therapy services during the pandemic. The main texts found were from the American Occupational Therapy Association (AOTA).\n\nThe technical notes found are responsible for clarifying doubts of occupational therapists in matters regarding COVID-19. They aim at being a quick communication channel between representative entities and professionals, in addition to demonstrating their position on governmental decisions.\n\nThe articles were separated into five thematic axes, namely: guidelines for occupational therapy services; quality of life during isolation; telehealth; social context; and possibilities of occupational therapy.\n\nThe publications of the guidelines for occupational therapy services (Table 1) have an important documentary role in assisting therapists in understanding measures to deal with the pandemic, addressing the main strategies implemented to treat the symptoms and consequences of COVID-19. The publications on occupational therapy are also important to stimulate reflections and decision making of professionals about the consequences of COVID-19 and social isolation in carrying out daily activities. From this theoretical framework, the concern of the representative entities is to provide information to offer several possibilities of how professionals should perform hospital, outpatient and home care for the treatment of the physical and psychological health of people with COVID-19.\n\nAOTA: American Occupational Therapy Association; OT: Occupational Therapy; WFOT: World Federation of Therapists\n\nOccupational; RENETO: National Occupational Therapy Teaching and Research in Occupational Therapy.\n\nOccupational therapy aims to weave support networks in the fight against COVID-19 that go beyond the hospital context in order to achieve balance and occupational justice in a harmonious way that promotes social and family satisfaction, and reduces stressful environments that provide panic, anxiety, and other distressing emotions. The current scenario offers occupational therapists the opportunity to contribute their knowledge through governmental and non-governmental programs that aim to mitigate the side effects of social isolation that directly affect people's occupational performance7.\n\nRegarding quality of life (Table 2), the studies found show the negative effects of the routine change in the subjects' lives, mainly due to the abrupt break and modification in their daily routine. This shows the need for reflection on and adaptation of habits to prevent social isolation. The English-language texts address aspects such as low quality of life due to decreased income, lack of sleep and unstructured routine. The authors allow us to observe financial losses, which part of the population are facing, as well as repercussions on mental health, work activities and physical health. Guidelines and reflections about a quality routine are the main points addressed which may help populations that are experiencing occupational disorders. The main suggestions for enhancing quality of life focus on structuring a good routine, including personal care, home care, work (in person or online), leisure activities, exercise, developing new hobbies, and receiving psychological support with professionals online. There is an emphasis on structuring a routine with schedules, pleasurable activities, exercise and home maintenance, for physical, mental and emotional health.\n\nWHOQOL: World Health Organization Quality of Life.\n\nThe articles in Portuguese deal with the issue of quality of life and isolation, emphasizing issues such as the professional identity of occupational therapists regarding their role in the pandemic, and discuss different isolation processes that may vary according to socioeconomic class and other contextual factors. They also point out the challenges of the academic community in developing strategies to continue undergraduate teaching of occupational therapy, in addition to providing psychosocial support to students.\n\nThe construction of the thematic axis of quality of life during isolation, made it possible to reaffirm the competence of the occupational therapist in identifying impairments in patient independence, setting treatment goals, increasing autonomy and performance capacities in the activities of daily life8.\n\nFinally, the articles compile actions taken by occupational therapists in order to promote a better quality of life during social isolation, such as reorganizing daily routine, promoting self-care, and providing support networks to help maintain mental and physical health.\n\nGiven the high degree of infection by COVID-19, telehealth, which is shown in Table 3, was one of the main strategies adopted to continue remote treatments to guarantee the safety of professionals and patients. The Canadian Association Of Occupational Therapy defines telehealth as the provision of health services through telecommunications, which may vary according to the user's skills9.\n\nAOTA: American Occupational Therapy Association; CAOT: Canadian Association of Occupational Therapy; ASD: Autistic Spectrum Disorder.\n\nThe articles addressing this theme involved occupational therapists’ reflections on the introduction of telehealth in their practice. When using remote means of communication, therapists need to improve their skills in order to identify patient needs in distant care. This might be achieved by the therapist paying greater attention to the tone of the patient's voice and their way of communicating for example. The use of telehealth in occupational therapy impacts the way therapists interact with their patients for appointments regarding a range of issues including mental health, hand and upper limb therapy, group therapy, the use of sensory diet as a form of intervention, and assessing elderly people with cancer.\n\nThe advantages of telehealth have the potential to optimize the time and costs of services, the possibility of expanding the reach of the health team in remote areas and the increased potential for rehabilitation. In addition to being more practical for the patient, call centers enables the care and monitoring of a greater number of patients. Telehealth could also be used in an educational program through daily dialogues that expand the understanding of patient’s health conditions and encourage them to make changes in their lives to make them healthier and more independent36.\n\nThe articles in Table 4 show occupational therapy actions in the social context, pointing out the inequalities observed in vulnerable populations in coping with COVID-19 due to the pre-existence of disadvantages in social and economic factors37.\n\nLGBTQIA+: Lesbian, Gay, Bisexual, Transgender, Queer, Intersex, Asexual/Aromantic/Agender\n\nOne of the groups cited is the LGBTQIA+ community. This community has faced daily situations of stigma, prejudice, social isolation, and increased mental health problems, due to the interruption of support groups and therapy aimed at mental health during the COVID-19 pandemic38. People living in the suburbs and with low incomes are also more affected, due to the lack of resources to obtain individual protective items and hand and object hygiene39. Many people have lost their jobs during the pandemic, which has compromised the basic livelihood of many families39. Also highlighted are the elderly, who are at risk of having severe symptoms and requiring hospitalisation. Although they are in this high risk group, many elderly people in Brazil have not obeyed the isolation guidelines and have continued to frequent places with crowds of people during the pandemic, such as markets and shopping centers39.\n\nThe study by Pereira et al.40 describes a strategy created by students and professionals in occupational therapy during the pandemic, which aimed to provide social support for these groups, not only in this moment of social isolation, but also for after the pandemic. This involved socio-educational support for children and youth in communities via distance learning, promoting the strengthening of socio-community ties and assisting the organization of collecting and donating basic supplies to poor communities in various regions of Brazil.\n\nFor possibilities of occupational therapy during the pandemic (Table 5), 12 papers were found divided into scientific articles, performance guides and technical notes that discuss mental health, assistive technology, hospital care, primary health care, worker health, and rehabilitation, written in all three languages.\n\nAOTA: American Occupational Therapy Association; ICU: Intensive care unit; PHC: Therapy in Primary Health Care.\n\nPrimarily, articles that dealt with hospital care included strategies that are being applied in the institutions, including training for the correct use of personal protective equipment (PPE), remote work strategies, reorganization of the units, individualized care, interventions with the patient’s family, priority screening and construction of informative booklets on the disease and protective measures for non-contamination. Some papers provided resources more directed to care in bed, such as postural information, how to communicate during therapy, and attention to clinical conditions, especially the respiratory system1.\n\nWith regard to mental health, contents were found that dealt with the impact of the pandemic and social isolation in particular. Some articles commented on changes in the mental health of professionals who are on the front lines. These studies pointed out ways to support health professionals since many of them have had to leave their homes and isolate themselves to avoid contaminating their families. In addition there has been an intensification in working hours due to the very high demand for health services worldwide for a small number of professionals on the front line. These variables were observed as potentially stressful factors for professionals41.\n\nThe assistive technology addressed in the publications refers to adaptations in the masks and respirators used to prevent the spread of COVID-19, focusing on the low cost and availability of equipment, guaranteeing the population's access to these tools2. As far as rehabilitation is concerned, the action guide found seeks to guide occupational therapy for patients discharged from the ICU after infection, prioritizing sequelae in the respiratory and musculoskeletal system that can affect occupational performance. The guide was based on the Person-Environment-Occupation (PEO) model to promote better patient follow-up and rehabilitation42.\n\nAs for the performance of professionals in primary health care, the study by Falcão, et al. carried out remote work at a family health unit (USF) and at a family health support center (NASF) with the objective of continuing the monitoring of mildly symptomatic patients. Health education groups, assessment and guidance in carrying out ADLs, and monitoring of mental health were created. In remote service, through the internet, they carried out environmental organization, listening and solving problems that patients reported, assistance in triage, urgency classification and order of care, including emergencies due to mental illness43.\n\nThe article by Barroso et al. discusses the teaching of occupational therapy students and reflects on the safety and health of workers during the pandemic period, addressing issues regarding legislation and worker rights. Work is considered a potential source of spreading the virus due to workplaces often not providing self-protection resources, such as protective masks, gloves and alcohol gel. There are also often shifts with large numbers of workers and inadequate distance between staff. In addition, many workers use public transport to get to work, and this is also a potential source of contagion. Therefore, for workers to guarantee their rights in having protection against catching COVID-19 at work, it is necessary to discuss the regulations and organizational measures of each active job, according to its level of exposure, thus guaranteeing the safety of workers44.\n\nSince this review searched for papers in three languages, the number of papers found was small compared to the initial expectation of the study. This can be considered a negative and aggravating point for the recognition of the profession, since occupational therapists are part of the essential team on the front line of the COVID-19 pandemic. Studies about the work and contributions of occupational therapy for the affected public are of great use to support discussions, and enable students and professionals to access information about what actions can be taken by therapists, as well as drawing attention to the profession, which as shown in this review, is important in the face of health emergencies.\n\n\nConclusion\n\nThis review found that both professionals and professional associations showed the concern to report and scientifically disseminate the experiences and guidelines of occupational therapists during the COVID-19 pandemic. The resulting 44 papers demonstrate the quick response of the profession to the challenges and barriers that have been imposed during this time, promoting scientifically based strategies to provide the best assistance to patients within the pandemic. The contributions addressed include positioning in bed, the humanization of care, and the restructuring of a daily routine after recovery of the infected person or just for those who are at home due to social distancing measures.\n\nThere were some limitations of the review such as the low number of papers found, the high concentration of papers from magazines and specific organizations, as well as the lack of preparation and visibility of the profession in public health emergency contexts. Further, this review had structural limitations due to the lack of qualitative and quantitative analysis. These analyses were not done due to the types of study included (technical notes, performance guides and reflection articles) which do not work for meta-analysis or qualitative evaluation. Another limitation is that we did not see any risk of bias, due to the lack of qualitative and quantitative analyses. Finally, the search time was short; we are aware that there was an accelerated production of new articles published after the end of the survey carried out by the authors.\n\nWe believe that this review serves as a basis for further research in this area, instigating students, researchers, professionals and other agents of occupational therapy to produce and discuss the profession's practices in public health emergencies with a focus on infectious agents like COVID-19.\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 2009 checklist for ‘Occupational therapy productions in times of pandemic: a systematic review of occupational therapeutic actions against the new coronavirus epidemic’. https://doi.org/10.6084/m9.figshare.13387904.v26\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\ndo Carmo GP, Nascimento JS, dos Santos TRM, et al.: Intervenções terapêutico-ocupacionais para pacientes com COVID-19 na UTI. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 397–415. [acesso em 13 abr 2020]. Publisher Full Text\n\nHarapan H, Itoh N, Yufika A, et al.: Coronavirus disease 2019 (COVID-19): A literature review. J Infect Public Health. 2020; 13(5): 667–673. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Federation of Occupational Therapist: Occupational Therapy and the COVID-19 Pandemic - information and resources. 2020. [acesso em 04 jun 2020]. Reference Source\n\nSilva CR, Fornereto APN, Paolillo AR, et al.: Terapia Ocupacional na universidade pública e ações de enfrentamento à Covid-19: singularidades e/nas multiplicidades. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 351–370. [acesso em 11 abr 2020]. Publisher Full Text\n\nSantos N, Brito J, Nascimento L, et al.: Plano de ação institucional de terapeutas ocupacionais de um hospital escola de Pernambuco frente a pandemia de COVID-19. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 389–396. [acesso em 18 abr 2020]. Publisher Full Text\n\nZanona A: PRISMA 2009 checklist.docx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13387904.v2\n\nMunõz AF: Reflexiones y acciones desde Terapia Ocupacional em la lucha contra el covid-19 durante el confinamiento social. Rev Interist Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 451–459. [acesso em 29 abr 2020]. Reference Source\n\nConselho Federal de Fisioterapia e Terapia Ocupacional (Coffito): Resolução nº 316, de 19 de julho de 2006. Dispõe sobre a prática de Atividades de Vida Diária, de Atividades Instrumentais da Vida Diária e Tecnologia Assistiva pelo Terapeuta Ocupacional e dá outras providências. Diário Oficial da república federativa do Brasil, Brasília, DF; 2006. [acesso em 30 mai 20]. Reference Source\n\nCanadian Association Of Occupacional Therapy (CAOT): Practical considerations for occupational therapists who are considering telehealth. Canadian Association Of Occupacional Therapy. Ottawa. 2020. [acesso em 24 mai 2020]. Reference Source\n\nAmerican Occupational Therapy Association: Decision Guide for Phased Reopening of Occupational Therapy Services during a Pandemic. 2020. [acesso em 13 abr 2020]. Reference Source\n\nAmerican Occupacional Therapy Association: Home Health Occupational Therapy—Decision Guide for COVID-19. 2020. [acesso em 13 abr 2020]. Reference Source\n\nAmerican Occupacional Therapy Association: COVID-19 Occupational Therapy Service Delivery Guide. 2020. [acesso em 13 abr 2020]. Reference Source\n\nCollege of Occupational Therapists of Ontario (COTO): Occupational Therapy Services During COVID-19. 2020. [acesso em 22 abr 2020]. Reference Source\n\nThe Phillipine Academy of Occupational Therapists: Interim Guides on the Practice of Occupational Therapy amidst the Coronavirus Disease (COVID-19) situation in the Philippines. 2020. [acesso em 7 mai 2020]. Reference Source\n\nMichel PS, Pineda RCS, Yao DPG, et al.: Shared voices of Filipino occupational therapists during the COVID-19 pandemic: reflections from an online forum. Boletim da Federação Mundial de Terapeutas Ocupacionais. 2020; 76(1): 60–64. [acesso em 10 mai 2020]. Publisher Full Text\n\nAgudelo A, Cedeño T, Guzmán SA, et al.: Reflexiones desde La Confederación Latino americana de Terapeutas Ocupacionales CLATO em tiempos de COVID-19. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 275–280. [acesso em 9 mai 2020]. Publisher Full Text\n\nBregalda MM, Correia RL, Amado CF, et al.: Ações da Terapia Ocupacional frente ao coronavírus: Reflexões sobre o que a Terapia Ocupacional não deve fazer em tempos de pandemia. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 269–271. Publisher Full Text\n\nWFOT, Omura KM: Posicionamento público: resposta da Terapia Ocupacional à pandemia do Covid-19/Publicstatement: OccupationalTherapy response tothe Covid-19 pandemic. Rev Interinst Bras Ter Ocup.. Rio de Janeiro. 2020; 4(3): 272–274. [acesso em 22 abr 2020]. Publisher Full Text\n\nAssociação Brasileira dos Terapeutas Ocupacional – ABRATO: Posicionamento técnico-político-científico da ABRATO frente ao COVID-19. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 281–289. Reference Source\n\nRENETO, de Oliveira Borba PL, Barreiro RG, et al.: Posicionamento da Rede Nacional de Ensino e Pesquisa em Terapia Ocupacional – RENETO frente a pandemia da Covid-19. Rev Interist Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 290–294. [acesso em 20 abr 2020]. Publisher Full Text\n\nWFOT, Omura KM, Carreteiro G: Declaração de posição telessaúde/Position statement telehealth. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 416–421. [acesso em 20 abr 2020]. Publisher Full Text\n\nCordeiro JJR: A comunicação dos terapeutas ocupacionais durante a pandemia da Covid-19/Occupational therapist’s communication during Covid-19’s pandemic. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 438–450. [acesso em 25 mai 2020]. Publisher Full Text\n\nda Silva TR, Mariotti MC, Bridi A: Aprendendo a lidar com as mudanças de rotina devido ao Covid-19: Orientações Práticas para Rotinas Saudáveis. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 519–528. [acesso em 17 mai 2020]. Publisher Full Text\n\nAcademia Nacional de Cuidados Paliativos: Terapia Ocupacional em cuidados paliativos na covid-19. 2020. [acesso em 7 abr 2020]. Reference Source\n\nSomeshwar H, Sarvaiya P, Desai S, et al.: Does social distancing during the lock down due to Covid-19 outbreak in Mumbai affect quality of life? Inter Journal Clin Biomed Research. Vijayapura, 2020; 1–4. Publisher Full Text\n\nHammell KW: Engagement in living during the COVID-19 pandemic and ensuing occupational disruption. CAOT. 2020; [acesso em 17 mai 2020]. Reference Source\n\nMynard L: Normal life has been disrupted: managing the disruption caused by COVID-19. OTAUS. 2020. [acesso em 22 mai 2020]. Reference Source\n\nda Silva DB: Terapia Ocupacional, cotidiano e pandemia COVID-19: inquietações acerca do ocupar o tempo-espaço/Occupational Therapy, Everday life and pandemic Covid-19: concerns about occupying time-espace. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 529–553. [acesso em 23 abr 2020]. Publisher Full Text\n\nCorrêa VAC, do Nascimento CAV, Omura KM: Isolamento social e ocupações/Social isolation and occupations. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 295–303. [acesso em 25 mai 2020]. Publisher Full Text\n\nTeixeira MR, Dahl CM: Recriando cotidianos possíveis: construção de estratégias de apoio entre docentes e estudantes de graduação em Terapia Ocupacional em tempos de pandemia/Recreating possible everday lifes: building support strategies between teachers and graduate students in occupational therapy in pandemic times. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 509–518. [acesso em 23 mai 2020]. Publisher Full Text\n\nAmerican Occupational Therapy Association: Occupational Therapy Telehealth Decision Guide. 2020. [acesso em 13 abr 2020]. Reference Source\n\nBuckinghamshire Heltcare: Children and Young People’s Service Occupational Therapy Services. 2020. [acesso em 7 mai 2020]. Reference Source\n\nMacêdo FDA, Lopes KAP, Lopes LAMR, et al.: Ações e experiências de terapeutas ocupacionais no contexto de pandemia da COVID-19/Occupational Therapists actions and expiriences in the COVID-19 pandemic context. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 318–333. [acesso em 8 abr 2020]. Publisher Full Text\n\nDiGiovanni G, Mousaw K, Lloyd T, et al.: Development of a telehealth geriatric assessment model in response to the COVID-19 pandemic. J Geriatr Oncol. 2020; 11(5): 761–763. [acesso em 19 abr 2020]. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Souza VRB: A atuação do terapeuta ocupacional com base na Teoria da Integração Sensorial na assistência de crianças com Transtorno do Espectro Autista (TEA) durante a pandemia do Covid-19/The occupational therapist actuation based on the Sensory Integration Theory in the care of children with Autistic Spectrum Disorder (ASD) during the Covid-19 pandemic. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 371–379. [acesso em 23 abr 2020]. Publisher Full Text\n\nMarques MR, Ribeiro ECC, Santana CS, et al.: Aplicações e benefícios dos programas de Telessaúde e Telerreabilitação: uma revisão da literatura. RECIIS. Rio de Janeiro. 2014; 8(1): 43–52. [acesso em 29 mai 2020]. Reference Source\n\nMalfitano APS, Da Cruz DMC, Lopes RE: Terapia Ocupacional em tempos de pandemia: Seguridade social e garantias de um cotidiano possível para todos. Cad Bras Ter Ocup. São Carlos. 2020. [acesso em 11 mai 2020]. Publisher Full Text\n\nCorreia RL, Corrêa M, Pedro R, et al.: Velhices dissidentes de gêneros e sexualidades: as ocupações coletivas frente a pandemia COVID-19. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 460–487. [acesso em 26 abr 2020]. Publisher Full Text\n\nBardi G, Bezerra WC, Monzeli GA, et al.: Pandemia, desigualdade social e necropolítica no Brasil: reflexões a partir da terapia ocupacional social. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 496–508. [acesso em 22 mai 2020]. Publisher Full Text\n\nPereira BP, Soares CRS, Galvani D, et al.: Terapia Ocupacional Social: reflexões e possibilidades de atuação durante a pandemia da Covid-19/Social Occupational Therapy - reflections and possibilities for action the COVID-19 pandemic. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 554–566. [acesso em 4 mai 2020]. Publisher Full Text\n\nIto A, Ishioka T: Exploring the impact of the COVID-19 pandemic on the mental health of rehabilitation therapists. J Rehabil Neurosci. inpress. 2020. [acesso em 18 mai 2020]. Publisher Full Text\n\nRoyal College of Occupational Therapists: A quick guide for occupational therapists: Rehabilitation for people recovering from COVID-19. London, 2020. [acesso em 16 mai 2020]. Reference Source\n\nBarroso BID, De Souza MBCA, Bregalda MM, et al.: Saúde do trabalhador em tempos de covid-19: reflexões sobre saúde, segurança e Terapia Ocupacional. Cad Bras Ter Ocup. São Carlos. 2020; 28(3). [acesso em 15 abr 2020]. Publisher Full Text\n\nda Conceição AF, da Silva AP, de Vargas LD, et al.: O acesso vertiginoso na aquisição de máscaras faciais e suas repercussões na saúde pública e de populações vulneráveis/The vertiginous access in the acquisition of face masks and their repercussions on pubic health and vulnerable populations. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 380–388. [acesso em 13 mai 2020]. Publisher Full Text\n\nLiu DCY, Koo TH, Wong JKK, et al.: Adapting re‐usable elastomeric respirators to utilise anaesthesia circuit filters using a 3D‐printed adaptor ‐ a potential alternative to address N95 shortages during the COVID‐19 pandemic. Anaesthesia. 2020; 75(8): 1022–1027. [acesso em 11 abr 2020]. Publisher Full Text\n\nAmerican Occupational Therapy Association: Inpatient Occupational Therapy—Decision Guide for COVID-19. 2020. [acesso em 13 abr 2020]. Reference Source\n\nBrettenny E: Metacognivite: 7 Steps For Thinking About The Coronavirus Pandemic. Estelle Brettenny Co. 2020. Acesso em: 6 mai 2020. Reference Source\n\nDa Silva MR, da Silva PC, Rabelo HD, et al.: A Terapia Ocupacional pediátrica brasileira diante da pandemia da COVID-19: reformulando a prática profissional/Brazilian pediatric Occupational Therapy before the COVID-19 pandemic: reformulating professional practice. Rev Interinst Bras Ter Ocup. 2020; 4(3): 422–437. [acesso em 20 abr 2020]. Publisher Full Text\n\nMiranda EFDS, Santos LBDS, dos Santos JM, et al.: Cotidianidades frente ao coronavírus em uma resiência em saúde: possibilidades construídas junto a terapeutas ocupacionais/“Quotidianity” in the face of the coronavirus in a health residence: possibilities built with occupational therapists. Rev Interinst Bras Ter Ocup. 2020; 4(3): 488–495. [acesso em 20 abr 2020]. Publisher Full Text\n\nFalcão IV, Jucá AL, Vieira SG, et al.: A Terapia Ocupacional na atenção primária a saúde reinventando ações no cotidiano frente as alterações provocadas pelo COVID-19/Occupational therapy in primary health care reinventing actions in everyday front the changes caused by COVID-19. Rev Interinst Bras Ter Ocup. Rio de Janeiro. 2020; 4(3): 333–350. [acesso em 8 abr 2020]. Publisher Full Text" }	[ { "id": "80278", "date": "04 May 2021", "name": "Camila Caminha Caro", "expertise": [ "Reviewer Expertise Occupational therapist graduated from UFSCarMaster and PhD in Occupational Therapy from PPGTO-UFSCarSpecialist in Neuroscience and Rehabilitation by FAMERP" ], "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 am very happy with the opportunity to review this article. The topic is very important for the occupational therapy, especially in this time of pandemic.\nThe article shows important information, but needs a better presentation of the results and discussion. I have some suggestions that are indicated below:\nTitle\nThe title is clear and good. I suggest deleting the word epidemic.\nAbstract\nThe abstract is clear, but I felt the necessity to describe the time considered for the search in the databases. I suggest you to include this information.\n\nIn the results I feel the necessity to inform the classifications of the articles (guidelines for occupational therapy services, quality of life, telehealth, social actions, and possibilities of occupational therapy during the pandemic).\nIntroduction\n\nThe introduction is clear and demonstrated the importance of the study. I suggest you write more about the different consequences of COVID-19 in the health, for example, in the memory, lost muscle strength, fatigue and tiredness, in moderate and severe cases, and the impact of this in the activities of daily living during and after hospitalization.\nMethods\nI suggest that you include a topic with criteria of exclusion that were showed in the Figure 1.\nSearch strategy section\nFirst paragraph - What are the websites of occupational therapy entities and organizations? It is necessary to describe where they are found.\n\nIn the second paragraph, I suggest you cite the reference of use PICO strategy.\n\nIn the last paragraph, I suggest you inform that neither time nor language was used in the FIRST TIME.\nData collection and analysis section\nWhat type of analysis is used? Simple? Descriptive?\nResults and discussion\nYou have a lot of results. I suggest that you create topics for presentation and discussion of the results.\n\nAll legends of the tables and figures need to indicate the number of articles.\n\nIn the second paragraph, it is necessary to indicate the number of studies of American Occupational Therapy Association (AOTA). It is important to present the references of the articles in the text. It is too important that you describe these performance guides.\n\nIn the third paragraph, it is necessary to present the number of studies. It is interesting that you describe some results of the technical notes here.\n\nIn the fourth paragraph, it is necessary to present the number of each type of study founded.\n\nIn the fifth paragraph it is necessary to present the number of publications about guidelines for occupational therapy services. Is too important to presents the main information of these articles and after discusses them. Where are from these articles? There are differences between the places?\n\nIn the seventh paragraph it is necessary to present the number of publications about quality of life during isolation. How many articles were writing in English? Where are from these articles? There are differences between the places? It is important to present the references of the articles in the text.\n\nEight paragraph - How many articles were written in Portuguese? Where are from these articles? There are differences between the places? It is important to present the references of the articles in the text.\n\nThe tenth paragraph has similar information of the eighth. It is possible put this information together.\n\nIn the eleven paragraph, it is necessary to present the number of publications about telehealth. Where are from these articles? There are differences between the places? It is important to present the references of the articles in the text.\n\nTwelfth paragraph - Where are from these articles? There are differences between the places? It is important to present the references of the articles in the text.\n\nIn the thirteenth paragraph, it is important to discuss the possible negative impacts of telehealth.\n\nIn the fourteenth paragraph, it is necessary to present the number of publications about occupational therapy actions in the social context. Where are from these articles? There are differences between the places? It is important to present the references of the articles in the text.\n\nFifteenth paragraph: Where are the articles about LGBTQIA+ community from? It is import to discuss the articles in terms of differences between countries.\n\nIn the sixteenth paragraph it is necessary to indicate the origin of the article.\n\nIn the seventeenth paragraph, it is necessary to present the number of articles in each classification. Is it a new classification? This classification is different of the presented in the first paragraph of Results and Discussion Section?\n\nEighteenth to Twenty-second paragraph - Where are these articles from? There are differences between the places? It is important to present the references of the articles in the text.\n\nTwenty-third paragraph: This paragraph must be in the Conclusion section.\n\nThere are differences between the countries in the tackling the pandemic? These differences have possible relations with the scientific publications of this rewiew? These discussions can help in the understanding the occupational therapy knowledge developed in different places.\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? Partly", "responses": [] }, { "id": "98271", "date": "01 Nov 2021", "name": "Chung-Ying Lin", "expertise": [ "Reviewer Expertise Occupational therapy", "psychosocial aspects" ], "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 applaud for the authors' efforts in conducting such an important topic regarding the effectiveness of occupational therapy during the era of COVID-19 pandemic. In general, the systematic review is well written and followed by the standard (i.e., the PRISMA guideline). The Results section is meaningful and have clear information that demonstrates the synthesized results. However, I believe that the systematic review can be improved with three minor comments from my side:\nIn the Introduction, I would like the authors to mention that occupational therapy can be applied to different aspects of human daily livings, including their sleep (Fan & Drumheller, 20211). This can strengthen the rationale of conducting this meaningful systematic review.\n\nI would suggest changing the title from \"the new coronavirus (COVID-19) epidemic\" to \"COVID-19 pandemic\". Given that the COVID-19 is spread over the world, epidemic cannot reflect the true situation of the severity of COVID-19.\n\nI would like to see a clear PICO in the present systematic review, especially I would like to see the outcomes in the authors' mind. It seems that the authors have several outcomes and quality of life is the most important one that the authors want to assess. However, this is not clearly stated in the systematic review.\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": [] } ]	1	https://f1000research.com/articles/10-75
https://f1000research.com/articles/10-74/v1	05 Feb 21	{ "type": "Research Article", "title": "Prevalence of pre-operative anxiety and associated factors among a group of women undergoing gynaecological surgeries at a single unit in a tertiary care hospital in Sri Lanka", "authors": [ "Madura Jayawardane", "Wedisha Gankanda", "Malsha Gunathilake", "Wedisha Gankanda", "Malsha Gunathilake" ], "abstract": "Background: Surgery-related anxiety is universal, leading to complications. The objective of this study was to assess the prevalence of pre-operative anxiety levels among a group of patients. Methods: A descriptive cross-sectional study of 64 women was conducted in a tertiary care hospital, Sri Lanka. Patients who underwent emergency surgeries, those with mental illnesses or those aged <18 years were excluded. Pre-operative assessment was done one day prior to the surgery using a self-administered Sinhala validated Amsterdam-Preoperative-Anxiety-and-Information-Scale (APAIS), Hospital Anxiety and Depression Scale (HADS) and Visual-Analogue-Scale (VAS). The APAIS consists of six questions which assess three anxiety components: anesthesia-related-anxiety (Sum A), surgery-related-anxiety (Sum S) and information-desire-component (Sum IDC). The combined score (Sum C) is given by the total of Sum A and Sum S. A Sum C of ≥11 indicates significant anxiety. Results: The mean age of participants was 38.03 years (SD=13.53 years). The mean total score of APAIS was 10.36 (4.06), of HADS was 5.734 (4.487) and of VAS was 3.156 (2.773). All scores were higher in participants <50 years. There were negative correlations between age and anxiety levels in VAS and APAIS scales; the Sum IDC in APAIS (r=-0.416, p=0.001) and VAS scores (r=-246, p=0.050) showed significant negative correlations. Excepting Sum IDC all APAIS, HADS and VAS scores were higher among the group without insurance; despite free healthcare. However only Sum S (t=-3.716, p=0.000) and Sum C (t=-2.281, p=0.026) in APAIS, HADS (t=-3.412, p=0.001) and VAS (t=-2.135, p=0.037) had statistically higher values. Anxiety scores were higher in the group that underwent minor surgeries but where not significantly related to education level, marital status, income, employment or living status. Conclusions: Pre-operative anxiety is common. Age <50 years, lacking insurance cover and undergoing minor surgeries are associated with increased pre-operative anxiety levels. Screening and appropriate interventions would be beneficial.", "keywords": [ "Pre-operative anxiety", "Surgery related anxiety", "Anxiety" ], "content": "Introduction\n\nAnxiety is an emotional state described as a vague, uneasy feeling, the source of which is often nonspecific. It is known to cause abnormal hemodynamics as a consequence of sympathetic, parasympathetic and endocrine stimulation1. Barlow described anxiety as a unique and coherent cognitive-affective structure within our defensive and motivational system2. At the base there is a sense of uncontrollability focused largely on possible future threats, danger, or other upcoming potentially negative events2. Anxiety can only be understood by taking into account some of its cognitive aspects, particularly because a basic aspect of anxiety appears to be uncertainty3. The uncertainty regarding these situations highlights a lack of control that contributes to feelings of anxiety and makes coping more difficult4.\n\nPatients awaiting surgery are more likely to develop anxiety in the pre-operative period. The study of qualitative aspects of anxiety reveals three distinct dimensions of preoperative fear: fear of the unknown, fear of feeling ill and fear for one’s life5. Pre-operative anxiety has a substantial negative impact on the overall outcome of surgeries. Among them, post-operative pain is the commonest complication6. Pre-operative anxiety has also been found to contribute to other post-operative problems such as nausea, vomiting, tachycardia, hypertension, and an increase in the risk of infection. Pre-operative anxiety in young children undergoing surgery is associated with a more painful postoperative recovery and a higher incidence of sleep related and other problems.\n\nStudies have shown that a large proportion of surgical patients experience considerable levels of pre-operative anxiety. This figure stands at around 60%–80% in the Western population7,8. A study conducted in Ethiopia has shown that 70.3% of patients have had anxiety during the pre-operative period7,9. A study done among a group of patients awaiting general surgeries in Sri Lanka showed that the prevalence of anxiety was 76.7%1.\n\nThere are certain identified factors which contribute to higher levels of pre-operative anxiety among patients, including female gender, younger age and absence of past surgical experience. The study conducted in Sri Lanka showed that females and those who have never undergone surgeries were more anxious than males and those who had previously undergone surgeries, respectively1.\n\nAs women are identified as a vulnerable group, assessing the prevalence and factors that contribute to pre-operative anxiety among them is important as a means of identifying high risk individuals, and to plan interventions that minimize anxiety and related complications.\n\nThe objective of this study was to assess the prevalence of pre-operative anxiety levels and related factors among a group of women awaiting elective gynaecological surgeries.\n\n\nMethods\n\nA descriptive cross-sectional study was conducted from 1/July/2018 to 1/December/2019 at the Professorial Gynaecology Unit, Ward 23, Colombo South Teaching Hospital. A total of 64 patients who were 18 and over years of age who underwent major elective gynaecological surgeries were included in the study. Patients who were admitted to the ward were checked for eligibility and once consented they were recruited into the study. Patients who underwent emergency surgeries, patients with mental illnesses or those aged below 18 years were excluded from the study sample. The sample size was determined using a non-probability sampling method where all subjects who fulfilled the criteria during the study period were enrolled into the study. Biasness of the study was controlled by excluding subjects who had previously undergone surgeries, those who had previously been exposed to the theatre environment and subjects with psychiatric disorders.\n\nThe data was collected by an independent research person who did not have any directly involvement in the patients’ care during their period of hospital admission for the surgery. Pre-operative assessment was done on the day prior to the surgery. The anxiety level was assessed using the Sinhala version of the self-administered Amsterdam Preoperative Anxiety and Information Scale (APAIS)10, Hospital Anxiety and Depression Scale (HADS)11 and Visual Analogue Scale (VAS)8. A pre-tested self-administered questionnaire was used to gather basic demographic data of the participants. All materials are provided as extended data12.\n\nThe APAIS10 is a self-administered questionnaire that is widely used to assess pre-operative anxiety. It consists of six questions which assess three components of anxiety; anesthesia-related anxiety (Sum A), surgery-related anxiety (Sum S) and information desire component (Sum IDC). Combined score (Sum C) is given by the total Sum of A and Sum S. Its Sinhala version has been validated in Sri Lanka1. In the Sinhala version, a Sum C of 11 or more indicates a significantly anxious patient. VAS is used to measure levels of anxiety. It is a 10 cm size graduated line in which the left end is marked as 0 (“no anxiety”), and the right end is taken as 10 (\"maximum anxiety”). The patients were advised to evaluate their own anxiety level and then mark it on the given line. The Hospital Anxiety and Depression Scale (HADS)11 is also a self-administered scale that consists of 7 questions. The total is scored out of 21. A score of 0–7 is considered as normal, 8–10 and 11–21 are considered as borderline and abnormal anxiety levels respectively.\n\nSPSS version 17 was used to analyze the data. Pearson's correlation analysis was used to assess the correlation between each tool. A paired t-test was used to compare the mean scores of anxiety in different scales with the socio-demographic and surgical factors (age, education level, and marital status, availability of insurance coverage, living status, income and type of surgery) for the two randomized samples. A p value < 0.05 was taken as significant.\n\nEthical approval was obtained from the Ethics Review Committee of the Faculty of Medical Sciences, University of Sri Jayewardenepura. Informed written consent was given by each participant. Identity and privacy of the data of the participants were handled with confidentiality accordingly.\n\n\nResults\n\nA total of 64 women were included in the study13. The mean age of the participants was 38.03 years (SD= 13.53 years, range- 20–83 years). The majority of them (56.3%) were awaiting major surgeries. Table 1 shows the basic demographic characteristics of the participants and the type of surgery they underwent.\n\nTable 2 summarizes the mean scores of three scales. According to the Sum C values in APAIS, 26 (40.6%) had significant pre-operative anxiety levels. According to the total score in HADS, 11 (17.2%), 6 (9.4%) and 47 (73.4%) had abnormal, borderline and normal levels of anxiety, respectively.\n\nThe correlation between information desire component (Sum IDC) and the total score (Sum C) was assessed using the Pearson's correlation. There was no significant correlation between the two scores (r=0.094, p=0.462).\n\nPearson's correlation was used to assess the correlations between Sum C in APAIS, HADS and VAS. All three showed significant correlation with each other; Sum C and HADS (r=0.481, p=0.000), Sum C and VAS (r=0.470, p=0.000), HAD and VAS (r=0.678, p= 0.000).\n\nAll the scores were higher in the <50 years of age category than in the older group (Table 3). Only the Sum IDC had a significant difference (t=2.875, P= 0.006). There were negative correlations between age and anxiety levels in the VAS and APAIS scales. But out of them, Sum IDC in APAIS (r=-0.416, p=0.001) and VAS scores (r=-246, p=0.050) showed significant negative correlations.\n\nThe data is represented as mean values.\n\n*p<0.05 (significantly different mean values).\n\nExcept Sum IDC, all scores in APAIS, HADS and VAS were higher among the group without an insurance. Out of the sub-scores, Sum S (t=-3.716, p=0.000) and Sum C (t=-2.281, p=0.026) in APAIS, HADS (t=-3.412, p=0.001) and VAS (t=-2.135, p=0.037) had statistically higher values.\n\nAll the anxiety scores were higher in the group that underwent minor surgeries. Out of all, Sum A (t=2.237, p=0.029), Sum IDC (t=4.788, p=<0.001) scores in APAIS and VAS (t=2.520, p= 0.014) showed significantly higher values.\n\nEducation level, marital status, income, employment and living status did not show significant associations with anxiety levels (p>0.05).\n\n\nDiscussion\n\nThe prevalence of pre-operative anxiety in western studies varies from 30–80%1. In this study, the prevalence of anxiety was 40.6%. However, a study performed among Sri Lankan general surgical patients using the same APAIS score and cutoff values has found a higher prevalence of 76.7% of pre-operative anxiety1. They also found that patients with higher information desire (Sum IDC) had more anxiety, whereas in the present study there was no significant correlation between the information desire and anxiety.\n\nThere were significant positive correlations among all three scales. Past studies, including the Sri Lankan study1,14–16, have identified various socio-demographic and surgery related factors contributing to pre-operative anxiety. In the Sri Lankan study the age, education, marital status, category of surgery (major/minor), income and having an insurance coverage were not associated with pre-operative anxiety but this study showed pre-operative anxiety scores of APAIS and VAS were negatively correlated with age1. Out of them, VAS score and the Sum IDC score of APAIS had significant negative correlations with age. Therefore, it is likely that the information desire and levels of pre-operative anxiety reduce with ageing. Furthermore, it was also identified that not having insurance had significantly higher scores in all three scales this is despite the healthcare being free in the study setting. Paradoxically, patients who were undergoing minor surgeries had significantly higher Information desire (Sum IDC) scores and VAS scores compared to those undergoing major surgeries. These findings were contradicting to the findings of former studies1. This may be due to less comprehensive informed consenting practiced for minor surgeries compared to major surgeries in the study setting. There were findings similar to previous studies such no significant associations between levels of pre-operative anxiety and education level, marital state, living alone or with family, employment status and income.\n\nAs this study is part of a clinical trial, prior experience in undergoing surgery or anaesthesia was an exclusion criterion. Therefore, the effect of prior experience on pre-operative anxiety was not assessed.\n\n\nConclusions\n\nOverall, 40.6% of patients undergoing elective gynecological surgeries suffer from anxiety. Age less than 50 years, not having an insurance coverage and undergoing minor surgeries increases the pre-operative anxiety levels in this group. Therefore, screening, early identification and appropriate interventions for those with the above risk factors are may be beneficial to reduce pre-operative anxiety levels.\n\n\nData availability\n\nHarvard Dataverse: Prevalence of pre-operative anxiety and associated factors among a group of women undergoing gynaecological surgeries at a single unit in a tertiary care hospital in Sri Lanka. https://doi.org/10.7910/DVN/KN3AUL13.\n\nThe project contains the following underlying data:\n\n- Prevelance of Pre-operative.tab (Dataset)\n\nHarvard Dataverse: Prevalence of pre-operative anxiety and associated factors among a group of women undergoing gynaecological surgeries at a single unit in a tertiary care hospital in Sri Lanka. https://doi.org/10.7910/DVN/4QMH3Z12.\n\nThe project contains the following extended data:\n\n- F1000_Prevalence of Pre-op_Extended data.docx (Data collection sheets)\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\nMatthias AT, Samarasekera DN: Preoperative anxiety in surgical patients - experience of a single unit. Acta Anaesthesiol Taiwan. 2012; 50(1): 3–6. PubMed Abstract \| Publisher Full Text\n\nBarlow DH: Unraveling the mysteries of anxiety and its disorders from the perspective of emotion theory. Am Psychol. 2000; 55(11): 1247–63. PubMed Abstract \| Publisher Full Text\n\nStrongman KT: The Psychology of Emotion: Theories of Emotion in Perspective. In: Behavioural and Cognitive Psychotherapy. Chichester, UK: Cambridge University Press, Wiley; 1997; 383.\n\nCraig KJ, Brown KJBA: Environmental factors in the etiology of anxiety. Psychopharmacology: the Fourth Generation of Progress. In: Environmental factors in the etiology of anxiety Psychopharmacology: the Fourth Generation of Progress. New York, NY: Raven Press; 1995; 1325–1339.\n\nKain ZN, Mayes LC, Caldwell-Andrews AA, et al.: Preoperative anxiety, postoperative pain, and behavioral recovery in young children undergoing surgery. Pediatrics. 2006 [cited 2020 Aug 19]; 118(2): 651–8. PubMed Abstract \| Publisher Full Text\n\nTrumper M: Bodily Changes in Pain, Hunger, Fear and Rage: An Account of Recent Researches into the Function of Emotional Excitement. Psychol Clin. 1930 [cited 2020 Aug 19]; 19(3): 100–101. Free Full Text\n\nNigussie S, Belachew T, Wolancho W: Predictors of preoperative anxiety among surgical patients in Jimma University Specialized Teaching Hospital, South Western Ethiopia. BMC Surg. 2014 [cited 2020 Aug 19]; 14(1): 67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKindler CH, Harms C, Amsler F, et al.: The visual analog scale allows effective measurement of preoperative anxiety and detection of patients' anesthetic concerns. Anesth Analg. 2000 [cited 2020 Aug 19]; 90(3): 706–12. PubMed Abstract \| Publisher Full Text\n\nBedaso A, Ayalew M: Preoperative anxiety among adult patients undergoing elective surgery: a prospective survey at a general hospital in Ethiopia. Patient Saf Surg. 2019 [cited 2020 Aug 19]; 13(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCelik F, Edipoglu IS: Evaluation of preoperative anxiety and fear of anesthesia using APAIS score. Eur J Med Res. 2018 [cited 2020 Aug 19]; 23(1): 41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStern AF: Questionnaire review the Hospital anxiety and Depression scale. Occup Med (Chic Ill). 2014 [cited 2020 Aug 19]; 64(1): 393–4. Reference Source\n\nJayawardane M, Gankanda WI, Gunathilake IAGMP: Prevalence of pre-operative anxiety and associated factors among a group of women undergoing gynaecological surgeries at a single unit in a tertiary care hospital in Sri Lanka. Harvard Dataverse. Harvard Dataverse, V1. 2021. http://www.doi.org/10.7910/DVN/4QMH3Z\n\nJayawardane M, Gankanda WI, Gunathilake IAGMP: Prevalence of pre-operative anxiety and associated factors among a group of women undergoing gynaecological surgeries at a single unit in a tertiary care hospital in Sri Lanka. Harvard Dataverse. 2021. Harvard Dataverse, V1, UNF:6:BaYrbsHy8UMdA2D69dFkkQ== [fileUNF]; 2021. http://www.doi.org/10.7910/DVN/KN3AUL\n\nMoerman N, van Dam FS, Muller MJ, et al.: The Amsterdam Preoperative Anxiety and Information Scale (APAIS). Anesth Analg. 1996 [cited 2020]; 82(3): 445–51. PubMed Abstract \| Publisher Full Text\n\nNishimori M, Moerman S, Fukuhara S, et al.: Translation and validation of the Amsterdam preoperative anxiety and information scale (APAIS) for use in Japan. Qual Life Res. 2002 [cited 2020 Aug 19]; 11(4): 361–64. PubMed Abstract \| Publisher Full Text\n\nApinya K, Siriporn P, Puchong L: Validity and reliability of the Amsterdam Preoperative anxiety and Information Scale (APAIS); Thai version in adult Thai pre-operative patients. Journal of Psychiatric Association of Thailand. 2009 [cited 2020 Aug 19]; 54: 86–9. Reference Source" }	[ { "id": "79007", "date": "23 Feb 2021", "name": "Chathurie Uththarika Suraweera", "expertise": [ "Reviewer Expertise Psychiatry" ], "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 good study.\n\nThe language needs to be corrected at places.\n\nExplain how the participants were checked for exclusion criteria. Justify excluding participants with mental illnesses. I suggest to re-visit and expand the results section. A higher level of interpretation of the results would have added more scientific rigour to the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "79008", "date": "22 Mar 2021", "name": "Henna Rather", "expertise": [ "Reviewer Expertise Obstetric and gynaecological 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\nA sound cross-sectional study.\n\nClearer description in tabular format of inclusion and exclusion criteria and how these were derived would be beneficial.\n\nThere are a few grammatical errors which need to be corrected.\n\nIt would be beneficial to provide the sample questionnaires/scales (possibly as an appendix) that were given to the patient to assess anxiety levels.\n\nAn expansion on results and discussion would be advantageous. It would be beneficial to expand the results obtained of other factors affecting anxiety listed in table 3, even if not statistically significant, particularly the link between pre-operative anxiety and information desire.\n\nIt would also make the article more comprehensive if, in the discussion, some comparison is made to conclusions found in other studies on pre-operative anxiety around the world.\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": "82932", "date": "12 May 2021", "name": "Thilina Jayarathne", "expertise": [ "Reviewer Expertise Biologics" ], "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 sound cross-sectional study. The scales used in this study are validated in the same setup and internationally. Pre-operative anxiety is a source of negative surgical outcome like prolonged recovery, nausea, vomiting, increased analgesia needs and post-operative infection, therefore this study fills several scientific gaps regarding the same. This study concludes pre-operative anxiety is common in a cohort of gynecological surgical patients in Sri Lanka whereas a similar study has proven higher prevalence in a cohort of general surgical patients appreciating the differences between specialties. The prevalence is also lower compared to worldwide data; further proving the value of studying regional differences. Paradoxical higher information desire in patients undergoing minor surgeries compared to major surgeries is an important finding which could be intervened. The results are well presented in tabular form, well discussed comparing with local and international studies.\nOverall, this study could be concluded as an impressive scientific study and I recommend it to be indexed without major alterations.\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/10-74
https://f1000research.com/articles/10-73/v1	04 Feb 21	{ "type": "Systematic Review", "title": "Optimal use of tocilizumab for severe and critical COVID-19: a systematic review and meta-analysis", "authors": [ "Cahyo Wibisono Nugroho", "Satriyo Dwi Suryantoro", "Yuliasih Yuliasih", "Alfian Nur Rosyid", "Tri Pudy Asmarawati", "Lucky Andrianto", "Herley Windo Setiawan", "Bagus Aulia Mahdi", "Choirina Windradi", "Esthiningrum Dewi Agustin", "Jonny Karunia Fajar", "Yuliasih Yuliasih", "Alfian Nur Rosyid", "Tri Pudy Asmarawati", "Lucky Andrianto", "Herley Windo Setiawan", "Bagus Aulia Mahdi", "Choirina Windradi", "Esthiningrum Dewi Agustin", "Jonny Karunia Fajar" ], "abstract": "Background: Several studies have revealed the potential use of tocilizumab in treating COVID-19 since no therapy has yet been approved for COVID-19 pneumonia. Tocilizumab may provide clinical benefits for cytokine release syndrome in COVID-19 patients. Methods: We searched for relevant studies in PubMed, Embase, Medline, and Cochrane published from March to October 2020 to evaluate optimal use and baseline criteria for administration of tocilizumab in severe and critically ill COVID-19 patients. Research involving patients with confirmed SARS-CoV-2 infection, treated with tocilizumab and compared with the standard of care (SOC) was included in this study. We conducted a systematic review to find data about the risks and benefits of tocilizumab and outcomes from different baseline criteria for administration of tocilizumab as a treatment for severe and critically ill COVID-19 patients. Results: A total of 26 studies, consisting of 23 retrospective studies, one prospective study, and two randomised controlled trials with 2112 patients enrolled in the tocilizumab group and 6160 patients in the SOC group, were included in this meta-analysis. Compared to the SOC, tocilizumab showed benefits for all-cause mortality events and a shorter time until death after first intervention but showed no difference in hospital length of stay. Upon subgroup analysis, tocilizumab showed fewer all-cause mortality events when CRP level ≥100 mg/L, P/F ratio 200-300 mmHg, and P/F ratio <200 mmHg. However, tocilizumab showed a longer length of stay when CRP <100 mg/L than the SOC. Conclusion: This meta-analysis demonstrated that tocilizumab has a positive effect on all-cause mortality. It should be cautiously administrated for optimal results and tailored to the patient's eligibility criteria.", "keywords": [ "Severe", "critically ill", "COVID-19", "tocilizumab" ], "content": "Introduction\n\nIn December 2019, a novel virus named Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) that causes Coronavirus Disease-19 (COVID-19) began to spread worldwide and it become a pandemic globally1. COVID-19 manifestation ranges broadly from mild symptoms to severe illness. Several studies probed multiple types of inflammatory cytokine levels and found higher levels of interleukin (IL)-1β, IL-1RA, IL-6, IL-7, IL-8, IL-10, IFN-γ, monocyte chemoattractant peptide-1, macrophage inflammatory protein (MIP)-1A, MIP-1B, granulocyte-colony stimulating factor, and tumor necrosis factor-alpha in severe COVID-19 patients2,3. COVID-19 causes severe illness due to activation of the cytokine cascade leading to cytokine release syndrome (CRS), which is delineated by systemic inflammation and multiple organ failure. Therefore, prompt strategies for treating CRS are essential for COVID-19 patients3–5.\n\nIL-6 is a proinflammatory cytokine that plays an essential role in CRS. Activation and secretion of IL-6 by infected monocytes, macrophages, and dendritic cells cause two main effects; a plethora effect on immune cells and the innate immune system, and increased vascular permeability due to secretion of vascular endothelial growth factor (VEGF), resulting in hypotension and acute respiratory distress syndrome3,5,6.\n\nTocilizumab, a humanized monoclonal antibody interleukin-6 receptor (IL-6R) inhibitor, is recommended by the National Health Commission of China for treating severe and critically ill patients with elevated IL-67. Recently, several case reports demonstrated tocilizumab could improve the clinical manifestations of seriously ill COVID-19 patients. Several retrospective case-control, single-armed studies and randomized clinical trials declared promising results of tocilizumab treatment in SARS CoV-2 infection. Nevertheless, some systematic reviews and meta-analyses showed an unclear risk of bias and reported debatable results about tocilizumab's benefit as a treatment6,8–13. We performed a systematic review and meta-analysis to research the risks and benefits of tocilizumab and investigate outcomes from different baseline criteria for administration of tocilizumab as a treatment for severe and critically ill COVID-19 patients.\n\n\nMethods\n\nWe conducted a systematic review and meta-analysis to examine optimal use and baseline criteria for administration of treatment with tocilizumab versus standard of care (SOC) in severe and critically ill COVID-19 patients using data published March to October 2020. All-cause mortality events, length of stay in hospital, and days until death (time to death after first intervention) were measured to determine the risks and benefits of tocilizumab treatment. The baseline criteria for using tocilizumab included physical findings and markers of inflammation such as C-reactive protein (CRP), PaO2 and FiO2 ratio (P/F ratio), lactate dehydrogenase (LDH), D-dimer, ferritin, IL-6, leucocyte, lymphocyte count, platelet count, and procalcitonin. We performed screening of several medical databases (PubMed, Embase, Medline, and Cochrane) to collect data and calculate the risk ratio (RR) and 95% confidence intervals (95% CI). This study used similar methods for the systematic review and meta-analysis to a previous study14, and was reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines accessed from the PRISMA website15.\n\nThe search strategy16, using medical subject headings (MeSH) terms, involved the use of a combination of the following keywords: (tocilizumab) OR (anti-IL-6 monoclonal antibody) OR (IL-6 blockade) OR (IL-6 receptor antagonist) AND severe AND critical ill AND (COVID-19) OR (novel coronavirus disease) OR (SARS-CoV-2). The search was performed by two authors (BAM and CW) in PubMed, Embase, Medline, and Cochrane (March 1st to October 31st 2020, last searched 2nd November 2020) and the language was limited to English. We selected 606 full text and free full text articles from PubMed, included all article types, then we excluded them based on the exclusion criteria of case reports, reviews, editorials, letters, duplicate records, and studies with incomplete data. From filter selection of clinical trials, meta analyses, randomized control trials and systematic reviews within one year we got 42 articles after removing 655 articles (see Figure 1).\n\nThe studies in the three searched databases were included based on the following criteria: (1) patient confirmed for SARS-CoV-2 infection; (2) patients treated with tocilizumab and compared with the SOC; and (3) complete data were provided for clinical outcomes. Exclusion criteria were (1) case reports, reviews, editorials, and letters; (2) duplicate records; and (3) studies with incomplete data.\n\nAll articles that qualified for inclusion according to the selection criteria were included in the analysis. Two independent investigators conducted the study assessment (BAM and CW). Two authors (BAM and EA) extracted necessary data from each included study including: first author, publication year, sample size, gender, baseline criteria for administration for tocilizumab, clinical outcomes of tocilizumab group and SOC group. Another consultant resolved any disagreement between the two investigators’ findings (ANR and TPA).\n\nWe performed a methodological quality assesment of the article using the Newcastle-Ottawa Scale (NOS) before study inclusion. NOS comprises several items including: patient selection (4 points), comparability of the groups (2 points), and ascertainment of exposure (3 points). Each study was interpreted to be low quality (scores <4), moderate quality (scores of 5–6), or high quality (scores ≥7)17. We only included moderate to high quality articles in the analysis. The study assessment was conducted by two independent investigators (CW and EA) using a pilot form. Another consultant resolved any disagreement between the two investigators’ findings (CWN and SDS).\n\nThe study outcomes were all-cause mortality events, length of stay in hospital, and days until death (time to death after first intervention), comparing SOC and tocilizumab. We performed subgroup analysis for those outcomes based on CRP level >100 mg/L, CRP level <100 mg/L, PaO2:FiO2 ratio (P/F ratio) 200–300 mmHg, and PaO2:FiO2 (P/F ratio) <200 mmHg.\n\nData were synthesized using RRs and mean differences (MDs), with 95% CIs. Significance of RRs was determined using the Z test (p<0.05 was considered statistically significant). They were assessed for heterogeneity and possibility of publication bias before calculating significancy. We used the Q test for evaluating the heterogeneity among the included studies. A random effect model was used if heterogeneity existed (p<0.10); if not, a fixed-effect model was adopted. For publication bias, we used Egger’s test and a funnel plot (p<0.05 was considered statistically significant).\n\nWe analyzed the data with Review Manager (RevMan, Cochrane, London, UK) version 5.4.1. Two authors (BAM and JKF) conducted statistical analysis and presented the results in a forest plot.\n\n\nResults\n\nWe obtained 697 qualifying studies, 655 of which were excluded after examining the titles and abstracts. We performed a review of the complete texts for 42 potential studies and 16 studies were then excluded because they were reviews (n=2); systematic review and meta-analyses (n=5); letters (n=1); single-center experiences (n=4); case reports (n=1); brief papers (n=1) or had incomplete data (n=2). Eventually, 26 papers met the inclusion criteria for our meta-analysis; these results are summarized in Figure 1. The characteristics of studies are described in Table 1. We have summarized the results of the outcomes in Table 2.\n\n**The dose and administration of tocilizumab are grouped into:\n\na. Category A: Intravenous Tocilizumab 8mg/kg bb up to 800 mg, added by a second dose after 12–24 hours\n\nb. Category B: Single dose intravenous Tocilizumab 400mg\n\nc. Category C: Single dose intravenous Tocilizumab 600mg\n\nd. Category D: 324 mg of Subcutaneous injections of tocilizumab\n\ne. Category E: not classified\n\nTCZ, tocilizumab; NOS, Newcastle-Ottawa Scale; CRP, C-reactive protein; LDH, lactate dehydrogenase; ICU, intensive care unit.\n\nNote, data were presents as mean ± SD or n [%], SOC, Standard of care; TCZ, tocilizumab; N, number of studies; CRP, C-reactive protein; RR, relative risk; MD, mean difference; pE, p Egger; PHet, p Heterogeneity; CI, confidence interval.\n\nThere is a significant difference between the SOC group and tocilizumab group (RR: 1.65; 95% CI = 1.37, 2.00) from all-cause mortality events (Figure 2) and days until death (time to death after first intervention) (MD: 6.03; 95% CI: 0.31, 11.76). There is no significant difference between the length of stay (MD: -2.05; 95% CI: -5.25, 1.16). All outcomes showed evidence of heterogeneity and the random effect model was adopted.\n\nA) All-cause mortality event; B) Subgroup CRP >100 mg/L; C) Subgroup PaO2:FiO2 200-300 mmHg; D) Subgroup PaO2:FiO2 <200 mmHg. SOC, standard of care; TCZ, tocilizumab; CRP, C-reactive protein; SD, standard deviation; CI, confidence intervals.\n\nThere is a significant difference in all-cause mortality events for patients with CRP level >100 mg/L (RR: 1.78; 95% CI: 1.35, 2.34); P/F ratio 200–300 mmHg (RR: 1.84; 95% CI: 1.35, 2.50); and P/F ratio <200 mmHg (RR: 1.44; 95% CI: 1.28, 1.63). For length of stay in hospital, CRP level <100 mg/L showed a significant difference (MD: -7.75; 95% CI: -10.31, -5.20) (Figure 3).\n\nSOC, standard of care; TCZ, tocilizumab; CRP, C-reactive protein; SD, standard deviation; CI, confidence intervals.\n\nWithin the subgroup analysis, evidence of homogeneity was found and we used the fixed effect model for all-cause mortality events for P/F ratio <200 mmHg and length of stay for CRP level ≥100 mg/L, CRP level <100 mg/L, and P/F ratio 200–300 mmHg. The other parameters were analyzed using the random effect model.\n\nWe assessed the possibility of publication bias using Egger’s test. There was no indication of publication bias (p<0.05) for all outcomes.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first meta-analysis investigating the optimal use of tocilizumab in severe and critically ill COVID-19 patients. The 26 studies analysed, mostly retrospective studies with only two clinical trials (Salvarini et al. and Somerset al.), suggest that treatment with tocilizumab gives fewer all-cause mortality events than the SOC18–43. Lan et al. showed that tocilizumab could not provide additional benefits for clinical outcomes of severe COVID-19, but the mortality rate was lower than the SOC, although this was not statistically different10. Studies from Kaye et al., Zhao, J et al., and Zhao, M et al., reported that tocilizumab showed a statistically significant reduction in mortality and fatality than the SOC, similar to our results9,11,13.\n\nNevertheless, hospital and ICU lengths of stay did not differ between tocilizumab and SOC20–26,31,32,35,40,43. Only one study (Eimer et al.) showed that length of stay in hospital on tocilizumab was shorter than the SOC and it was able to shorten the duration of use of a ventilator. However, for the variable days until death, intervention with tocilizumab resulted in a shorter duration until death than the SOC due to secondary infections after tocilizumab treatment20.\n\nSelection criteria from included studies for using tocilizumab treatment for COVID-19 mostly included similiar clinical manifestations but baseline laboratory parameters varied. Clinical manifestations for tocilizumab treatment eligibility were frequency of respiration ≥30 breaths/min and peripheral capillary oxygen saturation (SpO2) <93% while breathing ambient air. Laboratory markers for tocilizumab treatment eligiblity were P/F ratio, CRP, ferritin, LDH and IL-6. In most studies, baseline criteria for administration of tocilizumab were level of CRP ≥100 mg/L (normal values <6 mg/L), ferritin ≥900 ng/mL (normal value <400 ng/mL), LDH >220 U/L, and P/R ratio 200–300 mmHg18–20,24,36,39,40,42. .However, several studies used baseline criteria for administration of tocilizumab of CRP <100 mg/L and P/F ratio <200 mmHg23,30–34,36,43,44.\n\nThe SMACORE study used baseline criteria for administration of tocilizumab of CRP >50 mg/l, procalcitonin <0.5 ng/mL and P/F ratio <300 mmHg in seriously ill COVID-19 patients. Tocilizumab was first administered at 8 mg/kg (up to a maximum 800 mg per dose) intravenously, repeated after 12 hours if no side effects were reported after the first dose. The result from this study was that tocilizumab administration did not reduce mortality rate or ICU admissions23.\n\nSimilar selection criteria were used by Masia et al.; the eligible participants had CRP >50 mg/l and tocilizumab was given at an initial dose of 600 mg intravenously for a weight of >75 kg or 400 mg when the weight was <75 kg. If their condition worsened, treatment was reevaluated following 24 hours. A second dose of tocilizumab (400 mg) was given if there was no clinical response. The result from this study was that tocilizumab administration significantly reduced the mortality rate32.\n\nIn the randomized trial by Salvarini et al., the selection criteria for tocilizumab treatment were P/F ratio of 200–300 mmHg. Tocilizumab was given intravenously at a starting dose of 8mg/kg until 800 mg within eight hours of randomization, and a second dose administered after 12 hours. This study showed no benefit on disease progression in the tocilizumab group compared with the SOC group42.\n\nAccording to the Moreno-Perez study, candidates for tocilizumab treatment had poor prognostic factors or worsening disease. One of indication for worsening condition was CRP level >100 mg/L or P/F ratio <200 mmHg34.\n\nOur subgroup analysis showed tocilizumab had a good result when CRP levels were ≥100 mg/L and P/F ratio was 200–300 mmHg or <200 mmHg. Administration of tocilizumab for CRP levels <100 mg/L did not reduce mortality and showed a longer length of stay in hospital.\n\nThere are various types of administration of tocilizumab treatment among studies. Tocilizumab can be administrated at a low dose (400 mg or 4 mg/kg) or high dose (800 mg or 8 mg/kg), as a single-dose and then continue with the second dose if clinical condition worsens in 24 hours (maximum 800 mg per dose), intravenously or subcutaneously.\n\n\nStrengths and limitations of the analysis\n\nMeta-analysis on this topic has not been previously conducted; only mortality events and ICU admissions have been reported by previous studies9–11,13. In our study, we evaluate all-cause mortality events, length of stay in hospital, and days until death (time to death after first intervention) and carry out subgroup analysis of baseline criteria for administration of tocilizumab treatment. This study has a larger sample size; 2112 patients in the tocilizumab group and 6160 patients in the SOC group.\n\nThe limitations of this study are that we didn’t perform subgroup analysis outcomes according to the dosage and route of administration tocilizumab and didn’t analyze secondary outcomes after tocilizumab treatment like bacterial or fungal infections, thrombotic events, major bleeding, or requirement of invasive mechanical ventilation requirement. The results of our study should be used carefully because most studies included were retrospective and only two were randomized clinical trials, since it has been difficult to perform randomized trial during this pandemic. A meta-analysis of more clinical trial data will provide a more precise result for tocilizumab treatment in severe and critically ill COVID-19 patients.\n\n\nConclusion\n\nOur study provides meaningful data regarding the effect of tocilizumab in severe and critically ill confirmed COVID-19 patients. Tocilizumab is a treatment option for severe and critically ill COVID-19 patients and it appears to reduce mortality events, especially when CRP level >100 mg/L, P/F ratio 200–300 mmHg, and P/F ratio <200 mmHg. However, tocilizumab should be used cautiously according to proper selection criteria to achieve optimal results and its use should be tailored according to the eligibility of the patients. Further studies are still required, especially regarding optimal dosage and administration route of tocilizumab in COVID-19 patients.\n\n\nData availability\n\nFigshare: Data systematic review and meta-analysis optimal use tocilizumab.zip. https://doi.org/10.6084/m9.figshare.13655894.v116.\n\nThis project contains the following underlying data:\n\n- TCZ_for_COVID-19.csv9\n\nFigshare: Data systematic review and meta-analysis optimal use tocilizumab.zip. https://doi.org/10.6084/m9.figshare.13655894.v116.\n\nThis project contains the following extended data:\n\n- PubMed and Cochrane search strategies (in JPG format)\n\nFigshare: PRISMA checklist for “Optimal use of tocilizumab for severe and critical COVID-19: a systematic review and meta-analysis”. https://doi.org/10.6084/m9.figshare.13655894.v116.\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 here to thank Prof. Dr. dr. Nasronudin, Sp. PD K-PTI for supporting us in completing this study.\n\n\nReferences\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe Q, Wang B, Mao J: The pathogenesis and treatment of the `Cytokine Storm' in COVID-19. J Infect. 2020; 80(6): 607–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahmati M: Cytokine-targeted therapy in severely ill COVID-19 patients: Options and cautions. Eurasian J Med Oncol. 2020; 4(2): 179–181. Publisher Full Text\n\nGarg S, Garg M, Prabhakar N, et al.: Unraveling the mystery of Covid-19 cytokine storm: From skin to organ systems. Dermatol Ther. 2020; 33(6): e13859. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore JB, June CH: Cytokine release syndrome in severe COVID-19. Science. 2020; 368(6490): 473–474. PubMed Abstract \| Publisher Full Text\n\nZhang C, Wu Z, Li JW, et al.: Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality. Int J Antimicrob Agents. 2020; 55(5): 105954. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo P, Liu Y, Qiu L, et al.: Tocilizumab treatment in COVID-19: A single center experience. J Med Virol. 2020; 92(7): 814–818. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu X, Han M, Li T, et al.: Effective treatment of severe COVID-19 patients with tocilizumab. Proc Natl Acad Sci U S A. 2020; 117(20): 10970–10975. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao J, Cui W, Tian BP: Efficacy of tocilizumab treatment in severely ill COVID-19 patients. Crit Care. 2020; 24(1): 524. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLan SH, Lai CC, Huang HT, et al.: Tocilizumab for severe COVID-19: a systematic review and meta-analysis. Int J Antimicrob Agents. 2020; 56(3): 106103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaye AG, Siegel R: The Efficacy of IL-6 Inhibitor Tocilizumab in Reducing Severe COVID-19 Mortality: A Systematic Review. medRxiv. 2020. Publisher Full Text\n\nMojtabavi H, Saghazadeh A, Rezaei N: Interleukin-6 and severe COVID-19: a systematic review and meta-analysis. Eur Cytokine Netw. 2020; 31(2): 44–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao M, Lu J, Tang Y, et al.: Tocilizumab for treating COVID-19: a systemic review and meta-analysis of retrospective studies. Eur J Clin Pharmacol. 2020; 1–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRohman MS, Purnamasari Y, Ilmawan M, et al.: Comparison of major bleeding in patients with acute coronary syndrome that underwent coronary artery bypass grafting treated with clopidogrel or ticagrelor: a systematic review and meta-analysis [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Res. 2020; 9: 99). Publisher Full Text\n\nMcInnes MDF, Moher D, Thombs BD, et al.: Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies: The PRISMA-DTA Statement. JAMA. 2018; 319(4): 388–396. PubMed Abstract \| Publisher Full Text\n\nSuryantoro SD: Data systematic review and meta-analysis optimal use tocilizumab.zip. figshare. Journal contribution. 2021. http://www.doi.org/10.6084/m9.figshare.13655894.v1\n\nStang A: Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses. Eur J Epidemiol. 2010; 25(9): 603–5. PubMed Abstract \| Publisher Full Text\n\nAlbertini L, Soletchnik M, Razurel A, et al.: Observational study on off-label use of tocilizumab in patients with severe COVID-19. Eur J Hosp Pharm. 2021; 28(1): 22–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiran N, Ip A, Ahn J, et al.: Tocilizumab among patients with COVID-19 in the intensive care unit: a multicentre observational study. Lancet Rheumatol. 2020; 2(10): e603–e612. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampochiaro C, Della-Torre E, Cavalli G, et al.: Efficacy and safety of tocilizumab in severe COVID-19 patients: a single-centre retrospective cohort study. Eur J Intern Med. 2020; 76: 43–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanziani LM, Trovati S, Brunetta E, et al.: Interleukin-6 receptor blocking with intravenous tocilizumab in COVID-19 severe acute respiratory distress syndrome: A retrospective case-control survival analysis of 128 patients. J Autoimmun. 2020; 114: 102511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapra R, De Rossi N, Mattioli F, et al.: Impact of low dose tocilizumab on mortality rate in patients with COVID-19 related pneumonia. Eur J Intern Med. 2020; 76: 31–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColaneri M, Bogliolo L, Valsecchi P, et al.: Tocilizumab for Treatment of Severe COVID-19 Patients: Preliminary Results from SMAtteo COvid19 REgistry (SMACORE). Microorganisms. 2020; 8(5): 695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Rossi N, Scarpazza C, Filippini C, et al.: Early use of low dose tocilizumab in patients with COVID-19: A retrospective cohort study with a complete follow-up. EClinicalMedicine. 2020; 25: 100459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEimer J, Vesterbacka J, Svensson AK, et al.: tocilizumab shortens time on mechanical ventilation and length of hospital stay in patients with severe COVID-19: a retrospective cohort study. J Intern Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGokhale Y, Mehta R, Karnik N, et al.: Tocilizumab improves survival in patients with persistent hypoxia in severe COVID-19 pneumonia. EClinicalMedicine. 2020; 24: 100467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuaraldi G, Meschiari M, Cozzi-Lepri A, et al.: Tocilizumab in patients with severe COVID-19: a retrospective cohort study. Lancet Rheumatol. 2020; 2(8): e474–e84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGupta S, Wang W, Hayek SS, et al.: Association Between Early Treatment With Tocilizumab and Mortality Among Critically Ill Patients With COVID-19. JAMA Intern Med. 2021; 181(1): 41–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIp A, Berry DA, Hansen E, et al.: Hydroxychloroquine and tocilizumab therapy in COVID-19 patients-An observational study. PLoS One. 2020; 15(8): e0237693. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKewan T, Covut F, Al–Jaghbeer MJ, et al.: Tocilizumab for treatment of patients with severe COVID-19: A retrospective cohort study. EClinicalMedicine. 2020; 24: 100418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlopfenstein T, Zayet S, Lohse A, et al.: Tocilizumab therapy reduced intensive care unit admissions and/or mortality in COVID-19 patients. Med Mal Infect. 2020; 50(5): 397–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasiá M, Fernández-González M, Padilla S, et al.: Impact of interleukin-6 blockade with tocilizumab on SARS-CoV-2 viral kinetics and antibody responses in patients with COVID-19: A prospective cohort study. EBioMedicine. 2020; 60: 102999. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMikulska M, Nicolini LA, Signori A, et al.: Tocilizumab and steroid treatment in patients with COVID-19 pneumonia. PLoS One. 2020; 15(8): e0237831. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreno-Pérez O, Andres M, Leon-Ramirez JM, et al.: Experience with tocilizumab in severe COVID-19 pneumonia after 80 days of follow-up: A retrospective cohort study. J Autoimmun. 2020; 114: 102523. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel K, Gooley TA, Bailey N, et al.: use of the IL-6R antagonist tocilizumab in hospitalized COVID-19 patients. J Intern Med. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPotere N, Di Nisio M, Rizzo G, et al.: Low-dose subcutaneous tocilizumab to prevent disease progression in patients with moderate COVID-19 pneumonia and hyperinflammation. Int J Infect Dis. 2020; 100: 421–424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuartuccio L, Sonaglia A, McGonagle D, et al.: Profiling COVID-19 pneumonia progressing into the cytokine storm syndrome: Results from a single Italian Centre study on tocilizumab versus standard of care. J Clin Virol. 2020; 129: 104444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamaswamy M, Mannam P, Comer R, et al.: Off-Label Real World Experience Using Tocilizumab for Patients Hospitalized with COVID-19 Disease in a Regional Community Health System: A Case-Control Study. medRxiv. 2020. Publisher Full Text\n\nRamiro S, Mustard RLM, Magro-Checa C, et al.: Historically controlled comparison of glucocorticoids with or without tocilizumab versus supportive care only in patients with COVID-19-associated cytokine storm syndrome: results of the CHIC study. Ann Rheum Dis. 2020; 79(9): 1143–1151. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRojas-Marte G, Khalid M, Mukhtar O, et al.: Outcomes in patients with severe COVID-19 disease treated with tocilizumab: a case-controlled study. QJM. 2020; 113(8): 546–550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRossotti R, Travi G, Ughi N, et al.: Safety and efficacy of anti-il6-receptor tocilizumab use in severe and critical patients affected by coronavirus disease 2019: A comparative analysis. J Infect. 2020; 81(4): e11–e17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalvarani C, Dolci G, Massari M, et al.: Effect of Tocilizumab vs Standard Care on Clinical Worsening in Patients Hospitalized With COVID-19 Pneumonia: A Randomized Clinical Trial. JAMA Intern Med. 2020; 181(1): 24–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomers EC, Eschenauer GA, Troost JP, et al.: Tocilizumab for treatment of mechanically ventilated patients with COVID-19. medRxiv. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel U, Xie JJ, Palmer Q, et al.: An Extraordinary Case of Extranodal Non-Hodgkin's Lymphoma Presenting as Common Bile Duct Mass. J Gastrointest Cancer. 2020; 51(1): 359–362. PubMed Abstract \| Publisher Full Text" }	[ { "id": "78921", "date": "05 Feb 2021", "name": "Lorenzo Cosmi", "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\nThe manuscript “Optimal use of tocilizumab for severe and critical COVID-19: a systematic review and meta-analysis\", is an exhaustive meta-analysis on studies that have evaluated the efficacy of treatment with tocilizumab in COVID-19. The meta-analysis is well performed, and the results are convincing and in line with the real life evidences, which suggest an efficacy of such a treatment in selected patients. The authors correctly underline that the treatment works in patients with high inflammatory status and with impairment of respiratory function (P/F ratio below than 300 mm Hg). The concept that tocilizumab improves the prognosis of severe COVID-19 patients, reducing hyper inflammation that contributes to vascular pulmonary damage is an important message for the scientific community.\nI have no particular concerns on this manuscript. I only suggest to quote in the introduction also a recent paper that focuses on the ability of tocilizumab to improve the lung perfusion in these patients (Salvati et al., 20201).\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "79722", "date": "02 Mar 2021", "name": "Wiwien Heru Wiyono", "expertise": [ "Reviewer Expertise Clinical Pulmonology" ], "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) Data extraction and quality assessment: Two independent investigators conducted the study assessment (BAM and CW). Two authors (BAM and EA) extracted necessary data. I think it is not necessary to inform that one acts as an independent investigator, while he is also a member of the authors who extracted the data. Its independence is questionable.\n\n2) Conclusion: Tocilizumab is a treatment option for severe and critically ill COVID-19 patients and it appears to reduce mortality events, especially when CRP level >100 mg/L, P/F ratio 200–300 mmHg, and P/F ratio < 200 mmHg.\nThe authors did not explain in the discussion that P/F ratio 200-300 mmHg and P/F ratio < 200 mmHg is actually different in mode.\n\nWhen the mode of both variables is ignored, why not just conclude: when P/F ratio < 300 mm Hg.\n\nClinicians need clear conclusions, the authors' conclusions about P/F ratio 200-300 mmHg and P/F ratio < 200mm Hg are confusing.\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": [ { "c_id": "6415", "date": "04 Mar 2021", "name": "Satriyo Dwi Suryantoro", "role": "Author Response", "response": "Dear Prof. Wiwien, 1) Thank you for the helpful and considerate advices. 2) To all intents and purposes, in this research we didn't particularly examine and conclude the effect of TCZ on P/F ratio less than 300, yet we figured out significantly good impact/effect of TCZ on patients with mild ARDS (P/F ratio 200-300 mmHg), as we have mentioned on page 9 of the manuscript." } ] }, { "id": "81031", "date": "11 Mar 2021", "name": "Zhongheng Zhang", "expertise": [ "Reviewer Expertise critical care 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 meta-analysis can provide updated evidence for an important clinical question; However, I have several comments:\nThe authors combined evidence from RCT and observational studies, which is not appropriate; the authors need to at least perform subgroup (sensitivity) analysis by restricting only high-quality trials.\n\nBayesian meta-analysis may be appropriate for down-weighing observational studies.\n\nMost studies are small in sample size; the limitation of small study effect should be addressed and discussed; this effect has been well characterized in literature (Zhang et al., 20131).\n\nMany observational studies may include adjusted effect size, try to combine this adjusted OR (RR) and to see whether this will change the current conclusion.\n\nMany interventional meta-analysis on COVID-19 have been published in the literature (Kim et al., 20202, Aziz et al., 20213, Zhao et al., 20204 and Kotak et al., 20205); the authors need to clarify how their study can add to the existing literature.\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": "6445", "date": "12 Mar 2021", "name": "Satriyo Dwi Suryantoro", "role": "Author Response", "response": "We would like to firstly express our gratitude for having our manuscript reviewed thoroughly. Best regards, Research team." } ] } ]	1	https://f1000research.com/articles/10-73
https://f1000research.com/articles/9-1308/v1	10 Nov 20	{ "type": "Software Tool Article", "title": "MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy", "authors": [ "Sébastien Tosi", "Lídia Bardia", "Maria Jose Barallobre", "Arrate Muñoz-Barrutia", "María Luisa Soto-Montenegro", "Julien Colombelli", "Lídia Bardia", "Maria Jose Barallobre", "Arrate Muñoz-Barrutia", "María Luisa Soto-Montenegro", "Julien Colombelli" ], "abstract": "We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.", "keywords": [ "3D stitching", "Lighsheet microscopy", "Tiled scan", "Mosaic", "ImageJ" ], "content": "Introduction\n\nA number of open source tools are available to stitch mosaics from optical microscopy 3D tiled scans1–4 but they all implement automated algorithms, potentially converging to a suboptimal solution. This is especially likely if the initial positions are far from the optimal positions, or if the data suffers from unexpected artifacts. Even worse, this situation can be difficult to detect in practice since these tools bring no or scarce support to check the results and correct for errors manually. Additionally, some software limits the size of the datasets1,2, or requires the data to be redundantly converted to ad-hoc formats3. Finally, none of these solutions handles both dual-side illumination and dual-side camera detection, two useful lightsheet microscopy5 modalities that can advantageously be combined (Figure 1, Left). We developed MosaicExplorerJ to address these shortcomings and bring a complementary alternative to ImageJ BigStitcher4, the reference in the field.\n\nLeft: Illuminating the sample and collecting the light from both sides enables to image a sample by lightsheet microscopy in the best conditions. Middle: Overlapping tiles (green, blue, red) from a 3D mosaic are shifted axially to compensate for the tilt of the lightsheet (here exaggerated around Y axis). Right: Reconstructed mosaics from both cameras are aligned before stacking their best section.\n\n\nMethods\n\nWhereas stitching the tiles of confocal microscopy datasets chiefly consists in compensating for the scanning head to sample stage tilt, stitching lightsheet microscopy datasets is compounded by the fact that the lightsheet is not necessarily perfectly collinear to the object plane of the detection objective. This can lead to 1) a distortion of the aspect ratio of the images (often negligible), 2) an apparent axial displacement of the tiles while moving across the mosaic. This second effect can be simply compensated by axially offsetting the 3D tiles accordingly (Figure 1, Middle), but additional lightsheet non-uniformity (or lateral misalignment) can lead to differences in the features visible in the regions of tile overlap; potentially weakening correlation based algorithms. To address these issues, MosaicExplorerJ does not implement any automated stitching but instead assists the user in visually aligning the tiles along their possible degrees of freedom. This is basically achieved by following a step-by-step procedure to compensate for the misalignments highlighted in the regions of overlap between adjacent tiles.\n\nImageJ/Fiji should be installed and MosaicExplorerJ run from ImageJ macro editor. If no other data is available, the software can be tested with the data provided.\n\nFirst, the mosaics from both cameras (and illumination sides) should be aligned independently before being aligned together. This first operation can be as simple as joining two matching features between two adjacent tiles (Figure 2B), while compensating lightsheet tilt (Figures 2C, 2D) and the axial wobbling of the motors forming the mosaics might respectively require to adjust the axial shift of the tiles forming the top left corner or the first row and first column of the mosaic. Dual-camera alignment includes a calibration step to compensate for discrepancy between the magnifications of both detection objectives (Figure 1, Right). Finally, the user can save the overall mosaic alignment for further inspection, or export the stitched dataset as a TIFF series. Several tile blending modes are available.\n\nA: Two mosaics (2×8 tiles each with a full overlap in the central column along Y) from a dual illumination side scan aligned with MosaicExplorerJ, the arrows show the directions of the light sheets (the view is turned and cropped to fit the figure). Scale bar: 1 mm. B: Zoomed region from the red square prior to adjusting the XY positions of the tiles but after axial shift (Z) correction. The user joins two matching features (white arrow) from two adjacent tiles. C: The XY positions of the tiles are adjusted based on the previous user input and for correct alignment matching features are highlighted in white in the region of overlap. D: Same view but prior to axial shift correction to compensate for lightsheet tilt: no matching features are apparent in the region of overlap. Scale bar: 100 µm.\n\nMosaicExplorerJ has been extensively tested by stitching several large lightsheet microscopy datasets acquired from diverse optically cleared biological samples (see Data availability).\n\n\nUse cases\n\nAll datasets were successfully aligned, each time in under 30 minutes. The results were checked visually by scrolling through the slices and ensuring that the alignment was correct in the regions of overlap between the tiles. The dataset Brain2_izq_2x8Mosaic_LeftSide_300GB was also aligned by BigStitcher, leading to similar results both visually and quantitatively (Extended Data Table S16). It took about 2h30 to process this dataset with BigStitcher (from 3D TIF tiles to computed alignment), excluding the conversion from TIFF series (the original format of this dataset) to 3D TIFF tiles. This time is expected to scale at least linearly with increasing dataset size for BigStitcher while alignment time in MosaicExplorerJ is relatively constant, and mostly conditioned by the degrees of freedom of the 3D tiles. After alignment, the exportation of the stitched images to TIFF series could be achieved in a comparable time with both tools.\n\n\nConclusion\n\nMosaicExplorerJ brings a complementary alternative to BigStitcher, and presents a number of advantages (Extended Data S2 and Table S26). No fiducials or detectable feature points are required, which makes the tool robust, versatile, and compatible with large optically cleared samples for which introducing fiducials is virtually impossible. The processing is fast, and terabyte-size datasets can be explored on the fly without conversion to an intermediate format, even on laptop computers with limited memory. Finally, dual-side camera detection is supported and all alignment steps are performed visually, which brings direct control and feedback both on the imperfections of the datasets and on the quality of the results, minimizing the risk of leaving coarse errors unnoticed.\n\n\nData availability\n\nA complete description of the datasets used to test MosaicExplorerJ, including sample preparation and imaging can be found in Extended Data S1. The full datasets are too large (1.5TB) to feasibly host on a data repository; however, datasets can all be accessed publicly on the IRB Barcelona Google Drive at: https://bit.ly/37iocrP.\n\nZenodo: MosaicExplorerJ F1000Research article extended data. https://doi.org/10.5281/zenodo.41560306.\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 and documentation are available from: https://github.com/SebastienTs/MosaicExplorerJ\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.41068487.\n\nLicense: GNU General Public License v3.0.", "appendix": "Acknowledgements\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology). MJB acknowledges the support of Jérôme Lejeune Foundation.\n\n\nReferences\n\nEmmenlauer M, Ronneberger O, Ponti A, et al.: XuvTools: free, fast and reliable stitching of large 3D datasets. J Microsc. 2009; 233(1): 42–60. PubMed Abstract \| Publisher Full Text\n\nhttps://imagej.net/Grid/Collection_Stitching_Plugin.\n\nBria A, Iannello G: TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics. 2012; 13: 316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHörl D, Rojas Rusak F, Preusser F, et al.: BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples. Nat Methods. 2019; 16(9): 870–874. PubMed Abstract \| Publisher Full Text\n\nHuisken J, Swoger J, Del Bene F, et al.: Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 2004; 305(5686): 1007–1009. PubMed Abstract \| Publisher Full Text\n\nTosi S, Bardia L, Colombelli J, et al.: MosaicExplorerJ F1000Research article extended data (Version 1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4156030\n\nTosi S: SebastienTs/MosaicExplorerJ: First_Release (Version 1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4106848" }	[ { "id": "74555", "date": "07 Dec 2020", "name": "Stephan Preibisch", "expertise": [ "Reviewer Expertise Image analysis", "image registration", "lightsheet" ], "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 MosaicExplorerJ, an ImageJ macro for manual alignment of tiled lightsheet acquisitions.\nI think it can be useful as it provides simple, editable source code. I personally found it quite hard to use, but more practice might make it more efficient. But more explanations of the parameters and keyboard shortcuts in the application would certainly be helpful (press F1 or similar).\nMost importantly, the paper should clearly state the limitations of the software and correct wrong statements about the comparison to other tools, in particular BigStitcher from my point of view.\nNot mentioned limitations (or implicitly assumed ones):\nall files need to have the same dimensions (also in z)\n\nonly 3D translation models are supported\n\nno chromatic aberration correction\n\nmaximal xy output size is 2^31 pixels, the limitation of ImageJ (around 47000x47000 pixels)\nIncorrect comparisons that should be corrected: 1) “As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly.”\nThat is the same in BigStitcher, conversion to HDF5 is not necessary, TIFFs can be virtually loaded and displayed instantaneously\n\n2) “but they all implement automated algorithms … Even worse, this situation can be difficult to detect in practice since these tools bring no or scarce support to check the results and correct for errors manually.”\nBigStitcher provides user friendly methods for manual & interactive alignment (3D translation): https://imagej.net/BigStitcher_manual_translation\n3) “Finally, none of these solutions handles both dual-side illumination and dual-side camera detection, two useful lightsheet microscopy”\nBigStitcher supports dual-side illumination and dual-side camera detection (e.g. Supplementary Figure 17)\n4) S2-Comparison with BigStitcher:\n3D TIFF tiles can be natively used through virtual loading, live display in BDV even. HDF5 conversion is optional (yet very useful)\n\n2D TIFF slices are also supported, but it's a quite hidden feature]\n\nBigStitcher supports Dual-camera support\n\nPhysical model constrained grid alignment is also possible, see link above, even interactive & manual\n\nAutomatic color highlighting of tiles mismatch can be done in BigStitcher\n\nBigStitcher additionally supports per-tile intensity correction using Affine 1D models\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6293", "date": "04 Feb 2021", "name": "Sebastien Tosi", "role": "Author Response", "response": "First of all, we thank the reviewer for his thorough comments and we would like to apologize for overlooking some of BigStitcher's features that we were genuinely unaware of. Also, as rightly pointed out by the reviewer, some limitations of MosaicExplorerJ were not discussed in the article. We have remedied this, as described point by point in our answer, and we hope that the features and the operating workflow of the two applications are now reflected more accurately in the fully updated Extended Data S2. > More explanations of the parameters and keyboard shortcuts in the application would certainly be helpful (press F1 or similar). We have tried our best to improve this aspect in MosaicExplorerJ (v1.5, now on GitHub): a help window can now be displayed by pressing F1, contextual information is provided in ImageJ status bar (and strategically in some of the dialog boxes), and the manual operation to perform axial adjustment of the tiles has been simplified. Finally, five video tutorials (bit.ly/2KY0BDN) were created to illustrate the main operations and to complement the documentation. > Not mentioned limitations (or implicitly assumed ones) of MosaicExplorerJ: 1. All files need to have the same dimensions (also in z)Actually, this is not strictly required as missing tiles or tiles missing Z slices (for 2D TIFF series mode) will not trigger an error but the corresponding images will just be displayed black. However, to answer this comment, we now state in the Abstract of the article (and in Extended Data S2) that the tiles should be organized on a regular 2D grid. 2. Only 3D translation models are supported Yes, only these operations are supported for a single camera dataset but 2D similarity (rotation, scaling, translation) is performed (after 3D translations) when registering the mosaics from two opposing cameras (as described in Figure 1). 3. No chromatic aberration correction We now explicitly state in Extended Data Table S2 that chromatic aberrations correction is not supported by MosaicExplorerJ (while it is supported by BigStitcher). 4. Maximal xy output size is 2^31 pixels, the limitation of ImageJ (around 47000x47000 pixels) We now explicitly state in Extended Data Table S2 that mosaics stitched by MosaicExplorerJ are limited to about 47.000 x 47.000 pixels (and eight channels) while BigStitcher sets no limit. We haven’t included this information in the text of the main article as we believe that it is not really limiting in practice. Incorrect comparisons that should be corrected: 1) “As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly.” That is the same in BigStitcher, conversion to HDF5 is not necessary, TIFFs can be virtually loaded and displayed instantaneously. We apologize for this misleading statement and we were unaware that tiles could be loaded as a set of 2D TIFF series in BigStitcher. Still, we believe that HDF5 conversion is implicitly required for efficient operations when handling TB datasets, both for interactive 3D viewing and to enable a practical processing time (especially when several rounds of tile placement / parameters optimizations have to be performed to get to satisfying results). We updated this paragraph of the text to reflect this idea, and we stated in Extended Data Table S2 that while not strictly required HDF5 conversion is desirable to speed up operations. 2) “but they all implement automated algorithms … Even worse, this situation can be difficult to detect in practice since these tools bring no or scarce support to check the results and correct for errors manually.” BigStitcher provides user friendly methods for manual & interactive alignment (3D translation): https://imagej.net/BigStitcher_manual_translation We were aware of this feature but, as far as we can tell, tiles are first automatically laid down to a regular grid (with fixed overlap), and then only individual tiles (or whole groups) can be interactively moved. This feature is certainly useful to coarsely set the initial positions of the tiles but not so much to finely adjust the mosaic (that would ultimately require to finely move tiles on a tile by tile basis). In turn, thanks to the predefined physical correction models, as few as two pairs of tiles need to be manually adjusted in MosaicExplorerJ to stitch a whole mosaic (with no further processing). We tried to reflect this more accurately in this paragraph and in Extended Data Table S2. 3) “Finally, none of these solutions handles both dual-side illumination and dual-side camera detection, two useful lightsheet microscopy” BigStitcher supports dual-side illumination and dual-side camera detection (e.g. Supplementary Figure 17) We apologize for this plainly wrong statement (we did not know that BigStitcher supported dual-sided cameras as the option is not listed in the image loader). We modified the text to exclude BigStitcher from the set of software not supporting the combination of these two modalities and we reflected these features in details in Extended Data S2 and Extended Data Table S2. 4) S2-Comparison with BigStitcher: 3D TIFF tiles can be natively used through virtual loading, live display in BDV even. HDF5 conversion is optional (yet very useful) 2D TIFF slices are also supported, but it's a quite hidden feature Extended Data Table S2 has been updated to reflect this. BigStitcher supports Dual-camera support Extended Data Table S2 has been updated to reflect this. Physical model constrained grid alignment is also possible, see link above, even interactive & manual For the mosaic stitching part only (excluding cameras registration), MosaicExplorerJ physical correction models include camera tilt (XY translations), lighshsheet tilt (linearly increasing Z translations) and motor axis axial wobble (XY separable Z translations). BigStitcher global tile layout only supports a regular grid with fixed overlap (not accounting for previously mentioned effects). In our view, this is useful to coarsely set the starting positions of the tiles prior to optimization but it is really practical to finely adjust the positions of the tiles of large mosaics with no further processing. We tried to reflect this more accurately in the text and in Extended Data S2. Automatic color highlighting of tiles mismatch can be done in BigStitcher Extended Data Table S2 has been updated to reflect this. MosaicExplorerJ supports alternate red/green (and cyan/yellow) while BigStitcher supports random colors. BigStitcher additionally supports per-tile intensity correction using Affine 1D models Extended Data Table S2 has been updated to reflect this." } ] }, { "id": "75056", "date": "11 Dec 2020", "name": "Francesco Pampaloni", "expertise": [ "Reviewer Expertise Light Sheet Microscopy", "3D cell biology", "stem cell and tumor organoids." ], "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\nOverview\nThe MosaicExplorerJ ImageJ macro-code described in the manuscript is a useful tool to stitch very large data sets recorded with light sheet microscopy. Indeed, the amount of data acquired in a typical session of light-sheet imaging of optically cleared organs (e.g. the mouse brain), reaches easily the TB range with up-to-date equipment. Therefore, user-friendly and straightforward data navigation and stitching software is needed by the scientific community. MosaicExplorerJ, at variance with automatic stitching algorithms such as BigStitcher, it is a fully manual tool, requiring the user manually perform all the alignment steps in order to compensate the tiles mismatch, e.g. the tiles axial shift arising from a tilted light sheet. Moreover, also at variance with BigStitcher, multi-tiff image stacks can be opened directly without conversion. I recommend the manuscript for idexing with minor changes and additions as described in the following remarks.\nRemarks\nThe dataset deposited by the authors on the IRB Barcelona server allows readily testing the macro. The macro runs from the macro editor. The dataset is quickly loaded into the main window. In order to properly upload the tiles, the different files have to be named sequentially following the explained rule. After opening the dataset, the alignment and stitching are performed by manually setting the shifting parameters (xy translation and axial alignment along z) shown in the control panel.\nIn general, a first-time user can achieve the stitching of the mosaic 3D dataset as described in the manuscript by following the steps outlined in the documentation available on GitHub. However, the learning curve is quite steep and some effort and time is necessary by the user in order to understand the working of the macro and to achieve the result. In order to allow for a smoother learning curve, I strongly recommend the authors to upload videos illustrating the stitching procedure for two datasets at increasing difficulty.\nParticularly, the following step described in the documentation (p. 3, end) is not clearly explained, and should be better illustrated with a further figure and in a video:\n“For linear correction, it is recommended to use the top left tile as reference and gradually tweak ZxCor from the tile to the right, until some matching features are apparent in the overlap region. Then ZyCor can be adjusted, this time by considering the tile below. The adjustment of the Z offsets can be performed by typing values in the Panel or by following the procedure described below for free correction.”\nI also recommend to include the documentation in the manuscript extended material as well, and not exclusively in GitHub, as the macro “instructions” are an essential part of the work.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? 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": [ { "c_id": "6294", "date": "04 Feb 2021", "name": "Sebastien Tosi", "role": "Author Response", "response": "Thank you for reviewing the article and for pointing out these weaknesses of the documentation. We have tried to substantially improve the documentation accordingly and we have created five video tutorials to help illustrate all the steps described in the documentation. Please see our detailed answers below. > In general, a first-time user can achieve the stitching of the mosaic 3D dataset as described in the manuscript by following the steps outlined in the documentation available on GitHub. However, the learning curve is quite steep and some effort and time is necessary by the user in order to understand the working of the macro and to achieve the result. In order to allow for a smoother learning curve, I strongly recommend the authors to upload videos illustrating the stitching procedure for two datasets at increasing difficulty. We have included five video tutorials that illustrate the main operations and complement the documentation. These videos are available from the GitHub repository and from the new Extended Data S3. > Particularly, the following step described in the documentation (p. 3, end) is not clearly explained, and should be better illustrated with a further figure and in a video: “For linear correction, it is recommended to use the top left tile as reference and gradually tweak ZxCor from the tile to the right, until some matching features are apparent in the overlap region. Then ZyCor can be adjusted, this time by considering the tile below. The adjustment of the Z offsets can be performed by typing values in the Panel or by following the procedure described below for free correction.” We have completely rewritten this part of the documentation and the new video tutorial S3-V2 is fully dedicated to explaining this critical step. > I also recommend to include the documentation in the manuscript extended material as well, and not exclusively in GitHub, as the macro “instructions” are an essential part of the work. We created a new Extended Data S3 with links to the documentation of the software and to the five new video tutorials. Both are also available from the GitHub repository." } ] } ]	1	https://f1000research.com/articles/9-1308
https://f1000research.com/articles/9-1400/v1	04 Dec 20	{ "type": "Brief Report", "title": "Timing of harvesting reverses the effect of twice cutting with ratoon rice", "authors": [ "Masato Oda", "Huynh Van Thao", "Chiem Nguyen Huu", "Huynh Van Thao", "Chiem Nguyen Huu" ], "abstract": "A new ratoon rice cropping method known as the SALIBU system, which uses the lower nodes of the stubble, is gradually spreading throughout the tropical regions. In the technology package, ‘cutting twice’ has a large effect on the number of ratoons (tillers), and interestingly, the effect changed from positive to negative with changes in the management conditions used to cultivate rice. A previous study investigated conditions including fertilizing, water management, and growth stage at harvesting; however, which of these conditions has the greatest effect on the effect of ‘cutting twice’ remains unclear. We performed a pot experiment to clarify which of the level of fertilizer or the delay of harvesting time affects the number of ratoons. The results showed that with a short delay in harvesting there were almost no ratoons, a long delay in harvesting resulted in many ratoons. By contrast, the single cutting results in ratoons. This indicates the negative effect of cutting twice occur due to earlier harvesting. However, adding fertilizer results in more ratoons, and the response to delayed harvesting showed a similar tendency; in short, no reverse effect occurred due to the level of fertilizer. Harvesting earlier (at the physiological maturation stage; 25% green husk) than normal is emphasized in the SALIBU technology package. However, our results show the ‘cutting twice’ has a negative effect on the number of ratoons when harvesting early.", "keywords": [ "Mekong Delta", "ratoon rice cropping", "SALIBU", "triple cropping rice", "Harvesting timing." ], "content": "Introduction\n\nThe cultivation of rice using ratooning has several advantages over conventional cultivation, including lower costs, lower labor intensity, and lower greenhouse gas emissions (Sacks, 2013; Safruddin, 2017). A new practice for ratooning rice crops using the lower nodes of rice stubble, known as the SALIBU system, is gradually spreading throughout the tropical regions (Fitri et al., 2019; Yamaoka et al., 2017). The practice involves harvesting most of the above-ground portion of the plant but leaves the growing shoots intact. The SALIBU method implements 10 additional management practices to conventional management of rice cultivation in Indonesia. (1) early harvesting, (2) pre-fertilization (at seven days before harvesting), (3) cutting twice (rice was harvested 25 cm above the ground, then cut again at 3–5 cm above ground), (4) late irrigation (irrigation was started on day 14 after harvesting), (5) hand weeding, (6) dividing (dividing hills into two or three tillers and replanting to fill the space), (7) pushing (pushing the rice plants into the soil if the root came up on the soil surface), (8) removing excess plants (to keep original plant density), (9) draining from day 29 to 43 after harvesting, and (10) draining for the above (5) to (9) treatments. However, this new methodology has yet to be well studied. Oda et al. (2019) evaluated the Cohens’ d effect size (Cohen, 1992) of each practice in the technology package and clarified that the process of cutting twice has a large effect. Interestingly, the effect can be reversed depending on the management conditions. There is a large positive effect when poor management conditions are used but a medium negative effect when standard management conditions of direct seeding triple-cropping rice in the Mekong Delta are employed. The poor management conditions consisted of not using fertilizer, continuous-flooding water management, and late harvesting. There an interaction between the management conditions and the practice of cutting twice; however, which of the three conditions was responsible for the interaction has not been clarified. We assumed that it was late harvesting. Therefore, we examined the relationship between delayed harvesting and the number of ratoons under conditions with and without fertilizer. The results showed that the interaction does arise from the delay in harvesting.\n\n\nMethods\n\nA relationship between delayed harvesting and the number of ratoons was analyzed. We harvested rice plants grown under conditions of both with and without fertilizer. Rice was harvested on the day when 75% of the seeds on the main stems turned yellow (days 0), and after on days 4, 8, and 14. The water management approach we adopted was alternative wet and dry (AWD). Note, SALIBU practices are not used in this experiment.\n\nThe experiment was conducted in a screen-house at Can Tho University, Can Tho city, Vietnam from October 2019 to February 2020. The climate is classified as tropical savanna (Aw) and the range of the day average temperature of October to February is 26.0 to 27.3°C (Climate-data.org). Containers of size 38 × 58 × 30 cm (l × w × h) were filled with silty clay soil (52% clay, 48% silt, <1% sand, originally collected from a farmer’s paddy filed in the Mekong Delta; 10°22' N, 105°58' E) to a depth of 20 cm. To remove excess nutrients, the soil was watered, stirred, and the supernatant water was removed. This procedure was conducted three times. Rice (Oryza sativa) seeds of variety ST24 (105 – 110 days provided by Can Tho University) were planted, two seeds per hill, with nine hills per pot (in a 3 × 3 arrangement). The application of fertilizer followed the standards used for direct seeding of triple-cropping rice in the Mekong Delta (AN Giang’s DARD, 2014; on day seven (27.6 kg ha–1 N, 45.2 kg ha–1 P2O5, 3.68 kg ha–1 K2O), 20 (36.7 kg ha–1 N), and 42 (27.6 kg ha–1 N, 3.68 kg ha–1 K2O) after seeding), or pots were left unfertilized. The water management approach used was alternative wetting and drying (AWD) between +3 and –10 cm manually. Irrigation was initiated on day 7 after planting and the pots were drained 10 days before harvesting. Rice was harvested on the day when 75% of the seeds on the main stems turned yellow (as used with the SALIBU method (Yamaoka et al., 2017); days 0), and after on days 4, 8, and 14. Rice was first harvested at 25 cm aboveground, then cut again at approximately 5 cm aboveground on day 7 after each harvesting. The harvested rice straw was cut into pieces approximately 5-cm long and scattered on the soil. Irrigation (AWD) was started on day 7 after the second cutting. The container locations were randomized with four replications in each, with and without fertilizer (32 pots in total). No fertilizer was applied for the ratoons.\n\nWe counted the number of panicles and weighed the seeds of whole pots. The maturity of seeds were measured for composite samples of replications by specific gravity (1.06) method. We counted the number of living leaves (being alive was defined as more than half of the leaf being green) at harvesting and the number of ratoons on day 45 after harvesting.\n\nWe plotted the relationship between the delay of harvesting and the number of ratoons. We showed statistical significance with a 95% confidence interval in the graph. We used Microsoft Excel 2016 formulas (average, stdev.s, and confidence) for statistical analysis.\n\n\nResults\n\nThe rice yields, maturity, and number of panicles were 0.66 (±0.04) kg m–2, 77.4 (±3.1) %, and 67.5 (±3.3) m–2 respectively. Not using fertilizer decreased those properties, but a significant difference was found in the number of panicles (t=0.047) only.\n\nThe application of fertilizer did not reverse the number of ratoons. There was a greater number of ratoons with fertilizer than without fertilizer; the response to delayed harvesting showed the same tendency (Figure 1a).\n\n•: fertilized, Δ: unfertilized. Bars represent 95% CI (n = 4). Rice was harvested on the day when 75% of the seeds on the main stem turned yellow (the standard SALIBU method; days 0), and after on days 4, 8, and 14. Rice was harvested at 25 cm aboveground first, then cut again at approximately 5-cm aboveground on day 7 after each harvesting.\n\nThe number of ratoons increased significantly with increasing delays in harvesting (Figure 1a). In particular, by day 4, there were almost no ratoons. Conversely, living leaves decreased continuously with delayed harvesting (Figure 1b). Finally, by day 14, there were no living leaves.\n\n\nDiscussion and conclusion\n\nOda et al. (2019) show cutting twice can have a large effect on the number of ratoons; however, the effect can be reversed by different management conditions, such as adding fertilizer, delaying harvesting, or changing the type of water management used. We performed a pot experiment to investigate the effects that the level of fertilizer and the delay in harvesting time had on the number of ratoons.\n\nOur results showed that the application of fertilizer does not reverse the effect of cutting twice on the number of ratoons. However, the timing of harvesting can reverse the effect of cutting twice on the number of ratoons. The number of ratoons continuously increased until the rice plants lost all of their living leaves. However, the second cutting conducted on the rice plant harvesting on days 0 resulted in no ratoons. By contrast, the single cutting normally gets ratoons. This means that cutting twice reduces the number of ratoons. Previous work also shows that cutting twice reduces the number of ratoons (Shiraki et al., 2020).\n\nHarvesting earlier than normal (at the physiological maturing stage) is emphasized in the SALIBU technology package; however, our results showed that the second cutting is not advantageous when harvesting at the recommended timing. This is a significant finding for ratoon rice cultivation. The practice of cutting twice originated the custom of conventional harvesting of which cutting height is high. That should be distinguished from using lower nodes of stubble. In other words, twice cutting is recognized as an essential practice in SALIBU (Fitri et al., 2019; Yamaoka et al., 2017) but twice cutting is unnecessary when we can cut rice plant stems near the soil surface at harvesting. The use of a second cutting can have a large effect on the number of ratoons, so further studies should be conducted.\n\n\nData availability\n\nFigshare: Salibu Effect 2. https://doi.org/10.6084/m9.figshare.13142720.v2 (Oda et al., 2020)\n\nThis project contains the following underlying data:\n\n- SALIBU2.xlsx (Ratoon and living leaves data)\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\nThis work was conducted as a joint research project of JIRCAS and CTU.\n\n\nReferences\n\nCohen J: A power primer. Psychol Bull. 1992; 112(1): 155–159. PubMed Abstract \| Publisher Full Text\n\nFitri R, Erdiman, Kusnadi N, et al.: SALIBU technology in Indonesia: an alternative for efficient use of agricultural resources to achieve sustainable food security. Paddy and Water Environment. 2019; 17: 403–410. Publisher Full Text\n\nOda M, Nguyen HC, Huynh VT: Evaluation of cropping method for perennial ratoon rice: Adaptation of SALIBU to triple-cropping in Vietnam [version 3; peer review: 3 approved, 1 approved with reservations]. F1000Res. 2019; 8: 1825. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOda M, Thao Nguyen T, Nguyen HC: Salibu Effect 2. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13142720.v2\n\nSacks EJ: Perennial rice: challenges and opportunities. In: Batello, C. ed. Perennial Crops for food security. FAO, 2013; 16–26. Reference Source\n\nSafruddin S: Effect of Layout Trimming And Urea Fertilizer Dose On Productivity Of Rice (Oryza sativa L.) Varieties Ciherang System Salibu (Ratoon Modification). AGRICULTURA. 2017; 103(3–4): 21–26. Reference Source\n\nShiraki S, Cho TM, Htay KM, et al.: Effects of the Double-Cutting Method for Ratooning Rice in the SALIBU System under Different Soil Moisture Conditions on Grain Yield and Regeneration Rate. Agronomy. 2020; 10(11): 1621. Publisher Full Text\n\nYamaoka K, Htay KM, Erdiman, et al.: Increasing Water Productivity through Applying Tropical Perennial Rice Cropping System (SALIBU Technology) in CDZ, Myanmar. In: The 23rd ICID Congress: Towards A New Green Revolution 2.0. International Commission on Irrigation and Drainage (ICID). 2017. Reference Source" }	[ { "id": "76277", "date": "08 Jan 2021", "name": "Syarifa Mayly", "expertise": [ "Reviewer Expertise Value added product (biochar)", "waste management", "drought stress", "climate change", "tropical agroecosystem", "nutrient management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses the relationship between timing of harvesting (delay harvesting 0, 4, 8, 14 days) and level of fertilizer (with and without fertilizer) on the number of ratoons from the rice crop that had cutting twice at 7 days after harvesting. The parameters of this research are (1). number of ratoons, (2) living leaves, (3) yield, (4) maturity, (6) panicle. Parameter (1) was measured 45 days after harvesting (after twice cutting practices). Parameters (2-6) were measured at harvesting main crop (before twice cutting practices).\nThere are a number of issues/statements that need to be clarified, however. Below are more specific comments by section:\n1. Abstract:\nThe abstract had contained information about general topics of study, previous study, problem of research addresses, results and conclusions. But the objective of study and analytical methods were not seen clearly. The statement that “the effect changed from positive to negative with changes in the management conditions used to cultivate rice” must be described more specifically and with enough data to justify the statement.\n\nThe statement that “By contrast, the single cutting results in ratoons”, how to justify this statement when there was no single cutting in this research and all of rice get twice cutting.\n\nAnd which data in this study can show whether or not there was a reverse effect of twice cutting on number of ratoons from the delay harvesting and level of fertilizer treatment?\n\n2. Introduction:\nThe introduction had contained information about salibu method; gap of previous study that is still unclear. But it needs more information about early planting and the factors which influenced the formation of ratoon like variety, etc. Why did you choose harvesting time at 75%, the seeds on the main stems turned yellow (Physiological maturity), why you do not choose 80% which is also used in some studies of ratoon rice?\n\nThe statement “The results showed that ...\", should be written in the results section.\n\n3. Methods:\nNeed more information about harvesting time at 75%; obtained on how many days after sowing? And regarding the statement “SALIBU practices are not used in this experiment”, it would be clearer to write down which of the 10 additional activities from the Salibu System were not carried out.\n\n4. Treatments:\nThe step of treatments was clear, but needs more information about the details of irrigation (AWD) at ratoon crop. Why don’t you use the same variety from the previous study that still have many that can be clarified?\n\n5. Measurements and Analysis:\nThis is clear.\n\n6. Results:\n- Effect of fertilizer: Which data can be supported to justify your statement (The application of fertilizer did not reverse the number of ratoons)?\n\nThe title of Figure 1 was considered for change which the living leaves were measured before cutting twice practices. Caption 1 needs to add more information about measured time from number of ratoons and living leaves.\n\n7. Discussion and Conclusion:\nData showing the reverse effect of twice cutting by timing of harvesting and level of fertilizer are not seen in the results section, so how to justify the statement there was a reverse effect of twice cutting by timing of harvesting?\n\nThe statement “The number of ratoons continuously increased until the rice plants lost all of their living leaves” showed that the growth of ratoons and living leaves occurs simultaneously, but actually living leaves was for main crop not ratoon crop. The large number of ratoons in delay harvesting was most likely because there were sufficient food reserves from stem for the growth of ratoons. Some literature suggests that the ratooning performances was affected by varieties, water and fertilizer management, and number of nodes.\n\nConclusions of this study are not written clearly.\n\n8. Data Availability:\nAnalysis is clear (Mean, STDEV, CI and t test).\n\nWhy was only the maturity parameter data not displayed in replication form like other parameters' data?\n\n9. Acknowledgement and References:\nThis is 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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6317", "date": "04 Feb 2021", "name": "Masato Oda", "role": "Author Response", "response": "Thank you very much for your valuable comments. We sincerely appreciate it. Please check the manuscript as we have revised it as follows. 1. Abstract: “the effect changed from positive to negative with changes in the management conditions used to cultivate rice” must be described more specifically and with enough data to justify the statement. - We revised. How to justify this statement when there was no single cutting in this research and all of rice get twice cutting. - We explained using literature. Which data in this study can show whether or not there was a reverse effect of twice cutting on number of ratoons from the delay harvesting and level of fertilizer treatment? - We explained. 2. Introduction: The gap of previous study that is still unclear. - We explained using existing studies. It needs more information about early planting. - not \"early planting\" that is conventional in the Mekong delta. We explained it in the “Methods” The factors which influenced the formation of ratoon like variety, etc. - We explained using existing studies. As for variety, we think that it is a future study so we just mentioned it in the “Discussion and conclusion”. Why did you choose harvesting time at 75%, the seeds on the main stems turned yellow (Physiological maturity), why you do not choose 80% which is also used in some studies of ratoon rice? - That is the conventional of the Mekong Delta triple rice cropping and of the recommendation of SALIBU practices. We described that in the “Methods”. The statement “The results showed that ...\", should be written in the results section. - Deleted. 3. Methods: More information about harvesting time at 75%; obtained on how many days after sowing? - On day 91, 95, 99, and 105. We added. “SALIBU practices are not used in this experiment”, it would be clearer to write down which of the 10 additional activities from the SALIBU System were not carried out. - We added the explanation. 4. Treatments: More information about the details of irrigation (AWD) at ratoon crop. - We added. Why don’t you use the same variety from the previous study that still has many that can be clarified? - Jasmine rice is photoperiod sensitive so it can use for getting ratoons but not practical. 6. Results: Effect of fertilizer: Which data can be supported to justify your statement (The application of fertilizer did not reverse the number of ratoons)? - We explained in the “Discussion and conclusion”. The title of Figure 1 was considered for change which the living leaves were measured before cutting twice practices. - We changed to “Rotoons and Living leaves”. Caption 1 needs to add more information about measured time from the number of ratoons and living leaves. - Thank you. We added. 7. Discussion and Conclusion: Data showing the reverse effect of twice cutting by timing of harvesting and level of fertilizer are not seen in the results section, so how to justify the statement there was a reverse effect of twice cutting by timing of harvesting? - We explained it using existing studies. The previous study provides the data. The statement “The number of ratoons continuously increased until the rice plants lost all of their living leaves” showed that the growth of ratoons and living leaves occurs simultaneously, but actually living leaves was for main crop not ratoon crop. The large number of ratoons in delay harvesting was most likely because there were sufficient food reserves from stem for the growth of ratoons. Some literature suggests that the ratooning performances was affected by varieties, water and fertilizer management, and number of nodes. - The best harvesting time for good ratooning is when the culms were still greenish because the growth of ratoon tillers depends on the carbohydrate reserves of the stubble (Fukui, 1988). We revised the explanation. Conclusions of this study are not written clearly. - We revised." } ] } ]	1	https://f1000research.com/articles/9-1400
https://f1000research.com/articles/10-70/v1	04 Feb 21	{ "type": "Data Note", "title": "rt-me-fMRI: a task and resting state dataset for real-time, multi-echo fMRI methods development and validation", "authors": [ "Stephan Heunis", "Marcel Breeuwer", "César Caballero-Gaudes", "Lydia Hellrung", "Willem Huijbers", "Jacobus F.A. Jansen", "Rolf Lamerichs", "Svitlana Zinger", "Albert P. Aldenkamp", "Marcel Breeuwer", "César Caballero-Gaudes", "Lydia Hellrung", "Willem Huijbers", "Jacobus F.A. Jansen", "Rolf Lamerichs", "Svitlana Zinger", "Albert P. Aldenkamp" ], "abstract": "A multi-echo fMRI dataset (N=28 healthy participants) with four task-based and two resting state runs was collected, curated and made available to the community. Its main purpose is to advance the development of methods for real-time multi-echo functional magnetic resonance imaging (rt-me-fMRI) analysis with applications in neurofeedback, real-time quality control, and adaptive paradigms, although the variety of experimental task paradigms supports a multitude of use cases. Tasks include finger tapping, emotional face and shape matching, imagined finger tapping and imagined emotion processing. This work provides a detailed description of the full dataset; methods to collect, prepare, standardize and preprocess it; quality control measures; and data validation measures. A web-based application is provided as a supplementary tool with which to interactively explore, visualize and understand the data and its derivative measures: https://rt-me-fmri.herokuapp.com/. The dataset itself can be accessed via a data use agreement on DataverseNL at https://dataverse.nl/dataverse/rt-me-fmri. Supporting information and code for reproducibility can be accessed at https://github.com/jsheunis/rt-me-fMRI.", "keywords": [ "Functional magnetic resonance imaging", "Real-time fMRI", "Multi-echo fMRI", "Neurofeedback", "Methods development", "Finger tapping", "Motor", "Emotion processing", "Amygdala", "Task", "Resting state" ], "content": "Background and summary\n\nReal-time functional magnetic resonance imaging (fMRI) is a brain imaging method where functional brain signals are acquired, processed, and used during an ongoing scanning session. Applications include real-time data quality control (Dosenbach et al., 2017), adaptive experimental paradigms (Hellrung et al., 2015), and neurofeedback (Sitaram et al., 2017). Neurofeedback is a cognitive training method where the real-time feedback signal is presented back to the participant to allow self-regulation of their blood oxygen level-dependent (BOLD) signal, prompting researchers to investigate it as an intervention for patients with neurological or psychiatric conditions. Work by Ros et al. (2020) and Haugg et al. (2020) show an absence of standardisation in experimental design and outcome reporting restricts the synthesis of evidence to determine the efficacy of fMRI neurofeedback. Further, it remains a major challenge to delineate the sources of variance in the brain and in neurofeedback signals and their eventual effects on neurofeedback training outcomes. Similar challenges exist for separating BOLD and non-BOLD variations and their influences on data quality, and subsequently on all real-time fMRI applications.\n\nIn recent work (Heunis et al., 2020), we investigated the available acquisition and processing methods for improving real-time fMRI signal quality, and identified an absence of methodological denoising studies and a need for community-driven quality control standards. Here, we aim to advance this process by curating a multi-echo fMRI dataset (rt-me-fMRI). It builds on known benefits of multi-echo fMRI for increasing BOLD sensitivity both in resting state and task fMRI (Dipasquale et al., 2017; Gonzalez-Castillo et al., 2016; Kundu et al., 2017; Moia et al., 2020; Olafsson et al., 2015). Potential benefits of multi-echo fMRI in the real-time context have been reported before (Marxen et al., 2016; Posse et al., 2001; Posse et al., 2003; Weiskopf et al., 2005), but real-time multi-echo processing methods remain underexplored. By releasing the rt-me-fMRI dataset, we aim to facilitate a community effort to advance the development of methods and standards in this domain.\n\nThe rt-me-fMRI dataset includes multi-echo resting state and task-based fMRI data from 28 healthy participants. Figure 1 provides an overview, including the task types: finger tapping, emotion processing, imagined finger tapping, and imagined emotion. Several factors influenced the experimental and acquisition protocols:\n\nAcquired data include anatomical MRI, resting state and task-based multi-echo fMRI, task responses and physiology data. The bottom row indicates the order and type of acquired MRI scans. Colour-coding separates the anatomical scan from functional set 1 and from functional set 2. Functional set 1 includes resting state, fingerTapping and emotionProcessing acquisitions, while functional set 2 includes resting state, fingerTappingImagined, and emotionProcessingImagined acquisitions.\n\nMulti-echo fMRI: To facilitate the development of real-time multi-echo methods, all functional acquisitions have multiple echoes. The first resting state run allows calculation of quantitative multi-echo parameters such as baseline T2* or S0 maps, which can in turn be used for echo combination during subsequent runs.\n\nTask and resting state: The motor cortex, amygdala, and visual system were selected as representative regions based on frequency of studies in fMRI and neurofeedback literature (Thibault et al., 2018), and tasks were selected to elicit appropriate BOLD responses. The fingerTappingImagined and emotionProcessing tasks respectively allow investigations into mental imagery and visual shape/face processing. Since these structures are located at distinct anatomical regions that experience different levels of noise (e.g. the amygdala suffers from more severe image dropout and physiological noise; Boubela et al., 2015), this allows investigation of spatially distinct effects of real-time denoising. Resting state scans allow comparison of the effects of processing steps in the absence and presence of a task.\n\nTemplate data: In real-time fMRI applications, anatomical and functional scans are typically acquired before the main session to generate registration, segmentation, and localisation templates. This assists real-time realignment and extraction of region-based signals, and minimises per-volume processing time.\n\nNo neurofeedback: To keep the setup applicable to a range of real-time scenarios without introducing additional confounds, no neurofeedback was provided. Instead, to approximate similar mental states, the second functional set of scans were structured as imagined versions of the first functional set. This is a common approach in neurofeedback training: amygdala neurofeedback participants have been asked to think about an emotional event in their past (e.g. Misaki et al., 2018; Young et al., 2014), while motor cortex neurofeedback participants have been asked to think about performing physical exercises (e.g. Subramanian et al., 2011).\n\nPhysiology data: To facilitate the development and exploration of real-time physiological denoising methods and their relation to multi-echo-derived data, cardiac and respiratory signals were acquired.\n\nThe rt-me-fMRI is available in BIDS format via the DataverseNL repository: https://dataverse.nl/dataverse/rt-me-fmri. A browser-based environment allows interactive exploration of the data quality and derivatives (https://rt-me-fmri.herokuapp.com/).\n\n\nMethods\n\nThe data described here was collected as part of a study for which ethics approval was granted by two ethics review boards. To confirm that the study protocol is in accordance with the Dutch national law on medical-scientific research conducted on human participants (see WMO: https://wetten.overheid.nl/BWBR0009408/2020-01-01), the medical ethical review board at the Máxima Medisch Centrum (Veldhoven, NL) granted ethics approval. Secondly, the local ethics review board at Kempenhaeghe Epilepsy Center (Heeze, NL; where the data was collected) approved the study protocol.\n\nAll participants provided informed and written consent to participate in the study and for their maximally de-identified data (also referred to as limited data) to be shared publicly under specific conditions (see below for GDPR considerations). Participants were provided with an electronic version of a \"Participant Information Letter\" which contained, in addition to standard information about the study protocol, clear information about their personal data privacy and the risks and benefits involved in sharing maximally de-identified versions of their data. They were asked to read it thoroughly and to discuss it with friends and family if they wished to do so. They were granted an opportunity to discuss any questions or concerns about their voluntary participation in the study with the lead researcher, both via email and in person. If they decided to continue with participation, participants signed the consent form and were provided with an electronic copy.\n\nThe dataset was collected, processed and shared in accordance with the European Union's General Data Protection Regulation (GDPR) as approved by Data Protection Officers (DPOs) at Kempenhaeghe Epilepsy Center (Heeze, NL) and the Eindhoven University of Technology. Of particular note is the procedure that was followed to enable sharing of the dataset under specific conditions that allow personal data privacy to be prioritised while adhering to FAIR data standards (\"findable, accessible, interoperable, reusable\"; see Wilkinson et al., 2016), with this being the first documented implementation. It followed from the collaborative effort of the Open Brain Consent Working Group (Bannier et al., 2021), a group of researchers, data experts, and legal practitioners that aim to provide globally standardised templates for informed consent and data privacy statements that allow for brain research data to be shared while prioritising personal data privacy. Steps to accomplish this include following best practices to de-identify brain images (e.g. removing personally identifiable information from image filenames and metadata and removing facial features from T1-weighted images), converting the data to BIDS format, employing a Data Use Agreement, and keeping participants fully informed about each of these steps and the associated risks and benefits. The Data Use Agreement can be accessed in this manuscript's GitHub repository: https://github.com/jsheunis/rt-me-fMRI.\n\nThe rt-me-fMRI dataset consists of MRI and physiology data from 28 healthy, right-handed (self-report) adults recruited from the local student population: 20 male, 8 female; age = 24.9 ± 4.7 (mean ± standard deviation). During recruiting, possible participants were excluded if they reported prior or current (at the time of the study) indications of neurological or psychiatric conditions, or any other standard contraindications for MRI scanning. 31 participants were initially recruited for the dataset, but three were excluded because of technical and administrative challenges. All anatomical scans were inspected by a trained radiologist and no incidental findings were reported.\n\nPreparation and instructions. A single experimenter interacted with all participants. Data for each participant was collected during a single scanning session of approximately 1 hour, preceded by a 30 min onboarding procedure and followed by a 15 min offboarding procedure. Onboarding included a tour of the scanner and related equipment, detailed instructions for the participant to follow during each scan, and time for additional questions.\n\nTo minimise participant motion during scans so as to improve spatial and temporal image quality, participants were asked to remain as still as possible inside the scanner. Additionally, a length of tape was fixed across the participants' foreheads to the stationary part of the head coil. This provided tactile feedback which has been demonstrated as a simple and effective way to reduce head motion during fMRI scanning (Krause et al., 2019).\n\nLights in the scanner room were dimmed during the experiment. Participants viewed instructions projected on a screen at the back of the scanner bore via a head coil-mounted mirror. For resting state functional scans, participants were instructed to keep their eyes open and fixate on the cross on the screen.\n\nExperimental design. All functional scans have 210 volumes and exactly the same sequence parameters. All task scans follow a block design with 10 volumes (i.e. 20 s) per block, and with blocks alternating between control and task conditions. All task designs start and end with a control condition. These block design aspects are depicted in Figure 2 below for all task runs. Take note that the depictions do not necessarily agree with the exact stimuli as seen by the participants, as the depictions below are purely illustrative.\n\nSubfigures include: (A) fingerTapping - right hand finger tapping, (B) emotionProcessing - matching shapes and faces, (C) fingerTappingImagined - imagined finger tapping, and (D) emotionProcessingImagined - emotional memory recollection. All designs follow a block paradigm with 10 volumes (i.e. 20 s) per block, and with blocks alternating between control and task conditions. All task designs start and end with a control condition. Color code: functional set 1 = Green; functional set 2 = Red. FT = finger tapping.\n\nFor the fingerTapping task, participants were instructed to execute finger tapping with their right hand by steadily tapping the tip of the thumb to the tip of each other finger in succession, reversing the tapping order until the end of the task block is reached. For the fingerTappingImagined task, participants were instructed to imagine doing exactly the same as in the actual finger tapping task, but without actually moving their right fingers. For the control condition during the fingerTappingImagined task, participants were asked to count backwards in multitudes of 7.\n\nThe emotionProcessing task was an adapted \"Hariri\" task from the emotion processing task used in the Human Connectome Project (Hariri et al., 2002; Manuck et al., 2007; Van Essen et al., 2012). Materials were implemented to suit the paradigm for this rt-me-fMRI dataset. During each 20 s task block, participants were presented with a task cue (3 s duration), followed by a trial with three pictures of faces where the participant had to select one of the bottom figures (left or right) that resembled the top one, by pressing a left or right button (2 s duration). The inter-trial interval was 1 s duration (see Figure 3). Each 20 s block had 6 trials. The same design timing was used for the control condition blocks, i.e. matching shapes, as for the trial condition blocks depicted in Figure 3. Participants used an MRI-compatible button box with their right hand to complete the task. Participants were asked to press the left button with their right index finger if selecting the bottom left image (shape or face) on the screen, and to conversely press the right button with their right middle finger if selecting the bottom right image.\n\nTimes are provided for the cue, trials and inter-trial interval during a single block (20 s) of the face matching condition. The same design timing was used for the control condition blocks.\n\nFor the emotionProcessingImagined task, participants were instructed prior to the scanning session to identify an emotional event in their past that involved a person or people, and to think about this event and also try to mentally experience the identified emotion during the task blocks. For the control condition during this mental emotion task, participants were asked to count backwards in multitudes of 9.\n\nParticipants were interviewed after the scanning session about their experiences during the MRI acquisition and the tasks. None reported detrimental issues with regards to their ability to focus on the task or with task-switching.\n\nTasks and instructions were programmed and presented to the participants using E-Prime Studio version 2.0.10.248. The programmed E-prime files used for each task (\".es2\" format), as well as all presented images for trials, conditions, cues and instructions (.jpg format), can be accessed in the supplementary code repository: https://github.com/jsheunis/rt-me-fMRI. The exact timing information for the presented material (for all functional runs) and the button presses (for emotionProcessing), as well as the actual button press responses, were exported from E-prime (in .dat and .txt format) at the end of each session1.\n\nMRI data was acquired on a 3 Tesla Philips Achieva scanner (software version 5.1.7) and using a Philips 32-channel head coil.\n\nAnatomical MRI. A single T1-weighted anatomical image was acquired using a 3D gradient echo sequence (T1 TFE) with scanning parameters: TR = 8.2 ms; TE = 3.75 ms; flip angle = 8˚; field of view = 240×240×180 mm3; resolution = 1×1×1 mm3; total scan time = 6:02 min.\n\nFunctional MRI. All six functional MRI scans were acquired using a multi-echo, echo-planar imaging sequence with scanning parameters: TR = 2000 ms; TE =  14, 28, 42 ms (3 echoes); number of volumes = 210 (excluding 5 dummy volumes discarded by the scanner); total scan time = 7:00 min (excluding 5 dummy volumes); flip angle = 90˚; field of view = 224×224×119 mm3; resolution = 3.5×3.5×3.5 mm3; in-plane matrix size = 64×64; number of slices = 34; slice thickness = 3.5 mm; interslice gap = 0 mm; slice orientation = oblique; slice order/direction = sequential/ascending; phase-encoding direction = A/P; SENSE acceleration factor = 2.5; parts of the cerebellum and brainstem were excluded for some participants to ensure full motor cortex and amygdala coverage.\n\nThe echo times, spatial resolution, and SENSE factor were tuned with the aim of improving spatial resolution and coverage while limiting the TR at maximum 2000 ms, including a maximum number of echoes, and keeping the SENSE factor low to prevent SENSE artefacts.\n\nPhysiology data acquisition parameters. Breathing fluctuations were recorded with the use of a pressure-based breathing belt strapped around the participant's upper abdomen. Heart rate was recorded using a pulse oximeter fixed to the participant's left index finger. Both of these recording devices were wired directly to the scanner, sampled at 500 Hz, synchronized internally to the start/stop pulses of each functional scan, and data were written to Philips's standard \"scanphyslog\" log file type.\n\nStandardization: Brain Imaging Data Structure. To adhere to FAIR data principles, the full dataset was curated into the standardized and community-maintained Brain Imaging Data Structure (BIDS; Gorgolewski et al., 2016). This involved the use of several software packages and custom scripts to assist in file format conversion and data structuring, as detailed below. A Jupyter notebook containing Python code and descriptions for each of the steps below can be accessed at the project's code repository: https://github.com/jsheunis/rt-me-fMRI.\n\nMRI data\n\nAnatomical and functional MRI data were converted from the Philips PAR/REC format to BIDS using the Python package bidsify (v0.3; https://github.com/NILAB-UvA/bidsify) This package has dcm2niix (v1.0.20190410; https://github.com/rordenlab/dcm2niix/releases/) as a dependency to convert the PAR/REC files to NIfTI. It also structures the data into the directory system specified by the BIDS standard.\n\nAnatomical files were additionally de-identified using pydeface (v2.0.0; https://github.com/poldracklab/pydeface/releases/tag/2.0.0; Gulban et al., 2019), which removes facial features from the T1w NIfTI image. Further anonymization steps included removing time and date stamps and any identifiable information related to the acquisition location or system from the files output from bidsify.\n\nSince PAR/REC files do not contain slice timing information, the converted NIfTI files did not contain it either. Slice timing information was calculated using available parameters and added with a script to the BIDS-specific JSON sidecar files.\n\nPhysiology data\n\nHeart rate and breathing traces were converted from the Philips \"scanphyslog\" format to BIDS format using the Python package scanphyslog2bids (v0.1; https://github.com/lukassnoek/scanphyslog2bids).\n\nTask presentation and response data\n\nPresentation timing, button presses and button press response timing information were all converted to the BIDS format using a combination of custom Python scripts and the convert-eprime package (v0.0.1; https://github.com/tsalo/convert-eprime/releases/tag/0.0.1; Salo, 2020).\n\nRaw data was preprocessed using the open source MATLAB-based and Octave-compatible fMRwhy toolbox (see Software availability for details). The basic anatomical and functional preprocessing pipeline applied to all data is depicted in Figure 4 below.\n\nSteps include: (1) defining a functional template image from the first resting state run; (2) mapping the anatomical image and atlas-based regions of interest to the functional template space; (3) estimating realignment parameters from the template echo time series, running slice timing correction, applying realignment parameters to all echo time series, and applying spatial smoothing, and (4) generating quality control metrics and visualizations for anatomical and functional data.\n\nAs a first step, the T1-weighted anatomical image was coregistered to the template functional image (task-rest_run-1_echo-2, volume 1) using SPM12's coregister/estimate functionality, which maximizes normalised mutual information to generate a 12 degree-of-freedom transformation matrix. Before resampling to the functional resolution, this coregistered T1-weighted image was segmented using tissue probability maps and SPM12's unified segmentation algorithm (Ashburner & Friston, 2005). This yielded subject-specific probability maps for gray matter, white matter, CSF, soft tissue, bone and air in the subject functional space. All of these probability maps were then resampled (using coregister/write) to the subject functional resolution. Masks were generated for gray matter, white matter, CSF, and the whole brain (a combination - logical OR after thresholding - of the previous three masks). These were overlaid on the coregistered and resampled T1w image below, to allow visual inspection of segmentation and registration quality.\n\nAnatomical regions of interest were then taken from the cytoarchitecture-based atlases in the SPM Anatomy Toolbox (Eickhoff et al., 2005). For the motor cortex, regions 4a and 4p were used. For the amygdala, regions LB, IF, SF, MF, VTM, and CM were used. For the fusiform gyrus, regions FG1, FG2, FG3, and FG4 were used. Regions of interest were transformed from MNI152 space to the subject functional space using SPM12 normalise/write, as well as the inverse transformation field that was saved as part of the segmentation procedure mentioned above. The regions of interest for this study include the left motor cortex (for the motor processing tasks), the bilateral amygdala (for the emotion processing tasks) and the fusiform gyrus (for the emotionProcessing task). These ROIs are overlaid on the coregistered and resampled T1-weighted image, to allow visual inspection of normalisation quality.\n\nFunctional data were preprocessed, starting with estimating realignment parameters for each functional time series using SPM12's realign/estimate, which performs a 6 degree-of-freedom rigid body transformation that minimizes the sum of squared differences between each volume and the template volume. Realignment parameters were estimated for the second-echo time series of each run. Then, slice timing correction was done with SPM12, which corrects for differences in image acquisition time between slices. Each echo time series of all functional runs were slice time corrected. 3D volume realignment followed, which applied spatial transformation matrices derived from the previously estimated realignment parameters to all echo time series of all functional runs. Both raw time series and slice time corrected time series were realigned. Lastly, all echo time series of all functional runs were spatially smoothed using a Gaussian kernel filter with FWHM = 7 mm (i.e. double the voxel size). Smoothing was performed on raw, slice time corrected and realigned time series data.\n\nNext, several signal time series were calculated or extracted for use as possible GLM regressors in functional task analysis, or for quality control. From the realignment parameters (3 translation and 3 rotation parameters per volume), a Volterra expansion yielded derivatives, squares and squares of derivatives (Friston et al., 1996). Framewise displacement (FD, Power et al., 2012) was also calculated from the realignment parameters, and volumes were marked as outliers based on different thresholds of, respectively, 0.2 mm and 0.5 mm. RETROICOR regressors (Glover et al., 2000) were generated from the cardiac and respiratory signals using the TAPAS PhysIO toolbox, which yielded 6 cardiac regressors, 8 respiratory regressors, 4 interaction regressors, and additionally a cardiac rate regressor (CR; the cardiac rate time series convolved with the cardiac response function; Chang et al., 2009) and a respiratory volume per time regressor (RVT; respiratory volume per time convolved with the respiratory response function; Birn et al., 2006; Birn et al., 2008). From the slice time corrected and realigned time series data (of all functional runs), signals were extracted per voxel and spatially averaged within the previously generated tissue masks to yield tissue compartment signals for gray matter, white matter, cerebrospinal fluid (CSF) and the whole brain.\n\nThe last set of preprocessing steps included calculation of image quality metrics and visualizations, using the BIDS-compatible fmrwhy_workflow_qc pipeline from the fMRwhy toolbox. Operations on functional time series data were all done on detrended (linear and quadratic trends) realigned data, except where otherwise specified. Temporal signal-to-noise ratio (tSNR) maps were calculated for all runs by dividing the voxel-wise time series mean by the voxel-wise standard deviation of the time series. Tissue compartment averages were then extracted from these tSNR maps. Percentage difference maps (from the time series mean) were calculated per volume for use in carpet plots (or gray plots).\n\n\nDataset validation\n\nThe full dataset was validated for BIDS compatibility with the use of the web-based \"BIDS validator\" tool (v1.5.4; available at https://bids-standard.github.io/bids-validator/). A log of the BIDS validator output can be found in the project's code repository: https://github.com/jsheunis/rt-me-fMRI.\n\nData acquisition and experimental protocol parameters for this study were reported according to the community-formulated COBIDAS guidelines (Nichols et al., 2017). A modular version of this information is available in the project's GitHub repository.\n\nImage and data quality of this dataset was assessed using the fMRwhy toolbox. This allowed quality to be assessed for raw and minimally (pre)processed versions of the data, and also for interim steps on which the validity of eventual study outcomes might depend. A BIDS-compatible workflow in the fMRwhy toolbox, fmrwhy_workflow_qc, runs initial preprocessing and quality control of the raw data and outputs a quality report per subject, which includes metrics and visualizations for anatomical and functional MRI data and for peripheral data.\n\nFor anatomical MRI:\n\nCoregistered T1w segmentations (gray matter, white matter, CSF, and a whole brain mask) were overlaid onto the subject functional space, for visual inspection of the registration and segmentation quality.\n\nCoregistered anatomical regions of interest (in this case the left motor cortex, bilateral amygdalae and bilateral fusiform gyri) were overlaid onto the subject functional space, for visual inspection.\n\nFor functional MRI (all runs):\n\nA summary table provides values for all runs per subject for mean framewise displacement (FD), total FD, FD outliers, and mean tSNR in all tissue compartments. This allows quick inspection per participant, but is better understood when referenced to the whole dataset.\n\nSeveral image montages were generated per run, including the time series mean, the standard deviation and the tSNR map. The time series mean gives a quick view of the general quality of the time series and can indicate spike or interference artefacts. The standard deviation map shows areas with high signal fluctuation that can often be related to movement (e.g. close to the eyes). The tSNR maps are useful for investigating general signal quality, to indicate signal dropout and comparing signal quality across regions.\n\nA carpet (time series) plot was generated per run, which displays voxel intensity in percentage signal change from the mean over time. The vertical axis (voxels) is either grouped per tissue type (compartment ordered) or ordered from top to bottom according to the voxel's time series correlation strength to the global signal. Signal traces above the carpet plot are also shown, including tissue compartment signals, respiration, heart rate, and framewise displacement. These plots are useful quality checking tools as they make it easy to visualise wide scale signal fluctuations across voxels, which can then be related visually to changes in physiological signals or subject movement.\n\nChecking the quality of the recorded cardiac and respiratory traces is made possible with images generated by TAPAS PhysIO during the process of calculating RETROICOR, CR and RVT regressors. Images include a plot of the temporal lag between derived heart beats within thresholds for outliers, and a plot showing the breathing belt amplitude distribution that can be inspected for unexpected shapes.\n\nAll functional quality metrics of the full dataset, generated by the fmrwhy_workflow_qc workflow, are summarised in Table S1 (Heunis, 2021). This includes, per run, mean framewise displacement, total framewise displacement, framewise displacement outliers (based on a conservative 0.2 mm threshold, and a liberal 0.5 mm threshold), and mean tSNR in all tissue compartments (grey matter, white matter, cerebrospinal fluid, whole brain). This allows possible data users to inspect the quality measures and to set personalised thresholds and exclusion criteria.\n\nFigure 5 below displays summarised quality metrics for the rt-me-fMRI dataset, and examples of single-subject quality images. Individual quality reports can be downloaded together with the dataset. An example of a full, single-subject report can also be viewed online: https://jsheunis.github.io/fmrwhy_sample_QCreport.html.\n\nSubfigures include group level summary plots (A and B) and examples of subject level quality metric figures (C and D): (A) Vertical distribution (violin) plots of framewise displacement per subject, covering all functional runs. sub-010, sub-020 and sub-021 show comparatively high means and more outliers. (B) Vertical distribution (violin) plots of mean grey matter temporal signal-to-noise ratio (tSNR) per functional run, covering all subjects. Head movement results in higher signal fluctuations and hence lower tSNR, which is exemplified in the circled high mover data points: sub-021 (blue) and sub-010 (green); (C) An axial slice montage of temporal signal signal-to-noise ratio. (D) A time series \"carpet plot\" showing the global, white matter, CSF, respiration, and cardiac signals, as well as the calculated framewise displacement time series; (B) an axial slice montage of temporal signal signal-to-noise ratio.\n\nThe slice timing corrected, 3D realigned and spatially smoothed Echo 2 time series of all task runs underwent individual- and group-level statistical analysis using a general linear model with SPM12. Task regressors included the main \"ON\" blocks for the fingerTapping, fingerTappingImagined, and emotionProcessingImagined tasks, and both the separate \"SHAPES\" and \"FACES\" trials for the emotionProcessing task. Regressors not-of-interest for all runs included six realignment parameter time series and their derivatives, the CSF compartment time series, and RETROICOR regressors (both cardiac and respiratory to the 2nd order, excluding interaction regressors, selected based on common implementation procedures in literature). Additional steps executed by SPM12 before beta parameter estimation include high-pass filtering using a cosine basis set and AR(1) autoregressive filtering of the data and GLM design matrix.\n\nContrasts were then applied to the single task-related beta maps for the fingerTapping, fingerTappingImagined, and emotionProcessingImagined tasks, and to the FACES, SHAPES, and FACES>SHAPES beta maps for the emotionProcessing task. Statistical thresholding, consisting of familywise error rate control with p < 0.05 and a voxel extent threshold of 0, was then applied on a per-subject basis to identify task-related clusters of activity. Unthresholded subject-level contrast maps were normalized to MNI152 space and then fed into a group-level one-sided t-test, for which the t-statistic maps were subsequently thresholded at p < 0.001 and an extent threshold of 20 voxels. Unthresholded individual- and group-level t-statistic maps can be accessed as a NeuroVault collection: https://neurovault.org/collections/XWDGUJHD/.\n\nFigure 6 below shows the resulting thresholded group t-statistic maps for all four task runs. Figure 6A clearly shows activity clusters in the left motor cortex and right cerebellum, as expected for a finger tapping task as well as a negative activation pattern in the default mode network. Figure 6C shows activation in the visual cortex commensurate with a face/shape matching task, specifically in the left and right fusiform gyri. Additional clusters are found in the amygdalae and hippocampi, as expected for an emotion processing task. For both imagined tasks, similar but weaker activation clusters are found in the expected regions (respectively the motor cortex in Figure 6B, and amygdalae in Figure 6D) but both wide scale activation patterns are consistent with mental tasks including imagery and memory recollection. Additionally, Figures 6B and 6D show negative activation patterns in the dorsal attention network. The activation results in Figure 6 are further evidenced by the resulting highest correlated terms when decoding the unthresholded group t-statistic images with the web-based Neurosynth tool (www.neurosynth.org, Yarkoni et al., 2011). Table 1 shows the resulting terms2.\n\nSubfigures include: (A) fingerTapping, (B) fingerTappingImagined, (C) emotionProcessing, and (D) emotionProcessingImagined (p<0.001, voxel extent=20). Images were generated with bspmview. Figure 6A clearly shows activity clusters in the left motor cortex and right cerebellum, as expected for a finger tapping task. Figure 6C shows activation in the visual cortex commensurate with a face/shape matching task, specifically in the left and right fusiform gyri. For both imagined tasks, similar but weaker activation clusters are found in the expected regions (respectively the motor cortex in Figure 6B, and amygdalae in Figure 6D) but both wide scale activation patterns are consistent with mental tasks including imagery and memory recollection.\n\nA core contribution of this rt-me-fMRI dataset lies in the multi-echo acquisition. Multi-echo fMRI samples multiple T2-weighted images at a range of echo times along the decay curve following a single transverse magnetic excitation, which theoretically allows the optimum BOLD contrast to be optimized for a range of baseline tissue T2 values. Subsequently, echo combination through weighted summation or averaging is a typical processing step that generally increases temporal signal-to-noise ratio and contrast-to-noise ratio and decreases signal drop-out in regions with high susceptibility artefacts and signal dropouts (Menon et al., 1993; Posse et al., 1999; Posse, 2012). Echoes can be combined using a variety of weights, including baseline voxelwise tSNR and T2* maps.\n\nFigure 7 and Figure 8 illustrate that such combination procedures improve tSNR and signal dropout, hence validating the use of multi-echo fMRI for improved quality data. Representative signal recovery is demonstrated in the tSNR maps of Figure 7 for a single run of a single subject, particularly by the blue and magenta arrows showing areas of signal dropout in the Echo 2 time series (including, respectively, the medial temporal and inferior temporal lobes, and the orbitofrontal lobe) and subsequent recovery in the combined time series. The light green arrows indicate substantial increases in tSNR in areas close to the bilateral temporal-occipital junction and towards the occipital lobe as the slices increase in a superior direction. Figure 8 shows distribution plots of the mean grey matter tSNR for the single (2nd) echo and two combined echo (tSNR-combined and T2-combined) time series, covering all functional runs and all subjects. The two combined echo time series clearly have improved tSNR values, increasing by ~30% from 85 (2nd echo) to 112 (tSNR-combined).\n\nTime series include: the 2nd echo time series (top row) and two combined time series (middle row = T2-combined; bottom row = tSNR-combined). Blue and magenta arrows indicate areas of signal dropout and recovery (including, respectively, the medial temporal and inferior temporal lobes, and the inferior frontal lobe). Light green arrows indicate areas with substantial increases in tSNR (in the lateral cortex and towards the anterior cortex as the slices increase in a superior direction). Combined multi-echo time series result in both substantially higher tSNR and signal recovery compared to Echo 2.\n\nDistributions are shown for three time series: Echo 2, tSNR-combined and T2-combined. A single distribution plot covers all subjects and all runs excluding rest_run-1. The two combined echo time series show clear increases in mean tSNR values.\n\nFurther benefits of multi-echo over conventional single-echo fMRI exist (see for example Caballero-Gaudes et al., 2019; Dipasquale et al., 2017; Gonzalez-Castillo et al., 2016; Lombardo et al., 2016; Moia et al., 2020; Olafsson et al., 2015), but such analyses are beyond the scope of this validation step and can be explored further with this publicly available dataset. In complementary work using this dataset we evaluate the use of several combination and T2-mapping procedures for both offline and real-time BOLD sensitivity (Heunis et al., 2020).\n\nTo be a possible participant in this study, individuals had to be healthy, right-handed volunteers with no prior or current (at the time of the study) indications of neurological or psychiatric conditions. They also had to report the absence of any other standard contraindications for MRI scanning. 32 participants were initially recruited for the study, and the datasets of three participants were excluded due technical and one due to administrative challenges. No further datasets were excluded, even in cases of more than average or severe motion (e.g. sub-010 and sub-021), since it was decided that such data could still be useful for future methods development or related insights. Table S1 (Heunis, 2021) provides a list of all functional quality metrics for all participants and runs, which allows possible data users to inspect the quality measures and to set personalised thresholds and exclusion criteria.\n\n\nData availability\n\nDataverseNL: rt-me-fMRI: A task and resting state dataset for real-time, multi-echo fMRI methods development and validation, https://doi.org/10.34894/R1TNL8 (Heunis, 2020).\n\nIn order to access the data, users must apply for access to the data via the repository (https://dataverse.nl/dataverse/rt-me-fmri) and agree to the Data Use Agreement (see https://github.com/jsheunis/rt-me-fMRI/blob/master/DUA.md). Users will be granted access to the data once they have consented to the terms of the Data Use Agreement.\n\nThe rt-me-fMRI dataset is available in BIDS format. This repository includes the raw BIDS data, descriptive metadata, and derivative data including quality reports.\n\nApart from the dataset README file, all core files are available in one of 3 formats: NIfTI, TSV and JSON. Functional and anatomical data are stored as uncompressed NIfTI files (with the \".nii\" extension), which contain image and header data and can be handled/viewed by all major neuroimaging analysis packages and programming languages. Tabular data such as participants, task events, response timing and physiology data are stored in tab-separated value text files (with the extension \".tsv\", or if compressed \".tsv.gz\") and can be handled by text or spreadsheet reading/editing software on all major operating systems, or alternatively by all major software programming languages. Metadata about the dataset, tasks, events and more are stored as key-value pairs in text-based JSON files (with the extension \".json\") that can be handled/viewed using all major software programming languages.\n\nAll data files are organised according to the BIDS convention for dataset participants, MRI data type (anatomical or functional), and derivatives, as depicted in Figure 9.\n\nThe top level directory includes metadata about the dataset, participants and task events, as well as a directory per participant and lastly a derivatives directory. The expansion of \"sub-001\" (top right) shows subdirectories \"anat\" and \"func\", each with neuroimages and metadata related to anatomical and functional scans, respectively. The expansion of the \"derivatives\" directory (bottom right) shows subdirectories \"fmrwhy-dash\" and \"fmrwhy-qc\". The former contains all derivative data required to run the interactive browser-based application accompanying this dataset. The latter includes a quality report per participant in HTML format.\n\nEach participant directory contains two subdirectories: \"anat\" and \"func\", respectively containing all anatomical and functional images and metadata. Different data types can be distinguished based on BIDS identifiers, e.g. \"_bold\" for functional and \"_T1w\" for anatomical MRI data. The full list of data acquisitions with their data types, descriptions, and formats are provided below in Table 2. Note that for functional data, each resting state and task run consists of three separate image files, one per echo (i.e. \"_echo-1_bold.nii\", \"_echo-2_bold.nii\", and \"_echo-3_bold.nii\"). JSON sidecar files accompany all BOLD and physiology data files on the participant level, while the accompanying JSON sidecar files for the four types of task event files are on the dataset level. Other files on the dataset level include the README, the dataset description (JSON) and the participant list (TSV).\n\nApart from the core dataset, rt-me-fMRI also includes derivative data in two subdirectories generated by the fMRwhy toolbox and related scripts: \"fmrwhy-qc\" and \"fmrwhy-dash\". The former results from the fmrwhy_workflow_qc pipeline and contains a subdirectory per participant, each in turn including subdirectories \"anat\" and \"func\". These directories contain NIfTI, TSV and PNG files of quality control outputs, which are all required for the HTML quality report contained in the \"report_[yyyymmddhhmmss]\" directory. These reports can be opened with all major Internet browsers. The \"fmrwhy-dash\" derivative directory contains (as TSV files) all data required to yield the interactive visualisations of the supplementary browser-based application provided with this dataset: https://rt-me-fmri.herokuapp.com/.\n\nTable S1 (Functional quality metrics for the rt-me-fMRI dataset) is available from: https://doi.org/10.5281/zenodo.4467479 (Heunis, 2021)\n\n\nSoftware availability\n\nAn interactive environment (https://rt-me-fmri.herokuapp.com/) was created alongside this study to allow users to interactively explore summaries of the data derivatives and quality control aspects.\n\nAll software scripts and self-developed tools used to prepare, preprocess and quality check the data are openly available at the project's code repository https://github.com/jsheunis/rt-me-fMRI. This includes instructions to download, extract, and understand the data; the data preparation script; the preprocessing script; the quality reporting script; and the script to reproduce the figures for this manuscript.\n\nArchived scripts as at time of publication: https://doi.org/10.5281/zenodo.4467479 (Heunis, 2021)\n\nLicense: MIT\n\nDependent software and toolboxes/packages used for these preparation, preprocessing and quality reporting steps include:\n\nPython 3.7+\n\nbidsify (v0.3; https://github.com/NILAB-UvA/bidsify)\n\nscanphyslog2bids (v0.1; https://github.com/lukassnoek/scanphyslog2bids).\n\ndcm2niix (v1.0.20190410; https://github.com/rordenlab/dcm2niix/releases/tag/v1.0.20190410)\n\npydeface (v2.0.0; https://github.com/poldracklab/pydeface/releases/tag/2.0.0; Gulban et al., 2019)\n\nconvert-eprime (v0.0.1; https://github.com/tsalo/convert-eprime/releases/tag/0.0.1; Salo, 2020)\n\nMATLAB R2016b or later (9.1.0.441655; The MathWorks Inc)\n\nfMRwhy (v.0.0.1, https://github.com/jsheunis/fMRwhy)\n\nSPM12 (r7771; https://github.com/spm/spm12/releases/tag/r7771)\n\nAnatomy Toolbox (v3.0; Eickhoff et al., 2005)\n\nbids-matlab (v.0.0.1, https://github.com/jsheunis/bids-matlab/releases/tag/fv0.0.1)\n\ndicm2nii (v0.2 from a forked repository; https://github.com/jsheunis/dicm2nii/releases/tag/v0.2)\n\nTAPAS PhysIO (v3.2.0; https://github.com/translationalneuromodeling/tapas/releases/tag/v3.2.0; Kasper et al., 2017)\n\nRaincloud plots (v1.1 https://github.com/RainCloudPlots/RainCloudPlots/releases/tag/v1.1; Allen et al., 2019)\n\nbspmview (v20180918; https://github.com/spunt/bspmview/tree/20161108; Spunt, 2016)", "appendix": "Acknowledgements\n\nA previous version of this article is available on bioRxiv: https://doi.org/10.1101/2020.12.07.414490\n\n\nFootnotes\n\n1 Note: for the majority of participants, the presentation timing for the emotionProcessing task was delayed by tens of milliseconds for each trial (planned versus actual timing). This resulted in the full task presentation running on for about 5 s after the scan acquisition stopped. This is not deemed a problem, mainly since the exact presentation time was captured and is available in the BIDS dataset. However, users should take note not to use the planned timing parameters as that would ignore the delay that occurred.\n\n2 Task names of the rt-me-fMRI dataset were selected based on the desired activation response for the given use cases, e.g. emotionProcessing to elicit a response in regions involving emotion processing, with the knowledge that the tasks might yield varied responses and have varied use cases. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "80216", "date": "10 Mar 2021", "name": "Klaus Mathiak", "expertise": [ "Reviewer Expertise Psychiatry", "neuroimaging", "statistics" ], "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 and document a dataset comprising functional and anotomical MRI data of 28 healthy young participants. In particular, functional data were recorded using multi-echo (triple) EPI imaging applying resting state, and (imagined) finger tapping and emotion processing. The aim is to make real-time processing software for multiecho EPI more assessable.\n\nThe data are important and useful. The data format accord to the current standard and should be easy to process with free and commercial software. The paradigms are well chosen, in particular with respect to Realtime imaging/neurofeedback, e.g. robust – finger tapping, clinically relevant – emotions processing, and the comparison with imagined stimuli to evaluate the effect of cognitive strategies. I miss in the report somehow the reference to real-time processing; the dataset by itself can be equally (or better) evaluated offline.\n\nI guess the inclusion of the Tedana ressource (https://tedana.readthedocs.io/en/latest/index.html) may help to complement the relevant citations and strategies for me evaluation.\n\nIndeed, in particular I suggest to extent the analysis to CNR measures which have higher relevance to assess BOLD sensitivity (e.g. Bhavsar et al., Neuroimage 2013, PMID: 239544881; which is also feasible with the presented standardized paradigms).\nPlease double check that acronyms are introduced before use: TSV, JSON.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "80222", "date": "25 Mar 2021", "name": "Christian Paret", "expertise": [ "Reviewer Expertise Clinical psychology", "real-time fMRI neurofeedback in mental disorder of emotion dysregulation" ], "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\nHeunis et al. provides an open, accessible fMRI dataset. The dataset features multi-echo fMRI data of five functional tasks, including both a motor and an emotional faces-viewing task that are matched with a motor imaginary and emotional imaginary task, respectively. The authors argue that the dataset is particularly useful for inspection of real-time data processing pipelines, because the tasks may represent critical aspects of a typical real-time fMRI neurofeedback study.\nI congratulate the authors for this fine piece of work, which resulted from careful and diligent preparation. The interactive interface to explore the data and its accessibility are remarkable and exceed the present work in the field.\nThere are a few minor questions I would like the authors to answer to complement the current manuscript version:\nIt says that 3 participants were excluded due to \"technical and administrative challenges\". It would be helpful to get a bit more information what kind of challenges the authors encountered and whether this was related to the open data-approach?\n\nWere any specific instructions given what kind of emotion participants should evoke during the emotionProcessingImagined task? Did the authors try to characterize emotional self-induction, e.g. by collecting interview data on imagination strategy and/or effective valence?\n\nIt might be helpful for readers with a limited technical understanding to provide an explanation of the SNR measure in layterms. Why is smaller SD, in general, related to higher SNR for fMRI BOLD?\n\nLooking at the group level T-maps (https://rt-me-fmri.herokuapp.com/pages/quality) I found spurious activations outside the brain. Usually, SPM would exclude such activations with an inclusive mask. Can you please comment on this observation?\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/10-70
https://f1000research.com/articles/10-69/v1	04 Feb 21	{ "type": "Research Article", "title": "Using DHS and MICS data to complement or replace NGO baseline health data: an exploratory study", "authors": [ "Peter R. Berti", "Milena Nardocci", "Minh Hung Tran", "Malek Batal", "Rebecca Brodmann", "Nicolas Greliche", "Naomi M. Saville", "Milena Nardocci", "Minh Hung Tran", "Malek Batal", "Rebecca Brodmann", "Nicolas Greliche", "Naomi M. Saville" ], "abstract": "Background: Non-government organizations (NGOs) spend substantial time and resources collecting baseline data in order to plan and implement health interventions with marginalized populations. Typically interviews with households, often mothers, take over an hour, placing a burden on the respondents. Meanwhile, estimates of numerous health and social indicators in many countries already exist in publicly available datasets, such as the Demographic and Health Surveys (DHS) and the Multiple Indicator Cluster Surveys (MICS), and it is worth considering whether these could serve as estimates of baseline conditions. The objective of this study was to compare indicator estimates from non-governmental organizations (NGO) health projects’ baseline reports with estimates calculated using the Demographic and Health Surveys (DHS) or the Multiple Indicator Cluster Surveys (MICS), matching for location, year, and season of data collection. Methods: We extracted estimates of 129 indicators from 46 NGO baseline reports, 25 DHS datasets and three MICS datasets, generating 1,996 pairs of matched DHS/MICS and NGO indicators. We subtracted NGO from DHS/MICS estimates to yield difference and absolute difference, exploring differences by indicator. We partitioned variance of the differences by geographical level, year, and season using ANOVA. Results: Differences between NGO and DHS/MICS estimates were large for many indicators but 33% fell within 5% of one another. Differences were smaller for indicators with prevalence <15% or >85%. Difference between estimates increased with increasing year and geographical level differences. However, <1% of the variance of the differences was explained by year, geographical level, and season. Conclusions: There are situations where publicly available data could complement NGO baseline survey data, most importantly when the NGO has tolerance for estimates of low or unknown accuracy.", "keywords": [ "DHS", "MICS", "surveys", "maternal and child health" ], "content": "Introduction\n\nNon-government and civil society organizations spend substantial time and resources collecting baseline data in order to plan and implement health interventions with marginalized populations, and to measure the impact of those interventions (Data for Impact, 2019). Typical methods involve baseline and endline household surveys, where the household residents are interviewed and asked a hundred or more questions about asset ownership, mother and child health, diet, health system access, and other topics of interest. The costs of these surveys vary depending on design, methods, sample size, survey length, and local context (Data for Impact, 2019), but in the authors’ experience tens of thousands of dollars is typical, and in some cases, much more. Depending on the number and nature of questions, interviews can be over an hour long, placing a burden on the respondents. In addition, the accuracy of the indicator estimates in NGO-led surveys may be insufficient for project design and monitoring purposes, due to relatively small sample sizes and the inherent high variability of the indicators of interest.\n\nMeanwhile, estimates of numerous health and social indicators in many countries already exist in publicly available datasets, such as the Demographic and Health Surveys (DHS), supported by USAID (U.S. Agency for International Development, 2018), and the Multiple Indicator Cluster Surveys (MICS), supported by UNICEF (UNICEF, 2020), and it is worth considering whether these could serve as estimates of baseline conditions. DHS/MICS provide standardized data collected using rigorous methods and large sample sizes, and datasets are available on request for free. They are designed to be representative at the national, regional and provincial level (but rarely at lower levels, such as district and village, where NGOs are working), and probably exclude homeless, institutionalized and nomadic populations (Carr-Hill, 2013). DHS/MICS are collected every three to ten years so there may up to ten-years gap between DHS/MICS data collection and the baseline conditions that the NGO wants characterized. Although some indicators’ descriptions have been modified and improved over time, caution is taken to ensure that data are directly comparable across countries, regions and years (Hancioglu & Arnold, 2013; UNICEF, 2020; U.S. Agency for International Development, 2018). DHS/MICS surveys are adapted to specific country needs and are conducted by well-trained interviewers who have access to tools and guidelines for quality assurance throughout (UNICEF, 2020; U.S. Agency for International Development, 2018).\n\nUsing publicly available data to complement or replace NGOs’ primary data collection for project baseline measures and project monitoring would save valuable resources, reducing the burden on data collectors and respondents alike. A few studies have compared estimates between DHS/MICS and NGO surveys. One found that they provided very different estimates of electricity and water access in Kenya, Tanzania, and Uganda (Carr-Hill, 2017), and a second found that DHS and a NGO-led survey provided similar estimates of several maternal and child health estimates in Rwanda (Langston et al., 2015). Other studies found that estimates of the market share of faith-based health care providers by DHS and NGO surveys in sub-Saharan Africa were within 5 to 50% of each other (Wodon et al., 2012), and the confidence intervals for the difference between Lot Quality Assurance Sampling (LQAS) and DHS district-level estimates were within +/-10% for 15 of 37 health indicators (Anoke et al., 2015). Therefore, no consensus exists on the potential for DHS/MICS to substitute NGO surveys.\n\nWe hypothesized that publicly available data can provide estimates of baseline conditions similar to those reported in NGO baseline reports when matched as closely as possible for location, year, and season of data collection. We tested this hypothesis by comparing indicator estimates from NGO reports with estimates calculated using DHS/MICS.\n\n\nMethods\n\nWe collected and retained a sample of 46 NGO baseline reports through a combination of internet search and personal contacts with Canadian and Vietnamese NGOs using the following selection criteria:\n\ni) household survey (n>100) which used valid methods and representative sampling to generate point estimates of maternal, newborn and child health indicators;\n\nii) conduced between 2005 and 2019;\n\niii) in a low- or middle-income country.\n\nThe baseline reports from NGOs working on maternal, newborn and child health covered 23 countries spanning South Asia (Bangladesh, India, Pakistan), Africa (Burkina Faso, Ethiopia, Ghana, Kenya, Liberia, Malawi, Mali, Mozambique, Nigeria, Senegal, South Sudan, Tanzania, Zambia), South/Central America (Bolivia, Honduras), the Caribbean (Haiti), and SE Asia (Laos, Myanmar, Philippines, Vietnam) (Table 1) (Berti, 2021). From the reports, we extracted: country name, NGO name, dates of data collection, population of study, inclusion/exclusion criteria, indicator name and definition, sample size (total and n for each indicator), and the indicator estimate (percentage and standard deviation (SD) if available).\n\n* 1st level represents village, town, locality or traditional authority; 2nd level: district or equivalent; 3rd level: province, state or equivalent; 4th level: region; 5th level: country.\n\nDHS: Demographic and Health Surveys; MICS: Multiple Indicator Cluster Surveys; NGO: non-governmental organization.\n\nWe also retained the location of data collection (e.g. country, region, province, district, or/and village) and geographical level. These geographical levels of data aggregation were defined as: (1) the smallest geographical subdivision in a country (village, town, locality, traditional authority); (2) district or district council (larger than a village but smaller than the third level); (3) province, state, department, county or district (if it refers to a division equivalent to province or state); (4) region (combining several units of level 3); (5) country level.\n\nWe matched 25 DHS and 3 MICS surveys (from Vietnam, Laos, and South Sudan) with 46 NGO baseline reports (Table 1). We used the most recent DHS/MICS survey carried out prior to the NGO baseline survey, with some surveys matching more than one NGO survey.\n\nIndicators from DHS/MICS were calculated following the methods recommended by DHS/MICS accounting for weighting and sample selection (Croft et al., 2018). Wherever possible, we used the methods employed by the NGO to create the matching DHS/MICS indicator. For instance, if the NGO baseline survey included women of reproductive age and their children aged 0-24 months living in the district of Homoine in Mozambique, we extracted the same sample from the DHS/MICS. In the absence of representative data from the same geographical level, we used DHS/MICS data from the next level up in the geopolitical hierarchy to match the lower level from the NGO. For instance, if data from the district of Homoine were not available in the DHS, we used data from the province of Inhambane (one level up).\n\nWe matched similar indicators from NGO baseline reports with DHS/MICS wherever available and excluded those that had no match in the DHS/MICS datasets. Table 2 provides an example of how the data were matched for the indicator “Woman received at least three antenatal care visits (ANC) during last pregnancy”.\n\n* 1st level represents village, town, locality or traditional authority; 2nd level: district or equivalent; 3rd level: province, state or equivalent; 4th level: region; 5th level: country.\n\nANC: antenatal care; DHS: Demographic and Health Surveys; MICS: Multiple Indicator Cluster Surveys; NGO: non-governmental organization.\n\nIn total there were 129 indicators (Table 3) from eight main groups including child anthropometry, child diet, child health, household characteristics, household wealth, maternal characteristics, maternal health, and WASH. We excluded estimates based on fewer than ten observations (n=64), in either the DHS/MICS or NGO data, retaining a total of 1,996 pairs of NGO-DHS/MICS indicators for analyses.\n\n* for a complete list of all the indicators see Table 2 in HealthBridge (2020).\n\nHH: household; WASH: Water, Sanitation, and Hygiene; DPT: diphtheria, pertussis and tetanus; ORS: oral rehydration salts; ORT: oral rehydration therapy; SBA: skilled birth attendant; ANC: antenatal care; PNC: postnatal care; TT: tetanus toxoid.\n\nAfter collating the data, we grouped similar indicators into 37 subgroups (Table 3) on the basis of whether they had similar definitions/concepts (e.g. stunting prevalence in different age groups). We refined the grouping by using scatterplots of the difference of estimates by year difference and geographical level difference to check if any indicators differed widely from others in the grouping. After assessing the indicators graphically, we separated “Diarrhea in the last two weeks: 0-5m” from the same indicator for other age groups since the differences of estimates were closer to zero for this age group than the others. We also separated “Household has a car” from the subgroup “Household has agricultural land/bike/phone” since car ownership was much lower than ownership of other assets.\n\nNGO versus DHS/MICS\n\nWe subtracted NGO from DHS/MICS estimates to calculate difference and absolute difference between estimates.\n\nTo compare data from NGO and DHS/MICS we used: same or different season of data collection; number of years difference between data collection (DHS/MICS year - NGO year); and number of geographical levels difference (DHS/MICS level - NGO level). If data collection spanned two years, for instance data collection started in 2013 and was completed in 2014, the year of data collection was coded as “2013.5”. Geographical level difference was calculated by subtracting the NGO level from DHS/MICS level. For example, we subtracted district level data available from the Mozambique NGO survey (level=2) from province level data collected in the DHS (level=3), making the geographical level difference one. We grouped geographical level differences as: no difference; one level difference; 2-3 levels difference.\n\nWe plotted how difference and absolute difference between DHS/MICS and NGO estimates varied with the indicator and indicator grouping. We used Analysis of Variance (ANOVA) to partition the variance of difference or absolute difference between estimates (DHS/MICS estimate - NGO estimate) by indicator, geographical level difference (as 0,1,2+), year difference (continuous), and season (same season, different season, season unknown).\n\nDHS versus DHS\n\nIn order to better understand the contribution of difference in methods employed in the different sources of survey data (DHS/MICS and NGO) to the resulting difference in estimates, we repeated the analyses used to compare DHS/MICS and NGO estimates but this time comparing DHS data from one country, year and geographical level to a different year and/or geographical level from the same country. The assumption is that the DHS methods are similar between years and geographical levels, whereas DHS/MICS and NGOs may use somewhat different methods. There is a level of discordance between DHS/MICS and NGO estimates, and there would also be discordance between two DHS estimates. The difference between DHS/MICS-NGO discordance and DHS-DHS discordance will not be due to difference in years, or geographical levels, but rather due to difference in methods.\n\nFor the DHS-DHS comparisons, we compiled DHS data from the seven countries that contributed the most pairs in the DHS/MICS-NGO dataset: Bangladesh, Ethiopia, Kenya, Malawi, Pakistan, Tanzania, and Zambia. Retaining the same indicators as in the DHS/MICS - NGO comparisons, we calculated estimates for different geographical levels, i.e. at the country level, and for each region, province and district available. For this analysis, we included district data to mimic the NGO data, even though these estimates are not always representative at this level in the DHS. We excluded indicators based on a sample size smaller than ten observations (n=26,539).\n\nWe matched DHS indicators from different cycles and geographical levels using different combinations mimicking the actual DHS/MICS-NGO scenarios: indicators from the same level but different years (Scenario 1), indicators from the same year but different levels (Scenario 2), and indicators from different years and levels (Scenario 3). To mimic the NGO data, we used data from the most recent cycle and the lower geographical levels, whereas to represent the comparative DHS data we used older DHS cycle and higher geographical level data. Using DHS data only, we were not able to simulate a scenario where DHS/MICS and NGO data were from the same year and geographical level. Table 4 provides an example of how we compared the estimates for an ANC indicator in Zambia using 31 pairs from DHS in the three scenarios for this one country. Repeating across all indicators and all countries yielded 109,251 pairs of DHS-DHS indicators.\n\n* 3rd level represents province level data and 5th level represents country-level data.\n\nWe calculated the difference and absolute difference between these pairs of estimates, mimicking the scenarios from the DHS/MICS-NGO data. Table 5 summarises the DHS cycles included as well as the geographical level comparison for each scenario in each of the seven countries.\n\nFinally, as with DHS/MICS vs NGO estimates, we used ANOVA to partition the variance of difference or absolute difference between DHS estimates by indicator, geographical level difference, and year difference. We did not include season in this analysis since most DHS data are collected during the same season within a country.\n\nWe simulated a situation where the only source of imprecision of the indicator’s measures would be from sampling error, in order to separate this known and estimable source of error from other sources of error that lead to differences in indicator estimates. The simulation samples from a \"true\" prevalence (p) of 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99%. We assumed an n of 500, which was a typical sample size of both DHS and NGO samples in our data set. We then generated a “Baseline Estimate 1” (to mimic the DHS/MICS estimates) by drawing randomly from a binomial distribution with mean n*p and variance np(1-p). A “Baseline estimate 2” (to mimic the NGO estimate) was generated in the same way, and the difference between the first and second estimate was calculated. We ran 1,000 iterations to estimate the distribution of the differences.\n\nIn order to investigate how absolute differences vary by the nature of the point prevalence estimates we used box plots to compare simulated, DHS-DHS and DHS/MICS-NGO absolute differences.\n\nAll data were compiled in Microsoft Excel 15 and analyzed with SAS 9.4.\n\nThis study respects current research ethics standards and it was approved by the Health Research Ethics Board of the Université de Montréal (CERSES-19-030-D).\n\n\nResults\n\nThe NGO reports often presented over 100 indicators in their baseline reports. On average, 18 of their indicators were also available in the DHS/MICS datasets. The estimate sample size for the NGO surveys ranged from 12 to 16,530 and from 10 to 98,446 for the DHS/MICS. Table 6 presents, by indicator subgroup, mean DHS/MICS and NGO percentage prevalence estimates, mean difference between pairs (DHS/MICS minus NGO) and percentage of differences falling within 5 and 20 percentage points. Some subgroups have mean difference close to zero, but almost all have at least some pairs that are widely different (not within 20%). Fifteen subgroups had positive (DHS<NGO) and 21 had negative (DHS>NGO) mean differences, but we identified no meaningful pattern in which indicators were negative and which were positive, and all the differences (except for consumption of vitamin A-rich foods) were within 1 standard deviation of 0.\n\nFigure 1 presents the scatterplots of NGO against DHS/MICS estimates by subgroup of indicators. For all subgroups, there was some correlation between the DHS/MICS and NGO estimates. Figure 2 shows the boxplot distribution of the mean difference between estimates by subgroup. The only subgroups that had all the pairs of indicators within ±20% were “Consumption of vitamin A-rich foods”, “Bottle fed yesterday”, “Diarrhea in the last two weeks: 0-5m”, “Diarrhea in the past two weeks: given more to eat”, and “Household has a car”. Other indicators that had most of their pairs within ±20% were “Household treats drinking water” and “Ever married”. All the indicators with the smallest differences between estimates had very low or very high prevalence (Table 6), except for “Consumption of vitamin A-rich foods” (that was based on only four pairs of estimates).\n\nAbbreviations: BF: breastfeeding; HH: household; HF: health facility; SBA: skilled birth attendant; ANC: antenatal care; PNC: postnatal care; DHS: Demographic and Health Surveys; MICS: Multiple Indicator Cluster Surveys; NGO: non-governmental organization.\n\nAbbreviations: Anthros: anthropometry indicators; HH: household; WASH: Water, Sanitation, and Hygiene; BF: breastfeeding; HF: health facility; SBA: skilled birth attendant; ANC: antenatal care; PNC: postnatal care; DHS: Demographic and Health Surveys; MICS: Multiple Indicator Cluster Surveys; NGO: non-governmental organization.\n\nTable 7 summarizes the absolute differences between DHS/MICS and NGO, and between DHS and DHS. They are summarized according to the similarity of data collection timing (year and season), geographical level, and sample size. Using the absolute difference enabled us to see the size of the difference without taking the direction into account. The absolute difference between DHS/MICS and NGO estimates increases as year difference increases, as geographical levels difference increase, and as sample sizes decrease. The differences between DHS and DHS show similar patterns in terms of broad geographical level, sample size, and ≥3.5 years versus 0 to 3 years’ time differences.\n\na For the DHS/MICS - NGO comparison, refers to the DHS/MICS data.\n\nb For the DHS - DHS comparison, refers to the DHS data from the higher geographical level and earlier survey year.\n\nc For the DHS/MICS - NGO comparison, refers to the NGO data.\n\nd For the DHS - DHS comparison, refers to the data mimicking the NGO (from the lower geographical level and more recent survey year).\n\nTable 8 shows the partition of variation results from DHS/MICS vs NGO and DHS vs DHS comparison. For DHS/MICS vs NGO about 15% of the variance was attributed to the indicator and less than 1% attributed to geographical level, year and season difference. For DHS vs DHS, geographical level and year account for more variation in absolute difference (1.25 and 4.5% respectively). However, in all cases, most (>82%) of the variance was unattributed, that is, it remained unexplained by the model.\n\nResults from all three comparisons, DHS/MICS - NGO, DHS - DHS, and Simulations, are shown in Figure 3 as boxplots of the absolute difference between estimates by the indicator reference value (the DHS estimate or the estimate simulating DHS). The distribution of absolute differences is similar between DHS/MICS - NGO and DHS - DHS, with DHS/MICS - NGO showing only a slightly larger spread. For all three types of comparisons, the distribution of the absolute difference between estimates is narrower in the extremes and larger when the reference value is between 35% and 65%. Since the simulated sampling error differences are small (range <10%), only a small proportion of the differences can be attributed to sampling error.\n\nAbsolute difference between estimates calculated as:\n\nSimulation: Simulated estimate 1 - Simulated estimate 2\n\nDHS vs DHS: DHS estimate - DHS mimicking the NGO estimate (lower geographical level, more recent year of data collection)\n\nDHS/MICS vs NGO: DHS/MICS estimate - NGO estimate\n\nReference value: DHS or the estimate mimicking DHS (higher geographical level, earlier year of data collection)\n\nAbbreviations: DHS: Demographic and Health Surveys; MICS: Multiple Indicator Cluster Surveys; NGO: non-governmental organization.\n\n\nDiscussion\n\nOur study showed that many indicators presented large differences between NGO and DHS/MICS estimates. Almost all indicators had at least some pairs that were widely different. Only about 33% of the pairs of indicators were within 5%, and about 80% of the pairs of indicators were within 20%. Agreement between indicators was higher when comparing indicators that had low or high prevalence (e.g. <15% or >85%), which is consistent with sampling theory, but throughout the prevalence range, the distribution of differences in the DHS/MICS-NGO and DHS-DHS comparisons is larger than that found from sampling error alone (reflected in the simulation distribution). An NGO could obtain an accurate estimate using DHS/MICS data for indicators with expected values close to 0% or 100%.\n\nWe had hoped that if DHS/MICS and NGO estimates were similar, then NGOs could forego baseline data collection and use as a substitute DHS/MICS estimates, or estimates from some other publicly available dataset instead, saving NGO time and money, and reducing respondent burden. While we cannot give a blanket recommendation that DHS and MICS could always replace NGO baseline surveys, there are at least some situations where DHS/MICS could be used to the NGO’s advantage: when the estimate is expected to be less than 15% or above 85%; when the indicator of interest is one of the few with consistent similarity between DHS/MICS and NGO estimates; and when the NGO has tolerance for estimates of low or unknown accuracy.\n\nWe had hypothesized that publicly available data can provide estimates of baseline conditions similar to those reported in NGO baseline reports when matched as closely as possible for location, year, and season of data collection. From the descriptive analyses, we found that as year difference increased, the mean difference between estimates slightly increased, and estimates derived from lower geographical levels (such as village or district from NGO and province for DHS/MICS) contributed to a higher mean absolute difference between estimates. In general, larger sample sizes were obtained at higher geographical levels and the larger the sample size (with their smaller sampling error) from DHS/MICS or NGO, the smaller the mean absolute difference between estimates. This meant that the advantage of geographical proximity is offset by the larger sampling error associated with small sample sizes. Whether the seasons of data collection were matched or different did not make a measurable difference to the similarity between estimates.\n\nHowever, the partition of variance analyses showed that DHS/MICS and NGO estimates differed, for the most part, in unpredictable ways, and geographical levels, years difference and seasons explained only a small part of the variation.\n\nWe hypothesize that large differences between estimates from NGO baseline reports and DHS/MICS data are due to three main reasons:\n\n(i) It is possible that NGOs’ estimates are collected from different populations with different underlying true values. NGOs often try to target lower wealth villages, and so baseline estimates may be worse off than the nationally representative DHS/MICS estimates. Note, however, that differences in household wealth indicators were small (e.g. “Household has electricity” 0.8% difference; “Household has a car” 0.2% difference). Additionally, the differences between DHS/MICS and NGO estimates might reflect actual changes over the years or across different geographical locations. Results from the analyses comparing data from the same source (DHS) but from different years and geographical levels also resulted in large differences between estimates.\n\n(ii) Different methods employed while sampling, collecting, processing and analyzing data might also have contributed to the differences between DHS/MICS and NGO estimates.\n\n(iii) Several indicators related to maternal and child health included in this study have not been validated and some have been shown to have low validity, such as maternal report of skilled birth attendance (Blanc et al., 2016). Inappropriate conflation of answer options and inconsistent coding and analysis of DHS surveys has also been documented (Footman et al., 2015). High measurement error can result in bias in unpredictable direction and dimension, resulting in large differences between estimates.\n\nWhatever the cause of the large differences between estimates was, it was not possible to know which of the data sources (DHS/MICS or NGO) provided the most accurate estimation of the true prevalence in the NGOs target populations. Furthermore, while we have been comparing DHS/MICS and NGO point estimates, these indicators are measured with error. The standard error (SE) for the DHS indicators is greater than 5% in eleven percent of the estimates. An estimate with a standard error of 5% will have a 95% confidence interval of ± 9.8%.\n\nOur analyses document and try to understand the large differences between NGO and DHS/MICS estimates. However, a study comparing DHS data to a small population-based survey from Rwanda showed that nine out of fifteen indicators related to maternal, newborn and child health were within a 10% difference (Langston et al., 2015). Similarly, in case studies from Nepal and Vietnam (HealthBridge, 2020) there were many indicators where the DHS/MICS and NGO estimates were similar. In Nepal 70% of indicators were within 20% of one another. Estimates for ANC, iron-folic acid uptake, vitamin A supplementation at 18-23 months and mobile ownership were similar while breastfeeding, child dietary diversity and tetanus vaccination in pregnancy differed widely. In contrast, in Vietnam NGO estimates for exclusive/continued breastfeeding and dietary diversity at 6-8 months were close to DHS, while others differed by >30%. Using secondary data may be useful, especially in situations of budget or mobility restraint, such as during the COVID-19 pandemic with limited data collection opportunities. However, use of DHS surveys may risk underestimating the scale of problems for poor and marginalised groups such as nomads or slum dwellers (Carr-Hill, 2017). When using DHS/MICS data, the user must keep in mind the potential differences between DHS/MICS and NGO estimates.\n\nThis study had some limitations. Most NGO data we used came from unpublished, not peer-reviewed reports created for internal use only. Indicators extracted from NGO reports were not necessarily consistent across all reports and often SDs or SEs were missing. Although, we matched the methods employed by the NGO as closely as possible in order to obtain the same indicators from DHS/MICS, some reports provided limited information concerning methods of data collection and analysis. Dates of and season of data collection were impossible to assess for eight reports. Assigning the geographical level of data from the NGO report was also challenging for some settings due to lack of contextual information. However, we were able to communicate with several NGOs in order to obtain supplementary information about the reports’ methods.\n\n\nConclusion\n\nOur hypothesis was that publicly available data can provide estimates of baseline conditions similar to those reported in NGO baseline reports when matched as closely as possible for location, year, and season of data collection. Our answer to this, in brief, is that publicly available data can be used, if the NGO is tolerant of imprecise estimates.\n\nWhile an NGO may use the evidence presented here to justify forgoing their own baseline survey, they should keep in mind that DHS and MICS provide estimates for only some of the indicators of interest to the NGO. On average, we estimated 18 of the NGO’s indicators using DHS/MICS, but NGOs were often reporting 100+ estimates. Furthermore, collecting data in the NGO working area can provide valuable insights for project design and implementation.\n\n\nData availability\n\nThis study used data owned by the DHS, the MICS and the NGOs that shared their baseline report. The DHS data can be downloaded at: https://www.dhsprogram.com, and the MICS data can be obtained at: https://mics.unicef.org. The DHS and MICS require registration and data access are only granted for legitimate research purposes.\n\nThe NGO reports were either available online on each NGO website or obtained by personal contact by email. The full list of NGO reports used in this study including report title, year of publication, organization name and how to access each report can be found at:\n\nHarvard Dataverse: Details on reports used in the Maxdata project. https://doi.org/10.7910/DVN/32FUQV (Berti, 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": "Acknowledgements\n\nWe thank all non-governmental organizations that shared their baseline reports with us, and USAID and UNICEF for making the Demographic and Health Surveys (DHS) and Multiple Indicator Cluster Surveys (MICS) data available. We thank Bana Salameh for assisting with extraction of data from the NGO reports.\n\n\nReferences\n\nAnoke SC, Mwai P, Jeffery C, et al.: Comparing two survey methods of measuring health-related indicators: Lot Quality Assurance Sampling and Demographic Health Surveys. Trop Med Int Health 2015; 20(12): 1756–1770. PubMed Abstract \| Publisher Full Text\n\nBerti P: Details on reports used in the Maxdata project. Harvard Dataverse 2021; V1. Publisher Full Text\n\nBlanc AK, Warren C, McCarthy KJ, et al.: Assessing the validity of indicators of the quality of maternal and newborn health care in Kenya. J Glob Health 2016; 6(1): 010405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarr-Hill R: Missing Millions and Measuring Development Progress. World Development 2013; 46(C): 30–44. Publisher Full Text\n\nCarr-Hill R: Improving Population and Poverty Estimates with Citizen Surveys: Evidence from East Africa. World Development 2017; 93: 249–259. Publisher Full Text\n\nCroft TN, Marshall AMJ, Allen CK: Guide to DHS Statistics 2018; ICF.\n\nData for Impact: Data for Impact: What’s the cost of evaluations and other surveys? — MEASURE Evaluation 2019. Reference Source\n\nFootman K, Benova L, Goodman C, et al.: Using multi-country household surveys to understand who provides reproductive and maternal health services in low- and middle-income countries: A critical appraisal of the Demographic and Health Surveys. Trop Med Int Health 2015; 20(5): 589–606. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHancioglu A, Arnold F: Measuring Coverage in MNCH: Tracking Progress in Health for Women and Children Using DHS and MICS Household Surveys. PLoS Med 2013; 10(5): e1001391. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealthBridge: Final Report: Maximizing use of existing data to strengthen program design, evaluation, and impact. HealthBridge; 2020. Reference Source\n\nLangston AC, Prosnitz DM, Sarriot EG: Neglected value of small population-based surveys: A comparison with demographic and health survey data. J Health Popul Nutr 2015; 33(1): 123–136. PubMed Abstract \| Free Full Text\n\nUNICEF: About Multiple Indicator Cluster Surveys (MICS). 2020Reference Source\n\nU.S. Agency for International Development: The Demographic and Health Survey (DHS) Overview. 2018. Reference Source\n\nWodon Q, Cong Nguyen M, Tsimpo C: Market share of faith-inspired and private secular health care providers in Africa: Comparing DHS and multi-purpose integrated surveys. 2012Reference Source" }	[ { "id": "78933", "date": "16 Mar 2021", "name": "Luay Basil", "expertise": [ "Reviewer Expertise I have been involved in several baseline and end-line surveys for projects in pubic health conducted in developing countries and have witnessed the pros and cons of conducting them. My engagement has been in the planning", "design", "data analysis and reporting phases. I have also used findings from the DHS/MICS to guide project design", "implementation and evaluation." ], "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 addresses an important issue tackled frequently by NGOs that struggle to decide whether to use existing data or collect their own at baseline. It follows a rigorous methodology and uses a good number of studies to draw conclusions from. Thank you for the excellent work. Below are some reflections, questions and suggestions.\nIntroduction: Statement in p3 “In addition, the accuracy of the indicator estimates in NGO-led surveys may be insufficient for project design and monitoring purposes, due to relatively small sample sizes and the inherent high variability of the indicators of interest.”\nSince the statement is in the introduction section, and since the paper shows later that sampling errors are not a factor for the studies that met the criteria of inclusion, how do we reconcile between both?\n\nA sample size of NGOs might be adequate for indicators such as antenatal care, postnatal care, delivery by skilled birth attendant, but not for illnesses among children under 5 years when we ask about children with symptoms in the past 2 weeks or when measuring the prevalence of early child marriage among adolescents since the age group is between 14 and 17, especially if the focus is on girls. Have such indicators been part of the simulations to see the potential errors by NGOs in sampling? Can the paper mention whether sample size by NGOs is a factor in such cases? Are such indicators the one that are in the small error of sampling in Figure 3? If so, would it be better for NGOs to use DHS/MICS data even when the surveys are a few years old or for a higher level of geography compared to their areas?\n\nIn addition to the challenges in sample size, sometimes the questionnaire in NGOs’ baseline surveys are not technically sound to measure a standard indicator. Has this been observed from the cases reviewed? If so, the paper could refer to that and add suggest using the questionnaire of DHS/MICS if the NGO is going to collect its own baseline data, especially since they are adjusted to local context.\nTable 4: Scenario 1 shows improvement for a geography over time but there is not much difference within the provinces or geographies in the same year. Scenario 2 shows there is not much difference between 3rd level and national level in the same survey. Scenario 3 shows estimates of a later study that is not much different from earlier (slight improvement) because the earlier performance was quite high at 88.5%. Question: The table illustrate an important point; to what level is it representative of all examined studies? It would be good to mention that. If it is not representative, could the paper cite another example where there are significant differences? Or, at least mention that there is, if this is the case. See related comment on Table 4 later.\nTable 6: Is there a need to explain in the methodology the rationale behind selecting 5% and 20% as the thresholds for comparison of differences? What would the picture be if the thresholds were 10% and 20%?\nDiscussion: P19 statement “In general, larger sample sizes were obtained at higher geographical levels and the larger the sample size (with their smaller sampling error) from DHS/MICS or NGO, the smaller the mean absolute difference between estimates. This meant that the advantage of geographical proximity is offset by the larger sampling error associated with small sample sizes.” One would expect that the NGO would calculate the adequate sample size required using a sample calculator (like the one of RADAR project). If the resources available would result in a sample size that is significantly less than the adequate one, should there be a recommendation that the NGO uses DHS/MICS data?\nP19 statement “It is possible that NGOs’ estimates are collected from different populations with different underlying true values. NGOs often try to target lower wealth villages, and so baseline estimates may be worse off than the nationally representative DHS/MICS estimates. Note, however, that differences in household wealth indicators were small (e.g. “Household has electricity” 0.8% difference; “Household has a car” 0.2% difference).” Since the DHS presents some findings by wealth quintiles, one would expect that findings from the lowest quintile could represent the areas NGOs work in and be close to those from NGOs data. Was comparison between indicators values from NGOs and the lowest wealth quintile from DHS made? If so, could you add a statement to reflect that?\nP 19 statement “Additionally, the differences between DHS/MICS and NGO estimates might reflect actual changes over the years or across different geographical locations. Results from the analyses comparing data from the same source (DHS) but from different years and geographical levels also resulted in large differences between estimates.” Table 4 for Zambia does not show large differences. Are there other studies that have that?\n\nConclusions: P 22 “Our hypothesis was that publicly available data can provide estimates of baseline conditions similar to those reported in NGO baseline reports when matched as closely as possible for location, year, and season of data collection. Our answer to this, in brief, is that publicly available data can be used, if the NGO is tolerant of imprecise estimates.” The paper also shows that NGO can use DHS/MICS when the values of the indicators are very high or very low.\nP 22 statement “While an NGO may use the evidence presented here to justify forgoing their own baseline survey, they should keep in mind that DHS and MICS provide estimates for only some of the indicators of interest to the NGO. On average, we estimated 18 of the NGO’s indicators using DHS/MICS, but NGOs were often reporting 100+ estimates. Furthermore, collecting data in the NGO working area can provide valuable insights for project design and implementation.” It would be good to expand on this in the discussion section. NGOs’ need to measure different outcome levels on knowledge, attitudes and practice; the first two guide project design, implementation and setting targets. NGOs also need to report on the different outcome levels to their donors. DHS/MICS focus more on practice/utilization of services and less on attitude and knowledge.\n\nAdditional point In a webinar presenting the paper in March 2, 2021, it was mentioned if an NGO wants to have a baseline so it can compare with the end-line, it can have a properly randomized and controlled end-line that can give good findings on the project’s impact. Could that be added to 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? 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": "7047", "date": "20 Sep 2021", "name": "Peter Berti", "role": "Author Response", "response": "Thank you very much for taking the time to review our manuscript and for these detailed and thoughtful comments. Please see below the answers to your comments. Introduction Comment 1: Statement in p3 “In addition, the accuracy of the indicator estimates in NGO-led surveys may be insufficient for project design and monitoring purposes, due to relatively small sample sizes and the inherent high variability of the indicators of interest.” Since the statement is in the introduction section, and since the paper shows later that sampling errors are not a factor for the studies that met the criteria of inclusion, how do we reconcile between both? Answer to Comment 1: Thanks for this comment. In the paper we show that the larger the sample size the smaller the difference between estimates (Table 7). However, we note that the advantages of larger sample size are often offset by being drawn from a higher geographical area, and so for an NGO working at the village level, they provide less specific information about the target village. Comment 2: A sample size of NGOs might be adequate for indicators such as antenatal care, postnatal care, delivery by skilled birth attendant, but not for illnesses among children under 5 years when we ask about children with symptoms in the past 2 weeks or when measuring the prevalence of early child marriage among adolescents since the age group is between 14 and 17, especially if the focus is on girls. Have such indicators been part of the simulations to see the potential errors by NGOs in sampling? Can the paper mention whether sample size by NGOs is a factor in such cases? Are such indicators the one that are in the small error of sampling in Figure 3? If so, would it be better for NGOs to use DHS/MICS data even when the surveys are a few years old or for a higher level of geography compared to their areas? Answer to Comment 2: As you note, sample size for a household survey is largest for household level indicators, smaller for populations that are subsets of households (such as children under 5 years) and smaller still for subsets of those subsets (such as children who have been ill in the past two weeks). Sampling error is inversely proportional to sample size and this relationship would hold across all indicators. There is nothing unique about those indicators, other than their smaller sample size. That said, a quick analysis shows that for those indicators that are drawn from subpopulations (in most cases regarding children who have had diarrhea in the previous two weeks), agreement between DHS/MICS and NGO estimates is somewhat lower, with only 65% of estimates falling within +/- 20 percentage points of each other Comment 3: In addition to the challenges in sample size, sometimes the questionnaire in NGOs’ baseline surveys are not technically sound to measure a standard indicator. Has this been observed from the cases reviewed? If so, the paper could refer to that and add suggest using the questionnaire of DHS/MICS if the NGO is going to collect its own baseline data, especially since they are adjusted to local context. Answer to Comment 3: We did not review the NGO reports for technical quality. However, in our experience, NGOs are usually quite good at finding and using recommended, validated tools when such tools exist. Often there is no single recommended, validated method though and this can result in differences in estimates. We addressed this in the discussion in points ii and iii. Comment 4: Table 4: Scenario 1 shows improvement for a geography over time but there is not much difference within the provinces or geographies in the same year. Scenario 2 shows there is not much difference between 3rd level and national level in the same survey. Scenario 3 shows estimates of a later study that is not much different from earlier (slight improvement) because the earlier performance was quite high at 88.5%. Question: The table illustrate an important point; to what level is it representative of all examined studies? It would be good to mention that. If it is not representative, could the paper cite another example where there are significant differences? Or, at least mention that there is, if this is the case. See related comment on Table 4 later. Answer to Comment 4: Note that Table 4 is just to show an example of how the calculations were done for the three scenarios. It is just for one indicator, so should not be over interpreted. It is not meant to be representative. We used ANC for the example because it is one of the most commonly reported indicators. For the meaning of the results, the reader should refer to the full report. However, we realized after reading your comment that we have neglected to report on the three different scenarios. While it is documented in the full report, the results of the three scenarios are not very different so we reported the three scenarios combined, which is the most similar to the DHS/MICS – NGO comparisons. Comment 5: Table 6: Is there a need to explain in the methodology the rationale behind selecting 5% and 20% as the thresholds for comparison of differences? What would the picture be if the thresholds were 10% and 20%? Answer to Comment 6: Those levels were arbitrarily chosen as very good (5%) and fair (20%) agreement. Because the differences are on a continuum, the cut-off of 10% yields, as you would expect, values between those for 5 and 20%. We in fact did this for the report (Table 4 in HealthBridge, 2020) (also for 15%) but we did not include it in this shorter paper. Discussion Comment 6: P19 statement “In general, larger sample sizes were obtained at higher geographical levels and the larger the sample size (with their smaller sampling error) from DHS/MICS or NGO, the smaller the mean absolute difference between estimates. This meant that the advantage of geographical proximity is offset by the larger sampling error associated with small sample sizes.” One would expect that the NGO would calculate the adequate sample size required using a sample calculator (like the one of RADAR project). If the resources available would result in a sample size that is significantly less than the adequate one, should there be a recommendation that the NGO uses DHS/MICS data? Answer to Comment 6: That is a good point and we agree with you. Comment 7: P19 statement “It is possible that NGOs’ estimates are collected from different populations with different underlying true values. NGOs often try to target lower wealth villages, and so baseline estimates may be worse off than the nationally representative DHS/MICS estimates. Note, however, that differences in household wealth indicators were small (e.g. “Household has electricity” 0.8% difference; “Household has a car” 0.2% difference).” Since the DHS presents some findings by wealth quintiles, one would expect that findings from the lowest quintile could represent the areas NGOs work in and be close to those from NGOs data. Was comparison between indicators values from NGOs and the lowest wealth quintile from DHS made? If so, could you add a statement to reflect that? Answer to Comment 7: That is another good point, thanks for your comment, but unfortunately, we did not do so. We will hopefully have the opportunity to address this question in future studies. Comment 8: P 19 statement “Additionally, the differences between DHS/MICS and NGO estimates might reflect actual changes over the years or across different geographical locations. Results from the analyses comparing data from the same source (DHS) but from different years and geographical levels also resulted in large differences between estimates.” Table 4 for Zambia does not show large differences. Are there other studies that have that? Answer to Comment 8: Table 4 is just an example. There were 9,024 matches in Scenario 1 (comparing the same indicator in the same geographical level at two points in time). Yes, some of these show apparent improvements, and some show apparent worsening, but part of the point is that these real secular changes may well be artifacts of method/sampling/random error. Conclusions Comment 9: P 22 “Our hypothesis was that publicly available data can provide estimates of baseline conditions similar to those reported in NGO baseline reports when matched as closely as possible for location, year, and season of data collection. Our answer to this, in brief, is that publicly available data can be used, if the NGO is tolerant of imprecise estimates.” The paper also shows that NGO can use DHS/MICS when the values of the indicators are very high or very low. Answer to Comment 9: We agree with you. Comment 10: P 22 statement “While an NGO may use the evidence presented here to justify forgoing their own baseline survey, they should keep in mind that DHS and MICS provide estimates for only some of the indicators of interest to the NGO. On average, we estimated 18 of the NGO’s indicators using DHS/MICS, but NGOs were often reporting 100+ estimates. Furthermore, collecting data in the NGO working area can provide valuable insights for project design and implementation.” It would be good to expand on this in the discussion section. NGOs’ need to measure different outcome levels on knowledge, attitudes and practice; the first two guide project design, implementation and setting targets. NGOs also need to report on the different outcome levels to their donors. DHS/MICS focus more on practice/utilization of services and less on attitude and knowledge. Answer to Comment 10: That is a very useful added detail. Thank you. Additional point Comment 11: In a webinar presenting the paper in March 2, 2021, it was mentioned if an NGO wants to have a baseline so it can compare with the end-line, it can have a properly randomized and controlled end-line that can give good findings on the project’s impact. Could that be added to the paper? Answer to Comment 11: We had that point in earlier versions but then felt it was too different from the main point of the paper. We are adding in here links to two blogs and a paper that we were drawing on when we made this point. By including it here, interested readers will be able to find it. 1. Alaka Holla, Are we over-investing in baselines? https://blogs.worldbank.org/impactevaluations/are-we-over-investing-baselines 2. David Evans, Advice for impact evaluations with government: Drop the baseline. https://davidevans.blog/2018/06/21/advice-for-impact-evaluations-with-government-drop-the-baseline/ 3. McKenzie D (2012) Beyond baseline and follow-up: The case for more T in experiments. Journal of Development Economics. 99(2):210-221 http://datacolada.org/wp-content/uploads/2015/06/5191-McKenzie-JDE-2012-Beyond-baseline-and-follow-up-the-case-for-more-t-in-experiments.pdf In a similar vein, I have put the links below to two papers on “shoestring evaluations”. 1. Bamberger M, Rugh J, Church M (2004) Shoestring Evaluation: Designing Impact Evaluations under Budget, Time and Data. Am J Evaluation 25(1): 5-37 http://www.managingforimpact.org/sites/default/files/resource/shoestring_evaluation.pdf 2. Can we trust shoestring evaluations? Martin Ravallion, 2013 http://documents.worldbank.org/curated/en/604591468332054439/pdf/WPS5983.pdf" } ] }, { "id": "78936", "date": "24 May 2021", "name": "Neff Walker", "expertise": [ "Reviewer Expertise Modeling", "program evaluation", "estimation procedures" ], "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 addresses a very interesting question but I must admit I remain unconvinced that the analyses presented here actually answer the question raised. From my perspective and that of the authors, the answer to this question is primarily related to how representative the data from the big surveys are of the program population. So for me, at one level the answer to this question is rather straightforward. If an NGO is working at provincial level and there has been a recent (in the last year) DHS or MICS survey that was sampled to be representative at the provincial level, then I would always use the DHS and MICS data as the baseline data forgoing the data collection by the NGO. Not only does this save time and energy, but my bias is that I believe the methods (e.g., sample size, mapping and household listing for sampling) used by major surveys like DHS and MICS are almost better than what an NGO would use. Of course, the answer to this question becomes less clear as the DHS or MICS survey data become less representative of the ideal program baseline, either in time or population. This is in large part what the analyses presented in this paper were all about.\nOn the time issue, would I still use the DHS data if it was two years old? In large part the answer to that question depends on how rapidly the indicators you are measuring change. If, for example, one was interested in measuring baseline values such as total fertility rate, household composition, wealth which change very slowly two or even five year period between the survey and the start of the NGO program would not be a big concern. However, for indicators that may change quickly, say coverage of some interventions like bednet ownership, vitamin A supplementation coverage which can quickly be scaled up with campaigns, data from a survey that is two or more years old would probably not provide a very accurate measure. While the analyses presented here did some work to look at this issue I am not really sure that it was captured in their analyses.\nOn population representation, using data from a very recent DHS survey that is sampled to be representative at a provincial level as baseline data for a program that covers 80% of the province may be okay. The data are not perfectly representative but unless there is extreme heterogeneity in the indicators of interest using the indicator values from the province probably provide a reasonable estimate of baseline coverage and therefore could replace a separate NGO-run survey. As with time, the key is how much variability is there within the population that was sampled for the DHS or MICS survey. This is hard to know, but clearly urban rural differences, ethnic mixes, topology all link to this. Again, I would think this would be a major issue that again, am not sure is captured in the current analyses.\n\nI think these are exactly the issues that the authors were seeking to address in their analyses so why do I feel like these results do not really help us much in finding the answer? In their analyses they matched DHS/MICS data to data from NGO’s and then looked at how well these points matched when they varies in time or population. One major issue for me revolves around the NGO survey data. The analytical approach basically assumes that NGO surveys produced the right answer (as they are for the correct population and for the right time) ignoring that methodological or procedural weakness in the NGO surveys (e.g., lack of household listing, mapping during sampling, small sample size, poor training and supervision of interviewers) may make their results far from a gold standard comparison. I think I would be happier with analyses that restricted the comparisons to NGO surveys where a review of the methods and procedures and the sample size of the survey make me more confident about the quality of the NGO estimates.\nThe second issue I have is the inclusion of data from DHS and MICS surveys that is broken down into smaller geographic region that was part of the sampling frame. Even if there are sufficient households at the district level, I am left wondering why we would expect the data to be very representative if that was not part of the sampling frame. I suppose that was part of the authors point of the analyses, but then we were not surprised that we found very weak correspondence between estimates of variables at the smaller geographic areas. I am a bit surprised the authors did not build a bit more on previous work on small area estimation that has tried to address many of the same issues focusing not just on differences due to population differences but also on techniques to adjust for these.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7048", "date": "20 Sep 2021", "name": "Peter Berti", "role": "Author Response", "response": "We thank the reviewer for these helpful comments. We agree with much of what the reviewer has offered, and we provide some clarification to other points. The reviewer observed that “The analytical approach basically assumes that NGO surveys produced the right answer”. Thanks for pointing this out. In earlier versions of the paper we were careful to note that we do not consider either the NGO or DHS/MICS to be “right”. We do not consider either the NGO estimates or the DHS/MICS estimates to be correct. Rather, the NGOs have used their reported estimates to characterize the baseline conditions and we are testing if they would have had similar characterization if they used the existing publically available data. Whether the NGO is “right” or not, those are the baseline estimates that they have used. For the scope of this study, we did not test if they were right or if DHS/MICS was right, but rather, would they have the same, or similar, baseline estimates if they used DHS/MICS. We agree that if we could isolate those NGO baseline surveys that used stronger methods and better trained teams would have lower measurement error, and so would be more similar to the DHS/MICS estimates. However, our research question was intended to address all NGO baseline reports, to determine if, in general, there was concordance between the estimates. Also, note that most of the NGOs were well resourced, international NGOs (see Table 1). We did test for an “NGO effect” but there was no observable impact of NGO on the concordance. Note also that we compared DHS to DHS estimates in different geographical levels and years and the absolute differences were similar to the ones observed when comparing DHS/MICS vs NGO (see Figure 3). We had a similar hypothesis to the reviewer. We expected that when the time between surveys was shorter, concordance would be higher. We also expected that some indicators would be much more concordant than others. However, our intuition was not consistent with the results, where we observed that most of the variation between estimates was not due to timing nor the indicator, but rather to unattributed error. We also expected that a closer match in location would have higher concordance, but again, this is not what we observed." } ] } ]	1	https://f1000research.com/articles/10-69
https://f1000research.com/articles/10-67/v1	03 Feb 21	{ "type": "Research Article", "title": "Parent-child signals identify candidate cancer driver genes", "authors": [ "Emilie Ann Ramsahai", "Vrijesh Tripathi", "Melford John", "Vrijesh Tripathi", "Melford John" ], "abstract": "Background: The DREAM Challenge evaluated methods to identify molecular pathways facilitating the detection of multiple genes affecting critical interactions and processes. Dysregulation of pathways by well-known driver genes is often found in the development and progression of cancer. We used the gene interaction networks provided and the scoring rounds to test disease module identification methods to nominate candidate driver genes in these modules. Method: Our algorithm calculated the proportion of the whole network accessible in two steps from each node in a combined network, which was defined as a 2-reach gene value. Genes with high 2-reach values were used to form the center of star cover clusters. These clusters were assessed for significant modules. Within these modules we identified novel candidate driver genes, by considering the parent-child relationship of well-known driver genes. Disturbance to such driver genes or their upstream parents, can lead to disruption of highly regulated signals affecting the normal functions of cells. We explored these parents as a potential source for candidate driver genes. Results: An initial list of 57 candidate driver genes was identified from 13 significant modules. Analysis of the parent-child relationships of well-known driver genes in these modules prioritized PRKDC, YWHAB, GSK3B, and PPP1CB. Conclusion: Our method incorporated the simple m-reach topology metric in disease module identification and its relationship with known driver genes to identify candidate genes. The four genes shortlisted have been highlighted in recent publications in the literature, which supports the need for further wet lab experimental investigation.", "keywords": [ "driver gene", "cancer", "module identification", "pathway analysis", "network biology", "topology", "reach metric", "signaling network" ], "content": "Introduction\n\nCancer is a disease of uncontrolled cell proliferation. Genetic mutations alter operations inside normal cells in ways that promote tumorigenesis. Receptors at the cell surface gather the signals from other cells, and funnel them into the cell. Signals are transmitted from upstream proteins and passed to downstream effector proteins. These mappings are represented in signaling networks, one of which was provided as a weighted directed graph by the DREAM Challenge. Dysregulation of these pathways by driver mutations is often found in the development and progression of cancer. Known driver genes, as listed in the Cancer Genome Census (CGC)1, within this signaling network provided points of reference. Disturbance to these driver genes or their upstream parents by mutations in either parent or child, can lead to disruption of highly regulated signals affecting the normal functions of cells. We explored these parents as potential candidate driver genes. We also considered driver gene parent protein products that were functionally related in physical protein-protein interaction (PPI) networks and co-expression networks as candidate driver genes.\n\nModule or community detection is a classical problem in social and computer network science2–4. Different methods yield communities that are either too large or too small to be easily understood. Biologically relevant modules and their relevance to a disease are poorly understood5. With the increased availability of biological network data, scientists are no longer solely relying on fixed pathways6,7. They are now seeking to expand on known pathways and discover novel pathways in the analysis of diseases. The challenge assessed our modules using a number of genome-wide association studies (GWAS). Each of our modules were scored for enrichment against 104 GWAS, using their PASCAL tool8. We then classified these novel disease pathways with CGC known driver genes as cancer modules. The signaling parent genes, which formed part of these novel cancer pathways, were nominated as candidate driver genes.\n\n\nMethods\n\nThe co-expression and physical Protein-Protein Interaction Network provided in Subchallenge 2 were combined. The networks were maintained as interaction lists, which proved to be more efficient than the memory demanding matrix manipulation. The network combination method employed was based on the system developed previously9. This network combination process considered the interaction existence and weighting in each of the individual networks before accepting it as part of the combined network. Linear regression was used in the calculation of the weighting in the combined network.\n\nThe individual networks are summarized in Table 1. These anonymized networks prevented any bias in the module identification process. Edges in the combined network were weighted and during the leaderboard stages this weight and its cutoff values were optimized to construct a final combined network with high-scoring edges. The clustering technique was employed on this combined network, while the signaling network was used to determine the parent-child relationships.\n\nThe number of nodes accessible within two steps as a proportion of the total nodes in the network, is defined as the 2-reach of that node. This 2-reach value was calculated for each node in the combined network. By repeatedly selecting two genes with the highest 2-reach values from the set of genes not yet assigned to a cluster, the complete network was decomposed. These two genes were the center of two separate clusters and their immediate neighbors chosen as members. To prevent overlapping, clusters were removed from the network, resulting in further fragmentation; such fragments give rise to clusters themselves as they may be within a range of 3–100 members. The largest remaining fragment was decomposed by repeating the 2-reach cluster formation process.\n\nThe DREAM Challenge assessed modules based on a collection of GWAS, which is superior to matching the predicted modules against well-known pathway databases or annotated information. The unique GWAS used in the assessment was not part of the construction of the networks provided. The Challenge used these GWAS and our modules as input to the PASCAL scoring tool8, which provided 104 p-values for each of our modules in the final submission. With these multiple p-values, for each of our modules, we were then required to perform a level of correction to control the False Discovery Rate (FDR) using the Benjamini-Hochberg procedure10, as some of the p-values less than 0.05 may have been by chance.\n\nSignificant modules (SM) with at least one driver gene from the CGC list were classified as cancer modules (CM). These cancer modules were a subset of the set of significant disease modules as depicted in Figure 1A. The list of genes in these cancer modules (CMgenes), were a source of potential driver genes. Additionally, from the signaling network in Table 1, we identified a list of parent genes (Pgenes) to well-known driver genes as another source of potential driver genes. Figure 1B highlights the intersection of these two lists. The CMgenes were based on the network module identification and GWAS scoring, while the Pgenes were based on the parent-child relationships with well-known driver genes in the signaling network. The overlap of these two lists provided the list of candidate driver genes based on a consensus.\n\n(A) The set of significant modules (SM) with well-known driver genes from the Cancer Genome Census list were identified as cancer modules (CM). (B) The intersection of the genes from the cancer modules CMgenes and the parents from the signaling network Pgenes highlight our first list of candidate driver genes.\n\nThe scripts and source code used to perform the analysis in this study are available as Extended data11.\n\n\nResults\n\nThe network formed by combing the co-expression network and the protein-protein interaction (PPI) network consisted of 14,200 genes and 824,528 edges. The removal of low-confidence edges resulted in the loss of 1,583 genes, 199 of which were unique to the co-expression network, 1,201 of which were unique to the PPI network, and 183 of which were common to both. The overlap of these two DREAM Challenge networks and the combined network is illustrated in Figure 2A.\n\n(A) Genes from Combined Network and the individual networks. (B) Genes from the signaling network, Cancer Genome Census (CGC) and their parents. PPI, protein-protein interaction.\n\nThe signaling network included 5,254 nodes and 21,826 edges. Of the 616 CGC driver genes 470 were present in this network. These driver genes were children of 1,721 parents, with TP53 having 154 (the highest number of parents); 63 other driver genes were orphans. All of the orphan driver genes were themselves parents in this signaling network. Figure 2B shows the overlap of the signaling network, the CGC driver genes and their parents. We also see that 306 of the CGC driver genes are both parents and children of other driver genes.\n\nOur 2-reach clustering algorithm produced 237 non-overlapping modules, ranging in size from 3 to 100, as shown in Figure 3A. Many of our modules were 50 genes in size, as this was the default module size in the cluster formation process. Our method also produced modules that were at the upper bound of 100 genes and the lower bound of 3 genes in size, a result of fragmentation of the network. The 237 modules extracted from the combined network included 4,682 genes, which was less than 33% of the total network, since only the largest fragment was further assessed for clusters.\n\n(A) All 237 modules. (B) 27 Significant modules with 13 cancer modules identified by solid circles.\n\nOn examination of the 104 multiple p-value scores provided by the DREAM Challenge for each of our modules, we found 27 significant modules existed, all shown in Figure 3B. The 13 solid circles are the CM, which contain known driver genes. It is difficult to get highly significant enrichment p-values for modules of a small size. Consider a module of size 4 that includes two significant genes. Even though 50% is certainly a clear enrichment, this could also occur by chance. In contrast, a module of size 100 that contains 50% significant genes would get an extremely small p-value. Of the 237 modules identified from our 2-reach method on the combine network, 27 were identified as significant at the 10% FDR, and 13 were cancer related as seen in Figure 3. There were different assessment stages during the challenge. Our best result was at the 2.5% FDR, where we were the fifth-best clustering method as identified in the Supplementary Figure 5 in the main paper12. Figure 4 shows the details with the performance of every method from the teams listed on the x-axis as compared to the highest scoring method from subchallenge 1. The first three teams with Bayes factor K less than three indicated their methods were better than this reference method.\n\nThe y-axis represents the number of significant (NS) modules identified in the final phase of subchallenge 2.\n\nThe 35 driver genes in these 13 cancer modules contained 84 unique parents from the signaling network. In total, 27 of the 84 parents were also driver genes, which left 57 as candidate driver genes. Many of the driver genes were also parents, but module 20, module 55, module 109, and module 143 contained driver genes that were not parents, since the “#Drivers in module” column value was larger than the number of red genes provided in Table 2. In the case of module 55, with as many as nine known driver genes, only five were parents in the signaling network. Module 143 has a unique condition of containing the single CGC driver gene HLA-A, which was not a parent, and therefore does not appear in Table 2. The presence of this driver gene, qualified module 143 to be a cancer module, but only the parents of other known driver genes were considered as candidate drivers. Each of the cancer modules CM and the candidate driver genes they contain are listed in Table 2; candidate genes are listed in black.\n\nRed: Known driver genes which are also parents; Black: Parents and possible candidate genes; Underlined: Parents in a parent-driver gene relationship in the given cancer module.\n\nAdditional analysis of the cancer modules further prioritized our initial list; we assessed the specific parent child relationships in the subgraphs using the parent genes for each of the modules in the signaling network. On examination of the 13 subgraphs, only three (modules 15, 19 and 55) had direct parent-child relationships, where the parent was listed in the module and the child was a CGC well known driver gene. In the case of module 15, there were five driver genes FBXW7, THRAP3, ABL1, FANCD2 and YWHAE, with 13 parent genes. The five driver genes were themselves parent genes, so we were left with eight parents to consider as potential candidate driver genes as listed in Table 2. Of the five driver genes in this module, only ABL1 had a parent from this same module in the signaling network – PRKDC, as seen in Figure 5. Similarly, module 19 showed the RAF1 driver gene had three parents in the signaling network, CDK4, YWHAB, and GSK3B. Module 55 has nine driver genes, but only five were parents. RB1 was the only driver gene with the neighboring parent PPP1CB from the signaling network. This process prioritized five parents, which are underlined in Table 2. They are neighbors of existing drivers in the cancer modules, and are parents to the same driver gene in the signaling network. Of these five, CDK4 is already present in the CGC list of known driver genes. The other four genes PRKDC, YWHAB, GSK3B, and PPP1CB are shortlisted.\n\nFive blue nodes are the known drivers. Only driver gene ABL1 is seen to have a parent which forms part of module 15.\n\n\nDiscussion/conclusion\n\nWe used three diverse genomic networks to reveal novel candidate genes from pathways underlying cancer: the physical PPI, the functional co-expression, and the signaling networks. Our method incorporated the simple m-reach topology metric in disease module identification. The reach measure has been shown to be useful in the key player problem13, and has been used to identify cancer driver genes14. Recent publications in the literature highlight the four candidate genes that were shortlisted. PRKDC has been shown to be associated with poor clinical outcome in gastric cancer patients15. Recent studies proposed the YWHA family is able to regulate a vast number of proteins involved in key cellular processes, with implications for tumorigenesis and cancer progression16. GSK3B is known to regulate epithelial-mesenchymal transition and cancer stem cell properties, and is a novel drug target for triple-negative breast cancer17. PPP1CB has been identified as a protein safeguarding nuclear integrity; altered nuclear shape is a defining feature of cancer cells18.\n\n\nData availability\n\nSynapse: Disease Module Identification DREAM Challenge. Synapse ID syn11944943.\n\nZenodo: Parent-child signals identify candidate cancer driver genes. http://doi.org/10.5281/zenodo.374080511.\n\nThis project contains the scripts and source code used to perform the analysis in this study.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author contributions\n\n\n\nER worked with the DREAM Challenge, downloading data, running analysis, preparing and submitting results. ER also wrote the manuscript, which the other authors reviewed, corrected and approved.\n\n\nReferences\n\nFutreal PA, Coin L, Marshall M, et al.: A census of human cancer genes. Nat Rev Cancer. 2004; 4(3): 177–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFortunato S: Community detection in graphs. Phys Rep. 2010; 486(3–5): 75–174. Publisher Full Text\n\nBishop JM: The molecular genetics of cancer. Science. (New York, NY). 1987; 235(4786): 305–11. Publisher Full Text\n\nNewman ME: The structure and function of complex networks. SIAM review. 2003; 45(2): 167–256. Publisher Full Text\n\nWang J, Zuo Y, Man YG, et al.: Pathway and network approaches for identification of cancer signature markers from omics data. J Cancer. 2015; 6(1): 54–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMitra K, Carvunis AR, Ramesh SK, et al.: Integrative approaches for finding modular structure in biological networks. Nat Rev Genet. 2013; 14(10): 719–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson DM, Huberman BA: A method for finding communities of related genes. Proc Natl Acad Sci U S A. 2004; 101(suppl 1): 5241–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLamparter D, Marbach D, Rueedi R, et al.: Fast and rigorous computation of gene and pathway scores from SNP-based summary statistics. PLoS Comput Biol. 2016; 12(1): e1004714. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamsahai E, Walkins K, Tripathi V, et al.: The use of gene interaction networks to improve the identification of cancer driver genes. PeerJ. 2017; 5: e2568. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the royal statistical society Series B (Methodological). 1995; 57(1): 289–300. Publisher Full Text\n\nRamsahai E, Tripathi V, John M: Parent-child signals identify candidate cancer driver genes.2020. http://www.doi.org/10.5281/zenodo.3740805\n\nChoobdar S, Ahsen ME, Crawford J, et al.: Assessment of network module identification across complex diseases. Nat methods. 2019; 16(9): 843–852. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorgatti SP: The Key Player Problem. Dynamic social network modeling and analysis: Workshop summary and papers. National Academies Press. 2003. Reference Source\n\nRamsahai E, Tripathi V, John M: Cancer driver genes: a guilty by resemblance doctrine. PeerJ. 2019; 7: e6979. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSotgia F, Lisanti MP: Mitochondrial biomarkers predict tumor progression and poor overall survival in gastric cancers: Companion diagnostics for personalized medicine. Oncotarget. 2017; 8(40): 67117–67128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore S, Järvelin AI, Davis I, et al.: Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer. Curr Opin Genet Dev. 2018; 48: 112–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaja GV: GSK3B regulates epithelial-mesenchymal transition and cancer stem cell properties and is a novel drug target for triple-negative breast cancer. 2017. Reference Source\n\nTakaki T, Montagner M, Serres MP, et al.: Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability. Nat commun. 2017; 8: 16013. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "83221", "date": "27 May 2021", "name": "Hilal Kazan", "expertise": [ "Reviewer Expertise cancer genomics", "statistical 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\nThe study extends a method that is originally proposed for finding network modules to cancer driver gene identification task. Cancer driver identification is a well-studied problem in bioinformatics. However, the manuscript does not have enough discussion on the existing literature. Methods such as Hotnet2, MEMCover, DriverNet can be cited.\n\nAlso, a graph theoretical measurement, betweenness centrality, has been recently used to find cancer driver genes (https://doi.org/10.1186/s12859-021-03989-w1). Since it is related to 2-reach concept, it’d be useful if it is included in the literature discussion.\nIn the current version of the study, it is very difficult to assess the ability of the proposed method for cancer driver identification task. Several existing methods that predict cancer driver genes using PPI and heat diffusion might be performing better than the current model.\nAlso there are several problems regarding the content of the manuscript:\nThe authors refer to DREAM challenge throughout the paper, however many DREAM challenges have been organized to date and it is not immediately clear which one they refer to. Referring to it as “Disease Module Identification DREAM challenge” would be better.\n\nAlso, the aim and details of this challenge should be better explained.\n\nIt is not clear what the authors mean by anonymity in the following sentence: “These ano-nymized networks prevented any bias in the module identification process. ”\n\nWhat’s the intuition behind selecting two genes simultaneously in The 2-reach center cluster formation?\n\nFigure 1A can be removed, Figure 3’s should be converted to histograms as module ids are not critical.\n\nFigure 4 can be removed. The performance of the cluster identification method in the DREAM challenge is not relevant in the current 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "91848", "date": "10 Nov 2021", "name": "Alexander Lachmann", "expertise": [ "Reviewer Expertise gene expression analysis", "microservice development", "enrichment analysis", "gene function prediction", "gene regulatory network reconstruction", "cloud computing", "single-cell 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 network construction is not completely explained. The paper cites a reference (9) that employs the same approach. I was not able to find an explanation in this paper either. Many questions are unanswered, which makes this step not reproducible. How are PPIs weighted, how are negative correlations weighted, how are signaling weighted. I did not find a mention of the source data for the three networks (signaling, PPI, correlation). Did the dream challenge provide the networks?\nThe 2-reach cluster formation is not clearly described. It is not clear to me how clusters are generated. The authors state that iteratively, two genes with the highest 2-reach are selected and form two clusters. Why are two genes selected at the same time and not one iteratively? Clusters are the direct neighbors of the selected genes, rather than the 2-reach neighborhood that was used as selection criteria. Defining clusters as direct neighbors of highly connected nodes will result in overlapping sets of genes. If two genes are selected at the same time, their resulting clusters can have overlap? In short, I would not be able to reproduce the procedure as described in the document.\nAccess to a GitHub repository would greatly help with a lot of the uncertainties of the algorithm.\nThe manuscript mentions that the genes are anonymized. How are the significant modules matched to well-known driver genes using Cancer Genome Census?\nThe document states the network consists of 824,528 edges. The network file in the supplement has 1,388,324 rows. It is mentioned that some edges are filtered, but the exact methodology of the filtering is not further discussed.\nThe clusters found are not characterized in any functional way. It would be interesting to see if there is any common biological theme to the genes in a module (e.g. perform enrichment analysis for biological processes)\nMinor: The paper refers to a Dream Challenge, but it is not made clear which dream challenge the authors refer to. (Disease Module Identification DREAM Challenge) A citation in the introduction is missing. In general, the provided data and the task of the challenge could be described more clearly.\nReference 11 pointing to the combined network is labeled unclearly. The columns of the matrix are (V3, tot, tot2, R, Q, S) and it is not clear what they mean.\nThe manuscript mentioned that the source code is posted at reference 11, but it is not.\nFigure 5 would probably be more informative if the nodes were labeled.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-67
https://f1000research.com/articles/10-66/v1	03 Feb 21	{ "type": "Research Article", "title": "New insights about the serine/threonine protein kinase substrates from Mycobacterium tuberculosis using molecular docking, quantum similarity analysis and DFT calculations", "authors": [ "Alejandro Morales-Bayuelo", "Jesús Sánchez-Márquez" ], "abstract": "Background: The protein kinases present in the human body have received a lot of attention because of the interest in their use as therapeutic targets. However, little is known about the protein kinases associated with tuberculosis. For these reasons, this research investigates a new point of view regarding the crystallized serine/threonine protein kinases Pkn A, B and G of Mycobacterium tuberculosis. Methods: The conformational analysis shows a DFG-in motif in Pkn B and G and a DFG-out motif in Pkn A. For all the protein kinases that have been studied, the gatekeeper residue is methionine. A study of the protein kinases with their ligands was also conducted to find new insights on the binding site with a series of ligands associated to protein kinases Pkn A, B and G through molecular docking. The residues with hydrogen bonds on the hinge zone of Pkn A are GLU96 and VAL 98, of Pkn B are GLU 93 and VAL 95 and of Pkn G are GLU233 and VAL235. Results: The results show the H-bond acceptor and H-bond donor sites on the hinge zone to all ligands, establishing a structural model of the ligands on the active site with two or three interactions in this zone. This interaction model was validated using density functional theory calculations (by means of net charges and images of the electrostatic potential) and molecular quantum similarity analysis, showing a high correlation between the electronic and steric effects in each ATP complex studied. Conclusions: In this work we can see that the interactions of the hinge zone are characterized by the key factor of one or two H-bonds acceptors and one H-bond donor in the ligands of this zone. The quantum similarity analysis shows good correlation between the steric and electronic effects in each ATP complex.", "keywords": [ "Mycobacterium tuberculosis", "Serine/Threonine Protein Kinases A", "B", "G", "Molecular Docking", "Molecular Quantum Similarity Analysis", "Electrostatic Interactions." ], "content": "Introduction\n\nProtein kinases (PKs) are a key in controlling proliferation and differentiation in eukaryotic cells in living organisms. They are enzymes that catalyze the protein phosphorylation process. An important reason to analyse the protein phosphorylation process is that it represents an attractive drug target in a variety of fatal diseases such as cancer1. The PKs present in the human body have been widely used in the field of drug design because they play an important role as therapeutic targets in these diseases. However, little is known about the serine/threonine protein kinases (STPKs) involved in tuberculosis. For this reason, we propose a study about crystalized STPKs Pkn A, B and G, to gain new insights in the mechanism of the phosphorylation process.\n\nTo date, one of the main questions in the tuberculosis field is ‘what are the protein substrates of the STPKs?’ According to Av-Gay et al.2, to deal with this topic we need to study the interactions of crystalized STPKs Pkn A, B and G with a particular series of ligands to each PK. The competitive ATP ligands used are a series of compounds for Pkn A reported by Sipos et al.3, ligands for Pkn B reported by Székely et al.4, Loughheed et al.5 and Chapman et al.6 and ligands for Pkn G reported by Sipos et al.3 and Pato et al.7. These ligands were selected with the goal of finding new insights into the inhibition of oxidative phosphorylation. Therefore, we are researching new pharmacological alternatives for this disease through the structural models of the ligands on the ATP sites.\n\nAnother aim of this study is to conduct molecular quantum similarity (MQS) analysis using a quantum similarity field to understand the electronic and structural patterns involved in stabilization of the active site of the inhibitors for each PK studied. Therefore, a hybrid quantum mechanics/molecular mechanics (QM/MM) approach was used to carry out a structure-activity relationship analysis of the inhibitor set for Pkn A, Pkn B and Pkn G.\n\n\nMethods\n\nThe crystal structures of Pkn A (PBD code: 4OW8, resolution: 2.03 Å8), Pkn B1 (PDB code: 3F69, resolution: 2.80 Å9(a)), Pkn B2 (PDB code: 1MRU, resolution: 3.0 Å9(b)), Pkn B3 (PDB code: 2FUM, resolution: 2.89 Å9(c)) and Pkn G (PBD code: 2PZI, resolution: 2.40 Å10) were selected for the preparation process using Schrödinger Suite 2014-1’s Protein Preparation Wizard module11, which refines the protein structure and optimizes the hydrogen bond (H-bond) network. The open-source software AutoDock Vina could also be used to perform these steps.\n\nProtonation states were identified using PropKa utility at a physiological pH12. After, restrained molecular minimization using the Impact Refinement (Impref) module13 was carried out, taking into account the heavy atoms restrained within a root-mean-square deviation (RMSD) of 0.18 Å from the initial coordinates.\n\nThe main characteristics of Pkn A and B consist of a transmembrane receptor with a tyrosine kinase domain protruding into the cytoplasm. For Pkn G, the unique multidomain topology of this PK reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively13,14.\n\nThe molecular datasets used for the docking study were reported by Sipos et al.4, Székely et al.5, Lougheed et al.6, Chapman et al.7 and finally Pato et al.8 (see Table 1–Table 6). To select this molecular dataset, the structural diversity and uniform distribution of IC50 was taken into account. Logarithmic IC50 (μM) (pIC50=−log IC50) was employed as the dependent variable instead of IC50. The 3D molecular structures of the compounds were drawn and optimized using Maestro15 and then prepared with the LigPrep module16, where ionization/tautomeric states were predicted at physiological pH conditions using Epik17. Afterwards, energy minimization was performed using Macromodel18 with the OPLS2005 force field. The open-source software AutoDock Vina could also be used to perform these steps.\n\naCompounds to Pkn A reported by Sipos et al.4\n\naCompounds to Pkn A reported by Sipos et al.4\n\nbThis compound has biological activity to Pkn B and G. Sipos et al.4\n\ncCompound to Pkn B reported by Székely et al.5\n\ndCompound to Pkn B reported by Loughheed et al.6\n\naCompounds to Pkn B reported by Chapman et al.7\n\naCompounds to Pkn B reported by Chapman et al.7\n\naCompounds to Pkn B reported by Chapman et al.7\n\naCompounds to Pkn G reported by Sipos et al.4\n\nbCompounds to Pkn G reported by Pato et al.8\n\nDocking studies were performed with the software Glide18 using standard precision (SP) mode and default parameters. The open-source software AutoDock Vina could also be used to obtain similar results. The docking grid was generated with standard protocol centered at the co-crystallized ligand. Due to that, the scaling factor for the van der Waals interactions is very important it was taken as 0.8. The radii of nonpolar protein atoms were selected to allow the binding of larger ligands. Further, the induced fit docking (IFD) workflow19 was also used to generate a good number of conformations of the receptor suitable for binding ligands that had strange orientations. It allows the protein to undergo side-chain movements, backbone movements, or both, upon ligand docking. The docking procedure has four steps where accuracy is ensured by Glide’s scoring function and Prime’s:\n\n(i) The first step for Glide docking is carry out into the rigid receptor to generate a good ensemble of poses.\n\n(ii) Analysis of the protein using Prime21. Taking into account side-chain prediction module followed by a structure energy minimization of each protein/ligand complex pose.\n\n(iii) Analysis through re-docking of the ligand into low energy induced-fit structures taking in account the previous step using default Glide settings (no vdW scaling).\n\n(iv) Finally, the binding energy is estimated using (IFDScore) by accounting for the docking energy (GScore), as well as receptor strain and some solvation terms (Prime energy).\n\nTo obtain the docking outcomes, the dynamics simulation of 100ns in vacuo using the GROMOS11 force field set was used on the protein–ligand complex, using Gromacs 5.1.220 to analyze the stability of the protein–ligand complexes22.\n\nIn recent studies, MQS methodology has shown good results in understanding steric and electronic effects of ligands23,24. The MQS field was introduced by Carbó and co-workers 30 years ago25–30 and has been used to understand the steric and electronic effect on the molecular sets. The mathematic relationship to obtain quantum similarity ZAB between two compounds A and B, with electron density ρA(r1) and ρB(r2) is defined using the minimization of the expression for the Euclidean distance as:\n\n\n\nTherefore, the Euclidean distance is the square root of the integral on the squared differences between the densities ρA(r1) and ρB(r2). In this context, ZAB represents the similarity measure between the electronic densities of the compounds A and B, and ZAA and ZBB are the self-similarity of compounds A and B31. The similarity index very often used is the Carbó index31. This index is expressed as follows:\n\n\n\nIn Equation 2, (Ω) represents an operator. A simple way to make quantum similarity measures (QSM)29–31 involving two density functions, in the most usual form, is expressed as an integral:\n\n\n\nIn this equation ρA(r1) and ρB(r2) are the density functions to quantum objects A and B, while Ω(r1,r2) is a positive operator that defines weight. The Dirac delta function: δ(r1 – r2) is used to obtain the overlap similarity measure and to measure the Coulomb similarity the Coulomb operator: \|r1 – r2\|-1 is used. These operators are the most useful for obtaining similarity comparisons between ligands.\n\nAn important factor when carrying out the QSM is an optimal molecular alignment. As the integrals associated to the QSM produce, in any case, real positive results, the relative position problem can be approached using a maximal QSM. An overlap QSM can be expressed according to the equation:\n\n\n\nIn this equation, it is implicitly supposed that ρB(r) is translated and rotated by the six possible ways: (T;φ) and are shown as explicit parameters in this integral31. This key factor was applied using the topo geometrical superposition algorithm (TGSA)31.\n\nThe calculations of this study were performed with the GAUSSIAN 09 program32. The open-source software AutoDock Vina could also be used to obtain similar results. The structures were optimized using the B3LYP functional (DFT exchange-correlation method)33,34 and the 6-31G(d,p) basis set35. The contour lines of the molecular electrostatic potential have been obtained with the GaussView program36.\n\n\nResults and discussion\n\nThe conformational analysis of the STPKs defines the active and inactive form. These forms are also known as the DFG-in and DFG-out states. They are distinguishable by a flip of the Asp and Phe orientation in the activation loop as well as conformational changes in the surrounding loops37. The main difference between DFG-in and DFG-out conformations is that the different position of the DFG residue in the “out” form results in the opening of an additional cavity, the allosteric site, which can be hydrophobic in nature according to Zuccotto et al.38. In Pkn A the Asp side chain is buried and the Phe side chain is flipped outside the pocket. Therefore, Pkn A becomes DFG-out. However, in the STPKs Pkn B1 (crystal: 1MRU), Pkn B2 (crystal: 2FUM) and Pkn B3 (crystal: 3F69), the Asp side chain is exposed and the Phe side chain is buried (DFG-in), allowing ATP binding. Pkn G has DLG (Asp-Leu-Gly) instead of DFG (see Figure 1); the conformation is DLG-in. An important residue of STPKs is the gatekeeper. Their size determines the access of small, medium and large inhibitors to the back cavity even from the DFG-in state. In these crystallized PKs, the gatekeeper residues are MET95 for Pkn A, MET92 for Pkn B1 (crystal: 3F69), Pkn B2 (crystal: 1MRU), Pkn B3 (crystal: 2FUM), and MET232 for Pkn G. The methionine is a medium gatekeeper residue, according to Zuccotto et al.38 and allows access to small, medium and large inhibitors. This characteristic is important because it increases the range of molecular diversity in the development of new inhibitors.\n\nDFG conformational analysis and gatekeeper residues to STPKs crystalized from Mycobacterium tuberculosis for (A) Pkn A, (B) Pkn B and (C) Pkn G. Hydrogen-bond-acceptor map (red mesh) for (D) Pkn A, (E) Pkn B and (F) Pkn G. Hydrogen-bond donor map (blue mesh) for (G) Pkn A, (H) Pkn B and (I) Pkn G. Hydrophobic map for (J) Pkn, (K) Pkn B and (L) Pkn G. Note: map sites were calculated using SiteMAP in Maestro. The open-source software AutoDock Vina could also be used to obtain similar results.\n\nIn Figure 1D, 1E and 1F we can see the hydrogen-bond-acceptor maps in red mesh. Pkn A shows a small red contour on the hinge zone (Figure 1D), a medium contour near to the DFG motif and a large contour near to the helix-αC and C-terminal loop. This is unlike Pkn B, which, using the crystal 3F69 (Figure 1E), shows only acceptor maps near to the DFG motif and in the C-terminal loop. Pkn G (Figure 1F) has large acceptor contours involving the hinge zone, DLG zone, helix-αC and C-terminal loop. Figure 1G, 1H and 1I shows the hydrogen-bond donor maps in blue mesh. In Pkn A (Figure 1G) we can see medium contours to hydrogen-bond donor on the hinge zone and C-terminal loop and a large contour near the DFG motif. In contrast, Pkn B (Figure 1H) shows only a large contour near to the DFG motif. Pkn G (Figure 1I) has small contours to hydrogen-bond donors along the hinge zone and near the DLG motif, helix-αC, C-terminal and N-terminal loops.\n\nAnalyses of hydrophobicity are shown in Figure 1J, 1K and 1L. Figure 1J shows a large contour of hydrophobicity for Pkn A near to the hinge zone and small contours on the C-terminal loop. Pkn B (Figure 1K) has only a small contour on the C-terminal loop. In contrast, Pkn G (Figure 1J) shows a large contour near the hinge zone, DLG motif and small contours near to the helix-αC and C-terminal loop. These physical-chemistry properties are important to describe the non-covalent stabilization on the active sites and selectivity of the ligands in the active pocket.\n\nTo obtain the docking outcomes, hydrogen bonds on the hinge zone and non-covalent interactions near the “gatekeeper door”, helix-αC, C-terminal and N-terminal were analyzed. An important insight are the non-covalent interactions. Theses interactions involved backbone, side chain hydrogen bonds and aromatic-aromatic interactions. On the other hand, the ligands with high scores have these characteristics in their non-covalent interactions. The ligands with lower scores have few to no interaction forces. Further, some of the higher scoring ligands forming hydrogen bonds and aromatic-aromatic interactions with the amino acid residues are related to the hinge zone.\n\nFigure 2 shows the docking interactions of the Pkn A inhibitors from Table 1. These compounds have groups with electron withdrawing resonance effects such as nitro, amine, carbonyl groups and electron donating groups such as methylene.\n\nInteractions on the hinge zone of compound 1 (A) and compounds 2-4 aligned (B), from Table 1.\n\nIn Figure 2A we can see the hydrogen bonds on the hinge zone to compound 1 (Table 1). This compound has two interactions on the hinge zone with the residues Glu96, Val98 of 2.29 Å and 2.54 Å, respectively. Another hydrogen bond can be formed with the residue Asn104 of 1.98 Å. Compounds 2, 3 and 4, see Figure 2B, have differences only in the substituent group, in the case of 3, this is a pyrrole ring and in 4, is the dimethyl-amine group. In this series, the most active compound is 2, with pIC50 -1.839. These compounds show two hydrogen bonds on the hinge zone with residues Glu96 and Val98 of 1.31 Å and 2.30 Å to 2, 1.71 Å and 2.35 Å to 3 and 1.81 Å and 2.21 Å to 4. Another hydrogen bond can also be formed with the residue Gly145 of 2.22 Å to 2, 1.81 Å to 3 and 1.95 Å to 4. From these results, we can see the key factor to the stabilization of the active site in this ligand set, with the main characteristic of one H-bond acceptor and H-bond donor in this zone.\n\nFigure 3 (left) and Figure 4 show, respectively, the atomic charges calculated by the CHelpG population and electrostatic potential analyses of compound 1 in some areas of special interest. The net charges obtained for H22, O24, O30 and O31 are consistent with the formation of hydrogen bonds shown in Figure 2A. The electrostatic potential surfaces shown in Figure 4 also justify the formation of hydrogen bonds with Glu96, Val98 and Asn104. Figure 3 (right) and Figure 5 show, respectively, the atomic charges and electrostatic potential of compound 4. The atomic charges of H27, H42 and H15 justify very well the formation of hydrogen bonds with Glu96, Val98 and Asn104 that can be seen in Figure 2B. An equivalent conclusion can be found from the electrostatic potential surfaces shown in Figure 5.\n\nThe compounds to Pkn B have groups with electron withdrawing resonance effects, such as carbonyl, hydroxyl, amine and methoxy groups, with big rings central to its stabilization on the active site. Figure 6 shows the docking results of the Pkn B inhibitors from Table 2–Table 5, grouped by series, taking into account their functional groups and structural similarities. Figure 6A shows compound 5, which has interactions in the hinge zone with the residues Glu93 (2.63Å) and Val95 (-CO: 2.46Å, -NH: 1.99Å, 2.16Å), see Table 7. Compound 5 has dual effects, interacting with Pkn B (pIC50: -0.585) and Pkn G (pIC50: 1.301), see Table 2. Figure 6B shows the interaction with compound 6, which has interactions with the residue Val95 (-CO: 1.31Å, -NH: 2.48Å). In these interactions the -NH is H-bond donor and the –CO is H-bond acceptor; these interactions can be related to the pIC50 of 0.569 for this compound.\n\nInteractions in the hinge zone with compound 5 (A); compound 6 (B); compounds 7, 8, 9 (C); compound 10 (D); compounds 11, 12, 17, 18, 19, 20 and 23-53 (E); compounds 13 and 22 (F); and compounds 14, 15, 16, 21 and 54-57 (G), from Table 2–Table 5.\n\nFigure 6C shows the interaction in the hinge zone to of compounds 7, 8 and 9. These compounds have two interactions in the hinge zone with the residue Val95. The most active compound of this series is 8 (pIC50: 0.585) with its interactions with the residue Val95 (-CO: 1.80Å, -NH: 2.15Å), see Table 7. Figure 6D shows the interactions of the compound 10; this compound has two interactions in the hinge zone with the residue Val95 (-NH: 2.33Å, -C=O: 2.09Å). Another H-bond happens with the residues Asp96 (1.71Å) Asp102 (-C=O: 2.42Å, -OH: 2.10Å) and Lys140 (2.01Å). The interactions with the two guanidine groups are through the aspartates. This is very interesting because these kinds of interactions could differentiate one kinase from another. Figure 6E shows the interactions in the hinge zone of the compounds 11, 12, 17, 18, 19, 20 and 23–53. This series of compounds have three H-bonds in the hinge zone with the residues Glu93 and Val95. The most active compound from this series is 52 (pIC50: 1.638). It has interactions with residues Glu93 (2.21Å) and Val95 (-CO: 2.33Å, -NH: 1.85Å), see Table 7. In this series there are two –CO H bond acceptors and one -NH H bond acceptor. Figure 6F shows the interactions in the hinge zone of the compounds 13 and 22; these compounds have two interactions in the hinge zone with the residue Val95 (-CO: 2.39Å, -NH: 2.42Å for 13 and -CO: 2.27Å, -NH: 1.90Å for 22), see Table 7. The most active compound is 22 (pIC50: 1.187). Finally, Figure 6G shows the interactions in the hinge zone of the compounds 14, 15, 16, 21 and 54–57. This series of compounds also has three interactions in the hinge zone with the residues Glu93 and Val95. The most active compound in this series is 57 (pIC50: 1.398), see Table 5.\n\nTaking in account the results from Table 7, the key factor to the stabilization of the active site of these ligands is one or two –CO (H bond acceptor) and one -NH (H bond acceptor) groups in this zone.\n\nFigure 7 shows the atomic charges calculated with the CHelpG population analysis (upper left) and the electrostatic potential (upper right and down) of compound 5 in some areas of special interest. The net charges obtained for H11, O13, H21 and H22 are consistent with the formation of hydrogen bonds shown in Figure 6A. The electrostatic potential of surfaces shown in Figure 7 also justify the formation of hydrogen bonds with Glu93 and Val95.\n\nFigure 8 shows the atomic charges calculated by CHelpG population analysis (left) and the electrostatic potential (right) of compound 6 in some areas of special interest. The net charges obtained for H40 and O41 are consistent with the formation of hydrogen bonds shown in Figure 6B. The electrostatic potential of surfaces shown in Figure 8 also justify the formation of hydrogen bonds with Val95.\n\nFigure 9 shows the atomic charges calculated by CHelpG population analysis (left) and the electrostatic potential (right) of compound 8 in some areas of special interest. The net charges obtained for N9 and H13 are consistent with the formation of hydrogen bonds shown in Figure 6C. The electrostatic potential of surfaces shown in Figure 9 also justify the formation of hydrogen bonds with Val95.\n\nFigure 10 shows the atomic charges calculated by CHelpG population analysis (left) and the electrostatic potential (upper right and lower) of compound 10 in some areas of special interest. The net charges obtained for O29, N31, N16, H64 and H76 are consistent with the formation of hydrogen bonds shown in Figure 6D. The electrostatic potential of surfaces shown in Figure 10 also justify the formation of hydrogen bonds with Val95 (O29 and N31), Lys140 (N16) and Asp102 (H64 and H76).\n\nFigure 11 shows the electrostatic potential generated by compound 13 in some areas of special interest. The electrostatic potential of surfaces shown in Figure 11 are consistent with the interactions shown in Figure 6F and justify the formation of hydrogen bonds of H34 and N36 with Val95.\n\nFigure 12 shows the atomic charges calculated with the CHelpG population analysis (left) and the electrostatic potential (right) of compound 15. The net charges obtained for H31, H38 and N39 are consistent with the formation of hydrogen bonds shown in Figure 3G. The electrostatic potential of surfaces shown in Figure 12 also justify the formation of hydrogen bonds with Glu93 (H38) and Val95 (H31 and N39).\n\nThe Pkn G inhibitors from Table 6 have a common structure and three interactions in the hinge zone with the residues Glu233 and Val 235 (see Figure 13).\n\nThe most active compound of this series is 66 (pIC50: 2.000). This compound has interactions with the residue Glu93 (2.59Å) and Val95 (-CO: 2.24Å, -NH: 2.17Å), see Table 8. Other compounds with good reactivity are 62, 63C1 and 63C2 (pIC50: 1.699). These compounds have interactions with the residues Glu93 (2.18Å), Val95 (-CO: 2.56Å, -NH:1.78 Å); Glu93 (2.36Å), Val95 (-CO: 2.18Å, -NH:1.90 Å) and Glu93 (2.10Å), Val95 (-CO: 2.22Å, -NH:1.89 Å), respectively. In these ligands the key factors are two –CO (H bond acceptor) and one -NH (H bond acceptor) groups for stabilizing the ATP active site.\n\naConformations with respect to the methyl in the cyclopropyl group.\n\nFigure 14 shows the atomic charges calculated by CHelpG population analysis (left) and the electrostatic potential (right) of compound 58. The net charges obtained for H16, O11 and H14 are consistent with the formation of the hydrogen bonds shown in Figure 13. The electrostatic potential of surfaces shown in Figure 14 also justify the formation of hydrogen bonds with Glu233 (H16) and Val235 (O11 and H14).\n\nThe MQS analysis was conducted to understand the correlation between the ATP ligands associated to each kinase studied and the stabilization process in the active site. This correlation allows postulation of the structural models of the ATP complexes.\n\nFor the Pkn A ligands (see Figure 15), the correlation between the steric and electronic effects is R2 =0,9487 using the coulomb and overlap operators to determine electronic and steric effects, respectively. This correlation shows the nature of the stabilization of these ligands on the active site and its non-covalent character.\n\nFigure 16 shows the correlation of Pkn B ligands (see Table 2–Table 5). This correlation shows the electronic and steric effects along the ligand set associated with the active site. The high values in the Carbó indexes give an idea about the ATP complex generated and stabilized.\n\nIn Figure 17 is shown the correlation between the steric and electronic effects associated with the ATP complex of Pkn G (see Table 6). This complex has a correlation of R2=0,7357. In this complex, the steric and electronic effects also are very relevant and the stabilization in the active site has a high non-covalent character.\n\nIn Figure 15–Figure 17 a strong correlation between the two operators used to determine electronic and steric effects is shown. These effects can be related to the stabilization process and non-covalent interactions. In this way, the quantum similarity analysis allows us study the ATP complex and understand the steric and electronic relationship along the ligand set.\n\n\nConclusions\n\nInadequate dosing and incomplete treatment regimens, coupled with the ability of the tuberculosis bacilli to cause latent infections that are tolerant of currently used drugs, have fueled the rise of multi-drug resistant tuberculosis. To deal with this problem, there is currently an increased interest in kinase inhibitors with the main aim of proposing new anti-tuberculosis drugs. For these reasons, in this work we conducted a study of the crystalized STPKs Pkn A, B1, B2, B3 and G. The conformational analysis shows the DFG-in state of Pkn B, G, and DFG-out state of Pkn A. Methionine is the gatekeeper residue in all the kinases studied.\n\nTo understand the interactions of the active site of the protein kinases from Mycobacterium tuberculosis, a docking study using the protein kinases Pkn A, B and G was conducted. In these protein kinases, the C-term domain is extracellular and N-term is cytosolic. The cytosolic domain is almost the same for all the kinases and the extracellular domain is different. Ligands bind to the kinase through the formation of two to three hydrogen bonds with the backbone residues of the hinge region connecting the kinase N- and C-terminal loops. The residues with hydrogen bonds on the hinge zone of Pkn A are GLU96 and VAL 98, of Pkn B are GLU 93 and VAL 95, and finally of Pkn G are GLU233 and VAL235. From these results, it is possible to see that the interactions of the hinge zone are characterized by the key factor of one or two H-bonds acceptors and one H-bond donor in the ligands of this zone. On the other hand, the DFT calculations performed with some of the most representative compounds have supported the previous conclusions. Finally, the quantum similarity analysis shows good correlation between the steric and electronic effects in each ATP complex. In this sense, the outcomes presented in this work can be used to develop new protein substrates of the STPKs and find new tuberculosis treatments.\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\nThanks to Fundación Universitaria Tecnológico Comfenalco (Cartagena, Colombia) for their support of this work. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "78802", "date": "24 Feb 2021", "name": "Hugo Fraga", "expertise": [ "Reviewer Expertise Tuberculosis", "Drug Development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough the scientific question is interesting - I don’t understand how the question stated in the introduction: “what are the protein substrates of the STPKs?” can be directly answered by studying ATP competitive inhibitors. We already know ATP is a binder - what we don’t know are the proteins that are targets for the kinases. Perhaps, by having specific inhibitors that would help in the identification of the targets but this is not stated in the introduction. Nevertheless, this does not reduce the interest of the study per se – to study the binding surface of putative kinase inhibitors is itself relevant. While this is a computational study, and the methodology and results look sound, I feel that some experimental data would be required to validate the exercise. It would be interesting to test if the residues identified – by mutating etc – do influence the IC50 of the compounds tested – but don’t know how easy is to express the proteins and if the researchers have the know-how to perform such a task.\nSomething is missing in the sentence: “The compounds to Pkn B have groups with electron withdraw- ing resonance effects, such as carbonyl, hydroxyl, amine and methoxy groups, with big rings central to its stabilization on the active site.”\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": "6383", "date": "24 Feb 2021", "name": "Ph. D. Alejandro Morales-Bayuelo", "role": "Author Response", "response": "Ok, thank you very much by the sugestions. PKs (or PK domains within a larger protein) are generally made up of two lobes separated by a large cavity capable of including the substrate and a smaller cavity capable of including Adenosine TriphosPhate (ATP). The N-terminal lobe is mainly made up of β sheets and a conserved helix called the “C-helix”. The C-terminal lobe is larger and is made up mostly of α-helices. In general, molecules that are designed with the intention of inhibiting PKs compete with ATP. For this reason, the inhibitors with higher activity has been selected inthis work for the PKs selected. It has been reported that there is a relationship between the characteristics found at the ATP binding site and the pharmacology associated with this site. In the current work, a characterization of the 3D structures of the PKs involved in tuberculosis was made. To study these protein kinases, the components that make up the ATP binding site were also taken into account, such as the hinge zone residues, gatekeeper residue, presence or absence of the domain (DFG) and position of the glutamate residue conserved in the C-helix. On the other hand, For the future, the experimental studies to extrapolate the conclusions are being consolidated. However, for the moment this study uses the experimental data of the activities and performs computational analyzes. Best regards __________________________________ Alejandro Morales-Bayuelo. Chemist. Molecular Physical Chemistry Ph. D. Molecular Physical-Chemistry Post-doctoral Molecular Biophysics Post-doctoral Academic Researcher. Cartagena de Indias, Colombia." } ] } ]	1	https://f1000research.com/articles/10-66
https://f1000research.com/articles/10-62/v1	02 Feb 21	{ "type": "Case Report", "title": "Case Report: Voriconazole induced refractory hyperkalemia and adrenal insufficiency", "authors": [ "Ezza Fatima Tariq", "Yusra Irshad", "Usman A. Khan", "Muhammad Shoaib", "Hajra Asif", "Yusra Irshad", "Usman A. Khan", "Muhammad Shoaib", "Hajra Asif" ], "abstract": "Voriconazole, an azole drug, inhibits cytochrome P450 dependent lanosterol 14-alpha-demethylase. It is a potent anti-fungal agent. Adverse effects include neurotoxicity, cardiac arrhythmias, electrolyte disturbances and adrenal insufficiency. Hyperkalemia is a rare adverse effect that has been described, but is not well reported, in the literature. We present a case of intractable hyperkalemia resulting from voriconazole use. A 47-year-old male presented with worsening shortness of breath, requiring mechanical ventilation within 24 hours of presentation. Diagnosis of acute respiratory distress syndrome was made. Empirical treatment with broad-spectrum antibacterial coverage resulted in no improvement. Voriconazole was added for anti-fungal coverage, causing treatment resistant hyperkalemia necessitating continuous renal replacement therapy (CRRT). Renal function remained normal. Stopping voriconazole restored normal potassium levels. CRRT was discontinued. Hyperkalemia caused by voriconazole occurs in less than 2% of cases. It is a potentially life-threatening side effect. Physicians should be aware of this association and seek alternative treatment when necessary.", "keywords": [ "Voriconazole", "Hyperkalemia. Adrenal Insufficiency", "Fungal infection", "Adverse effects." ], "content": "Introduction\n\nVoriconazole is a second-generation azole anti-fungal drug. One side effect of Voriconazole is interference with CYP 450 as it is a potent inhibitor of CYP 450.This can cause interactions with other drugs. Other adverse effects documented, include hormonal, cardiac, neurotoxic effects, and electrolyte imbalance1,2. In our literature review we found a case report of adrenal insufficiency due to concomitant use of voriconazole and glucocorticoids3, a case series of hyponatremia associated with voriconazole use2 and a case of iatrogenic Cushing syndrome associated with concomitant use of voriconazole and budesonide4. Hyperkalemia, in the light of the literature review is a rare side effect associated with voriconazole. Our case is noteworthy as in our patient the adrenal insufficiency and hyperkalemia manifested in absence of use of any other accompanying drug that could cause hyperkalemia. We present a case of hyperkalemia caused by voriconazole in a hospitalized patient. The likely cause of hyperkalemia in our patient is adrenal deficiency due to inhibition of adrenal enzymes.\n\n\nCase presentation\n\nA 47-year-old white male, retired trailer driver by occupation, with known Crohn’s disease and hypertension presented with productive cough and worsening shortness of breath over three weeks in August 2020. On physical exam, patient was febrile with a temperature of 100-degree F, slightly tachypneic, with a respiratory rate of 24 per minute; blood pressure was 96/89 and he had right lower zone bronchial breathing on auscultation. The patient was admitted and initially started on piperacillin-tazobactam 4.5 gm. IV q6 hr, azithromycin 500 mg IV daily, vancomycin 1gm IV BID, and oseltamivir 75 mg BID. However, his respiratory status worsened over the first 48 hours and required transfer to the intensive care unit on the second day of hospitalization with septic shock and acute respiratory distress syndrome. The patient was intubated and mechanically ventilated and therapeutic paralysis induced. The patient was administered rocuronium IV at the dose of 8mcg/kg/min. Patient was supported by ventilator with volume assisted control. He required two vasopressors, epinephrine (0.3 mcg/kg/min) and nor-epinephrine (12 mcg/kg/min) as IV infusion to maintain his blood pressure. The patient failed to show improvement, therefore, voriconazole 240 mg IV BID was added on the fifth day of hospitalization. Following this, the patient started developing electrolyte abnormalities with rising potassium (Figure 1 and Table 1),and falling sodium (Figure 2 and Table 2). While the patient’s creatinine remaining stable (Figure 3 and Table 3).\n\nThe patient serum potassium level was noted to increase following voriconazole addition to the drug regimen. Discontinuing voriconazole shows normalization of potassium levels, establishing that voriconazole was the offending agent.\n\nHyponatremia trend shown after voriconazole administration; along with finding of hyperkalemia suggests voriconazole induced adrenal insufficiency.\n\nThe trends in creatinine levels were seen to be within normal range, reflecting adequate renal function.\n\nFor the rising potassium, immediate medical management with intravenous furosemide 80mg, rectal sodium polystyrene sulfonate 60 gm., and intravenous regular insulin 7 units were given, with 50 ml of 50% dextrose, continuous albuterol 2.5 mcg nebulization, and intravenous sodium bicarbonate 50 mEq intravenous push .However, this failed to improve the hyperkalemia. His urine output during this period was within normal range. Due to the intractable hyperkalemia, continuous renal replacement therapy (CRRT) was initiated. The patient was also started on intravenous hydrocortisone 60 mg every six hours and fludrocortisone 0.2 mg. The voriconazole was discontinued, the patient’s electrolyte abnormalities normalized and the CRRT was stopped in less than 48 hours. Fludrocortisone was discontinued in 48 hours as well. The patient continued to improve and was extubated on day 11 of hospitalization. He was discharged to a specialist nursing facility on day 14. His follow up chemistry showed Na+ levels of 139 mEq/L (Normal range: 135 mEq/L– 145 mEq/ L), K+ levels of 4 mEq/L (Normal range:3.5mEq/L–5.5mEq/L), HCO3 levels of 20 mEq/L ( Normal range: 23mEq/L – 30mEq/L) , Creatinine level of 1.3 mg/dl (Normal range: 0.7– 1.5 mg/dL).\n\n\nDiscussion\n\nAmphotericin B had been the drug of choice for mycosis up to the development of azole drugs. Azoles have the convenience of being administered orally and have a safer side effect profile compared to Amphotericin B5. Azoles are synthetic compounds and are further classified into two groups. The imidazole sub-group includes miconazole and ketoconazole. The triazole sub-group includes itraconazole, fluconazole, and voriconazole5.\n\nVoriconazole has a potent antifungal effect. Voriconazole inhibits ergosterol synthesis in the fungal cell wall by inhibiting the 14-alpha demethylase enzymes hence causing eradication of fungal infection. Voriconazole leads to many drug-drug interactions because it causes inhibition of CYP 450 enzymes. Many drugs are metabolized by CYP450 enzymes hence voriconazole causes their level to increase in blood if used concomitantly.\n\nVoriconazole is a relatively new, second-generation addition to the triazole anti-fungal family. It is both a substrate and an inhibitor of the enzymes of cytochrome P450 family, including CYPC19, CYPC9, and CYP3A4, thus leading to a number of adverse effects4.\n\nSide effects in human, caused by inhibition of CYP 450 include decreased synthesis of many adrenal and gonadal enzymes. This can lead to adverse effects including gynecomastia and adrenal insufficiency5. Voriconazole also decreases enzyme function in the kidney and liver6.\n\nOther adverse effects include liver toxicity; hormonal effects like alopecia, decreased libido, and impotence2. Electrolyte imbalances like hyponatremia2, hypokalemia, and adrenal insufficiency have been previously reported1. Certain side effects associated with voriconazole itself include neurotoxicity, cardiac arrhythmias, pancreatitis phototoxic reactions, and periostitis1.\n\nSevere adverse effects due to drug interaction of steroids2, and tacrolimus7 are noted with concomitant use of azole drugs. The azole potentiates the drug effect by CYP 450 inhibition. There have been cases of increased steroid effect causing Cushing disease due to potentiated effect and later adrenal insufficiency due to hypothalamic-pituitary-adrenal (HPA) axis suppression3,4,8. Due to inhibition of CYP 450 as a side effect of voriconazole, the metabolism of tacrolimus may be decreased, causing high levels of tacrolimus in blood which causes hyperkalemia8.\n\nOur patient presented with shortness of breath, was diagnosed with acute respiratory distress syndrome and was treated with broad-spectrum antibiotics; he was also prescribed voriconazole for antifungal coverage. As voriconazole was initiated, the patient developed hyperkalemia and hyponatremia. Creatinine was noted to be within normal limits during hospitalization. The patient was treated on the lines of adrenal insufficiency. Voriconazole, when removed, resulted in clinical improvement. Sodium and potassium levels improved, establishing that the adrenal insufficiency had been due to an adverse reaction to voriconazole. In our case, adrenal insufficiency was treated on clinical suspicion. Voriconazole was confirmed as the causative agent because removing voriconazole relieved the patient’s symptoms.\n\nVoriconazole can cause adrenal insufficiency by directly inhibiting the adrenal enzymes. In case of concomitant use of steroids, HPA suppression can be the reason for adrenal insufficiency. The diagnosis of adrenal insufficiency is made by measuring ACTH level. The cortisone challenge test helps differentiate between primary and secondary adrenal insufficiency8.\n\nOur case, has limitations as the diagnosis was made on clinical suspicion. However, in the absence of a history of steroid use, direct adrenal enzyme inhibition due to voriconazole is the most likely cause. The diagnosis is supported by the fact that removal of voriconazole resulted in an improvement in the patient’s condition.\n\n\nConclusion\n\nHyperkalemia resulting from voriconazole use is an uncommon adverse effect occurring in less than 2% of cases but it is important because of the serious and potentially life-threatening consequences. It should be kept in mind whenever initiating treatment and an alternative treatment sought if deemed necessary. Moreover, electrolyte levels should be monitored when initiating voriconazole. Our case report underscores the need for case control studies to establish this association.\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 was obtained from the patient.", "appendix": "Acknowledgements\n\nThe case has been previously reported as conference abstract at American Society of Nephrology (ASN), Kidney Week, San Diego, 2018. https://www.asnonline.org/education/kidneyweek/archives/\n\n\nReferences\n\nBenitez LL, Carver PL: Adverse Effects Associated with Long-Term Administration of Azole Antifungal Agents. Drugs. 2019; 79(8): 833–853. PubMed Abstract \| Publisher Full Text\n\nKim K, Lee S, Lee S, et al.: Voriconazole-associated severe hyponatremia. Med Mycol. 2012; 50(1): 103–105. PubMed Abstract \| Publisher Full Text\n\nDuman A, Fulco P: Adrenal Insufficiency With Voriconazole and Inhaled/Intranasal Corticosteroids: Case Report and Systematic Review. J Pharm Pract. 2016; 30(4): 459–463. PubMed Abstract \| Publisher Full Text\n\nJones W, Chastain C, Wright P: Iatrogenic Cushing Syndrome Secondary to a Probable Interaction Between Voriconazole and Budesonide. Pharmacotherapy. 2014; 34(7): e116–e119. PubMed Abstract \| Publisher Full Text\n\nWood A, Como J, Dismukes W: Oral Azole Drugs as Systemic Antifungal Therapy. N Engl J Med. 1994; 330(4): 263–272. PubMed Abstract \| Publisher Full Text\n\nOates J, Wood A, Sonino N: The Use of Ketoconazole as an Inhibitor of Steroid Production. N Engl J Med. 1987; 317(13): 812–818. PubMed Abstract \| Publisher Full Text\n\nChang H, Lee N, Ko W, et al.: Voriconazole inhibition of tacrolimus metabolism in a kidney transplant recipient with fluconazole-resistant cryptococcal meningitis. Int J Infect Dis. 2010; 14(4): e348–e350. PubMed Abstract \| Publisher Full Text\n\nAlbert S, DeLeon M, Silverberg A: Possible association between high-dose fluconazole and adrenal insufficiency in critically ill patients. Crit Care Med. 2001; 29(3): 668–670. PubMed Abstract \| Publisher Full Text" }	[ { "id": "81346", "date": "23 Mar 2021", "name": "Taseen Syed", "expertise": [ "Reviewer Expertise Gastroenterology and Transplant Hepatology" ], "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 was a pleasure reviewing this interesting case report written by Tariq, EF et. al \"Voriconazole induced refractory hyperkalemia and adrenal insufficiency\". Authors have reported a rare side effect of refractory hyperkalemia secondary to Voriconazole. It was interesting to see the downward trend of potassium levels and resolution of adrenal insufficiency after stopping Voriconazole. Such case reports are important to be published as physicians should be aware of such rare side effects from these commonly used medications in the ICU setting.\nMinor revisions:\nAuthors can take out the date when patient presented.\n\nTables and figures are essentially showing the same information. I would recommend authors use only graphs as these are showing exactly what happened with electrolyte abnormalities, and the downward trend after starting voriconazole.\n\nFollowing needs reference: \" Voriconazole has a potent antifungal effect. Voriconazole inhibits ergosterol synthesis in the fungal cell wall by inhibiting the 14-alpha demethylase enzymes hence causing eradication of fungal infection. Voriconazole leads to many drug-drug interactions because it causes inhibition of CYP 450 enzymes.\"\n\nI hope these minor revision can be done by the authors to improve the content of the case report.\nThank you.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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": "128395", "date": "28 Mar 2022", "name": "Ming-Jen Chan", "expertise": [ "Reviewer Expertise Nephrology", "onco-nephrology", "critical nephrology", "acute kidney injury", "electrolytes" ], "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 demonstrate a case of voriconazole-induced refractory hyperkalemia and adrenal insufficiency. The manuscript is well written. However, I still have some questions about the manuscript.\n\nThe patient was diagnosed by the authors with adrenal insufficiency. Please provide the diagnosis data for the patient (e.g. cortisol level, at what time in the day, and ACTH data) because the treatment of the underlying disease of Crohn's disease may require budesonide, the adrenal insufficiency may easily result from the Crohn's disease treatment related side effect. Please provide the long treatment regiment of Crohn's disease (at what dose and for how long).\n\nTables 1 to 3 and Figures 1 to 3 seem redundant. All these figures and tables may be integrated into one table or figure, marking the timing of insult of voriconazole.\n\nThe time course of voriconazole infusion with hyperkalemia is compatible. However, for the diagnosis of etiology of hyperkalemia, the workup is not yet completely presented in the manuscript. The authors should provide more data. For example, is there shifting in this patient (ex. rhabdo, hemolysis), what is the TTKG or urine fraction of excretion of potassium? Also, renin aldosterone levels had been associated with voriconazole-related hyperkalemia - is renin/ aldosterone data available in such a patient? I sincerely ask for a discussion in the manuscript. Additionally, acid-base status is required for making such a diagnosis, I suggest the authors make a table to integrate relevant data for the diagnosis etiology for hyperkalemia (including serum/urine/acid-base/endocrine data). It is difficult to convince the reader that hyperkalemia is mediated by inhibition of CYP450 if these data are not available. As previous reports have postulated inhibition of 11β-hydroxysteroid dehydrogenase1.\n\nAlso, a basic fluid status evaluation should be provided in the manuscript due to the setting of hyponatremia. Urine sodium and osmolarity could be provided if available for better determination of etiology for critically ill patients.\n\nIn the discussion, paragraphs 2-4, the authors mentioned CYP450, is there any medication the patient used during ICU admission that would cause drug-drug interaction and enhance such effect?\n\nIn the conclusion section, they describe less than 2% of voriconazole use results in hyperkalemia. Please add references and consider adding them to the discussion section. The conclusion stretching out of the discussion and case part is not recommended.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/10-62
https://f1000research.com/articles/9-1055/v1	27 Aug 20	{ "type": "Software Tool Article", "title": "VIRdb 2.0: Interactive analysis of comorbidity conditions associated with vitiligo pathogenesis using co-expression network-based approach", "authors": [ "Priyansh Srivastava", "Mehak Talwar", "Aishwarya Yadav", "Alakto Choudhary", "Sabyasachi Mohanty", "Samuel Bharti", "Priyanka Narad", "Abhishek Sengupta", "Priyansh Srivastava", "Mehak Talwar", "Aishwarya Yadav", "Alakto Choudhary", "Sabyasachi Mohanty", "Samuel Bharti" ], "abstract": "Vitiligo is a disease of mysterious origins in the context of its occurrence and pathogenesis. The autoinflammatory theory is perhaps the most widely accepted theory that discusses the occurrence of Vitiligo. The theory elaborates the clinical association of vitiligo with autoimmune disorders such as Psoriasis, Multiple Sclerosis and Rheumatoid Arthritis and Diabetes. In the present work, we discuss the comprehensive set of differentially co-expressed genes involved in the crosstalk events between Vitiligo and associated autoimmune disorders (Psoriasis, Multiple Sclerosis and Rheumatoid Arthritis). We progress our previous tool, Vitiligo Information Resource (VIRdb), and incorporate into it a compendium of Vitiligo-related multi-omics datasets and present it as VIRdb 2.0. It is available as a web-resource consisting of statistically sound and manually curated information. VIRdb 2.0 is an integrative database as its datasets are connected to KEGG, STRING, GeneCards, SwissProt, NPASS. Through the present study, we communicate the major updates and expansions in the VIRdb and deliver the new version as VIRdb 2.0. VIRdb 2.0 offers the maximum user interactivity along with ease of navigation. We envision that VIRdb 2.0 will be pertinent for the researchers and clinicians engaged in drug development for vitiligo.", "keywords": [ "Vitiligo", "Database", "Comorbidity Network", "Differential Genes", "co-expressed Genes" ], "content": "Introduction\n\nVitiligo is a long-term pigmentary disorder of ambiguous origins. It is a multifactorial disease described by a plethora of theories1. As stated in neural theory, the dysfunction of the sympathetic nervous system affects melatonin production which leads to depigmentation2. The oxidative stress hypothesis states that vitiliginous skin is the result of the overproduction of reactive oxygen species such as H2O2 leading to the demolition of melanocytes that results in depigmentation3. The zinc-α2-glycoprotein (ZAG) deficiency hypothesis exploits the integral role of ZAG, which acts as a keratinocyte derived factor responsible for rapid melanocyte production4. Absence of ZAG results in the detachment of melanocytes from epidermis which leads to vitiligo4. Chronic hepatitis C virus and autoimmune hepatitis have strong union with vitiligo and account for viral origins5. According to Intrinsic theory, melanocytes may have natural defects such as abnormal rough endoplasmic reticulum or deficiency of growth factors which could lead to melanocyte apoptosis6. The Autoinflammatory theory is the most widely accepted causation supported by strong evidence7. The hypothesis is mainly based on the clinical association of vitiligo with several other autoimmune disorders like psoriasis (PS), multiple sclerosis (MS) or rheumatoid arthritis (RA)8–11.\n\nVitiligo Information Resource (VIRdb) is home to statistically significant and manually curated information about vitiligo12. In our previous work, we integrated drug-target and systems-based approaches to generate a comprehensive resource for vitiligo-omics. Data stored in VIRdb is linked with major public databases making it a cross-functional database12–17. It is an encyclopedic resource consisting of 129 differentially expressed genes (DEGs) in different phenotypes of vitiligo12. It also holds 40 curated protein targets along with their natural ligands that are derived through virtual screening12. In the present work, we aim to provide a comprehensive set of DEGs involved in the crosstalk events of vitiligo and associated autoimmune disorders (PS, MS, RA). We further investigated the interactions of the gene through comorbidity gene-gene interaction network (GGI). All data have been synthesized using standardized differential-expression pipelines. The data presented in the present study have been integrated on the VIRdb along with the previously published datasets. The paper also discusses the major updates and expansions in the VIRdb and delivers the new version as VIRdb 2.018.\n\n\nResults\n\nThe expression set for each disease (vitiligo, MA, RA and PS) constituted expression values of 27,338 probe IDs that belong to the Affymetrix GPL570 platform. After differential expression analysis, 834 differential genes were expressed in vitiligo (Lesional, Peri-lesional and Non-lesional together) out of which 639 genes were over-expressed and 195 genes were under-expressed (Extended data, Appendix-I). In RA samples a total of 938 genes were expressed, out of which 422 genes were over-expressed and 516 genes were under-expressed in the diseased condition (Extended data, Appendix-I). For MS 1783 differentially expressed genes were filtered in which 710 genes were over-expressed and 1073 genes were under-expressed (Extended data, Appendix-I). In PS, 4016 differentially expressed genes were filtered out in which 2088 were over-expressed and 1928 were under-expressed (Extended data, Appendix-I).\n\nPearson’s correlation analysis on DEGs (vitiligo) produced 397 pairs that were positively correlated with each other (Extended data, Appendix-II19). Interestingly, we found none of the gene-pairs to be under-expressed in vitiligo except the FKBP5-CUL7. In PS there were a total of 1089 positively correlated pairs out of which the 45 pairs were showing under-expression and 1044 pairs were showing over-expression (Extended data, Appendix-II19). A skewness towards over-expressed pairs can be seen in the pairs positively correlated with PS. Pearson’s correlation testing of MS-DEGs generated 767 positively correlated pairs, with 623 under-expressed pairs and 144 over-expressed pairs (Extended data, Appendix-II19). Testing of RA-DEGs produced the least number of positively correlated pairs, i.e. 411, in which 301 pairs were under-expressed and 110 pairs were over-expressed (Extended data, Appendix-II19). It showed skewness towards the under-expressed pairs.\n\nPS shared 26 differentially expressed (positive co-expressed) genes with vitiligo. In total, 23 differentially expressed genes showed similar expression (Over-expression) both in the vitiligo and PS (Figure 1a). However, ZNF395, INTS6 and BBX showed an opposite expression in PS (Figure 1a). MS and vitiligo shared 37 differentially expressed (positive co-expressed) genes, all of which showed contradictory expressions except SPEN, NEAT1, MIR612, MALT1 and KMT2A (similar expression) (Figure 1b). RA shared 24 differentially expressed (positive co-expressed) genes with the vitiligo and showed randomness in the expression (Figure 1c).\n\n(a) Positively correlated DEGs common between vitiligo and PS; (b) Positively correlated DEGs common between vitiligo and MS; (c) Positively correlated DEGs common between vitiligo and RA.\n\nThe GGI network of the intersecting positively correlated DEGs of vitiligo and PS holds 38 nodes with 93 edges denoting the co-expression, physical interactions and pathway relationships mined from literature (Extended data, Appendix-IV19) (Figure 2a). GeneMania’s algorithm also fetched 12 intermediary genes to connect the shared 26 differentially expressed (positive co-expressed) genes (Extended data, Appendix-IV19). the network for vitiligo and MS holds 51 nodes and 219 edges mined from literature, having 14 intermediary genes (Extended data, Appendix-IV19) (Figure 2b). The GGI network of RA and vitiligo constituted 33 nodes and 470 edges, out of which 9 nodes (genes) were intermediary genes that were fetched by GeneMania’s algorithm (Extended data, Appendix-IV19) (Figure 2c).\n\n(a) GGI Network for common positively correlated DEGs between vitiligo and PS. (b) GGI Network for common positively correlated DEGs between vitiligo and MS. (c) GGI Network for common positively correlated DEGs between vitiligo and RA.\n\nMechanical annotation of the genes from the GGI network of vitiligo and PS shows the highest expressivity in chromosome 14. Chromosome 18-21 doesn’t express at all (Extended data, Appendix-V19) (Figure 3). Genes of the GGI network of vitiligo and MS were expressed mainly on chromosomes 1,7 and 12 (Extended data, Appendix-V19) (Figure 3). Nodes from the GGI network of vitiligo and RA showed no expression through chromosomes 2, 8-10, 15, 16, 18, 21 and 22 (Extended data, Appendix-V19) (Figure 3). Expression was not seen through chromosome Y in any case (Extended data, Appendix-V19) (Figure 3). Intersecting genes of the GGI network of vitiligo and PS were mostly (23/36) enrichment with “Cellular protein metabolic process” as a biological process (Extended data, Appendix-VI19). MS and vitiligo network was enriched in “Regulation of innate immune response” (8/50) as a biological process (Extended data, Appendix-VI19). “RNA binding” (24/32) was enriched as the biological process for the nodes of the RA and vitiligo network (Extended data, Appendix-VI19).\n\nRed bars show the distribution of the intersecting DEGs between vitiligo and MS. Green bars show the distribution of the intersecting DEGs between vitiligo and PS. Blue bars show the distribution of the intersecting DEGs between vitiligo and RA.\n\nVIRdb 2.018 offers an engaging user-interface along with interactive visualizations. Data descriptors have been added to the browsing interface for effortless navigation through the data (Figure 4a)12. The JSmol visualizer of the protein profiles has been optimised for quick visualizations (Figure 4b)12. The profiles have cross-connectivity to other databases through their respective Accession IDs. The user can now visualize the protein structure in various styles (i.e. cartoon, ribbons, etc.) using JSmol options. The downloadable structure has been already prepared for molecular docking procedures (i.e. removed the water, ions, chargers, ligands and minimized with OPLS 2005) (Figure 4b)12. The natural leads section has been reduced to the top 50 computational hits against the protein targets (Figure 4c)12. The user can browse through fewer compounds before setting up wet lab experimentations (Figure 4c)12. Intersecting positively co-expressed DEGs between vitiligo and other conditions (PS, RA and MS) is the new addition to the database (Figure 5b). The section consists of four columns which are connected to GeneCards via gene symbols. The section holds expression status (Overexpression/Underexpression) of the positively co-expressed DEGs between vitiligo and associated conditions (Figure 5b). The GGI networks which are made using the shared positively co-expressed DEGs between vitiligo and other conditions can be viewed in the network gallery section (Figure 5c). The networks are highly interactive and can be simulated with mouse clicks.\n\n(a) The browsing section has been redesigned with descriptors for easy navigation through the database. (b) JSmol has been optimized for various visualization styles which can be enabled using right-click. (c) The Natural lead section has been reduced to the top entries based on the significant Glide-Scores.\n\n(a) Revamped home page of the VIRdb 2.0 with all the section tabs for easier navigation; (b) Intersection section displays the positively co-expressed DEGs across PS, vitiligo, MS and RA. Gene symbols are cross-connected with GeneCards db. (c) Network gallery section with D3-force layout. It offers a highly interactive network visualization of the comorbidity networks.\n\nNetwork Gallery and Intersecting Gene sections have been added with cross-connectivity and D3 force-directed graph visuals.\n\n\nMethods\n\nRaw datasets from the microarray studies for RA (GSE56649), PS (GSE14905), MS (GSE21942), and vitiligo (GSE65127) were downloaded from the Gene Expression Omnibus (GEO)20. To maintain the uniformity of the downstream analysis, experiments with Affymetrix GPL570 platform were taken into consideration. Expression values were extracted from the raw CEL files using the affy (version 1.66.0) library in R version 3.621. Expression datasets were normalized using the robust multichip averaging method for background corrections22. After the normalization, the IQR method was used with a standard cut-off of 0.5 to remove the low expression values from the datasets using genefilter (version 1.70.0) library23. Each dataset was divided into two groups based on the phenotype of the samples, i.e. “Experiment” (disease phenotypes) and “Control” (healthy phenotypes). Specifically, for vitiligo expression set, “Experiment” groups constituted of lesional, peri-lesional and non-lesional vitiligo samples together. Scripts used for computational analysis of data generated through microarray experiments are available on Github: https://github.com/pnarad/Micro-Array-Data-Analysis24.\n\nStandard linear models’ library limma (version 3.44.3) was used to perform the differential expression analysis25. The t-test was performed with each gene expression value to examine variations across groups (“Experiment” vs “Control”). Benjamini Hochberg's false discovery rate was computed to filter the significant multiple testings26. P-value cut-off of 0.05 and adjusted-P-value (FDR) cut-off of 0.03 was used to filter the significant test results. Log of fold change (LogFC) across groups was calculated alongside with standard “limma” functions to explore the expression status of the differentially expressed probes ids. Probe Ids with LogFC < -0.5 were annotated as underexpressed and those with LogFC > 0.5 were annotated as overexpressed in “Experiment” samples and the remaining probe ids were dropped (i.e. -0.5 < LogFC < 0.5).\n\nThe respective feature dataset that contains the gene symbol mapping for the probe id fetched from the GEO using “GEOquery”27. The differentially expressed probe ids were mapped to their respective gene symbols programmatically28. The gene symbols that were mapped to multiple probe ids with different expression status (i.e. ambiguity in over/under-expression) were removed using dplyr (version 1.0.1) methods29. The gene symbols which were mapped on multiple probe ids with uniform expression (i.e. uniformity in over/under-expression) were averaged over the calculated parameters (i.e. P-value, adjusted P-value, average expression etc.). The final datasets constituted unique differentially expressed genes in “Experimental” samples of all the four diseases.\n\nThe correlated pairs (similar expressions across the samples) were formed within the disease groups (i.e. MS, RA, PS and vitiligo) individually. Initially, the differentially expressed gene symbols were reverse mapped to their respective probe ids for extraction of expression values. Pearson’s correlation test was performed to compute the correlation coefficient. The correlation matrix was filtered for correlations having a correlation coefficient > ±0.9. The probe IDs were re-mapped to the gene symbols along with LogFC. The negatively correlated pairs were removed using the LogFC (i.e. different expression status of gene in a pair), and only positive correlated differential pairs were taken for further analysis30. Cytoscape (version 3.8) was used to create four individual co-expression networks using the differentially expressed and positively correlated gene pairs31. The co-expression networks for MS, RA and PS were individually intersected with the co-expression network of vitiligo for evaluation of commonly expressed genes across diseases.\n\nThe intersecting genes were fed to GeneMania for edge-reconstruction of the comorbidity gene interaction networks (i.e. vitiligo with RA, vitiligo with MS and vitiligo with PS)32. The intersecting gene symbols were joined using annotated edges of GeneMania algorithm. We dropped “predicted” edges and selected the rest of the edges which were sourced from the literature. The networks were examined using Cytoscape and the largest connected subnetworks were isolated.\n\nThe gene symbols from each dataset were fed to the g:Profiler server for gene ontologies33. The Benjamini–Hochberg false discovery rate of 0.03 was chosen with an annotated domain scope for enrichment analysis. Finally, network nodes were annotated using the biomaRt (version 2.44.1) data mining tool for chromosome number, start base-pair and stop base-pairs34. The three comorbidity network datasets were mapped with their respective LogFC for meaningful insights\n\nThe previous schema of the database was updated with the new addition of Network Gallery and Intersecting Gene’s sections (Figure 3). Bootstrap objects were added for styling along with HTML 5 and CSS in the front-end stack. JavaScript was added for dynamic visualizations of JSmol viewer and D3 force-directed networks based on Verlet integration35–37.\n\nThe VIRdb 2.018 is available online as an open-source database. The user can access the database through any platform (Linux, Mac OS, or Windows). There are no specific system requirements to browse the database. The user can browse through different sections using the easy to use interface.\n\nThe database is implemented in various sections. Intersection section displays the positively co-expressed DEGs across PS, vitiligo, MS, and RA. Gene symbols are cross connected with GeneCards db. Network gallery section of the database offers a highly interactive network visualization of the comorbidity networks. The JSmol viewer has been optimized for various visualization styles which can be enabled using right-click. The user can visualize the structure in various styles such as cartoons, ribbons etc. The Natural lead section has been reduced to the top entries based on the significant Glide-Scores.\n\n\nDiscussion\n\nThe present study discusses the comorbidities of the vitiligo with known associative disorders based on auto-inflammatory theory8–11. With the aid of statistical procedures, we found significant DEGs that show correlated expressions in vitiligo, PS, MS and RA. ZNF395 which activates a subset of ISGs including the chemokines CXCL10 and CXCL11 in keratinocytes was co-expressed between the PS and vitiligo38. CXCL10 and CXCL11, are expressed in skin keratinocytes and are involved in the development of proinflammatory skin diseases such as vitiligo38. ZNF395 was over-expressed in our dataset of positively co-expressed DEGs from vitiligo samples and was under-expressed in samples of PS (Extended data, Appendix-III19).ZNF395 has direct associations with Huntington’s disease and might be a crucial biomarker in vitiligo-PS auto-immune progression as well39. CSNK1A1, which was expressed in all the conditions showed under-expression in MS and therefore could be used as a biomarker for MS (Extended data, Appendix-III19). It is also interesting to see that most of the shared DEGs in PS and RA show uniform expression like vitiligo. However, the expressions of DEGs shared with MS show contradictory expressions, which points towards a weaker association between vitiligo and MS. The presented comorbidity networks hold many new DEGs which are shared among the auto-immune diseases. These DEGs can be explored for their cross-talks events as supported by auto-inflammatory theory7.\n\nVIRdb 2.018 integrates statistically significant DEGs that could be responsible for crosstalk events between vitiligo, PS, MS and RA. VIRdb 2.0 incorporates all the attributes of the previous version and projects them in a more user-friendly database. The intersecting DEGs with other diseases (PS, MS and RA) are projected with their expression status in diseases. VIRdb 2.0 also utilizes a network approach to project positively co-expressed genetic interaction networks of the intersecting differentially expressed genes. VIRdb 2.0 also offers the maximum user interactivity with the D3 layouts. The users can now visualize the GGI networks and minimized protein structs directly on the database. The visualization tools (JSmol and D3-simulator) allow switching between various poses. The datasets can be downloaded in a zipped archive with few clicks from the download section. Thus, VIRdb 2.0 will be pertinent for the researchers and clinicians engaged in drug development and genomics of vitiligo.\n\n\nConclusion\n\nWe present VIRdb 2.0, intersecting differential networks that could be responsible for cross-talks events between vitiligo, PS, MS and RA. The presented networks are designed using the common positively co-expressed DEGs in the disease. The VIRdb 2.0 inherits all the previous datasets of VIRdb and incorporates new datasets that are discussed in the present study. Future versions will include data submission capabilities and functional-omics perspectives.\n\n\nData availability\n\nFigshare: VIRdb 2.0: Interactive analysis of comorbidity conditions associated with vitiligo pathogenesis using co-expression network-based approach. https://doi.org/10.6084/m9.figshare.12776468.v119.\n\nThis project contains the following extended data:\n\nAppendix-I (Differential Genes).xlsx. (Results of the differential expression analysis.)\n\nAppendix-II (Positive Correlations).xlsx. (Results of the Pearson’s correlation testing.)\n\nAppendix-III: (Intersection results of the differential genes.)\n\nAppendix-IV (Networks).xlsx. (Network files of the GGI networks constructed through GeneMania.)\n\nAppendix-V (Annotations).xlsx. (Results of the gene annotation using the BioMart Data Mining tool.)\n\nAppendix-VI (Ontologies).xlsx. (Results of Gene Ontology enrichment analysis.)\n\nScripts used for data analysis are available from: https://github.com/pnarad/Micro-Array-Data-Analysis.\n\nArchived scripts at time of publication: https://doi.org/10.5281/zenodo.397563824.\n\nLicense: MIT License.\n\n\nSoftware availability\n\nVIRdb 2.0 is available at: https://vitiligoinfores.com/.\n\nSource code available from: https://github.com/pnarad/VIRdb.\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.397563418.\n\nVIRdb license: Creative Commons Attribution 4.0 International.\n\nSource code license: Creative Commons Zero \"No rights reserved\" data waiver.", "appendix": "References\n\nMalhotra N, Dytoc M: The Pathogenesis of Vitiligo. J Cutan Med Surg. 2013; 17(3): 153–172. PubMed Abstract \| Publisher Full Text\n\nKotb El-Sayed M, Abd El-Ghany A, Mohamed R: Neural and Endocrine Pathobiochemistry of Vitiligo: Comparative Study for a Hypothesized Mechanism. Front Endocrinol (Lausanne). 2018; 9: 197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe Y, Li S, Zhang W, et al.: Dysregulated autophagy increased melanocyte sensitivity to H2O2-induced oxidative stress in vitiligo. Sci Rep. 2017; 7: 42394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBagherani N: The Newest Hypothesis about Vitiligo: Most of the Suggested Pathogenesis of Vitiligo Can Be Attributed to Lack of One Factor, Zinc-α 2-Glycoprotein. ISRN Dermatol. 2012; 2012: 405268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman R, Hasija Y: Exploring vitiligo susceptibility and management: a brief review. Biomedical Dermatology. 2018; 2: 20. Publisher Full Text\n\nTaieb A: Intrinsic and Extrinsic Pathomechanisms in Vitiligo. Pigment Cell Res. 2000; 13(Suppl 8): 41–47. PubMed Abstract \| Publisher Full Text\n\nEzzedine K, Eleftheriadou V, Whitton M, et al.: Vitiligo. Lancet. 2015; 386(9988): 74–84. PubMed Abstract \| Publisher Full Text\n\nSchallreuter KU, Lemke R, Brandt O, et al.: Vitiligo and Other Diseases: Coexistence or True Association? Hamburg study on 321 patients. Dermatology. 1994; 188(4): 269–275. PubMed Abstract \| Publisher Full Text\n\nBirlea S, Fain P, Spritz R: A Romanian Population Isolate With High Frequency of Vitiligo and Associated Autoimmune Diseases. Arch Dermatol. 2008; 144(3): 310–316. PubMed Abstract \| Publisher Full Text\n\nDiallo A: Halo Nevi Association in Nonsegmental Vitiligo Affects Age at Onset and Depigmentation Pattern. Arch Dermatol. 2012; 148(4): 497–502. PubMed Abstract \| Publisher Full Text\n\nRamagopalan S, Dyment D, Valdar W, et al.: Autoimmune disease in families with multiple sclerosis: a population-based study. Lancet Neurol. 2007; 6(7): 604–610. PubMed Abstract \| Publisher Full Text\n\nSrivastava P, Choudhury A, Talwar M, et al.: VIRdb: a comprehensive database for interactive analysis of genes/proteins involved in the pathogenesis of vitiligo. PeerJ. 2020; 8: e9119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M: KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMering C: STRING: a database of predicted functional associations between proteins. Nucleic Acids Res. 2003; 31(1): 258–261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSafran M, Dalah I, Alexander J, et al.: GeneCards Version 3: the human gene integrator. Database (Oxford). 2010; 2010: baq020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBairoch A: The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res. 2000; 28(1): 45–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZeng X, Zhang P, He W, et al.: NPASS: natural product activity and species source database for natural product research, discovery and tool development. Nucleic Acids Res. 2017; 46(D1): D1217–D1222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSrivastava P: pnarad/Micro-Array-Data-Analysis: VIRdb: Micro-Array-Data-Analysis (Version v.2.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3975634\n\nSengupta A, Narad P: VIRdb 2.0: Interactive analysis of comorbidity conditions associated with vitiligo pathogenesis using co-expression network-based approach. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12776468.v1\n\nBarrett T, Edgar R: Gene Expression Omnibus: Microarray Data Storage, Submission, Retrieval, and Analysis. Methods Enzymol. 2006: 411: 352–369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGautier L, Cope L, Bolstad B, et al.: affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics. 2004; 20(3): 307–315. PubMed Abstract \| Publisher Full Text\n\nIrizarry R: Exploration, normalization, and summaries of high-density oligonucleotide array probe level data. Biostatistics. 2003; 4(2): 249–264. PubMed Abstract \| Publisher Full Text\n\nGentleman R, Carey V, Huber W, et al.: Genefilter: methods for filtering genes from high-throughput experiments. 2015; 1(1).\n\nBharti S: pnarad/VIRdb: Source code of the database:VIRdb (Version v.2.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3975638\n\nRitchie M, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen S, Feng Z, Yi X: A general introduction to adjustment for multiple comparisons. J Thorac Dis. 2017; 9(6): 1725–1729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavis S, Meltzer PS: GEOquery: a bridge between the Gene Expression Omnibus (GEO) and BioConductor. Bioinformatics. 2007; 23(14): 1846–1847. PubMed Abstract \| Publisher Full Text\n\nIntroduction to data.table. Cran.r-project.org. Published in 2020. Accessed July 10, 2020. Reference Source\n\nIntroduction to dplyr. Cran.r-project.org. Accessed July 10, 2020. Reference Source\n\nvan Dam S, Võsa U, van der Graaf A, et al.: Gene co-expression analysis for functional classification and gene-disease predictions. Brief Bioinform. 2018; 19(4): 575–592. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMostafavi S, Ray D, Warde-Farley D, et al.: GeneMANIA: a real-time multiple association network integration algorithm for predicting gene function. Genome Biol. 2008; 9 Suppl 1(Suppl 1): S4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaudvere U, Kolberg L, Kuzmin I, et al.: g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019; 47(W1): W191–W198. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmedley D, Haider S, Durinck S, et al.: The BioMart community portal: an innovative alternative to large, centralized data repositories. Nucleic Acids Res. 2015; 43(W1): W589–W598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHerráez A: Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006; 34(4): 255–261. PubMed Abstract \| Publisher Full Text\n\nBostock M: Force-Directed Graph. Observablehq.com. Accessed July 18, 2020. Reference Source\n\nRozmanov D, Kusalik PG: Robust rotational-velocity-Verlet integration methods. Phys Rev E Stat Nonlin Soft Matter Phys. 2010; 81(5 Pt 2): 056706. PubMed Abstract \| Publisher Full Text\n\nSchroeder L, Herwartz C, Jordanovski D, et al.: ZNF395 Is an Activator of a Subset of IFN-Stimulated Genes. Mediators Inflamm. 2017; 2017: 1248201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNopoulos PC: Huntington disease: a single-gene degenerative disorder of the striatum. Dialogues Clin Neurosci. 2016; 18(1): 91–98. PubMed Abstract \| Free Full Text" }	[ { "id": "70478", "date": "21 Sep 2020", "name": "Dinesh Gupta", "expertise": [ "Reviewer Expertise Translational Bioinformatics", "Systems biology", "database", "and algorithm 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\nVIRdb2.0 is an integrative database for vitiligo research, integrating it's links to Psoriasis, Multiple Sclerosis, Rheumatoid Arthritis, and Diabetes. The database release 2.0 is developed with additional features over the previous version. Summarily, the database is a collection of differentially co-expressed genes involved in the crosstalks with Vitiligo and associated autoimmune disorders. This looks like a good resource for researchers interested in Vitiligo research. Few concerns and suggestions, which the authors should address:\nThe manuscript should illustrate a few specific examples to demonstrate the utility of the database, apart from showing the overall capabilities.\n\nThe database site should include a tutorial for users, demonstrating the database tools and searches.\n\nA new page to be created to highlight \"what's new in this release\".\n\nIt will be nice if the authors could include gene regulatory networks too, involving the role of common TFs in the related diseases.\n\nIt will be nice if some data on the druggability of a few common hubs could be highlighted too.\n\nFew logical search options should be considered to make the database search more useful.\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": "6307", "date": "01 Feb 2021", "name": "Abhishek Sengupta", "role": "Author Response", "response": "Dear Sir, We thank you for the generous and concise comments on our manuscript. We have incorporated the suggestions and changes have been added to the manuscript text also. Q.1 The manuscript should illustrate a few specific examples to demonstrate the utility of the database, apart from showing the overall capabilities. As per the suggestion, we have incorporated a case study at the Home page of the database, which demonstrates the use of the database to construct an interaction network for the intersecting genes between Rheumatoid Arthritis and Vitiligo and further use Cytoscape tool to import and analyse the network using mCODE app as a demonstration of the utility of the database. Q.2 The database site should include a tutorial for users, demonstrating the database tools and searches. A short tutorial has been incorporated at the Home page highlighting the sections and the process of retrieval from the sections. Complete code for accessing the database is already available at GitHub. Q.3 A new page to be created to highlight \"what's new in this release\". A new page has been created to highlight \"what's new in this release\" as advised. Q.4 It will be nice if the authors could include gene regulatory networks too, involving the role of common TFs in the related diseases. A network gallery is available under the “Browse Section”. The visualization of the network of the intersecting genes with D3-Fore is available. These networks have been generated using GeneMania plugin from Cytoscape. Further, the analysis was also done to elaborate the Gene Ontology for the intersecting DEGs, the data is available in Appendix-VI. Further, we aim to build more towards the on this in our future studies. Q.5 It will be nice if some data on the druggability of a few common hubs could be highlighted too. We have incorporated ADMET studies for the compounds that we have used for the purpose of screening and the excel files are also available in the “Downloads Section” Q.6 Few logical search options should be considered to make the database search more useful. Since, this is a static database, for now, we will incorporate the logical search options in our next version of the database/tool." } ] }, { "id": "75389", "date": "04 Jan 2021", "name": "Mitesh Dwivedi", "expertise": [ "Reviewer Expertise Immunogenetics", "disease biology", "vitiligo", "autoimmunity" ], "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 presented by Srivastava et al. (2020) is a unique approach to elucidate the link between Vitiligo and associated autoimmune disorders through a set of differentially co-expressed genes involved in their pathogenesis. For this, authors have designed a valuable tool called VIRdb 2.0: an integrative database that can be utilized by researchers and clinicians for drug development for vitiligo. Authors have well explained the need and rationale for developing this new software tool. This database has been connected to KEGG, STRING, GeneCards, SwissProt, & NPASS. The different datasets can be easily downloaded by the user such as protein target information, differential expression of genes, docking results with natural compounds, and interaction network. In addition, VIRdb 2.0 also has a separate blog section where researchers can write interesting and updated informative blogs. Overall, the software seems to be user friendly, though certain information and functionalities about submission of data by other researchers working in the fields of Vitiligo and its associated diseases is lacking in this version. The below are a few suggestions and concerns which authors should consider and implement in the future version of the VIRdb 2.0. Conceptual concerns:\nThough VIRdb 2.0 has included information on certain autoimmune disorders associated with Vitiligo (i.e. Psoriasis, Multiple Sclerosis and Rheumatoid Arthritis); it is lacking a few other important autoimmune diseases which are frequently associated with vitiligo such as autoimmune thyroid disease, adult-onset type 1 diabetes mellitus, pernicious anemia, systemic lupus erythematosus, and Addison disease.1,2\n\nFurther, in the study, it is not clear which type of Vitiligo has been taken into account. It would be interesting and rather more significant if authors could integrate the different types of Vitiligo into consideration. Since the disease pathology varies between different types of Vitiligo as well as disease activity can further be considered in such DEG and gene interaction studies.\n\nThe demographic data of such patients can be linked along with the details of the type of disease, disease onset, duration of disease, VASI score, disease activity, etc. These data would be more meaningful with respect to the treatment aspect as well and can be very well correlated with the gene expression data in Vitiligo as well as associated autoimmune diseases.\n\nAnother important aspect to confirm in the present study is that: Did the data used by the authors for the differentially expressed genes in each disease were validated with respect to medication? Importantly, whether the expression data obtained from the patients differentiate who were on treatment or without treatment? Because treatment would surely affect the gene expression in these patients. Hence, it would be good if the authors could segregate and take the data accordingly.\n\nAuthors must avoid the term ‘auto-inflammatory disease’ for Vitiligo. Vitiligo is an autoimmune disease. The autoinflammatory theory mentioned here is not the correct term to use and it must be ‘autoimmune theory’. [The autoinflammatory theory is perhaps the most widely accepted theory that discusses the occurrence of Vitiligo.]\n\nTechnical and utility concerns:\nThe software should have features to generate clustered heat maps for the selected DEGs in vitiligo, psoriasis, RA and MS.\n\nThe software should also provide an option to compare DEG in lesional skin vs non-lesional skin of vitiligo patients.\n\nSimilarly, the software should provide an option to compare DEG within different types of vitiligo, for example, generalized vitiligo vs localized vitiligo.\n\nThe software should provide features to generate boxplots for the expression values for genes comparing expression between vitiligo (lessional, non-lessionals) and the different disease conditions (vitiligo, psoriasis, RA, MS).\n\nIt would be good to add features in the software to search the levels of a particular gene from the database.\n\nThe software should add the data of DEGs within particular cells/tissue and features to compare the DEG within different cell types/tissues.\n\nThe software should provide an option to carry out a meta-analysis of a particular gene in all the disease conditions (vitiligo, psoriasis, RA, MS), which would be beneficial to understand their role in the cross talk between the different disease conditions (vitiligo, psoriasis, RA, MS).\n\nThe software should have features to link the DEG to their respective signaling pathways, which will be crucial to pinpoint the role of the altered gene expression in these conditions.\n\nThe software should have an option to compare DEG of the available genes for drug treated group vs non-treated group.\n\nThe authors should provide video tutorials for the different features of the software, which will be helpful for first time users to easily navigate through the user interface.\n\nFigures in the article could be provided with good resolution. The labeling and annotation in the figures are not visualized clearly (especially in Fig. 1, 2, 4 & 5C).\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": [] }, { "id": "75274", "date": "11 Jan 2021", "name": "Animesh A Sinha", "expertise": [ "Reviewer Expertise Immunogentics" ], "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 overall well written paper comparing transcriptional datasets in vitiligo to other autoimmune disease datasets.\nBuilding on previous work, the authors further develop their software for integrative analysis.\nComments:\nIntro: the Specific goals of the study and the previous work by the authors relevant to this study should be further explained.\n\nIt is not clear that all published and available datasets for VL and the other autoimmune diseases have been incorporated into the program. A Table of all relevant studies should be provided in the manuscript or supplemental data.\n\nThe site itself would benefit from tutorials and a better search function.\n\nSome more extensive data on disease intersecting genes and functional pathways should be included.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6308", "date": "01 Feb 2021", "name": "Abhishek Sengupta", "role": "Author Response", "response": "Dear Sir, We thank you for the generous and concise comments on our manuscript. We have incorporated the suggestions and changes have been added to the manuscript text also. Q.1 Intro: the Specific goals of the study and the previous work by the authors relevant to this study should be further explained. As per the suggestion, we have incorporated a few lines about the specific goals of the study as well as the explained our previous work briefly. We have given the reference to our previously published paper to be explored in more detail. Q.2 It is not clear that all published and available datasets for VL and the other autoimmune diseases have been incorporated into the program. A Table of all relevant studies should be provided in the manuscript or supplemental data. We have mentioned in our previous paper the dataset that was used for Vitiligo and the inclusion and exclusion criterion for all the available datasets. To briefly answer your question, Gene Expression Omnibus was manually queried using “(“vitiligo” (MeSH Terms) OR vitiligo (All Fields)) AND (“humans” (MeSH Terms) OR “Homo sapiens” (Organism) OR homo sapiens (All Fields))”. The returned results were manually curated for microarray experiments having healthy controls against affected individuals only for both the VL and other autoimmune diseases that we have considered in our work. As no plausible Our primary focus was on microarray data and no plausible data was available having an adequate amount of data for analysis so other autoimmune diseases were excluded. We aim to work on more data and include more information in the further prospects of this project. Q.3 The site itself would benefit from tutorials and a better search function. A short tutorial has been incorporated at the Home page highlighting the sections and the process of retrieval from the sections. Complete code for accessing the database is already available at GitHub. A new page has been created to highlight \"what's new in this release\". Q.4 Some more extensive data on disease intersecting genes and functional pathways should be included. A network gallery is available under the “Browse Section”. The visualization of the network of the intersecting genes with D3-Fore is available. These networks have been generated using GeneMania plugin from Cytoscape. Further, the analysis was also done to elaborate the Gene Ontology for the intersecting DEGs, the data is available in Appendix-VI. Further, we aim to build more towards the on this in our future studies. ADMET studies have also been included." } ] } ]	1	https://f1000research.com/articles/9-1055

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